PDF Advances in Immunology First Edition Alt Ebook Full Chapter

Advances in immunology First Edition
Alt
Visit to download the full and correct content document:
https://textbookfull.com/product/advances-in-immunology-first-edition-alt/
More products digital (pdf, epub, mobi) instant
download maybe you interests ...
Advances in Comparative Immunology Edwin L. Cooper
https://textbookfull.com/product/advances-in-comparative-
immunology-edwin-l-cooper/
Avian Cellular Immunology First Edition Jagdev M.

Sharma
https://textbookfull.com/product/avian-cellular-immunology-first-
edition-jagdev-m-sharma/
Stem Cell Research : Hope or Hype? 1st Edition Alt
https://textbookfull.com/product/stem-cell-research-hope-or-
hype-1st-edition-alt/
Advances in Clinical Chemistry Volume 66 First Edition

Makowski
https://textbookfull.com/product/advances-in-clinical-chemistry-
volume-66-first-edition-makowski/
Advances in Clinical Chemistry Volume 68 First Edition
Makowski
https://textbookfull.com/product/advances-in-clinical-chemistry-
volume-68-first-edition-makowski/
Advances in Parasitology, Volume 91 First Edition David

Rollinson
https://textbookfull.com/product/advances-in-parasitology-
volume-91-first-edition-david-rollinson/
Advances in Groundwater Governance First Edition Karen

G. Villholth
https://textbookfull.com/product/advances-in-groundwater-
governance-first-edition-karen-g-villholth/
Tumor Immunology, 1st Edition Schreiber
https://textbookfull.com/product/tumor-immunology-1st-edition-
schreiber/
Case Studies in Veterinary Immunology 1st Edition

Laurel Gershwin (Author)
https://textbookfull.com/product/case-studies-in-veterinary-
immunology-1st-edition-laurel-gershwin-author/
ASSOCIATE EDITORS
K. Frank Austen
Harvard Medical School, Boston, Massachusetts, USA
Tasuku Honjo
Kyoto University, Kyoto, Japan
Fritz Melchers
University of Basel, Basel, Switzerland
Hidde Ploegh
Massachusetts Institute of Technology, Massachusetts, USA
Kenneth M. Murphy
Washington University, St. Louis, Missouri, USA
Academic Press is an imprint of Elsevier
225 Wyman Street, Waltham, MA 02451, USA
525 B Street, Suite 1800, San Diego, CA 92101-4495, USA
The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK
125 London Wall, London, EC2Y 5AS, UK
First edition 2016
© 2016 Elsevier Inc. All rights reserved
No part of this publication may be reproduced or transmitted in any form or by any means,
electronic or mechanical, including photocopying, recording, or any information storage and
retrieval system, without permission in writing from the publisher. Details on how to seek
permission, further information about the Publisher’s permissions policies and our
arrangements with organizations such as the Copyright Clearance Center and the Copyright
Licensing Agency, can be found at our website: www.elsevier.com/permissions.
This book and the individual contributions contained in it are protected under copyright by
the Publisher (other than as may be noted herein).
Notices
Knowledge and best practice in this field are constantly changing. As new research and
experience broaden our understanding, changes in research methods, professional practices,
or medical treatment may become necessary.
Practitioners and researchers must always rely on their own experience and knowledge in
evaluating and using any information, methods, compounds, or experiments described
herein. In using such information or methods they should be mindful of their own safety and
the safety of others, including parties for whom they have a professional responsibility.
To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors,
assume any liability for any injury and/or damage to persons or property as a matter of
products liability, negligence or otherwise, or from any use or operation of any methods,
products, instructions, or ideas contained in the material herein.
ISBN: 978-0-12-804799-6
ISSN: 0065-2776
For information on all Academic Press publications

visit our website at http://store.elsevier.com/
CONTRIBUTORS
Hisashi Arase
Department of Immunochemistry, Research Institute for Microbial Diseases, and Laboratory
of Immunochemistry, WPI Immunology Frontier Research Center, Osaka University, Suita,
Osaka, Japan
Ameya Champhekar
Department of Microbiology, Immunology, and Molecular Genetics, University of
California Los Angeles, Los Angeles, California, USA
Huai-Chia Chuang
Immunology Research Center, National Health Research Institutes, Zhunan, and Research
and Development Center for Immunology, China Medical University, Taichung,
Taiwan, ROC
Marie-Dominique Filippi
Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s
Research Foundation, and University of Cincinnati College of Medicine, Cincinnati,
Ohio, USA
Hidetoshi Inoko
GenoDive Pharma Inc., Atsugi, Kanagawa, Japan
Alain Lamarre
Immunovirology Laboratory, Institut national de la recherche scientifique (INRS), INRS-
Institut Armand-Frappier, Laval, Quebec, Canada
Pascal Lapierre
Immunovirology Laboratory, Institut national de la recherche scientifique (INRS), INRS-
Institut Armand-Frappier, Laval, Quebec, Canada
Satoko Morishima
Division of Hematology, Fujita Health University School of Medicine, Toyoake, Aichi,
Japan
Yasuo Morishima
Division of Epidemiology and Prevention, Aiichi Cancer Center Research Institute,
Chikusa-ku, Nagoya, Japan
Armstrong Murira
Immunovirology Laboratory, Institut national de la recherche scientifique (INRS),
INRS-Institut Armand-Frappier, Laval, Quebec, Canada
Ellen V. Rothenberg
Division of Biology & Biological Engineering, California Institute of Technology, Pasadena,
California, USA
Takehiko Sasazuki
Institute for Advanced Study, Kyushu University, Higashi-ku, Fukuoka, Japan
#
Advances in Immunology, Volume 129 2016 Elsevier Inc. ix
ISSN 0065-2776 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-804799-6.09986-X
x Contributors
Luis J. Sigal
Thomas Jefferson University, Department of Microbiology and Immunology, Philadelphia,
Pennsylvania, USA
Tse-Hua Tan
Immunology Research Center, National Health Research Institutes, Zhunan; Research and
Development Center for Immunology, China Medical University, Taichung, Taiwan,
ROC, and Department of Pathology & Immunology, Baylor College of Medicine, Houston,
Texas, USA
Jonas Ungerbäck
Division of Biology & Biological Engineering, California Institute of Technology, Pasadena,
California, USA, and Department of Clinical and Experimental Medicine, Experimental
Hematopoiesis Unit, Faculty of Health Sciences, Link€oping University, Link€oping, Sweden
Xiaohong Wang
Department of Pathology & Immunology, Baylor College of Medicine, Houston, Texas,
USA
CHAPTER ONE
Rheumatoid Rescue of Misfolded

Cellular Proteins by MHC Class II
Molecules: A New Hypothesis for
Autoimmune Diseases
Hisashi Arase*,†,1
*Department of Immunochemistry, Research Institute for Microbial Diseases, Osaka University, Suita,
Osaka, Japan
†
Laboratory of Immunochemistry, WPI Immunology Frontier Research Center, Osaka University, Suita,
Osaka, Japan
1
Corresponding author: e-mail address: arase@biken.osaka-u.ac.jp
Contents
1. Introduction 2
2. Transport of ER-Misfolded Proteins to the Cell Surface by MHC Class II Molecules 3
2.1 MHC Class II Molecules Induce Cell Surface Expression of Misfolded MHC Class I
Molecules 3
2.2 MHC Class II Molecules Function as a Molecular Chaperon to Transport
Misfolded Cellular Protein to the Cell Surface 5
3. Function of Protein Antigens Presented by MHC Class II Molecules 6
3.1 MHC Class II Molecules Present Protein Antigens to B Cells 6
3.2 Misfolded Cellular Proteins Rescued from Protein Degradation by MHC Class II
Molecules Might Be Pathogenic 6
3.3 Aberrant MHC Class II Expression on Autoimmune-Diseased Tissues 7
4. Misfolded Proteins Presented on MHC Class II Molecules Are Targets for
Autoantibodies in Autoimmune Diseases 9
4.1 IgG Heavy Chain Presented on MHC Class II Molecules Is a Specific Target for
Autoantibodies in RA 9
4.2 β2-Glycoprotein I Associated with MHC Class II Molecules Is a Specific Target
for Autoantibodies in Antiphospholipid Syndrome 10
5. Susceptibility to Autoimmune Diseases Is Associated with the Affinity of Misfolded
Proteins for MHC Class II Molecules 11
5.1 MHC Class II Alleles and Autoimmune Disease Susceptibility 11
5.2 Autoantibody Binding to Misfolded Protein/MHC Class II Complex Is
Associated with Autoimmune Disease Susceptibility 12
6. Involvement of Misfolded Protein–MHC Class II Molecule Complexes in
Autoantibody-Mediated Pathogenicity 12
6.1 Pathogenesis of Autoantibodies in RA and APS 12
6.2 B Cell Removal Are Effective Treatment for Autoimmune Diseases 13
Advances in Immunology, Volume 129 # 2016 Elsevier Inc. 1

http://dx.doi.org/10.1016/bs.ai.2015.09.005
2 Hisashi Arase
7. Misfolded Cellular Proteins Rescued from Degradation by MHC Class II Molecules

May Abrogate Immune Tolerance 14
7.1 Misfolded Proteins Associated with MHC Class II Molecules as “Nonself”-
Antigens 14
7.2 Misfolded Protein Complexed with MHC Class II Molecules as Primary
Autoantigens for Autoantibodies 16
8. Misfolded Proteins Presented on MHC Class II Molecules as a Therapeutic Target for
Autoimmune Diseases 17
9. Concluding Remarks 18
Acknowledgments 19
References 19
Abstract
Misfolded proteins localized in the endoplasmic reticulum are degraded promptly and
thus are not transported outside cells. However, misfolded proteins in the endoplasmic
reticulum are rescued from protein degradation upon association with major histocom-
patibility complex (MHC) class II molecules and are transported to the cell surface by
MHC class II molecules without being processed to peptides. Studies on the misfolded
proteins rescued by MHC class II molecules have revealed that misfolded proteins asso-
ciated with MHC class II molecules are specific targets for autoantibodies produced in
autoimmune diseases. Furthermore, a strong correlation has been observed between
autoantibody binding to misfolded proteins associated with MHC class II molecules
and the autoimmune disease susceptibility conferred by each MHC class II allele. These
new insights into MHC class II molecules suggest that misfolded proteins rescued from
protein degradation by MHC class II molecules are recognized as “neo-self” antigens by
immune system and are involved in autoimmune diseases as autoantibody targets.
1. INTRODUCTION
Specific major histocompatibility complex (MHC) class II alleles affect
susceptibility to many autoimmune diseases. Recent genome-wide associa-
tion studies have confirmed that the MHC class II loci are the genes most
strongly associated with susceptibility to many autoimmune diseases, includ-
ing rheumatoid arthritis (RA). Because MHC class II molecules present pep-
tide antigens to helper T cells, an abnormal helper T cell response has been
considered to be the main cause of MHC class II gene-associated autoim-
mune diseases. However, the pathogenic peptide antigens associated with
the autoimmune disease susceptibility conferred by each MHC class II allele
have not yet been identified; therefore, it remains unclear how the MHC
class II gene controls susceptibility to autoimmune diseases. On the other
A New Hypothesis for Autoimmune Diseases 3
hand, our recent analyses of MHC class II molecules have revealed that pro-
teins misfolded in the endoplasmic reticulum (ER) are transported to the cell
surface without being processed to peptides upon association with MHC
class II molecules ( Jiang et al., 2013). Furthermore, misfolded proteins pres-
ented on MHC class II molecules appear to be involved in autoimmune dis-
ease susceptibility as specific targets for autoantibodies ( Jin et al., 2014;
Tanimura et al., 2015). These novel functions of MHC class II molecules
provide new insights into the molecular mechanism underlying autoim-
mune diseases that will help us answer questions regarding why autoanti-
bodies against autoantigens are produced in patients with autoimmune
diseases and why MHC class II genes are strongly associated with suscepti-
bility to many autoimmune diseases.
2. TRANSPORT OF ER-MISFOLDED PROTEINS TO THE

CELL SURFACE BY MHC CLASS II MOLECULES
2.1 MHC Class II Molecules Induce Cell Surface Expression
of Misfolded MHC Class I Molecules
The MHC class I molecule comprises a trimolecular complex that includes a
heavy chain, β2-microglobulin, and a peptide. MHC class I molecules are
not expressed on the cell surface in the absence of β2-microglobulin or trans-
porter associated with antigen processing (TAP), the latter of which trans-
ports proteasome-derived peptides to the ER where they are acquired by
MHC class I molecules, indicating that both β2-microglobulin and peptide,
are required for the cell surface expression of MHC class I molecules. On the
other hand, some unique monoclonal antibodies (mAbs) that are specific for
human MHC class I molecules lacking β2-microglobuin and peptide anti-
gens, such as HC10 and L31, have been identified (Giacomini et al., 1997;
Stam, Spits, & Ploegh, 1986). These mAbs do not recognize normal MHC
class I molecules associated with β2-microglobulin and peptide antigens
(Sibilio et al., 2005). Interestingly, the epitopes recognized by these mAbs
are localized on the α1- and α2-domains of MHC class I, which are not
located within the β2-microglobulin-binding region, suggesting that these
mAbs recognize a certain unique conformation of MHC class I molecules
that is induced in the absence of β2-microglobulin and peptide antigens
(Arosa, Santos, & Powis, 2007). These incomplete MHC class
I molecules fail to achieve a correct conformation and are not expressed
on the cell surface. However, certain cells, such as B cell lines, are recognized
4 Hisashi Arase
by these mAbs specific for unusual or misfolded MHC class I molecules lac-
king β2-microglobulin and peptides. This phenomenon suggests the exis-
tence of a molecular chaperon that transports misfolded MHC class
I molecules lacking β2-microglobulin and peptides to the cell surface.
Expression cloning to identify molecules that would permit expression of
MHC class I proteins on the cell surface unexpectedly revealed that MHC
class II molecules induce the cell surface expression of unusual or misfolded
MHC class I molecules ( Jiang et al., 2013). Upon further analysis, some
MHC class II alleles induced misfolded MHC class I expression on the cell
surface but others did not, and this difference depended on the amino acid
residues present within the peptide-binding groove of the MHC class II
molecule. Furthermore, the MHC class II-induced expression of misfolded
MHC class I molecules was almost completely blocked by a peptide cova-
lently attached to MHC class II molecules. Indeed, some MHC class
II-positive cells express misfolded MHC class I molecules that are recog-
nized by HC10 or L31 mAbs, and the direct association of MHC class
I molecules with MHC class II molecules is detectable in these cells. There-
fore, MHC class II molecules appear to be involved in the expression of
misfolded MHC class I molecules on the cell surface (Fig. 1).
Figure 1 Misfolded major histocompatibility complex (MHC) class I expression facili-

tated by MHC class II molecules. MHC class I molecules normally are expressed in asso-
ciation with β2-microglobulin and peptide antigens (right). In the absence of peptide
antigens or β2-microglobulin, MHC class I molecules are not folded correctly and are
not expressed on the cell surface. However, when the misfolded MHC class
I molecule is associated with an MHC class II molecule in the endoplasmic reticulum,
it is directly transported to the cell surface by the MHC class II molecule without under-
going peptide processing (middle).
2.2 MHC Class II Molecules Function as a Molecular Chaperon

to Transport Misfolded Cellular Protein to the Cell Surface
The cell surface expression of misfolded MHC class I molecules by MHC
class II molecules raised the possibility that other misfolded proteins might
also be transported to the cell surface by MHC class II molecules. Hen egg
lysozyme (HEL) is a well-characterized secreted protein, the correct folding
of which requires S–S bonds (Ohkuri, Ueda, Tsurumaru, & Imoto, 2001).
When a mutant HEL in which two cysteine residues were substituted with
alanine was co-expressed with MHC class II molecules, the mutant HEL
protein, which was neither secreted nor expressed on the cell surface, was
induced on the cell surface in the presence of MHC class II molecules
( Jiang et al., 2013). Furthermore, the full-length HEL protein was
co-precipitated with MHC class II molecules. These findings, together with
the analyses of MHC class I molecules, suggest that ER-misfolded proteins
are transported to the cell surface by MHC class II molecules upon associ-
ation with their peptide-binding grooves. In other words, MHC class II
molecules function as a molecular chaperon to transport ER-misfolded pro-
teins to the cell surface.
The presentation of whole proteins by MHC class II molecules may seem
unusual because it is widely accepted that MHC class II molecules present short
peptide antigens. However, several papers have reported the association of
large proteins with the peptide-binding grooves of MHC class II molecules
(Aichinger et al., 1997; Anderson, Swier, Arneson, & Miller, 1993; Busch,
Cloutier, Sekaly, & Hammerling, 1996; Lechler, Aichinger, & Lightstone,
1996). When MHC class II molecules were expressed with an invariant chain
lacking the endosomal localization signal, the invariant chain was directly trans-
ported to the cell surface in association with MHC class II molecules (Anderson
et al., 1993). In addition, association of MHC class II molecules with large pro-
teins was observed in the absence of invariant chain (Aichinger et al., 1997;
Busch et al., 1996). Therefore, the transport of ER-misfolded proteins to
the cell surface is an intrinsic function of MHC class II molecules. In addition,
antigens captured by the endocytic pathway form large molecular complexes
with MHC class II molecules in antigen-presenting cells (Castellino,
Zappacosta, Coligan, & Germain, 1998). These observations suggest that
MHC class II molecules exhibit the capacity to present large molecular antigens
derived not only from misfolded proteins in the ER but also from antigens cap-
tured by the endocytic pathway. However, the immunological functions of
large proteins associated with MHC class II molecules were not extensively
analyzed; therefore, these functions remained unclear.
6 Hisashi Arase
3. FUNCTION OF PROTEIN ANTIGENS PRESENTED BY

MHC CLASS II MOLECULES
3.1 MHC Class II Molecules Present Protein Antigens to
B Cells
The presentation of whole proteins instead of peptides by MHC class II mol-
ecules suggests that MHC class II molecules might be involved in an as yet
unknown immune response. B cells expressing a high-affinity antigen recep-
tor can be stimulated with soluble antigens. However, these antigens must be
associated with certain cell surface molecules to stimulate B cells with low-
affinity antigen receptors, such as those expressed on naı̈ve B cells
(Batista & Harwood, 2009; Qi, Egen, Huang, & Germain, 2006). The
presentation of whole proteins by MHC class II molecules suggests that these
proteins might be involved in B cell activation. Indeed, B cells expressing a
low-affinity antigen receptor against HEL protein can be stimulated with
HEL protein presented on MHC class II molecules but not by soluble
HEL protein alone ( Jiang et al., 2013). This indicates that MHC class II mol-
ecules might be directly involved in the antigen-specific B cell response.
3.2 Misfolded Cellular Proteins Rescued from Protein

Degradation by MHC Class II Molecules Might Be
Pathogenic
The invariant chain, which is associated with newly synthesized MHC class
II molecules, transports MHC class II molecules to endolysosomal compart-
ments, where they acquire peptide antigens (Germain, 2011). However, the
affinities of MHC class II molecules for the invariant chain are known to
differ in an MHC class II allele-dependent manner (Davenport et al.,
1995). It is possible that MHC class II molecules will preferentially associate
with misfolded proteins rather than the invariant chain if the former has a
stronger affinity for MHC class II molecules than the latter. Indeed, the effi-
ciency of the invariant chain to block the association of misfolded proteins
with MHC class II molecules differs in an allele-dependent manner, as
described above ( Jiang et al., 2013; Jin et al., 2014; Tanimura et al.,
2015). As most misfolded proteins do not possess a lysosomal targeting sig-
nal, MHC class II molecules associated with misfolded proteins instead of
invariant chain are directly transported to the cell surface without going
to the endolysosomal compartments.
The folding of a newly synthesized protein is a complex process that con-

stitutively generates significant amounts of misfolded proteins. In certain
types of cells, more than half of all newly synthesized proteins are folded
incorrectly (Meusser, Hirsch, Jarosch, & Sommer, 2005). However, these
newly synthesized misfolded proteins typically are promptly degraded in
the cells through various pathways such as ER-associated degradation
(ERAD) and therefore not transported outside the cells (Meusser et al.,
2005). Accordingly, immune cells are not exposed to these misfolded pro-
teins. Whereas both the primary structures and conformations of antigens are
involved in antibody recognition, only the primary structures of antigens are
involved in T cell recognition because T cell receptor recognizes short pep-
tide antigens presented on MHC molecules. Therefore, unlike T cells, it is
possible that some B cells do not acquire tolerance to misfolded cellular pro-
teins. If these misfolded cellular proteins are rescued from protein degrada-
tion by MHC class II molecules and subsequently transported extracellular,
B cells might recognize these proteins as “neo-self”-antigens and initiate an
antibody response (Fig. 2).
3.3 Aberrant MHC Class II Expression on Autoimmune-

Diseased Tissues
More than 30 years ago, it was reported that autoimmune-diseased tissues
aberrantly expressed MHC class II molecules (Bottazzo, Pujol-Borrell,
Hanafusa, & Feldmann, 1983). Unlike normal thyroid tissues, tissues from
patients with Graves’ disease or Hashimoto’s thyroiditis aberrantly express
MHC class II molecules. Similar aberrant MHC class II expression was reported
in various tissues affected by autoimmune diseases such as RA, type I diabetes,
primary biliary cirrhosis, and psoriasis (Ballardini et al., 1984; Feldmann et al.,
1988; Gottlieb et al., 1986). Because particular MHC class II alleles are associ-
ated with autoimmune disease susceptibility, this aberrant MHC class II expres-
sion in autoimmune-diseased tissues was considered to be involved in the
pathogenicity of autoimmune diseases. Indeed, nonimmune cells, such as
endothelial cells, strongly express MHC class II molecules in response to stim-
ulation from cytokines such as IFN-γ ( Jaffe et al., 1989; Pober et al., 1983;
Todd, Pujol-Borrell, Hammond, Bottazzo, & Feldmann, 1985). However,
these nonimmune cells do not express costimulatory molecules required for
the induction of T cell responses, such as CD80 or CD86. T cells that recognize
antigens presented on MHC class II molecules in the absence of costimulatory
signals are likely to become anergic (Appleman & Boussiotis, 2003). Therefore,
aberrant MHC class II expression on nonimmune cells has been considered a
8 Hisashi Arase
Figure 2 Rheumatoid rescue of misfolded proteins by major histocompatibility com-

plex (MHC) class II molecules. In steady state, MHC class II expression is restricted to spe-
cific immune cells such as dendritic cells and B cells. However, MHC class II molecules are
expressed on most cells following stimulation with certain cytokines such as IFN-γ,
which is produced in response to infection or inflammation. The invariant chain asso-
ciates with nascent MHC class II molecules and blocks the association of MHC class II
molecules with endoplasmic reticulum (ER)-misfolded proteins. However, the affinities
of various MHC class II molecules for the invariant chain differ due to allelic polymor-
phism of MHC class II genes. If the avidity of an MHC class II molecule for a misfolded
protein is higher than that for the invariant chain, it is possible that misfolded proteins,
rather than the invariant chain, will bind to MHC class II molecules. As the misfolded
proteins do not contain an endolysosomal-targeting signal, they are transported
directly to the cell surface by MHC class II molecules. Thus, MHC class II molecules func-
tion as a molecular chaperon to rescue ER-misfolded proteins from protein degradation.
Because immune cells normally are not exposed to misfolded proteins and may there-
fore be intolerant to them, misfolded proteins rescued by MHC class II molecules may be
recognized as “neo-self”-antigens and thus induce autoantibody production. In this
way, misfolded proteins rescued from protein degradation by MHC class II molecules
may be involved in the pathogenesis of autoimmune diseases as autoantibody targets.
consequence of the inflammation elicited by autoimmunity rather than a cause

of autoimmune disease, and the pathophysiological function of this aberrant
MHC class II expression in autoimmune-diseased tissues has not been exten-
sively analyzed. Unlike T cells, B cells do not require costimulatory signals to
respond to antigens, although they require T cell help for Ig class switching.
Given this difference between T cells and B cells, it is possible that misfolded
proteins rescued by aberrantly expressed MHC class II molecules can stimulate
B cells to produce autoantibodies.
4. MISFOLDED PROTEINS PRESENTED ON MHC CLASS II

MOLECULES ARE TARGETS FOR AUTOANTIBODIES IN
AUTOIMMUNE DISEASES
4.1 IgG Heavy Chain Presented on MHC Class II Molecules
Is a Specific Target for Autoantibodies in RA
Disease-specific autoantibodies are produced in many autoimmune disorders.
Some of these autoantibodies are directly involved in the pathogenicity of
autoimmunity. Therefore, it is important to identify the target molecules rec-
ognized by autoantibodies in order to understand the pathogenicity of auto-
immune diseases. If misfolded proteins rescued from protein degradation by
MHC class II molecules are targets for autoimmune diseases, autoantibodies
may recognize these proteins presented on MHC class II molecules.
Rheumatoid factor (RF) is a well-known autoantibody discovered
approximately 75 years ago. RF is specific for denatured, but not native,
IgG and is detected in approximately 80% of patients with RA (Dorner,
Egerer, Feist, & Burmester, 2004). Because RF titers are well correlated with
the clinical symptoms of RA, RF remains an important diagnostic indicator
of RA. However, the natural target antigens that induce RF production
remain undefined. In addition, it remains unclear why most patients with
RA are RF positive.
Antibodies comprise a heavy chain and a light chain; the heavy chain is
not secreted or expressed on the cell surface in the absence of the light chain.
However, the heavy chain alone can be expressed well on the cell surface in
the presence of MHC class II molecules ( Jin et al., 2014). Furthermore, IgG
presented on MHC class II molecules is recognized by autoantibodies from
patients with RA. More importantly, the IgG heavy chain presented on
MHC class II molecules was recognized by autoantibodies from patients
with RA but not by those from non-RA patients, including those positive
for RF. This suggests that the IgG heavy chain presented on MHC class II
molecules is more specific for autoantibodies from RA patients when com-
pared with traditional RF detected by immobilized IgG. This finding impli-
cates the IgG heavy chain, when presented on MHC class II molecules, as a
major target for RA autoantibodies.
10 Hisashi Arase
4.2 β2-Glycoprotein I Associated with MHC Class II Molecules Is

a Specific Target for Autoantibodies in Antiphospholipid
Syndrome
Antiphospholipid syndrome (APS) is an autoimmune disorder associated
with thrombosis and pregnancy complications (Wilson et al., 1999).
Although autoantibodies associated with APS were initially characterized
by reactivity to phospholipids such as cardiolipin, recent analyses have rev-
ealed that these autoantibodies are directed mainly against the phospholipid-
associated β2-glycoprotein I (Bas de Laat, Derksen, & de Groot, 2004; Galli,
Barbui, Zwaal, Comfurius, & Bevers, 1993; McNeil, Simpson,
Chesterman, & Krilis, 1990). β2-glycoprotein I forms a circular structure
in sera that is linearized upon binding to phospholipids, thus exposing cryp-
tic autoantibody epitopes on β2-glycoprotein I (Agar et al., 2010; de Laat,
Derksen, van Lummel, Pennings, & de Groot, 2006). However, it has
remained unclear whether phospholipid-bound β2-glycoprotein I is a nat-
ural target for autoantibodies and is involved in the pathogenesis of APS. In
an analysis of the association between β2-glycoprotein I and MHC class II
molecules, which was similar to that described earlier for the IgG heavy
chain, intact β2-glycoprotein I was also found to be presented on MHC
class II molecules on the cell surface. Furthermore, β2-glycoprotein
I presented on MHC class II molecules was recognized by APS autoanti-
bodies (Tanimura et al., 2015). Anti-β2-glycoprotein I Ab and anti-
cardiolipin Ab titers are used clinically to diagnose APS, although some
patients with clinical manifestations of APS do not have detectable these
Abs. On the other hand, more than 80% of patients express autoantibodies
against β2-glycoprotein I presented on MHC class II molecules (Tanimura
et al., 2015). This suggests that β2-glycoprotein I, when presented on MHC
class II molecules, is a major target antigen for autoantibodies in patients with
APS (Fig. 3).
The presence of autoantibodies against autoantigens associated with MHC
class II molecules suggests that these autoantigens associate with MHC class II
molecules in certain tissues. Indeed, complexes of the IgG heavy chain or β2-
glycoprotein I with MHC class II molecules were detected on synovial mem-
branes from patients with RA or uterine decidual tissues from patients with
APS, respectively. Similar to the analyses of autoantibodies from patients with
RA and APS, autoantibodies associated with other autoimmune diseases also
specifically recognize autoantigens complexed with MHC class II molecules
(Hui Jin, Ryosuke Hiwa, Satoko Morikami, Noriko Arase, & Hisashi Arase,
unpublished observation). Therefore, complexes of misfolded proteins with
Figure 3 Recognition of β2-glycoprotein I (β2GPI) presented on MHC class II molecules

by antiphospholipid autoantibodies. Native β2GPI forms a circular structure in sera that
is not recognized by antiphospholipid autoantibodies. When associated with a phos-
pholipid such as cardiolipin, β2GPI forms a linear structure that appears to expose cryp-
tic autoantibody epitopes. However, autoantibodies from some APS patients do not
recognize phospholipid-associated β2GPI. On the other hand, β2GPI is also expressed
on cell surfaces in association with MHC class II molecules. In addition, more than
80% of APS patients possess autoantibodies against β2GPI presented on MHC class II
molecules, suggesting that β2GPI presented on MHC class II molecules may be a major
target antigen for autoantibodies in antiphospholipid syndrome.
MHC class II molecules appear to be major targets of autoantibodies in many

autoimmune diseases.
5. SUSCEPTIBILITY TO AUTOIMMUNE DISEASES IS

ASSOCIATED WITH THE AFFINITY OF MISFOLDED
PROTEINS FOR MHC CLASS II MOLECULES
5.1 MHC Class II Alleles and Autoimmune Disease
Susceptibility
Particular MHC class II gene alleles are strongly associated with susceptibil-
ity to many autoimmune diseases. Extensive analyses of RA susceptibility
according to HLA-DR alleles have suggested that specific amino acid resi-
dues in the peptide-binding groove of HLA-DR are associated with suscep-
tibility to RA (Raychaudhuri et al., 2012). Therefore, certain peptide
antigens are thought to be involved in autoimmune diseases. However, pep-
tide antigens that could explain the susceptibility to autoimmune diseases
conferred by each MHC class II allele have not been identified, and there-
fore, the molecular mechanism underlying the control exerted by particular
12 Hisashi Arase
MHC class II alleles over susceptibility to autoimmune diseases remains

unknown (Raychaudhuri et al., 2012).
5.2 Autoantibody Binding to Misfolded Protein/MHC Class II

Complex Is Associated with Autoimmune Disease
Susceptibility
In an analysis of autoantibody binding to IgG heavy chains presented on
HLA-DR molecules encoded by various alleles, a strong correlation was
observed between autoantibody binding to IgG heavy chains presented
on HLA-DR and the RA susceptibility conferred by each HLA-DR allele
( Jin et al., 2014). The invariant chain only partially blocks the association of
IgG heavy chains with RA-susceptible HLA-DR, but strongly blocks the
association of IgG heavy chains with RA-resistant HLA-DR. Autoanti-
bodies fail to bind IgG heavy chains presented on RA-resistant HLA-DR
in the presence of the invariant chain. Thus, the IgG heavy chain is the first
molecule associated with the susceptibility to RA conferred by each HLA-
DR allele. Because IgG heavy chain associated with MHC class II molecules
comprises a specific RA autoantibody target, it is possible that the IgG heavy
chain–MHC class II molecule complex is involved directly in RA pathoge-
nicity as an autoantibody target.
Similarly, the presentation of self-antigens on MHC class II molecules
encoded by disease-susceptible alleles has been observed in APS
(Tanimura et al., 2015). APS-susceptible HLA-DR efficiently presents
β2-glycoprotein I, and autoantibodies preferentially bind to this complex
even in the presence of the invariant chain. Therefore, differences in auto-
immune disease susceptibility among the different MHC class II alleles might
be explained by different efficiencies of autoantigen presentation by MHC
class II molecules.
6. INVOLVEMENT OF MISFOLDED PROTEIN–MHC CLASS

II MOLECULE COMPLEXES IN AUTOANTIBODY-
MEDIATED PATHOGENICITY
6.1 Pathogenesis of Autoantibodies in RA and APS
Autoantibodies that are produced in most autoimmune diseases are involved
in the pathogenicity of some of these diseases. For example, myasthenia
gravis, Graves’ disease, and pemphigus are representative autoimmune dis-
eases caused by autoantibodies that exert blocking, activating, and destruc-
tive functions, respectively. In addition, the adoptive transfer of serum IgG
from RA patients to mice lacking FcγRIIB, an inhibitory Fc receptor, has
been shown to induce arthritis, suggesting that autoantibodies play an impor-

tant role in the pathogenesis of RA, although the target antigens for these
pathogenic autoantibodies remain undefined (Petkova et al., 2006). Autoan-
tibodies also play a crucial role in some RA mouse models such as K/BxN
mice and the type II collagen-induced arthritis model. In K/BxN mice, auto-
antibodies against glucose-6-phosphate isomerase are responsible for arthritis
development (Matsumoto et al., 2002; Matsumoto, Staub, Benoist, & Mathis,
1999). The adoptive transfer of anti-glucose-6-phosphate isomerase autoan-
tibodies from K/BxN mice to healthy mice induces arthritis. Similarly, anti-
type II collagen autoantibodies induced via immunization with type II colla-
gen directly mediate arthritis. Similar to the autoantibodies in K/BxN mice,
these anti-type II collagen autoantibodies induce arthritis when transferred to
healthy mice (Griffiths & Remmers, 2001). Therefore, autoantibodies appear
to play an important role in the pathogenesis of arthritis not only in RA mouse
models but also in patients with RA.
APS autoantibodies are directed mainly against the serum lipoprotein
β2-glycoprotein I and are thought to be involved in the pathogenesis of
APS. However, it remains unclear how these autoantibodies against
β2-glycoprotein I induce thrombosis or pregnancy complications, as the
protein is not expressed on the surface of healthy blood vascular endothelial
cells. In addition, it remains unknown why some patients mainly exhibit
thrombosis and others predominantly develop pregnancy complications,
despite detecting similar autoantibodies in both groups of patients. Endothe-
lial cells strongly express MHC class II molecules upon IFN-γ stimulation
( Jaffe et al., 1989; Pober et al., 1983). Indeed, aberrant MHC class II expres-
sion has been observed in uterine decidual tissues from patients with APS.
More importantly, complexes of β2-glycoprotein I with MHC class II mol-
ecules have been detected in uterine decidual tissues from patients with APS
(Tanimura et al., 2015). These observations suggest that aberrantly expressed
MHC class II molecules on endothelial cells could be targeted by autoanti-
bodies, possibly leading to thrombosis in peripheral vessels or the uterus.
Therefore, in addition to the presence of autoantibodies, aberrant MHC
class II expression might play an important role in autoantibody-mediated
pathogenesis.
6.2 B Cell Removal Are Effective Treatment for Autoimmune

Diseases
The removal of B cells via the administration of an anti-CD20 mAb
(rituximab) has been clinically approved as an effective treatment for RA
( Jacobi & Dorner, 2010). Anti-CD20 mAb treatment is also effective for
14 Hisashi Arase
other autoimmune diseases such as APS (Erkan, Vega, Ramon, Kozora, &
Lockshin, 2013), systemic lupus erythematosus (Anolik et al., 2004), myas-
thenia gravis (Sieb, 2014), Graves’ disease (Heemstra et al., 2008), and pem-
phigus (Ahmed, Spigelman, Cavacini, & Posner, 2006; Joly et al., 2007).
Furthermore, anti-B lymphocyte stimulator (BlyS) mAb (Belimumab) that
decreases autoantibody producing B cells is effective for some autoimmune
diseases such as SLE (Navarra et al., 2011). Because B cells are involved in
antibody production as well as antigen presentation and cytokine secretion,
B cell depletion may affect various aspects in autoimmunity. B cell depletion
by anti-CD20 mAb has been reported to correlate with a decrease in auto-
antibody levels as well as clinical manifestation, suggesting that autoantibody
producing B cells play an important role in the pathogenicity of autoim-
mune diseases (Cambridge et al., 2006; Thurlings et al., 2008). However,
it is difficult to directly test the pathogenicity of human autoantibodies from
autoimmune patients in mice because of species differences in autoantigens.
Therefore, the pathogenicity of human autoantibodies has been demon-
strated only in the context of some autoimmune diseases such as Graves’ dis-
ease, myasthenia gravis, and pemphigus. As autoantigen–MHC class II
molecule complexes are targeted by autoantibodies, mice expressing both
human autoantigens and human MHC class II molecules would be useful
for testing the pathogenesis of human autoantibodies.
7. MISFOLDED CELLULAR PROTEINS RESCUED FROM

DEGRADATION BY MHC CLASS II MOLECULES MAY
ABROGATE IMMUNE TOLERANCE
7.1 Misfolded Proteins Associated with MHC Class II
Molecules as “Nonself”-Antigens
As described above, misfolded proteins, when complexed with MHC class II
molecules, are specific targets for the autoantibodies produced in autoim-
mune diseases. In addition, a strong association has been observed between
autoantibody binding to IgG heavy chains presented on MHC class II mol-
ecules and the RA susceptibility conferred by each HLA-DR allele. These
observations suggest that self-antigens presented on MHC class II molecules
are involved in the pathogenicity of autoimmune diseases by serving as tar-
gets for autoantibodies. In cases involving aberrantly induced or increased
MHC class II expression in response to infection or inflammation in which
ER-misfolded proteins have a stronger affinity for MHC class II molecules
than the invariant chain, misfolded cellular proteins associate with MHC
class II molecules and are subsequently transported to the cell surface.
The prompt degradation of ER-misfolded proteins through various cellular

mechanisms in steady state (Meusser et al., 2005) prevents the exposure of
these proteins to immune cells. Therefore, ER-misfolded proteins aber-
rantly transported to the cell surface by MHC class II molecules might appear
as “neo-self ”-antigens and induce an abnormal immune response (Fig. 2).
Indeed, XBP-1, a transcription factor induced by the unfolded protein
response, was cloned originally from plasma cells in synovial membranes of
patients with RA (Iwakoshi et al., 2003), suggesting high levels of unfolded
protein production in these cells. Autoantibodies against citrullinated pro-
teins are also generated in RA. Interestingly, citrullination is known to cause
protein misfolding (Tarcsa et al., 1996). Therefore, citrullination-induced
conformational changes in proteins might augment the association of auto-
antigens with MHC class II molecules. Furthermore, MHC class II expres-
sion is strongly increased in the synovial membranes of patients with RA
(Feldmann et al., 1988; Klareskog, Forsum, Scheynius, Kabelitz, &
Wigzell, 1982). Because plasma cells expressing low levels of MHC class
II molecules produce large amounts of IgG, misfolded IgG heavy chains
might associate with MHC class II molecules encoded by RA-susceptible
alleles in certain conditions that induce upregulated MHC class II expression
on plasma cells; the resulting complexes could induce production of auto-
antibody against IgG. Similarly, β2-glycoprotein I is mainly produced in
hepatocytes, which do not express MHC class II molecules in steady state.
If APS-susceptible MHC class II molecules that preferentially bind to β2-
glycoprotein I are expressed on hepatocytes in response to inflammation
or infection, β2-glycoprotein I will associate with these molecules and thus
trigger autoantibody production.
Certain misfolded proteins may associate constitutively with MHC
class II molecules on B cells or dendritic cells. However, autoantibodies
against these misfolded proteins are not produced in steady state; immune
cells are exposed constitutively to these complexes, a process that appears
to have induced tolerance. Indeed, transgenic mice expressing MHC class
II molecules on pancreatic β cells did not exhibit β cell autoimmunity
(Lo et al., 1988; Sarvetnick, Liggitt, Pitts, Hansen, & Stewart, 1988).
Because MHC class II expression is not inducible in these transgenic mice,
even if certain β cell-specific misfolded proteins are presented on MHC
class II molecules, the proteins associated with MHC class II molecules
are always presented to immune cells and may not be recognized as
“neo-self ”-antigens. Analyses of mice harboring an inducible MHC class
II transgene might provide valuable information about the function of aber-
rantly expressed MHC class II molecules.
16 Hisashi Arase
The CIITA transcription factor is involved in the expression of both MHC

class II molecules and the invariant chain (Reith, LeibundGut-Landmann, &
Waldburger, 2005). Therefore, the invariant chain is expressed in most
MHC class II-expressing cells, where it blocks the binding of ER-misfolded
proteins to MHC class II molecules. However, MHC class II and invariant
chain gene transcription is regulated differentially, and thus the expression of
these molecules is not always equivalent (Paul et al., 2011). In addition, the
affinity of MHC class II molecules for the invariant chain differs depending
on the encoding MHC class II allele (Patil et al., 2001). Therefore, the amounts
of misfolded proteins, invariant chain, and MHC class II molecules, as well as
the MHC class II allele, seem to determine the efficiency of the association
between misfolded proteins and MHC class II molecules.
7.2 Misfolded Protein Complexed with MHC Class II Molecules

as Primary Autoantigens for Autoantibodies
It remains unclear how autoantibodies specific for autoantigens presented on
MHC class II molecules are produced. Given the presence of specific auto-
antibodies against these complexes, it is likely that the complexes themselves
induce autoantibody production. However, it is uncertain whether the self-
antigens presented on MHC class II molecules initiate local antibody
responses in nonlymphoid tissues because the germinal center usually is
required for antibody responses (Klein & Dalla-Favera, 2008). On the other
hand, cell surface MHC class II molecules are known to be released from
cells as exosomes (Thery, Ostrowski, & Segura, 2009) and have been
detected as such in serum (Almqvist, Lonnqvist, Hultkrantz, Rask, &
Telemo, 2008; Karlsson et al., 2001; Taylor, Akyol, & Gercel-Taylor,
2006). Therefore, when self-antigens are expressed in complex with
MHC class II molecules in certain tissues, the complexes may be released
from the cells as exosomes, which might subsequently induce the produc-
tion of specific antibodies against the complexes in lymphoid tissues.
Most autoantibodies are detected using self-antigens immobilized on plates
or microbeads, suggesting that MHC class II molecules might not be required
for autoantibody recognition. However, protein immobilization causes signif-
icant conformational changes. For example, RF does not bind native IgG in
sera but does bind immobilized IgG. Similarly, autoantibodies against β2-
glycoprotein I do not bind native β2-glycoprotein I in sera but do bind to
β2-glycoprotein I when immobilized on negatively charged plates. In addi-
tion, most autoantibodies can detect target antigens when assayed by Western
blot analysis, indicating that they recognize denatured forms of autoantigens.
These observations are compatible with the fact that autoantibodies are
directed against misfolded proteins presented on MHC class II molecules.
Autoantibodies from most patients with APS recognize β2-glycoprotein
I presented on MHC class II molecules, whereas only some patients
possess autoantibodies against plate-bound β2-glycoprotein I. Therefore,
β2-glycoprotein I presented on MHC class II molecules seems to be the pri-
mary target antigen for APS autoantibodies. Most autoantibodies are the
result of a somatic hypermutation process that increases the affinity for auto-
antigens (Rajewsky, 1996). Because somatic hypermutation is not observed
in naı̈ve B cells, the antigen specificities of autoantibodies that are
engineered in vitro to restore the codons present in the germline Ig gene will
provide information about the original antigens that stimulated naı̈ve B cells
to produce autoantibodies. A recent analysis indicated that germline-
reverted autoantibodies from pemphigus patients did not recognize the
autoantigen desmoglein-3, suggesting that this autoantigen did not induce
autoantibody production (Di Zenzo et al., 2012). However, some
germline-reverted autoantibodies still recognize autoantigens when pres-
ented on MHC class II molecules (Hui Jin & Hisashi Arase, unpublished
observation). Therefore, autoantigens presented on MHC class II molecules
might be the primary target antigens that induced autoantibody production.
Although most autoantibodies are directed against denatured autoantigens,
some autoantibodies recognize native autoantigens. Because epitope spread-
ing affects antibody diversity, it is possible that autoantibodies raised against
misfolded proteins presented on MHC class II molecules might have
acquired reactivity against native autoantigens through epitope spreading.
The molecular mimicry of autoantigens by microbial antigens is also
involved in the production of some autoantibodies (Munz, Lunemann,
Getts, & Miller, 2009). Considering that the antigenicity of misfolded auto-
antigens is more similar to that of native autoantigens than of microbial anti-
gens, misfolded proteins presented on MHC class II molecules may possibly
be involved in the production of autoantibodies against native autoantigens.
8. MISFOLDED PROTEINS PRESENTED ON MHC CLASS II

MOLECULES AS A THERAPEUTIC TARGET FOR
AUTOIMMUNE DISEASES
As misfolded proteins aberrantly rescued from protein degradation by
MHC class II molecules might be involved in the pathogenesis of autoim-
mune diseases, blocking the association of misfolded proteins with MHC
18 Hisashi Arase
class II molecules would be a good candidate treatment for autoimmune dis-

eases. The aberrant MHC class II expression observed in autoimmune-
diseased tissues seems to result from stimulation by cytokines such as
IFN-γ. Blocking the MHC class II expression induced by cytokine stimu-
lation would therefore effectively treat autoimmune diseases (Miller,
Maher, & Young, 2009). HMG-CoA reductase inhibitors, or statins, are
among the drugs used to reduce serum cholesterol levels. Statins have been
reported to reduce inflammation in some autoimmune diseases such as RA,
APS, and Sj€ ogren’s syndrome (Greenwood, Steinman, & Zamvil, 2006;
Khattri & Zandman-Goddard, 2013). Although the exact mechanisms
remain unclear, statins appear to reduce IFN-γ-induced MHC class II
expression on human endothelial cells in vitro (Kwak, Mulhaupt, Myit, &
Mach, 2000; Youssef et al., 2002). The anti-inflammatory function of statins
might also include inhibiting the association of misfolded proteins with
MHC class II molecules. The development of a strong and specific inhibitor
with which to block the association of misfolded proteins with aberrantly
expressed MHC class II molecules represents a new target in autoimmune
disease therapy.
9. CONCLUDING REMARKS
The MHC class II locus is the gene most strongly associated with suscep-
tibility to many autoimmune diseases. Extensive analyses of misfolded proteins
aberrantly rescued from protein degradation by MHC class II molecules have
not only revealed that these proteins are specific targets for autoantibodies but
have also suggested that autoantibody binding to these proteins may explain the
autoimmune disease susceptibility conferred by certain MHC class II alleles.
Therefore, aberrant MHC class II expression on certain tissues or cells might
induce autoimmune disease. An understanding the molecular factors that
induce aberrant MHC class II expression would be quite important to an under-
standing of the causes of autoimmune diseases. On the other hand, the physi-
ological functions of the aberrantly expressed MHC class II molecules on
nonimmune cells remain unclear. If aberrant MHC class II expression on non-
immune cells is solely responsible for diseases involvement, the pathway by
which MHC class II is expressed on nonimmune cells will be lost in the course
of evolution. Although MHC class II molecules expressed on nonimmune cells
cannot evoke helper T cell responses because of the lack of costimulatory mol-
ecule expression, these aberrantly expressed MHC class II molecules might
confer certain benefits to maintain homeostasis. Further analyses of the
misfolded proteins rescued from protein degradation and transported to the cell
surface by MHC class II molecules will reveal currently unknown mechanisms
of immunity in both normal and disease situations.
ACKNOWLEDGMENTS
We thank Prof. Lewis L. Lanier for critical reading of our manuscript. This work was partially
supported by JSPS KAKENHI Grant Numbers (15K15131, 15H02545, 26117714 and
24115005), the Practical Research Project for Allergic Diseases and Immunology from
Japan Agency for Medical Research and development, AMED, The Naito Foundation,
The Tokyo Biochemical Research Foundation, The Uehara Memorial Foundation and
Terumo Life Science Foundation.
REFERENCES
Agar, C., van Os, G. M., Morgelin, M., Sprenger, R. R., Marquart, J. A., Urbanus, R. T.,
et al. (2010). β2-glycoprotein I can exist in 2 conformations: Implications for our under-
standing of the antiphospholipid syndrome. Blood, 116, 1336–1343.
Ahmed, A. R., Spigelman, Z., Cavacini, L. A., & Posner, M. R. (2006). Treatment of pem-
phigus vulgaris with rituximab and intravenous immune globulin. The New England
Journal of Medicine, 355, 1772–1779.
Aichinger, G., Karlsson, L., Jackson, M. R., Vestberg, M., Vaughan, J. H., Teyton, L.,
et al. (1997). Major histocompatibility complex class II-dependent unfolding, transport,
and degradation of endogenous proteins. The Journal of Biological Chemistry, 272,
29127–29136.
Almqvist, N., Lonnqvist, A., Hultkrantz, S., Rask, C., & Telemo, E. (2008). Serum-derived
exosomes from antigen-fed mice prevent allergic sensitization in a model of allergic
asthma. Immunology, 125, 21–27.
Anderson, M. S., Swier, K., Arneson, L., & Miller, J. (1993). Enhanced antigen presentation
in the absence of the invariant chain endosomal localization signal. The Journal of Exper-
imental Medicine, 178, 1959–1969.
Anolik, J. H., Barnard, J., Cappione, A., Pugh-Bernard, A. E., Felgar, R. E., Looney, R. J.,
et al. (2004). Rituximab improves peripheral B cell abnormalities in human systemic
lupus erythematosus. Arthritis and Rheumatism, 50, 3580–3590.
Appleman, L. J., & Boussiotis, V. A. (2003). T cell anergy and costimulation. Immunological
Reviews, 192, 161–180.
Arosa, F. A., Santos, S. G., & Powis, S. J. (2007). Open conformers: The hidden face of
MHC-I molecules. Trends in Immunology, 28, 115–123.
Ballardini, G., Mirakian, R., Bianchi, F. B., Pisi, E., Doniach, D., & Bottazzo, G. F. (1984).
Aberrant expression of HLA-DR antigens on bileduct epithelium in primary biliary
cirrhosis: Relevance to pathogenesis. Lancet, 2, 1009–1013.
Bas de Laat, H., Derksen, R. H., & de Groot, P. G. (2004). β2-glycoprotein I, the playmaker
of the antiphospholipid syndrome. Clinical Immunology, 112, 161–168.
Batista, F. D., & Harwood, N. E. (2009). The who, how and where of antigen presentation to
B cells. Nature Reviews. Immunology, 9, 15–27.
Bottazzo, G. F., Pujol-Borrell, R., Hanafusa, T., & Feldmann, M. (1983). Role of aberrant
HLA-DR expression and antigen presentation in induction of endocrine autoimmunity.
Lancet, 2, 1115–1119.
Busch, R., Cloutier, I., Sekaly, R. P., & Hammerling, G. J. (1996). Invariant chain protects
class II histocompatibility antigens from binding intact polypeptides in the endoplasmic
reticulum. The EMBO Journal, 15, 418–428.
20 Hisashi Arase
Cambridge, G., Leandro, M. J., Teodorescu, M., Manson, J., Rahman, A., Isenberg, D. A.,
et al. (2006). B cell depletion therapy in systemic lupus erythematosus: Effect on
autoantibody and antimicrobial antibody profiles. Arthritis and Rheumatism, 54,
3612–3622.
Castellino, F., Zappacosta, F., Coligan, J. E., & Germain, R. N. (1998). Large protein frag-
ments as substrates for endocytic antigen capture by MHC class II molecules. Journal of
Immunology, 161, 4048–4057.
Davenport, M. P., Quinn, C. L., Chicz, R. M., Green, B. N., Willis, A. C., Lane, W. S.,
et al. (1995). Naturally processed peptides from two disease-resistance-associated HLA-
DR13 alleles show related sequence motifs and the effects of the dimorphism at position
86 of the HLA-DRβ chain. Proceedings of the National Academy of Sciences of the United
States of America, 92, 6567–6571.
de Laat, B., Derksen, R. H., van Lummel, M., Pennings, M. T., & de Groot, P. G. (2006).
Pathogenic anti-β2-glycoprotein I antibodies recognize domain I of β2-glycoprotein
I only after a conformational change. Blood, 107, 1916–1924.
Di Zenzo, G., Di Lullo, G., Corti, D., Calabresi, V., Sinistro, A., Vanzetta, F., et al. (2012).
Pemphigus autoantibodies generated through somatic mutations target the desmoglein-3
cis-interface. Journal of Clinical Investigation, 122, 3781–3790.
Dorner, T., Egerer, K., Feist, E., & Burmester, G. R. (2004). Rheumatoid factor revisited.
Current Opinion in Rheumatology, 16, 246–253.
Erkan, D., Vega, J., Ramon, G., Kozora, E., & Lockshin, M. D. (2013). A pilot open-label
phase II trial of rituximab for non-criteria manifestations of antiphospholipid syndrome.
Arthritis and Rheumatism, 65, 464–471.
Feldmann, M., Kissonerghis, A. M., Buchan, G., Brennan, F., Turner, M., Haworth, C.,
et al. (1988). Role of HLA class II and cytokine expression in rheumatoid arthritis. Scan-
dinavian Journal of Rheumatology. Supplement, 76, 39–46.
Galli, M., Barbui, T., Zwaal, R. F., Comfurius, P., & Bevers, E. M. (1993). Antiphospholipid
antibodies: Involvement of protein cofactors. Haematologica, 78, 1–4.
Germain, R. N. (2011). Uncovering the role of invariant chain in controlling MHC class II
antigen capture. Journal of Immunology, 187, 1073–1075.
Giacomini, P., Beretta, A., Nicotra, M. R., Ciccarelli, G., Martayan, A., Cerboni, C.,
et al. (1997). HLA-C heavy chains free of β2-microglobulin: Distribution in normal
tissues and neoplastic lesions of non-lymphoid origin and interferon-γ responsiveness.
Tissue Antigens, 50, 555–566.
Gottlieb, A. B., Lifshitz, B., Fu, S. M., Staiano-Coico, L., Wang, C. Y., & Carter, D. M.
(1986). Expression of HLA-DR molecules by keratinocytes, and presence of Langerhans
cells in the dermal infiltrate of active psoriatic plaques. The Journal of Experimental Med-
icine, 164, 1013–1028.
Greenwood, J., Steinman, L., & Zamvil, S. S. (2006). Statin therapy and autoimmune disease:
From protein prenylation to immunomodulation. Nature Reviews Immunology, 6,
358–370.
Griffiths, M. M., & Remmers, E. F. (2001). Genetic analysis of collagen-induced arthritis in
rats: A polygenic model for rheumatoid arthritis predicts a common framework of cross-
species inflammatory/autoimmune disease loci. Immunological Reviews, 184, 172–183.
Heemstra, K. A., Toes, R. E., Sepers, J., Pereira, A. M., Corssmit, E. P., Huizinga, T. W.,
et al. (2008). Rituximab in relapsing Graves’ disease, a phase II study. European Journal of
Endocrinology, 159, 609–615.
Iwakoshi, N. N., Lee, A. H., Vallabhajosyula, P., Otipoby, K. L., Rajewsky, K., &
Glimcher, L. H. (2003). Plasma cell differentiation and the unfolded protein response
intersect at the transcription factor XBP-1. Nature Immunology, 4, 321–329.
Jacobi, A. M., & Dorner, T. (2010). Current aspects of anti-CD20 therapy in rheumatoid
arthritis. Current Opinion in Pharmacology, 10, 316–321.
Jaffe, E. A., Armellino, D., Lam, G., Cordon-Cardo, C., Murray, H. W., & Evans, R. L.
(1989). IFN-γ and IFN-α induce the expression and synthesis of Leu 13 antigen by cul-
tured human endothelial cells. Journal of Immunology, 143, 3961–3966.
Jiang, Y., Arase, N., Kohyama, M., Hirayasu, K., Suenaga, T., Jin, H., et al. (2013). Transport
of misfolded endoplasmic reticulum proteins to the cell surface by MHC class II mole-
cules. International Immunology, 25, 235–246.
Jin, H., Arase, N., Hirayasu, K., Kohyama, M., Suenaga, T., Saito, F., et al. (2014). Auto-
antibodies to IgG/HLA class II complexes are associated with rheumatoid arthritis sus-
ceptibility. Proceedings of the National Academy of Sciences of the United States of America, 111,
3787–3792.
Joly, P., Mouquet, H., Roujeau, J. C., D’Incan, M., Gilbert, D., Jacquot, S., et al. (2007).
A single cycle of rituximab for the treatment of severe pemphigus. The New England Jour-
nal of Medicine, 357, 545–552.
Karlsson, M., Lundin, S., Dahlgren, U., Kahu, H., Pettersson, I., & Telemo, E. (2001). “Tol-
erosomes” are produced by intestinal epithelial cells. European Journal of Immunology, 31,
2892–2900.
Khattri, S., & Zandman-Goddard, G. (2013). Statins and autoimmunity. Immunological
Research, 56, 348–357.
Klareskog, L., Forsum, U., Scheynius, A., Kabelitz, D., & Wigzell, H. (1982). Evidence in
support of a self-perpetuating HLA-DR-dependent delayed-type cell reaction in rheu-
matoid arthritis. Proceedings of the National Academy of Sciences of the United States of America,
79, 3632–3636.
Klein, U., & Dalla-Favera, R. (2008). Germinal centres: Role in B-cell physiology and
malignancy. Nature Reviews. Immunology, 8, 22–33.
Kwak, B., Mulhaupt, F., Myit, S., & Mach, F. (2000). Statins as a newly recognized type of
immunomodulator. Nature Medicine, 6, 1399–1402.
Lechler, R., Aichinger, G., & Lightstone, L. (1996). The endogenous pathway of MHC class
II antigen presentation. Immunological Reviews, 151, 51–79.
Lo, D., Burkly, L. C., Widera, G., Cowing, C., Flavell, R. A., Palmiter, R. D., et al. (1988).
Diabetes and tolerance in transgenic mice expressing class II MHC molecules in pancre-
atic beta cells. Cell, 53, 159–168.
Matsumoto, I., Maccioni, M., Lee, D. M., Maurice, M., Simmons, B., Brenner, M.,
et al. (2002). How antibodies to a ubiquitous cytoplasmic enzyme may provoke
joint-specific autoimmune disease. Nature Immunology, 3, 360–365.
Matsumoto, I., Staub, A., Benoist, C., & Mathis, D. (1999). Arthritis provoked by linked
T and B cell recognition of a glycolytic enzyme. Science, 286, 1732–1735.
McNeil, H. P., Simpson, R. J., Chesterman, C. N., & Krilis, S. A. (1990). Anti-phospholipid
antibodies are directed against a complex antigen that includes a lipid-binding inhibitor
of coagulation: β2-glycoprotein I (apolipoprotein H). Proceedings of the National Academy
of Sciences of the United States of America, 87, 4120–4124.
Meusser, B., Hirsch, C., Jarosch, E., & Sommer, T. (2005). ERAD: The long road to
destruction. Nature Cell Biology, 7, 766–772.
Miller, C. H., Maher, S. G., & Young, H. A. (2009). Clinical use of interferon-γ. Annals of the
New York Academy of Sciences, 1182, 69–79.
Munz, C., Lunemann, J. D., Getts, M. T., & Miller, S. D. (2009). Antiviral immune responses:
Triggers of or triggered by autoimmunity? Nature Reviews Immunology, 9, 246–258.
Navarra, S. V., Guzman, R. M., Gallacher, A. E., Hall, S., Levy, R. A., Jimenez, R. E.,
et al. (2011). Efficacy and safety of belimumab in patients with active systemic lupus
erythematosus: A randomised, placebo-controlled, phase 3 trial. Lancet, 377, 721–731.
Ohkuri, T., Ueda, T., Tsurumaru, M., & Imoto, T. (2001). Evidence for an initiation site for
hen lysozyme folding from the reduced form using its dissected peptide fragments. Protein
Engineering, 14, 829–833.
22 Hisashi Arase
Patil, N. S., Pashine, A., Belmares, M. P., Liu, W., Kaneshiro, B., Rabinowitz, J.,
et al. (2001). Rheumatoid arthritis (RA)-associated HLA-DR alleles form less stable
complexes with class II-associated invariant chain peptide than non-RA-associated
HLA-DR alleles. Journal of Immunology, 167, 7157–7168.
Paul, P., van den Hoorn, T., Jongsma, M. L., Bakker, M. J., Hengeveld, R., Janssen, L.,
et al. (2011). A Genome-wide multidimensional RNAi screen reveals pathways control-
ling MHC class II antigen presentation. Cell, 145, 268–283.
Petkova, S. B., Konstantinov, K. N., Sproule, T. J., Lyons, B. L., Awwami, M. A., &
Roopenian, D. C. (2006). Human antibodies induce arthritis in mice deficient in the
low-affinity inhibitory IgG receptor Fc gamma RIIB. The Journal of Experimental
Medicine, 203, 275–280.
Pober, J. S., Gimbrone, M. A., Jr., Cotran, R. S., Reiss, C. S., Burakoff, S. J., Fiers, W.,
et al. (1983). Ia expression by vascular endothelium is inducible by activated T cells
and by human γ interferon. The Journal of Experimental Medicine, 157, 1339–1353.
Qi, H., Egen, J. G., Huang, A. Y., & Germain, R. N. (2006). Extrafollicular activation of
lymph node B cells by antigen-bearing dendritic cells. Science, 312, 1672–1676.
Rajewsky, K. (1996). Clonal selection and learning in the antibody system. Nature, 381,
751–758.
Raychaudhuri, S., Sandor, C., Stahl, E. A., Freudenberg, J., Lee, H. S., Jia, X., et al. (2012).
Five amino acids in three HLA proteins explain most of the association between MHC
and seropositive rheumatoid arthritis. Nature Genetics, 44, 291–296.
Reith, W., LeibundGut-Landmann, S., & Waldburger, J. M. (2005). Regulation of MHC
class II gene expression by the class II transactivator. Nature Reviews. Immunology, 5,
793–806.
Sarvetnick, N., Liggitt, D., Pitts, S. L., Hansen, S. E., & Stewart, T. A. (1988). Insulin-
dependent diabetes mellitus induced in transgenic mice by ectopic expression of class
II MHC and interferon-gamma. Cell, 52, 773–782.
Sibilio, L., Martayan, A., Setini, A., Fraioli, R., Fruci, D., Shabanowitz, J., et al. (2005).
Impaired assembly results in the accumulation of multiple HLA-C heavy chain folding
intermediates. Journal of Immunology, 175, 6651–6658.
Sieb, J. P. (2014). Myasthenia gravis: An update for the clinician. Clinical and Experimental
Immunology, 175, 408–418.
Stam, N. J., Spits, H., & Ploegh, H. L. (1986). Monoclonal antibodies raised against dena-
tured HLA-B locus heavy chains permit biochemical characterization of certain HLA-C
locus products. Journal of Immunology, 137, 2299–2306.
Tanimura, K., Jin, H., Suenaga, T., Morikami, S., Arase, N., Kishida, K., et al. (2015). β2-
glycoprotein I / HLA class II complexes are novel autoantigens in antiphospholipid syn-
drome. Blood, 125, 2835–2844.
Tarcsa, E., Marekov, L. N., Mei, G., Melino, G., Lee, S. C., & Steinert, P. M. (1996). Protein
unfolding by peptidylarginine deiminase. Substrate specificity and structural relationships
of the natural substrates trichohyalin and filaggrin. The Journal of Biological Chemistry, 271,
30709–30716.
Taylor, D. D., Akyol, S., & Gercel-Taylor, C. (2006). Pregnancy-associated exosomes and
their modulation of T cell signaling. Journal of Immunology, 176, 1534–1542.
Thery, C., Ostrowski, M., & Segura, E. (2009). Membrane vesicles as conveyors of immune
responses. Nature Reviews Immunology, 9, 581–593.
Thurlings, R. M., Vos, K., Wijbrandts, C. A., Zwinderman, A. H., Gerlag, D. M., &
Tak, P. P. (2008). Synovial tissue response to rituximab: Mechanism of action and iden-
tification of biomarkers of response. Annals of the Rheumatic Diseases, 67, 917–925.
Todd, I., Pujol-Borrell, R., Hammond, L. J., Bottazzo, G. F., & Feldmann, M. (1985). Inter-
feron-γ induces HLA-DR expression by thyroid epithelium. Clinical and Experimental
Immunology, 61, 265–273.
Wilson, W. A., Gharavi, A. E., Koike, T., Lockshin, M. D., Branch, D. W., Piette, J. C.,
et al. (1999). International consensus statement on preliminary classification criteria
for definite antiphospholipid syndrome: Report of an international workshop. Arthritis
and Rheumatism, 42, 1309–1311.
Youssef, S., Stuve, O., Patarroyo, J. C., Ruiz, P. J., Radosevich, J. L., Hur, E. M.,
et al. (2002). The HMG-CoA reductase inhibitor, atorvastatin, promotes a Th2 bias
and reverses paralysis in central nervous system autoimmune disease. Nature, 420, 78–84.
CHAPTER TWO
Mechanism of Diapedesis:
Importance of the
Transcellular Route
Marie-Dominique Filippi1
Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Research Foundation,
Cincinnati, Ohio, USA
University of Cincinnati College of Medicine, Cincinnati, Ohio, USA
1
Corresponding author: e-mail address: marie-dominique.filippi@cchmc.org
Contents
1. Introduction 26
2. Leukocyte Interactions with the Endothelium 28
2.1 Docking Structures and Crawling 29
2.2 Leukocyte Invasive Protrusions 32
2.3 Paracellular Diapedesis 33
2.4 Transcellular Diapedesis 34
3. Signaling Mechanism 36
3.1 Signaling in Leukocyte Transmigration 36
4. Factors Favoring Transcellular Migration 45
4.1 The Role of the Endothelium Environment 45
4.2 Neutrophil-Intrinsic Control of Transcellular Migration 46
5. Why Does Transcellular Migration Exist? 46
6. Conclusion 47
Acknowledgments 48
References 48
Abstract
The neutrophil transmigration across the blood endothelial cell barrier represents the
prerequisite step of innate inflammation. Neutrophil recruitment to inflamed tissues
occurs in a well-defined stepwise manner, which includes elements of neutrophil
rolling, firm adhesion, and crawling onto the endothelial cell surface before trans-
migrating across the endothelial barrier. This latter step known as diapedesis can occur
at the endothelial cell junction (paracellular) or directly through the endothelial cell
body (transcellular). The extravasation cascade is controlled by series of engagement
of various adhesive modules, which result in activation of bidirectional signals to neu-
trophils and endothelial cells for adequate cellular response. This review will focus on
recent advances in our understanding of mechanism of leukocyte crawling and diape-
desis, with an emphasis on leukocyte–endothelial interactions and the signaling
Advances in Immunology, Volume 129 # 2016 Elsevier Inc. 25

http://dx.doi.org/10.1016/bs.ai.2015.09.001
26 Marie-Dominique Filippi
pathways they transduce to determine the mode of diapedesis, junctional or non-

junctional. I will also discuss emerging evidence highlighting key differences in the
two modes of diapedesis and why it is clinically important to understand specificity
in the regulation of diapedesis.
1. INTRODUCTION
Neutrophils are the first line of cellular defense against invading
microorganisms and play a central role in innate immunity and inflammatory
processes (Ley et al., 2007; Phillipson & Kubes, 2011). These white blood
cells circulate into the blood stream but must cross the endothelial barrier to
reach inflamed tissues. This rapid migration from the blood to site of infec-
tions is critical for pathogen elimination and tissue repair in response to acute
inflammation. However, when uncontrolled, excessive accumulation of
activated neutrophils into tissue leads to tissue damage during hyper-
inflammatory disorders, including acute lung injury, multiple organ failure
syndrome, vascular inflammation, or arthritis.
The initial step of the inflammatory response is a reorganization of the
endothelial cell surface to capture floating neutrophils. The release of inflam-
matory cytokines and bacteria-derived peptides stimulates the upregulation
of adhesive molecules, on the endothelial luminal surface, which locally pro-
mote weak and transient adhesive interactions between neutrophils and the
endothelium, known as “rolling.” The deposition of chemokines on the
endothelial luminal surface, then, triggers the activation of leukocyte
integrins that promote their firm adhesion and arrest via interactions with
their ligand counterpart expressed on the endothelial surface. Subsequently,
the activated neutrophils further respond to chemokines and undergo a
drastic cell shape change from round to flat and highly polarized—defining
a cell “front” and “rear or uropod.” The adoption of this polarized shape
permits the cells to migrate or crawl on the endothelial lumen surface to
find a nearby site to cross the endothelial cells lining the blood vessels. This
latter process is called diapedesis. Once passed the endothelial barrier,
the cells must cross the pericyte layer within the venular basal membrane
before to reach the inflamed interstitial tissues. This sequence of events rep-
resents the paradigm of the extravasation cascade and is summarized in Fig. 1
(Ley et al., 2007; Muller, 2011; Nourshargh et al., 2010). Although
long debated, it is now accepted that leukocytes can breach the endothelial
barrier by two distinct routes (Carman et al., 2007; Feng et al., 1998;
Another random document with
no related content on Scribd:
evade the glance of those cool, grey eyes, trusting as they seemed.
The firm mouth and the set of the jaw told of a character that would
not be amenable to sudden panic. Captain Marven was obviously a
soldier. How could it be that he was carrying despatches for the
Foreign Office? What if he were mistaken? The thought startled the
young man from his reverie to hear his query answered.
Captain Marven was speaking to him, and he had missed the
opening remark. "It's unusual for me to find one of our own set as
companion on my frequent journeyings," he said, "though, of course,
one does run up against friends, occasionally."
"You travel a lot then?" asked Guy mechanically.
"It is the business of a King's messenger," answered Marven
drily.
"I didn't know," remarked Guy in genuine surprise, while the
thought flashed into his mind that the despatches must be important
indeed, since they had not been entrusted to the care of one of the
ordinary officials of the Foreign Office. "I had no idea that you
belonged to that select body."
"Nearly twenty years in the service, my boy," answered Marven.
"If I were a motor-car my mileage would be considered something
extraordinary, but being only a man——"
He ended the comment with a laugh.
Guy echoed it.
"I understood that you were——" He was about to add, "a man
about town like myself," but checked himself. Marven laughed and
finished the sentence. "A good-for-nothing idler like yourself, eh,
Hora? No," he continued. "I was once. Indeed, to my eternal regret, I
left the army when I ought to have been thinking seriously of it as a
profession. But I had everything I asked of life then, and I rather
chafed at my duties. Later"—a shadow passed over his face—"I felt
a need to do something which would keep me away from thoughts
which—I wanted some work with movement in it, and, having plenty
of influential friends, I found myself a superior sort of postman."
Almost the elder man had been betrayed into a confidence. Guy,
realising this, again felt a thrill of distaste for the task he had in hand.
Then he wondered if the thoughts from which the King's messenger
desired to escape were in any way connected with the events which
were responsible for the Commandatore's hatred. After all, this man
was the enemy of his, Guy Hora's father. The enemy through whose
actions Lynton Hora had found himself in conflict with the world. Guy
hardened his heart. Most possibly the genial mask of friendship
which Captain Marven wore was the ordinary disguise of hypocrisy.
Guile should be met with guile.
It was with a manner as frank and a smile as confiding as his
own that Marven found his questions responded to.
Indeed, he was genuinely interested in the young man. He had
already discovered at their first meeting that he was of an
intelligence beyond the ordinary. He had observed that Guy had
been strongly attracted by the niece whom his wife was chaperoning
for her first London season, and he thought that it would be well if he
could know more of her admirer. But, beyond all that, Captain
Marven had observed what Guy's younger eyes had missed. He saw
in the younger man something which reminded him of himself in his
own youthful days. A likeness of feature, of carriage, of manner. The
belief that one day the son, his only child, would be restored to him
had been an abiding one. It had been partly responsible for his
adoption of his present profession. Somewhere his son was growing
into manhood. Any day he might meet him.
He made the most of his wide knowledge of the world to secure
the young man's confidence, and flattered himself that he had
succeeded. He was quite unaware that Guy, though wondering at his
curiosity, was responding to his advances in furtherance of his own
objects. Guy talked freely of his boyhood. He spoke with filial
affection of his father, and of the mother he could just remember,
and, as he revealed the particulars of his parentage, Captain
Marven's half-formed hopes withered, and he told himself that his
expectancy had given to the young man a family likeness which had
no existence outside his own imagination. So the train rushed on
through the night, bearing father and son, sitting face to face, yet
unrevealed, and while the son plotted to rob his father of the
despatches which honour bade him guard with his life, the father
looked at him again and again, saddened with the thought of another
hope shattered.
The train sped on, stopping now and again to pick up the mails.
Conversation languished. Guy had decided on his course of action.
He determined to try a plan which if unsuccessful, would leave him
time for a second attempt, a plan, the feasibility of which, he had
frequently discussed with his mentor, Lynton Hora.
He surprised Captain Marven in a yawn. "Getting sleepy?" he
asked.
Marven smiled. "I don't sleep to-night until my journey is ended."
"What martyrdom to duty," scoffed Guy. "I am almost inclined to
save you from yourself."
Marven looked at him questioningly.
"By demonstrating the power of hypnotism," explained Guy
briefly.
"Oh!" Captain Marven laughed. "I am afraid you would not find
me an amenable subject," he said.
"I don't know," replied Guy. "It is astonishingly easy to induce
hypnotic sleep."
Marven smiled doubtingly. He held the belief that hypnotism was
a mere vulgar device of charlatans to impose upon the gullible. He
expounded his views to Guy at length. The young man, stimulating
opposition, by assertion and counter-assertion, at last declared that if
Captain Marven would only lend himself to the experiment he would
speedily prove to his satisfaction that there was substantial grounds
for belief in the realty of hypnotic force.
Captain Marven, serenely satisfied of his invulnerability and
amused at Guy's obstinacy, professed his willingness to submit to
any experiment Guy might suggest. Guy appeared to hesitate at the
prompt acceptance. He declared that he was unprovided with the
necessary materials. Marven chaffed him, seeing in his reluctance
doubt in his own beliefs. Guy remembered that he had in his
handbag a small electric torch with a reflector attached which might
serve the purpose of the mesmeric disc commonly used. He
produced it.
Marven, lounging comfortably in his corner, was bidden to divest
his mind of all thought and gaze intently upon the glowing point of
light. With a keen sense of the absurdity of the proceeding, the
King's messenger conscientiously endeavoured to obey the
instructions given him. Silence reigned in the compartment, for two,
three, four minutes. Then Guy rose from his corner, and stood over
his companion, every nerve quivering with the intensity of his
purpose. He laid his hand lightly on Marven's head.
"You are beginning to feel sleepy," he said.
Marven did not contradict him. Already his brain had wandered
far afield from the thoughts which had employed it when he had first
fixed his gaze on the brilliant point of light. He had forgotten that he
was the subject of an experiment. He was dreaming again, dreaming
that his son was found, and that they were going home together. It
was such a pleasant dream that he would not raise his eyes lest it
should be shattered, as such dreams had always been shattered
before. Perhaps he was feeling sleepy. Well, what of it? Surely there
was no reason why he should not sleep?
"You are feeling very sleepy," said Guy. He was watching
Marven's eyes closely, and observed that their lids drooped heavily.
"You will not keep awake much longer," he said.
The point of light seemed suddenly enlarged. It filled Marven's
field of vision. No, he would not be able to keep awake much longer.
"Yes, I am very sleepy," he replied. The voice was mechanical.
Guy's heart bounded triumphantly. His experiment was
succeeding beyond his wildest anticipations. At the most he had
hoped to be able to gain the opportunity to instil a suggestion into a
half-drugged consciousness, which would facilitate his obtaining
possession of the despatches, but now it seemed that he was on the
point of entirely subjugating his companion's consciousness.
"You must close your eyes," he said.
Marven obeyed.
"Stand up," commanded Guy.
Marven raised himself to his feet, but his eyes remained closed.
"You cannot lift your right arm," said Guy.
Some remnant of will yet remained. The hypnotised man strove
to raise his arm. But the effort was a failure, the arm half-lifted from
the elbow dropped again.
"Sit down and sleep comfortably until I awaken you,"
commanded Guy.
Marven obeyed. He lay back in his corner. His eyes were
closed, his breathing calm and even. To all appearances he was
asleep. Guy seated himself and gazed at the man opposite. Was his
experiment indeed successful, or was Marven merely shamming with
a view of ridiculing his efforts? He inclined to the former belief, yet
the ease with which Marven had succumbed was in itself suspicious.
It would be easy to apply a test.
Guy rose, and, drawing aside the overcoat which Marven wore,
deliberately unbuckled the strap upon which the despatch case was
slung. The Captain never stirred. He was really asleep. Guy looked
at his watch. In another ten minutes the train would be stopping at
Cambridge. Yes, he had just time to carry out the initial stage of his
plan. From his handbag he produced a mass of stiff modelling clay,
and with it he took two impressions of the seals which secured the
fastenings of the case. Then he placed the case and the impressions
in his own bag. Already he was beginning to enjoy the
consciousness of success.
The train ran into Cambridge railway station. Guy leaned over to
his sleeping companion.
"You must have a cup of coffee if you are to keep awake," he
said.
The Captain's eyes opened, but they were dull, unseeing.
The train stopped. Guy alighted and Marven followed him to the
refreshment room. Guy ordered coffee. Marven drank his
mechanically.
"We must return," said Guy. "The coffee will not keep you awake
long."
The guard was standing at the door when they re-entered their
compartment, and he closed it deferentially. Evidently he was aware
of Captain Marven's identity. Guy was glad that he had taken the risk
of awakening Marven; otherwise the railway official's acquaintance
might have proved troublesome.
The train rolled out again into the night.
"You will sleep again," said Guy soothingly.
The Captain composed himself in his corner, and Guy forgot
him. Seconds only passed before the despatch case was open
before him, seals broken, lock picked, and the papers it contained
lying in his hand. He groaned as he saw the voluminous character of
the document. Then his eyes brightened as he caught sight of the
précis of the contents attached. A fountain pen and paper were at his
hand, and a copy of the précis was soon in his possession. Then
watch in hand he read the more lengthy despatch. Ten minutes had
passed since leaving Cambridge and another ten would bring the
train to Ely. He returned the papers to the case. With deftness, born
of much practice, the lock was turned again. With a spirit lamp the
wax of the seals was melted and the seal reimpressed by aid of the
carefully oiled clay impression already hardened sufficiently for the
purpose. With a sigh of satisfaction Guy laid the case on the seat.
But the sigh died away. Looking up he was astounded to find that
Captain Marven had awakened, and was standing erect and with
outstretched hand was pointing to the case.
There came upon Guy the impulse for instant flight. But whither?
The impulse did not remain long in possession of him. He saw that
Marven was striving to speak and could not find words, and at the
same moment he realised that his victim was only partially
awakened from the hypnotic state. Collecting all his faculties for a
supreme effort Guy faced him.
"You cannot stand up," he said. "You cannot awaken for two
minutes yet."
The Captain hesitated. He seemed to be fighting against an
overpowering desire. But insistently Guy repeated his commands,
and with a despairing gesture, Marven sank back once again into his
corner.
Guy waited no longer. The moment Marven was quiescent, he
rebuckled the case to the strap, and disposed it beneath the
Captain's coat. Then he clapped his hands smartly before the
sleeping man's face and cried loudly, "Wake up, Captain Marven,
wake up."
Marven came back gradually from his trance. He rubbed his
eyes, he looked wonderingly about him.
"What have you to say about hypnotism now?" asked Guy.
Captain Marven looked puzzled.
"I—I don't quite understand. Do you mean to say that I have
slept?"
The train ran into Ely Station.
"Hullo! Where are we? At Cambridge already?" asked Marven.
Guy laughed. "This is Ely," he answered.
"Ely?" repeated the Captain incredulously.
"Yes, Ely," answered Guy. "Don't you remember stopping at
Cambridge and going with me to the refreshment room for a cup of
coffee to keep you awake?"
"Stopping at Cambridge," repeated Marven more incredulously
than before.
"It is a fact," answered Guy, and he added, "I hope you will
forgive me having given you so practical an exposition of the fact
that there is something in hypnotism after all."
Captain Marven did not answer immediately. He seemed to be
struggling for recollection. "I have no remembrance of that, but——"
By an instinctive gesture his hand sought the case beneath his coat.
His face lightened as he felt it there. "I've been dreaming,
nevertheless," he said. "I seem to remember that something in my
charge was in your possession, and that although I struggled to
regain possession of it I could not do so."
"I suppose it was the last impulse of your will to combat the
effort I was making to gain control of it," remarked Guy, secretly
congratulating himself that he had been in time to prevent Marven's
complete awakening before the case had been returned to him.
"No doubt that is the explanation," replied Marven, rising and
throwing open the carriage door. He stepped on to the platform.
"Are you not going to stretch your legs, Hora?" he asked. "We
have twenty minutes here."
Guy joined him, and they paced the platform together. They
chatted on indifferent topics. Then Captain Marven suddenly sprang
a personal question.
"By the way, Hora," he said, "if it's not impertinent to ask, what
made you choose this infernally slow train to come to this part of the
world?"
Guy had long had a lie prepared.
"Laziness," he answered lightly. "I missed the earlier one which
would have taken me on to Hunstanton, where I have an
appointment to-morrow morning. I found that I should be able to get
on from Lynn in time to keep it, and so here I am. I'm glad I missed
the fast train, as it has happened. I hate travelling alone."
Captain Marven made no reply. Guy could see that he was not
satisfied, but he gave no hint that he had observed anything, and,
when the journey was resumed, he kept up an easy flow of talk until
Lynn was reached.
Then he bade Captain Marven good-bye, and, if the King's
Messenger had any suspicion regarding the companion of his
journey, there was no hint of it conveyed in his parting greeting.
CHAPTER VII
MERIEL MAKES AN IMPRESSION
Captain Marven was certainly not at ease in his own mind in

regard to the experience on his journey. He seemed to remember
awaking, and seeing in Guy's possession the despatch box that he
carried. Yet in view of the fact that it was still buckled to the strap, the
suspicion seemed absurd. As soon as he was alone he carefully
examined the despatch case. The seals were to all appearances
intact. He missed none of his personal belongings, and was
ashamed of the suspicion which prompted his hurried search
through his pockets. Really he had no reason to suspect Guy Hora
of any ulterior object in hypnotising him. He recalled all the
circumstances. At his own invitation Guy had travelled with him, he
himself had pressed his companion to make the experiment. He had
lost consciousness while Guy was sitting opposite him, and when he
had regained it, Guy was still opposite him, and certainly did not
exhibit the slightest trace of trepidation. But what had happened in
the forty minutes interval which had elapsed between the losing and
regaining of consciousness. Guy had told him that they had alighted
at Cambridge and had drunk a cup of coffee at the refreshment
room. But was that all? He could not banish the thought of the dream
which had come to him during that hypnotic slumber, the vision of
Guy Hora bending over the despatch case doing something to the
seals, and of himself standing powerless to interfere with him. Was it
only a dream? Yet it were foolish to suppose otherwise. What could
the despatches he carried contain of interest to the wealthy young
man about town, who seemed to him so like what his own son might
have been? It was a puzzle which he could not elucidate, though it
engaged his attention during his long motor drive to the royal
residence; it was present in his thoughts when he placed the case in
the hands of the Secretary of State, who, warned by telegraph of his
coming, was waiting up for him, and he could not escape it even
when he himself sought repose. It was the first thought in his mind
when he awoke the next morning.
If he had been gifted with clairvoyant powers, he would not have
needed to flog his brains for a solution. Could he have seen Lynton
Hora gloating over the transcription of the cypher telegram which
had reached him that morning from Guy, Captain Marven would
have had an inkling of the truth. Could he later have seen Lynton
Hora sending off wires to brokers at Paris, Berlin, and New York,
could he have heard Lynton Hora instructing his London stockbroker
over the telephone, he would have been still nearer a solution.
Finally, could he have heard the Foreign Secretary's remarks later in
the day, when a detailed account of an unfortunate incident in the
South Pacific, in which a German gunboat and a British cruiser were
concerned, appeared in an evening paper, he would have known
that in some mysterious manner the information contained in the
despatches he had carried had become known to the world.
Yet, though he knew not, nor could have known, any of these
things, yet the vague uneasiness that there had been intention in
Guy's presence on the train was not easily dispelled. It begot in him
a still greater desire to learn something more of the young man
whose face haunted his thoughts, and when he returned to town the
next morning, carrying with him the answers to the stolen
despatches, it was with the fixed intention of cultivating Guy's
acquaintance, if the opportunity offered.
The chance he desired was not long presenting itself. A week
had not elapsed before they met again. The meeting was a purely
casual one. They recognised each other at the opera. Guy occupied
a stall and the Captain was with his wife and niece in a box. At the
end of the first act Guy left his seat. The Captain met him in the
foyer.
"Been trying any more experiments in hypnotism?" was
Marven's greeting.
Guy shook his head and laughed. "I haven't been upon any
railway journeys with unbelievers," he replied jestingly. He met the
Captain's glance fearlessly, and again Marven felt ashamed of his
suspicions.
"I was just coming to pay my respects to Mrs. Marven and Miss
Challys," continued Guy.
The Captain turned to accompany him. "You need not tell the
ladies how easily you bowled me out," he remarked. "I have been so
blatantly cynical on the subject always that I should never hear the
last of it."
"Why, certainly not," answered Guy, smiling. The Captain's
attitude dispelled the last chance of his being connected in any way
with the acquisition of the information which, when it had been made
public, had created the most intense excitement. Clearly the Captain
wished no one to know that he had allowed himself to be placed in
an hypnotic trance while on duty, and Guy, for his own protection,
was more than ready to fall in with the suggestion to keep silence
concerning the episode.
"After all, it is a purely personal matter of absolutely no concern
to anyone but our two selves," he added, as they reached the box.
Conveying his thanks with a nod, Marven opened the door, and,
as Guy entered, all thought of the Captain passed from his mind.
Only once previously had he met Meriel Challys, but the impression
which she had produced upon him was one that would not easily be
eradicated. He had first seen her floating towards him on some
booby's arm at Lady Greyston's dance. Who the booby was and
what the booby looked like Guy had not the faintest idea. He had no
time to spare a glance for anyone else, once his eyes had rested on
her face. But the man on whose arm she leaned so lightly must of
necessity be a booby. Any mere man could be nothing but a booby
in comparison with any personality so exquisitely ethereal as that
given by nature to Meriel Challys. Guy had determined to take the
place of that booby himself. He had enquired amongst his friends
until he had obtained the introduction he desired. He had received
one waltz and he had sat out one square dance in the plumbago
bower at the end of the long gallery. He would never forget the fair
picture she made, draped in maiden garb of white, her dazzling skin
and hair framed in the tender blues of the drooping blossoms. It had
been an effort for him to speak to so distractingly beautiful a subject,
and it had been only the fear lest she should think him moonstruck
which had forced him to unlock his lips. No wonder that the
despoiling of Mr. Flurscheim's treasure-house had been postponed.
Indeed, had the treasures been spread before him at that moment,
Guy would have passed them by unnoticed.
That first impression of instinctive reverence had not endured. A
fervid disciple of Hora's cynical philosophy could not remain for long
in a state of ecstatic adoration of one of the sex provided "for the
relaxation of the warrior."
But now, entering again into her presence, his first impression
was revived with redoubled intensity. Again he saw her coming
towards him. He remembered the Tennysonian line which flashed
into his memory at first sight of her:
"Robed in white samite, mystic, wonderful."
No, that did not do justice to Meriel Challys, and yet it might,
perchance, describe her in some moods, he thought; and though
there was nothing mystic in her mood when Guy had been
introduced, yet the swift impression was mayhap nearer the truth
than the later one.
Mrs. Marven greeted him warmly. They chatted a little while,
then, as Guy turned to the girl, she became a silent observer. The
family likeness which her husband had observed in Guy was equally
apparent to herself, but she had not given words to the vague
thoughts which had risen up in her mind when she had first seen him
at Lady Greyston's dance. Such thoughts were best crushed down at
once—they only invited disillusionment. Not for worlds would she
have suggested to her husband that the young man had awakened
in her the crushed down maternal instinct. The mere hint would have
made him uneasy. She even dreaded lest he should have
recognised for himself certain physical resemblances which certainly
existed between her husband and Guy. She had no idea that he had
recognised them, and was as loth as her husband himself to give
words to her imaginings, for precisely the same reasons which
imposed silence upon him. Each in point of fact had felt the
promptings of the parental instinct, and each in tenderness for the
partner of his and of her sorrows and joys forebore to awaken by a
word the memory of the great sorrow which had early cast its cloud
over their married life.
Lynton Hora had always been hopelessly wrong when he had
imagined that but for the rivalry of Marven he would have won the
affection of Beatrice Challys. So far as he was concerned she had
always been heart-whole. But from the day she had met Dick
Marven there had only been one mate for her in the whole wide
world. The affection had been mutually bestowed. Marven lived for
his wife, even as she lived for him. In each of their thoughts the one
object for consideration was their alter ego. Marven would have
sacrificed anything to save his wife a momentary pang, physical or
mental. She would have inflicted any torture upon herself that he
might be spared even momentary uneasiness. But this kindly
consideration for each other's feelings did not always prove
unexceptionable in practice. Impalpable, diaphanous, there was a
barrier of reserve ever between them from the day their first born
and only child had been mysteriously spirited away. In so far Hora's
scheme for revenge had been successful in action. It had shadowed
the lives of both the parties upon whom his blow had fallen, and,
though in tender consideration for each other, his action had brought
them closer together, yet it had also operated in placing between
them the barrier of a subject they dared not discuss.
The maternal instinct, deprived of its natural outlet, of necessity
had found another vent. Robbed of her son, Mrs. Marven had taken
to her heart a brother's child, who had been orphaned early in life.
The girl had repaid the devotion lavished upon her, and, if Mrs.
Marven could never quite banish the regret for her lost son, she
would always have heartily admitted that Meriel Challys had brought
sunshine again into her life. Meriel was all that a daughter could be,
and a very tender sympathy existed between her and her aunt.
Naturally the latter could not have failed to observe the
admiration which Guy Hora exhibited toward Meriel. It was patent in
his every glance, and he made no effort to conceal it. He
approached Meriel with the proud assurance of the man who feels
that he has the right to lay the tribute of his admiration at the feet of
the woman whom he admires, and is not ashamed of letting the
whole world perceive upon whom his choice had fallen.
Mrs. Marven liked the attitude, which was so different to that of
many of the admirers whom Meriel had already, in the course of her
first season, gathered in her train. It awakened memories, too, of the
days when Richard Marven had wooed her. His face had worn the
same unconscious air of adoration. Almost Mrs. Marven's heart
stood still, as she watched Guy's face as he stood leaning forward
looking into Meriel's eyes, answering her smiles with his own; the
likeness was so vivid to the memory she bore in her heart. She
would have liked to step forward and claim him. Her heart cried out
"My son, my son," but the words died on her lips. She crushed them
back, though the effort left her lips pale.
Meriel, looking towards her, noticed the pallor and with a hasty,
"You are ill, Auntie," was at her side.
"I find the theatre a little close," murmured Mrs. Marven. "If we
might have the door of the box open a little." She refused all
suggestions that they should leave the opera house, and, before the
attempts to persuade her were completed, the curtain was rung up,
the lights were lowered, and the opera proceeded.
Guy remained in the box. Mrs. Marven sat back in the shadow
studying his face intently. When the second act ended, she had
regained full control of her emotions, and could make light of the
supposed indisposition with which she had apparently been
attacked. She pressed Guy to remain in the box for the remainder of
the evening, and he was nothing loth to accept the invitation.
There, then, he remained, unconscious that he was the centre
of interest to the two elder members of the party, unstirred by any
instinctive emotion towards them, and with every thought, every
faculty, strained to make the most of the flying minutes which gave
him Meriel's company. He was unable to solace himself with the
assurance that she responded to the emotions which were aroused
in himself by her presence. She was purely natural, no spice of
affectation spoiled her glance or her smile. Indeed, at times she
seemed to be unconscious even of his existence, enthralled in the
melody of the music, responsive only to the rhythm of the throbbing
strings. Guy could hardly understand her rapture. Romeo et Juliette,
even with world-famed artists to sing the title rôles, had already
ceased to awaken him to passionate wonder. The naïveté of the
girl's enjoyment surprised him, and he wished that she might for
once have forgotten the music for him.
Yet, if he had possessed the power to peer into the girl's heart,
Guy would have found that his own image was already stamped
there; that it was his presence which was the inspiration to enjoy;
that, unknowingly, imperceptibly, the tiny seed of love had been
planted in her maiden bosom, needing but the passage of the hours
before it should spring up into the perfect plant of a pure woman's
first love. But, though Meriel knew not of the implantation, she was
conscious of the additional thrill in her delight. The whole world was
now become perfect to her. She thought the sense of beatitude was
the product of the sensuous melody of the opera.
There was no discordant note anywhere. The evening promised
to pass in joy unalloyed. A fly dropped into the ointment. She looked
up and met the glance of Hildebrand Flurscheim. To her it was at first
the mere casual glance of a complete stranger from a near box. But
there was something compelling in it. When she glanced in his
direction again he was still gazing steadily at her. She drew herself
back into the shadow. But the exhilaration of spirit had passed. She
felt compelled to look again in the direction of the stranger. He was
still watching. When the act was ended, she turned almost petulantly
to Guy.
"Can you tell me who that man is? The Jew staring at us so
persistently from the fifth box on the right?" she asked.
Guy glanced in the direction she indicated, but before he
answered Captain Marven spoke:
"The fifth?" he said. "That's Flurscheim, the chap who lost the
Greuze the other day. It's reported," he added meditatively, "that he
has sworn that he will devote the whole of the rest of his life if
necessary to tracking down the rascal who has robbed him of his
picture."
There was a smile on Guy's face as he answered: "He doesn't
expect to find it at Covent Garden, does he?"
Despite the smile, however, he was conscious of a vague sense
of uneasiness at the persistent scrutiny which Flurscheim was
bestowing upon the box. There are times when the fear of being
found out bears a marvellous similarity to the prickings of
conscience, and certain inward twitches which he felt made him
supremely uncomfortable. Hitherto he had always been able to
justify his actions by means of Lynton Hora's philosophy, but now he
realised in a flash that he would not longer be satisfied by
justification in his own eyes. Could he justify his deeds to the
satisfaction of the pure-browed girl by whose side he stood? Would
she accept the doctrine that since virtue consisted in a thirst for
danger and a courage for the forbidden, the highest virtue was to be
found in the breaking of laws? He knew instinctively that she would
not, that in her eyes he would appear no heroic figure engaged in
single combat against a host of enemies, but the despicable figure—
a furtive, cringing, creeping figure—the despicable figure of a thief.
He left the box with his thoughts in a turmoil. He scarcely
observed the particular warmth of the farewells with which both Mrs.
Marven and Captain Marven took leave of him. He hardly noticed
that Meriel's hand had lingered a moment in his grasp. He felt that
he must be assured that Flurscheim had not been watching him.
CHAPTER VIII
A SUCCESSFUL SPECULATION AND ITS RESULT
Lynton Hora felt that Fate had dealt generously with him when it
made Captain Marven the bearer of the despatch case which Guy
had so ingeniously rifled of its secrets. Guy's success had supplied
him with all the information necessary to bring off a magnificent coup
on the Stock Exchange. He had speculated heavily for the fall in the
securities of both countries. He knew that publication of the
information he had in his possession would make his operation
successful. He had not scrupled to publish that information, though
its disclosure before its discussion between the chancelleries of the
two nations concerned brought two great powers to the verge of war.
It found its way into the newspapers, and while the press of two
countries breathed defiance, Hora laughed in his sleeve, cynically
declaring that no farce is equal to the farce of civilisation, since
nations are as ready to fly at each other's throats as any street curs.
And while the newspapers snarled and patriots in beer houses and
at street corners sang national anthems, Hora watched the prices of
securities, and when the crisis reached its acutest stage, he bought
in his bear and found himself possessed of what to the majority of
men would have been a sufficient provision against the future.
But Guy's successful action had done more than lead to the
swift accumulation of a fortune. Hora had not for a moment
anticipated the long arm of coincidence would be stretched to such
purpose as to make the father the bearer of the despatches which
the son had stolen. It was the crowning stone in the carefully built
arch which was to bear the superstructure of his revenge.
Hora felt almost superstitiously inclined regarding the
coincidence. He did not learn of the identity of the messenger until
Guy's return from Lynn, and, as soon as it was made known to him,
he saw how the fact could be made to serve his purpose. Naturally
he said nothing of that ulterior purpose to either Guy or to Myra. He
was merely full of praises for his pupil. He declared himself to be so
satisfied that he expressed his intention of making the young man
master of his own actions for the future. "You have enabled me to
provide for myself, Guy," he said. "It is now my turn to provide for
you, so that if at any time you desire to retire from your profession
you will be at liberty to do so."
Guy had ridiculed the suggestion, but Hora had insisted. "Who
knows what may happen in the future," he said. "It will be well for
you to have money at your own disposal. You may quarrel with me.
To-day sons always quarrel with their fathers. Sometimes they rob
them, and if fathers were wise they would always see that their sons
are spared the necessity."
Guy laughed. He felt that he was not of the commonplace world
of sons who robbed their fathers, but he gave way to Hora's whim.
He instructed a broker on his own account, though the operation was
not in his own name. Hora had suggested that it would be just as
well if for the occasion Guy borrowed another surname. So the letter
of introduction he forwarded to Hora's broker introduced "my young
friend Guy Marven, upon whose information I am acting, and who,
wishing to speculate on his own account, I am prepared to guarantee
up to £10,000." Accordingly it was as Guy Marven that he signed in
due course the transfer notes of stock which came to hand, and it
was with the name of Guy Marven that he endorsed the cheque
which he paid into his own banking account as the result of the
speculation.
He had wondered that Hora should have selected the name of
Marven and had even suggested the inadvisability of making use of
it since Jones, Brown, or Robinson would have been equally useful if
the necessity existed to use another than his own. To him it seemed
that the use of the name Marven might excite remark. "Exactly why I
wish you to use it," Hora had answered. "If anyone who was aware
that Marven carried those despatches should hear that a Marven
had been speculating for the fall, it would not be remarkable if they
should arrive at the conclusion that he had looked inside his case
himself."
"That seems rather rough on Captain Marven," remarked Guy. It
seemed a mean deed, and Hora had not trained him to take delight
in mean deeds.
"Isn't he my enemy, and therefore yours? You have no reason, I
suppose, for treating him with more consideration than all the rest of
the world?" demanded Hora.
Guy did not answer. He was tempted to reply with a direct "no,"
but he had never yet lied to Hora. Yet he could not bring himself to
confide to the Commandatore the reason why he would have
extended consideration to Captain Marven. He had no wish to hear
the Commandatore's biting cynicisms applied personally to Meriel.
The idea seemed sacrilegious. He was relieved to find that Hora
made no comment on this silence. But the Commandatore had not
failed to observe it, and remembering at the same time how Guy had
nearly let slip the opportunity for securing the Greuze in the
seductive companionship of these same "pleasant people," he
realised that the time had come for him to discover what the
attraction was. Of course he guessed. The miniature set in the snuff-
box which Guy had expressed a desire to retain had not escaped his
notice.
"The inevitable feminine," he said sneeringly to himself. But he
knew that the inevitable feminine has always to be reckoned with
where a young man is concerned, and generally where an old man is
concerned also. It behooved him to know something of this new
factor, which might materially affect his plans. But Guy must have no
suspicion that he was under observation. Hora's mind jumped to an
expedient whereby his object would be secured without subjecting
himself to inconvenience. He determined to make the suggestion
which had occurred to him forthwith.
"There's another matter which I have been giving thought to,
Guy. Don't you think that it is about time you took some chambers for
yourself?"
Guy was more than startled by the suggestion thus sprung upon
him. He had always been left such perfect freedom that there had
never been the slightest temptation in the thought of possessing a
domicile of his own.
"Why, Commandatore?" he asked. "I am perfectly comfortable at
home here."
"You will always be at home here, I hope," replied Hora, "but at
the same time there is much to be said in favour of the course I am
proposing. Now listen, Guy. I will say nothing of the extra freedom
which will be yours."
"I could not have more than I enjoy at present," remarked Guy.
Hora shrugged his shoulders. "Men are men," he said. "You may
be having visitors who might hardly like to come here. Eh?"
Guy smiled.
"I see it is possible," continued Hora, smiling in his turn. "Then
there are others whom I might not wish to meet. The men of your
own years with whom you mix, with whom you must mix if you are to
keep your position, will probably be often dropping in on you. I am
getting of an age, Guy, when I might find their presence, I will not say
distasteful, but just a little wearisome. At chambers of your own you
will be much freer in that respect."
"There are my clubs," objected Guy.
Hora waved the objection aside.
"Then there is the additional reason of our joint safety to be
considered," continued Hora meditatively. "It seems to me that if you
are located in a place of your own we shall be provided with another
strategic centre from which to carry on operations. You see it might
be important for the safety of both or either of us that we should not
be living under the same roof."
"Certainly, I see your point there," replied Guy, and he became
thoughtful.
"Think it over, Guy," said Hora, and he left the young man.
Guy thought it over, and was surprised when he did so that the
idea proved so attractive. It was not that he was ungrateful. He could
not believe that the daily companionship of Hora was becoming
distasteful, and yet there was certainly relief in the thought that he
should be apart from the man whom he called father. How greatly
this thought was due to the impression that Meriel Challys had
produced upon him he did not appreciate, but the knowledge that if
he were settled in a home of his own he might perhaps escape
taking part in any plans for revenge which Hora might be weaving
about Captain Marven, was certainly a powerful consideration.
When, therefore, Hora broached the subject again after dinner
that night, Guy was quite prepared, nay, even eager, to fall in with his
views. Hora was more than ever convinced that some unknown
factor was influencing his adopted son.
The only voice raised in protest was Myra's.
"You—you are going away? Why?" she asked.
There was a tremor in her voice which made Hora intervene
hastily.
"It is my wish, Myra," he said. The woman shivered at the
menace of his tone. But it produced the result Hora desired. She
quelled the emotion that struggled for utterance, and listened in
silence while Hora reiterated the reasons which he had already given
to Guy.
But though dumbly acquiescent she did not believe in Hora's
statement as to the motives which animated him, and when, after
Guy had left them, she was alone with Hora, she returned again to
the subject.
"Why are you sending Guy away?" she asked. "Is it to avert
danger from yourself?" There was scorn in her tone. Hora made no
attempt to avert the threatened emotional storm.
"Have I ever feared danger?" he asked sneeringly.
"I've never known you to face it. I don't know anything about
your feelings," she replied.
Hora looked at her. She had never dared so to speak to him
before, and he knew that she must be greatly moved to so provoke
his anger.
"You know nothing," he replied calmly. "A woman is utterly
unable to comprehend a masculine point of view."
"A woman cannot live in the same house with a man and fail to
know something of his character, and I know something of yours,
Commandatore."
He shrugged his shoulders in amused surprise.
"You find me an interesting study, Myra?" he drawled. He could
not have said anything more calculated to arouse Myra's
tempestuous spirit.
"Interesting?" she exclaimed. "I don't know about that. You don't
interest me in the least. I—I—hate you."
"Dear me!" replied Hora equably. "I might have expected you to
do so. Perhaps I have, for there's undoubtedly wisdom in the
suggestion that one does not pluck figs from thistles, though the
saying is somewhat hackneyed."
"You need not throw my origin in my teeth, Commandatore," she
replied. "I am sufficiently conscious of it, and as for gratitude—well, I
often think I should have been far better off in the slums from which
you picked me. I should have been happy enough. The precious
education you have given me has only enabled me to realise my
own impossibility. You have given me knowledge, and with it the
capacity for suffering."
"Like your mother Eve," responded Hora quietly, "you longed for
the apple and blame the serpent."
"Eve was no child, and I was a child when you gave me the
apple," she answered more quietly.
"That is true," replied Hora. "Possibly I did wrongly. I should
have left you to bloom in your own soil. You would have been
overblown by this time, Myra—some drunken ruffian's doxy—taking
your weekly beating without a whimper and seeing heaven in a
quartern of gin."
"Better that—better that—than——" She paused.
"Well?" asked Hora. She muttered something sullenly, but in so
low a tone that the words did not reach his ears.
He continued, "You were asking me why I think it well that Guy
should have a home of his own. I have given you my reasons. I
really should have thought you were intelligent enough to realise
their force."
"I don't believe in them," she flashed out. "Oh, I know you better
than that, Commandatore. I know your subtle methods. You have
some other end in view. What it is I know not, but I am sure it means
danger to Guy." She had been moving restlessly backwards and
forwards, but now she paused and faced Hora. "I tell you,
Commandatore, that if anything happens to Guy you shall not
escape. I swear it."
Despite his apparent unconcern Hora was impressed by the
latent passion in her tone. Almost he regretted that he had not left
the beautiful flower to be choked by the weeds from which he had
plucked it. But he remembered that he had one means of controlling
Myra which would necessarily prove effective. He rose from the chair
where he had been sitting. "If anything happens to Guy, I don't
suppose I shall care much what happens to myself," he replied
gravely. "Is he not my son?"
"Your son?" replied Myra. "I doubt it, Commandatore. No father
would ever have brought up a son as you have brought up Guy.
Besides, there is nothing of you in him. I know you both. There's not
a feature alike, and the difference between your thoughts and
actions and his is just as strongly marked."
"You are talking nonsense, Myra," he said harshly, far more
perturbed than he cared to confess at the discovery by which the
woman's intuition had arrived at the truth.
"You may call it nonsense," she continued wildly, "but there's no
blood of yours in Guy's veins——"
Hora checked her. He laid his hand on her shoulder, and though
she made an effort to throw off his grip he held her tightly and thrust
her down into an easy chair.
"You little fool," he said, "you don't know what you are talking
about. Like all women in love you let your imagination run away with
you."
She still struggled ineffectually to escape from his grasp while
he continued. "Suppose I had other motives than those I have given
to you and Guy for desiring that he should live apart from us for a
little while, have I ever by any chance done anything which could
bring danger upon him? Did it never occur to you that one of my
motives had connection with yourself?"
She ceased to struggle. Her hands dropped limply by her side.
"I might have known, I might have known," she cried; "and yet,
Commandatore, you promised me that I should have my chance."
Hora saw that he had won the battle. He loosed his grip of
Myra's arm and returned to his chair.
The girl rose and following him dropped on her knees before
him. The tears were in her eyes and her bosom rose and fell
stormily. "You promised me I should have my chance,
Commandatore. Don't send Guy away. I cannot bear it. Indeed—
indeed, I cannot bear it." Sobs choked her utterance.
Hora allowed her to weep awhile. Then he laid his hand gently
on her bowed head.
"Poor little woman," he said, "if you could only see an inch
beyond your own nose, you would realise that I am giving you your
chance now."
She looked up incredulously, but with a dawning hope flushing
through her tears. "I don't understand how——" she began.
"You have been too near Guy," he remarked. "He has never
realised your value. At a little distance he will be more clear-sighted.
A woman is like any other work of art. Her beauties are invisible
unless seen in proper perspective. While you live in the same house
he will never realise that you may be more than a sister."
"Then you think——" she asked.
"I am sure of it," replied Hora decisively. "It is to give you the
chance you desire that I have persuaded Guy to live apart from us
awhile, for that and in order to discover something about a rival
whom I suspect you may have."
CHAPTER IX
CONCERNING A GREAT MAN'S VEXATION
When the carriage containing the Great Man rolled up to the

door on the south side of Downing Street the constable stationed
there stiffened himself preparatory to saluting. But his salute passed
unnoticed. The door swung open, but the commissionaire got not so
much as a glance.
"There's trouble brewing in China," said the worthy hall porter to
his circle of cronies that same evening in the private bar of the "Lord
Palmerston," "you mark my words if there isn't."
"More likely to be with Germany. That's the right place to look for
trouble," asserted one of the listeners.
"Of course, you would know better than me," replied the worthy
janitor. "You read the papers every day, so you can't be wrong."
There was unutterable scorn in his tone as he referred to the press.
"And what's wrong with the papers?" enquired the interlocutor in
an aggrieved voice. "I suppose they know as much about things as
you do, any day."
The commissionaire pursed his lips, blew his nose, and finished
his beer before he found words to convey his answer.
"The newspapers! The newspapers are common liars!" he
answered, "and ought to be hanged as such. No—don't you go for to
speak up for 'em—George Jenkins—we know all about the papers in
our department."
Jenkins did not subside immediately.
"I presume Sir Gadsby takes you into 'is confidence—not to say
asks your advice occasional?" he asked sarcastically.
"He might do worse than ask the advice of a man who's fought
in h'all four quarters of the globe and 'as the right to wear six medals
and twelve bars," interrupted another listener propitiatingly.
"So he might," replied the commissionaire smiling genially, "but
that's neither here nor there. If you want to know why I say it's China,
I don't mind telling you." He glanced round to see that no outsiders
were within earshot and dropped his voice into a confidential
whisper.
"Four secretaries have I known since I went to our place, and I
studies 'em until I comes to read 'em like books, and Sir Gadsby
Dimbleby is one of the easiest volumes I've ever had to study. I know
'is German face an' 'is Russian face as well as I know my own. This
morning when he come in I could read China in 'is heye as plain as if
it were in the biggest print. You mark my words there's trouble
brewin' in China."
The oracle had spoken, and as is often the case with oracles
which have not been primed with facts, the utterance was as wide of
the mark as it could well be.
Sir Gadsby Dimbleby's brain had not been occupied in the least
degree with Chinese affairs as he passed the portals of the Foreign
Office. He was troubling about something which had happened much
nearer home, a subject which had been pigeonholed in one of the
compartments of his brain until the crisis in the relations with
Germany caused by the premature disclosure of the unfortunate
incident in the South Seas should have passed. That storm had
been safely weathered, and H. M. Secretary of State for Foreign
Affairs bethought himself of the promise he had made to himself to
discover how the information had leaked out. To him it seemed
uncommonly like treachery on the part of someone, and being by
birth, education, and natural instinct a thorough English gentleman,
the idea even of having to suspect anyone in the great department of
which he was the head of treachery was odious to him. So the Great
Man passed, heedless of the salutes of officials, to the uttermost
sanctum where the Permanent Secretary sat with his fingers on the
strings which directed British policy all over the world, peacefully
enjoying a matutinal cigar while glancing over the précis of a verbose
despatch, prepared by his own secretary, from the Governor of a
wind-swept rock in the South Atlantic concerning the deadly damage
likely to be done to British interests by ceasing to imprison a garrison
in such an out-of-the-way spot.
The Permanent Secretary looked up as the Great Man entered
and wondered. He was at a loss to understand the reason for the
knitted brow. The political horizon was clear and nothing could
happen without his knowledge. He guessed that the reason was
domestic. He knew that Lady Gadsby had a will of her own, and that
even Great Men are human enough to be annoyed by feminine
displeasure.
But Sir Gadsby soon undeceived him. He fidgetted about
uncomfortably for a minute or two, irritably asking questions about
minor matters which had recently engaged his attention before he
plunged into the subject which filled his thoughts.
"Look here, Markham," he said, "that German business in the
South Pacific is still worrying me."
The Permanent Secretary looked at him in surprise. "There's no
need to worry, Dimbleby," he answered, "that storm's blown over all
right."
"That's not the point," snapped Sir Gadsby. "What I want to
know is how that information leaked out in the newspapers. Have
you any suspicions?"
The Great Man frowned at the Permanent Secretary, and the
Permanent Secretary frowned at the Great Man. The Permanent
Secretary was silent so long that the Great Man continued, "I hate to
think that any of our people can have been so lost to all sense of
decency and honour, yet what on earth can I think? You have told
me yourself that our despatch must have been tapped somehow."
"I have puzzled over the matter as much as yourself," said the
Permanent Secretary slowly, "and am as much at a loss as yourself
to account for the information leaking out. It seems to me that there
are only two persons whom it is possible to suspect."
"And those?" queried the Great Man eagerly.
"Are our own two selves," was the answer.
Despite his irritation the Great Man laughed at the whimsicality
of the idea, but he became grave again rapidly.
"If that's the case, we must bring a different order of intelligence
to bear upon the problem. What do you say to asking the assistance
of Scotland Yard, Markham?
"I've not much belief in Scotland Yard," replied the Permanent
Secretary, "but still there can be no harm in our giving them the
opportunity of investigating the affair. They may discover a clue
which will assist us in coming to some conclusion."
"Then send over a messenger at once, Markham," said the
Great Man energetically. "I shall never feel safe if the contents of
despatches are to leak out in this manner. Ask the Commissioner to
send over his most intelligent officer—no, ask them to send Kenly. I
know he's got common sense—to make a delicate investigation. We
will see him together." Then he stalked off to his own room and
worked off his irritation in preparing sarcastic answers to
inconvenient questions in Parliament, of which notice had been
given him by members of the Opposition Party. Consequently he had
recovered his accustomed urbanity when the Permanent Secretary,
accompanied by Detective Inspector Kenly, was ushered into his
presence. He greeted the newcomer heartily.
"Glad the Commissioner could spare you, Kenly. There's
nothing like having a man to undertake confidential work whom one
knows from experience one can trust. I suppose Sir Everard
Markham has told you what we want you to do?"
The Permanent Secretary interrupted with a hasty negation.
"Oh, well, the matter will not take long to explain," continued the
Great Man. "Correct me if I am wrong, Markham."
The Permanent Secretary nodded and handed a cigar case to
the chief.

PDF Advances in Immunology First Edition Alt Ebook Full Chapter

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

PDF Advances in Immunology First Edition Alt Ebook Full Chapter

Uploaded by

Copyright:

Available Formats

Advances in immunology First Edition

Advances in Comparative Immunology Edwin L. Cooper

Avian Cellular Immunology First Edition Jagdev M.

Stem Cell Research : Hope or Hype? 1st Edition Alt

Advances in Clinical Chemistry Volume 66 First Edition

Advances in Parasitology, Volume 91 First Edition David

Advances in Groundwater Governance First Edition Karen

Tumor Immunology, 1st Edition Schreiber

Case Studies in Veterinary Immunology 1st Edition

First edition 2016

© 2016 Elsevier Inc. All rights reserved

For information on all Academic Press publications

Rheumatoid Rescue of Misfolded

Advances in Immunology, Volume 129 # 2016 Elsevier Inc. 1

7. Misfolded Cellular Proteins Rescued from Degradation by MHC Class II Molecules

2. TRANSPORT OF ER-MISFOLDED PROTEINS TO THE

Figure 1 Misfolded major histocompatibility complex (MHC) class I expression facili-

2.2 MHC Class II Molecules Function as a Molecular Chaperon

3. FUNCTION OF PROTEIN ANTIGENS PRESENTED BY

3.2 Misfolded Cellular Proteins Rescued from Protein

The folding of a newly synthesized protein is a complex process that con-

3.3 Aberrant MHC Class II Expression on Autoimmune-

Figure 2 Rheumatoid rescue of misfolded proteins by major histocompatibility com-

consequence of the inflammation elicited by autoimmunity rather than a cause

4. MISFOLDED PROTEINS PRESENTED ON MHC CLASS II

4.2 β2-Glycoprotein I Associated with MHC Class II Molecules Is

Figure 3 Recognition of β2-glycoprotein I (β2GPI) presented on MHC class II molecules

MHC class II molecules appear to be major targets of autoantibodies in many

5. SUSCEPTIBILITY TO AUTOIMMUNE DISEASES IS

MHC class II alleles over susceptibility to autoimmune diseases remains

5.2 Autoantibody Binding to Misfolded Protein/MHC Class II

6. INVOLVEMENT OF MISFOLDED PROTEIN–MHC CLASS

been shown to induce arthritis, suggesting that autoantibodies play an impor-

6.2 B Cell Removal Are Effective Treatment for Autoimmune

7. MISFOLDED CELLULAR PROTEINS RESCUED FROM

The prompt degradation of ER-misfolded proteins through various cellular

The CIITA transcription factor is involved in the expression of both MHC

7.2 Misfolded Protein Complexed with MHC Class II Molecules

8. MISFOLDED PROTEINS PRESENTED ON MHC CLASS II

class II molecules would be a good candidate treatment for autoimmune dis-

Advances in Immunology, Volume 129 # 2016 Elsevier Inc. 25

pathways they transduce to determine the mode of diapedesis, junctional or non-

Captain Marven was certainly not at ease in his own mind in

"Robed in white samite, mystic, wonderful."

When the carriage containing the Great Man rolled up to the

You might also like