866
IL-22 (10 ng/ml), interferon (IFN)-β (1,500 units/ml), and combination of them.
After further 24 hour, the cells were harvested and transcriptional activity of REG
Iα was measured by luciferase assay.
Results: We found that IL-6 stimulation significantly enhanced the REG Iα
promoter activity in human NS-SV-DC cells and A5 rat salivary ductal cells.
Treatment with neither IL-8, IL-22, nor IFN-β changed the transcriptional activity
of REG Iα. To identify the regions necessary for activation of REG Iα promoter
by IL-6, progressive deletions of the REG Iα promoter were performed. Deletion
analysis revealed that the region of -141 to -117 of the REG Iα gene was
responsible for the promoter activation by IL-6. This region contains a consensus
sequence for signal transduction and activation of transcription (STAT). Site-
directed mutations of STAT-binding site in the region of REG Iα significantly
attenuated promoter activation by IL-6.
Conclusions: The present study showed that REG Iα transcription in salivary
ductal cells was stimulated by IL-6. Our study also suggested STAT3 bound
the consensus sequence of REG Iα promoter and regulated transcription in
ductal epithelial cells in response to IL-6 stimulation. It was suggested that
overexpression of REG Iα protein in salivary ductal cells is dependent on IL-
6/STAT pathway and IL-6/STAT dependent REG Iα induction may play a role in
the pathogenesis of SS.
References:
[1] Yoshimoto K et al. Clin Exp Immunol 2013; 174: 1-9.
Disclosure of Interest: None declared
DOI: 10.1136/annrheumdis-2014-eular.2813
AB0190 PATIENTS WITH ANTIPHOSPHOLIPID ANTIBODIES AND
RECURRENT PREGNANCY LOSS: 3’ UNTRANSLATED REGION
(3’UTR) POLYMORPHISMS OF THE HLA-G GENE AS A
POSSIBLE LINK BETWEEN INNATE IMMUNOLOGY AND
AUTOIMMUNITY
V. Canti 1 , G. Amodio 2,3 , M. Castiglioni 4 , L. Maggio 4 , S. Gregori 2,3 ,
P. Rovere-Querini 1,5 . 1 Department of Immunology, San Raffaele Hospital; 2 San
Raffaele Telethon Institute for Gene Therapy; 3 Division of Regenerative
Medicine, Stem Cells and Gene Therapy; 4 Obstetrics and Gynecology, San
Raffaele Hospital; 5 Università Vita-Salute S. Raffaele, Milano, Italy
Background: Recurrent pregnancy loss (RPL) occurs in 2-4% of fertile couples.
The cause of the miscarriages remains in half of the cases unknown. A defective
establishment of immune tolerance towards fetal antigens might be involved.
HLA-G is a non-classical class I HLA molecule, which is preferentially expressed
by extravillous trophoblast cells at the maternal-fetal interface. Polymorphism of
the coding sequence of the HLA-G gene is limited. In contrast, the noncoding 3’
untranslated region (3’UTR) of the gene is highly polymorphic and 14bp ins/del
polymorphism plays an important role in the post-transcriptional regulation of
HLA-G expression and might influence its function.
Objectives: To analyze whether 3’UTR HLAG polymorphisms might be associated
with RPL in patients with or without antiphospholipid antibodies (aPL).
Methods: Blood samples and clinical data were collected from 132 healthy donors
and 65 women with RPL (defined as two of more consecutive miscarriages),
prospectively followed at the “Pathologic Pregnancy Unit” outpatient clinic of San
Raffaele Hospital, Milan, Italy from 2002 to 2012 during a further pregnancy.
We evaluated and comparatively studied the insertion(+) or deletion(−) of 14
bp in the 3’UTR and the association between 3’UTR haplotypes of HLA-G and
the presence(aPL-pos) or absence(aPL-neg) of aPL(anticardiolipin, anti beta2
glycoprotein IgG and IgM or LLAC). Genetic and serological data were correlated
with the pregnancy outcomes.
Results: A heterozygous genotype(+/−) was detected in 58.8% (30/51) aPL-neg
RPL patients versus 42.9% (6/14) aPL-pos patients; 25.5% (13/51) aPL-neg
versus 35.7% (5/14) aPL-pos had a homozygous deletion genotype(−/−); 15.7%
(8/51) aPL-neg versus 21.4% (3/14) aPL-pos had a homozygous insertion(+/+).
In the control group 47.7% of the subjects (63/132) were (+/−), 30.4% (40/132)
were (−/−) and 21.9% (29/132) were (+/+). The frequency of the UTR-8(−/−)
haplotype in healthy donors was significantly lower than in patients (6.8%; 9/132:
p<0.05). In contrast, we did not observe any significant association between the
clinical phenotype and any other HLA-G haplotype. We are currently evaluating
whether the presence of the UTR-8 haplotype is associated with the pregnancy
outcomes in aPL-pos and aPL-neg patients. Preliminary data on a little group of
aPL-pos-UTR-8 haplotype show a higher incidence of RPL (66.7%, 2/3) than in
aPL-pos-UTR-different haplotype (20%, 2/10).
Conclusions: The non-coding 3’UTR HLA-G region is significantly more frequent
in RPL women when compared with the control group. The possible influence of
this finding on the pathogenic potential of aPL in pregnancy is being investigated.
References:
[1] Wang X. et al;Tissue Antigens. 2013
[2] Shankarkumar U. et al; J Hum Reprod Sci. 2011
Disclosure of Interest: None declared
DOI: 10.1136/annrheumdis-2014-eular.4307
Scientific Abstracts
AB0191 GENE AND PROTEIN EXPRESSION PROFILE OF PERIPHERAL
BLOOD IN PRIMARY SJOGREN’S SYNDROME PATIENTS
Y. Cheng 1 , C. Huang 2 . 1 Rheumatology and Immunology Department;
2
Rheumatology Department, Beijing Hospital, Beijing, China
Background: Sjogren syndrome (SS) is a chronic, multisystemic autoimmune
disease characterized by lymphoplasmocytic infiltration of the salivary. Until now,
its pathogenesis and diagnosis remain enigmatic. As a multisystem disease, it
involves joints, hematology, lymphocyte organ, pulmonary, kidney, liver and other
organs. Different organ involvements evolve from differential impression of genes
and proteins.
Objectives: To identify gene expression profile in peripheral blood mononuclear
cell (PBMC)of patients with Sjogren’s syndrome (SS) compare it to healthy
volunteers, and then verify serum protein level of identified genes in peripheral
blood and analyze the correlation between level of those serum protein and
disease activity parameters.
Methods: SS patients with leucopenia and 3healthy volunteers were included.The
cRNA probes prepared for total RNA were hybridized with three identical gene
chips.The difference of gene expression of each patient and volunteers were
compared. According to statistical analysis and pathway analysis, different
expression genes were identified. Serum protein levels of two different expressed
genes weredetected by enzyme-linked immunosorbent assay (ELISA) in 40 SS
patients, 40 Rheumatoid arthritis (RA) patients, 24 Osteoarthritis (OA) patients
and 40 healthy controls. Independent sample t-test was applied. Correlation
analysis was done to investigate the relationship between two serum protein
levels and disease activity parameters of SS, including number of white blood
cells, organ involvement, ESSDAI score, levels of ESR, C-reactive protein (CRP),
Rheumatoid Factor (RF), immunoglobin G (IgG), immunoglobin A (IgA) and
immunoglobin M (IgM).
Results: Significant difference in the expression of 82 genes could be detected
between patients and volunteers. Among these,45 were upregulated, and 37
were downregulated. Statistical difierence was calculated between patients
and volunteers especially in the following two pathways: the complement and
coagulation pathways (including C2, PROS1, F2R and SEPPING1) and the
cytokine-cytokine receptor interaction pathway (including FASLG, MPL, CCL20
and CXCL2) (P<0.01).Further study identified that CXCL2, CCL20 levels were
significantly higher in SS patients than in healthy controls (P<0.01). CCL20 level
was significantly correlated with number of white blood cells and level of CRP,
while negatively with pulmonary interstitial diseases, and positive correlation was
observed between CXCL2 level andnumber of white blood cells.
Conclusion: This study has identified distinct gene expression profiles in
PBMC from patients with SS patients with hematology involvement and healthy
volunteers. Among which, CXCL2 and CCL20 play a part in the development of
SS and their determination may benefit evaluation of disease activity.
Disclosure of Interest: None declared
DOI: 10.1136/annrheumdis-2014-eular.5314
AB0192 PROOF OF CONCEPT, SELF-ORGANIZED CRITICALITY
THEORY OF AUTOIMMUNITY: AUTOANTIBODY-INDUCING CD4
T CELL THAT CAUSES SLE BELONGS TO CD45RB-CD122-PD1+
SUBSET
Y. Miyazaki, K. Tsumiyama, S. Shiozawa. Department of Medicine, Rheumatid
Diseases Unit, Kyushu University Hospital, Beppu, Japan
Background: A critical question in elucidating the pathogenesis of SLE or
autoimmunity would be how autoreactive clones emerge and expand in the host.
According to the prevailing view of autoimmune disease, autoreactive clones may
come from either clones that had escaped negative selection in the thymus or
clones that have been reactivated from tolerance. However, clones that would
emerge by either process would be restricted in their antigen specificity and
apparently could not account for the broad T cell receptor (TCR) repertoire
and the more than 100 distinct autoantibody specificities found in SLE (Ref
1). We have proposed an alternative novel theory, called the self-organized
criticality theory, which explains that systemic autoimmunity necessarily takes
place when the host’s immune system is overdriven by repeated exposure to
antigen, reaching levels that surpass the immune system’s stability-limit, i.e.,
self-organized criticality (Ref 2). We found that a CD4 T cell subset which has
passed thru TCRα but not TCRβ revision is generated at peripheral lymphoid
organ spleen after heavily repeated immunization with any antigen, and we named
this CD4 T cell an “autoantibody-inducing CD4 T cell” (aiCD4 T cell). This aiCD4 T
cell not only induced B cells to generate a large variety of autoantibodies, but also
promoted final differentiation of CD8 T cells into cytotoxic T lymphocytes (CTL)
via antigen cross-presentation, leading to the tissue injuries identical to those
seen in SLE. This aiCD4 T cell is functionally indispensable for the pathogenesis
of SLE.
Objectives: We report our recent findings on the distinct cell surface markers that
characterize and identify aiCD4 T cells to be CD45RBlo 122lo and programmed
cell death-1 (PD-1)-positive.
Methods: BALB/c mice were repeatedly immunized with OVA. To investigate
gene expression profiles of aiCD4 T cell, we performed microarray analysis of
CD45RBlo 122lo CD4 T cell after immunization 12x with OVA. CD45RBlo 122lo
CD4 T cells were isolated referring to PD-1 expression, and these fractionated