Lipid Vesicle Size Determines the Th1 or Th2

Response to Entrapped Antigen

This information is current as
of December 7, 2019.
James M. Brewer, Laurence Tetley, James Richmond, Foo Y.
Liew and James Alexander
J Immunol 1998; 161:4000-4007; ;
http://www.jimmunol.org/content/161/8/4000

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Lipid Vesicle Size Determines the Th1 or Th2 Response
to Entrapped Antigen1

James M. Brewer,2*§ Laurence Tetley,† James Richmond,‡ Foo Y. Liew,§ and

James Alexander*
Understanding the factors that control the differential induction of Th1 and Th2 responses is a key immunologic objective with
profound implications for vaccination and immunotherapy of infectious and autoimmune diseases. Using Ag formulated in lipid
vesicles prepared from nonionic surfactants, we describe a novel mechanism influencing the balance of the Th1 or Th2 response.
Our results indicate that inoculation of BALB/c mice with vesicles with a mean diameter >225 nm preferentially induces Th1
responses, as characterized by increased titers of IgG2a in plasma and elevated IFN-g production by lymph node cells. However,
preparation of the same quantity of Ag in vesicles with mean diameter of <155 nm induces a Th2 response, as identified by IgG1
in the absence of IgG2a production and increased lymph node IL-5 production. Although large (>225 nm) vesicles could induce

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IL-12 production, smaller vesicles (<155 nm) could not. However, small vesicles did induce higher levels of IL-1b production by
macrophages than larger vesicles. The role of IL-12 in this response was confirmed in IL-12-deficient mice, whose spleen cells failed
to produce IFN-g following in vivo priming with Ag prepared in large vesicles. Our results therefore indicate that macrophages
respond to endocytosis of large or small vesicles by producing different patterns of cytokines that can subsequently direct the
immune response toward a Th1 or a Th2 phenotype. The Journal of Immunology, 1998, 161: 4000 – 4007.

T he reciprocal nature of the regulation of cell-mediated and
humoral immune responses was first proposed in the early
1970s as a result of studies with chemically modified Sal-
monella flagellin (1). More recently, control of this relationship
has been attributed to mutually antagonistic subsets of CD41 Th
lymphocytes. Activation of the Th1 subset is associated with the
production of IFN-g and IL-2 and the development of a classical
cell-mediated immune response, such as delayed-type hypersensi-
tivity, whereas activation of the Th2 subset and the subsequent
production of cytokines such as IL-4, IL-5, IL-6, and IL-10 are
associated with the development of classical humoral immune re-
sponses (2). Accordingly, the generation of a predominantly Th1
response is essential for the development of a protective immune
response against obligate intracellular organisms such as Leishma-
nia major (3), while the induction of a predominately Th2 response
is more appropriate for the effective control of certain helminth
infections (4). Although the available evidence indicates that Th1-
and Th2-type cells develop from a common precursor (5), the el-
ements that influence the preferential expansion of one subset
rather than the other remain ill defined. Postulated factors include
the population of accessory cells presenting the Ag (6, 7) and the
presence of different costimulatory molecules (8) and cytokines in
the cell microenvironment (9, 10). Using Ag formulated in lipid
*Department of Immunology, University of Strathclyde, Glasgow, Scotland; †Divi-
sion of Infection and Immunity, Institute of Biomedical and Life Sciences, Joseph
Black Building, University of Glasgow, Glasgow, Scotland; ‡Department of Pathol-
ogy, Glasgow Royal Infirmary, Glasgow, Scotland; and §Department of Immunology,
University of Glasgow, Western Infirmary, Glasgow, Scotland
Received for publication December 11, 1997. Accepted for publication June 17, 1998.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by a Wellcome Trust Career Development Fellowship (to
J.M.B.).
2
Address correspondence and reprint requests to Dr. James M. Brewer, Department
of Immunology, University of Glasgow, Western Infirmary, Glasgow, Scotland G11
6NT. E-mail address: j.m.brewer@clinmed.gla.ac.uk
vesicles (11–13), we have identified a novel mechanism by which
the balance of the Th1/Th2 response to an Ag can be influenced.
Our data indicate that vesicles with a mean diameter $225 nm
preferentially induce Th1-type responses, while the same quantity
of Ag entrapped in vesicles with a mean diameter #155 nm in-
duces Th2-type responses, as characterized by in vivo Ab subclass
production and in vitro cytokine production. Further studies have
revealed that the macrophage appears to play the central role in
orchestrating this effect.
Materials and Methods
Lipid vesicle preparation
All glassware was heated at 180°C for 5 h to inactivate endotoxin, and
autoclaved Elgastat Ultra High Purity water (Elga, Bucks, U.K.) was used
to prepare solutions. Vesicles were prepared under aseptic conditions by
the methods described previously (12) and were tested as endotoxin neg-
ative by the Limulus amoebocyte assay (Sigma, Poole, U.K.) Briefly, 150
mmol of 1-monopalmitoyl glycerol, cholesterol, and dicetyl phosphate
(Sigma) were mixed in a 15-ml Pyrex test tube in the molar ratio 5:4:1, and
then heated at 130°C in a dry-block (Grant Instruments, Cambridge, U.K.)
until melted. Vesicles were formed when 2.5 ml of aqueous buffer (PBS;
pH 7.4) was added, and the resulting suspension was vortexed vigorously
for 1 min. After shaking the suspension at 60°C for 2 h, OVA (grade V,
Sigma) was entrapped by freezing the Ag vesicle mixture in liquid nitrogen
and thawing to 60°C five times. After an additional 2 h of shaking at 60°C,
vesicle preparations were extruded through decreasing pore size polycar-
bonate filters (Costar, Bucks, U.K.) at 60°C in a thermobarrel extruder
(Lipex Biomembranes, Vancouver, Canada) as described previously (14).
After removing nonentrapped Ag by centrifuging at 100,000 3 g for 45
min, the protein concentrations of the vesicle suspensions were determined
using a modified ninhydrin assay (15). Protein concentrations in the various
vesicle preparations were then adjusted so all inoculations contained the
same quantity of protein.
Electron microscopy
Small samples (;10 ml) of the extruded vesicle preparations were sand-
wiched between the cleaned surfaces of pairs of copper support plates
(Balzers, Furstentum, Leichtenstein). The vesicle suspension was then
flash-frozen by immersion in liquid propane at 2180°C and then trans-
ferred under liquid nitrogen into a fracturing device. After fracturing at
290°C under a vacuum of 4 3 1026 Torr, the samples were shadowed

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00

The Journal of Immunology
immediately with evaporated platinum/carbon at 45° to the fracture surface
and strengthened by coating with carbon evaporated at 90° to the fracture
surface. The vesicle preparations were removed from the replicas by se-
quential washing with acetone/distilled water solutions of decreasing ace-
tone concentration. After washing the replicas several times in distilled
water, they were collected onto copper grids, dried, and examined by trans-
mission electron microscopy.
Animals and inoculations
Female BALB/c mice were in-house bred at the University of Strathclyde
and used when they were 8 to 10 wk old. Groups of five mice were inoc-
ulated s.c. with 0.1 ml of vesicle suspension containing 100 mg of OVA in
PBS or prepared in vesicles. Inoculations were repeated after 2 wk, and
blood was sampled for Ab determination 2 wk subsequently. For lymph
node cytokine assays, groups of five animals were inoculated in each foot-
pad with 10 ml of OVA (10 mg) in PBS or prepared in vesicles. Inguinal
and popliteal lymph nodes were collected 10 to 14 days later, although
previous studies using later time points have produced similar results (12),
consistent with the observation that cytokine profiles following inoculation
with Ag prepared in adjuvant are rapidly induced and stable (16).
IL-12 (p40)-deficient BALB/c mice (17) were donated by Dr. J. Ma-
gram, (Hoffmann-La Roche, Nutley, NJ). These mice and control mice
were bred and maintained at the Central Research Facility, University of
Glasgow (Glasgow, Scotland). Groups of five mice were immunized s.c.
with 10 mg of OVA in vesicles, and appropriate booster doses were ad-
ministered 2 wk later. Spleens were collected after an additional 4 wk.
Plasma Ab determination
ELISAs were performed as described previously (11) to detect Ag-specific
IgG, IgG1, and IgG2a in plasma. Briefly, flat-bottom polystyrene plates
(Dynatech, Alexandria, VA) were coated overnight at 4°C with 100 ml of
OVA (64 mg/ml in PBS; pH 9.0), and following blocking (18), 100-ml
samples of plasma serially diluted in PBS/Tween were added to duplicate
wells and incubated for 1 h at 37°C. One hundred microliters of HRP3-
conjugated goat anti-mouse IgG, IgG1, or IgG2a (Southern Biotechnology
Associates, Birmingham, AL) was added to each well at dilutions of
1/8000, 1/8000, and 1/800, respectively, in 75% PBS/25% sheep serum
(v/v). Substrate solution was prepared by addition of 5 ml of hydrogen
peroxide and 250 ml of 6 mg/ml tetramethylbenzidine in dimethylsulfoxide
to 25 ml of 0.1 M sodium acetate solution, pH 5.5. The enzyme-substrate
reaction was stopped by addition of 50 ml of 10% sulfuric acid (v/v), and
the absorbance at 450 nm was measured on a Titer-Tek Multiskan (Flow
Laboratories, Irvine, U.K.). Results are expressed as end-point dilutions
where the end point is determined as the final plasma dilution that yields a
higher absorbance than a negative control plasma sample included in the
assay. Comparisons between groups were performed using the Mann-Whit-
ney U test.
Lymphocyte cultures
Pairs of draining inguinal and popliteal lymph nodes from each mouse
were aseptically removed 10 to 14 days after footpad inoculation and were
placed in RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/ml
penicillin, 100 mg/ml streptomycin, 0.05 mM 2-ME, and 10% FCS
(Life Technologies, Paisley, U.K.). Individual cell suspensions were pre-
pared by gently teasing the lymph nodes from each mouse apart with for-
ceps. Alternatively, spleen cell suspensions were prepared as described
above, but a RBC depletion step was included, as described previously
(19). Following centrifugation at 200 3 g for 10 min, cells were resus-
pended in 0.5 ml of medium. Viable cells were enumerated in a hemocy-
tometer by trypan blue exclusion, and cell suspensions were adjusted to
5 3 106 cells/ml. Aliquots of cell suspension (100 ml) containing 5 3 105
cells were added to 96-well flat-bottom tissue culture plates (Costar), and
triplicate 100 ml/well aliquots of Con A (5 mg/ml) or OVA (1000 mg/ml)
were added as appropriate. We and others have demonstrated that these
levels of OVA are necessary to induce proliferation and cytokine produc-
tion by spleen and lymph node cells (12, 20 –22). Cell supernatants were
removed and stored at 270°C for cytokine analysis following 60 h of
culture at 37°C in 5% CO2, when optimal cytokine levels are achieved as
described previously (22).
Determination of lipid vesicle uptake by flow cytometry
The ability of B220-positive spleen cells and J.774 macrophages to accu-
mulate fluorescence-labeled lipid vesicles was determined by flow cytom-
3
Abbreviation used in this paper: HRP, horseradish peroxidase.
4001
etry. Briefly, naive spleen cells were prepared as described above and in-
cubated for various periods of time with FITC-dextran alone (m.w.,
20,000; Sigma) as a pinocytic marker (23) or with FITC-dextran prepared
in vesicles of different sizes. After incubation, cells were washed and stored
on ice before staining with phycoerythrin-labeled anti-B220 (PharMingen,
San Diego, CA) and subsequent analysis. Uptake of FITC-dextran is ex-
pressed as the mean fluorescence intensity of B220-positive cells. Uptake
of 1,19-dioctadecyl-3,3,39,39-tetramethylindocarbocyanine perchlorate
(Molecular Probes, Eugene, OR)-labeled lipid vesicles (24) by J.774 mac-
rophages was performed as described above, although the fluorescence
intensities were corrected for surface-bound vesicles by subtracting the
fluorescence intensity at time zero.
Peritoneal macrophage cultures
Resident cells were harvested from the peritoneum of normal, female
BALB/c mice in 5 ml of ice-cold RPMI 1640 supplemented with 2 mM
L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% FCS.
The cells were pooled, centrifuged at 200 3 g for 5 min, and resuspended,
and cell numbers were enumerated as described above. Cell suspensions
were adjusted to 2 3 106 cells/ml, and 100 ml/well aliquots of cell sus-
pension containing 2 3 105 cells were added to 96-well flat-bottom tissue
culture plates (Costar). Macrophage-enriched cultures were prepared by
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allowing cells to adhere to the tissue culture plates for 4 h at 37°C in 5%
CO2 and subsequently removing the cells in suspension and washing with
two changes of complete medium. Groups of five wells were then treated
with extruded vesicles (100 mg lipid/well) with sizes of either 560 or 155
nm containing Ag (50 mg/well). Control wells containing either medium
alone (n 5 5) or 0.25 mg of LPS from Salmonella abortus equii (Sigma)
per well (n 5 5) were also included with each experiment. Experiments
performed by us and others (25) have shown that optimum IL-12 produc-
tion occurs after 18 h of incubation at 37°C in 5% CO2; therefore, super-
natants were removed at this time point to fresh 96-well plates and were
stored at 270°C for cytokine analysis.
Cytokine assays
Levels of cytokines (IL-2, IL-5, and IFN-g) were determined in cell culture
supernatants by capture ELISA under the conditions described previously
(12). Reagents for IL-1b and IL-4 analysis were purchased from Genzyme
(Cambridge, MA) and used according to the manufacturer’s directions.
IL-12 reagents were generously donated by Dr. Horst Bluethmann, Roche
(Basel, Switzerland). Briefly, flat-bottom polystyrene plates were coated
with 50 ml of IL-1b, IL-4, and IL-12 (p70) neutralizing mAbs (B122,
11B11, and 9A5, respectively) at predetermined concentrations. One hun-
dred-microliter samples of supernatants and standards were added in du-
plicate to wells, and detection was performed using biotinylated polyclonal
anti-mouse cytokine Abs (IL-1b and IL-4) or HRP-labeled mAb (IL-12;
POD-5C3). Alkaline phosphatase-streptavidin conjugate (PharMingen)
was used at a dilution of 1/2000, and substrate (paranitrophenyl-phospha-
tase, 1 mg/ml; Sigma) in glycine buffer (0.1 M; pH 10.4) was added. Ab-
sorbances were read at 405 nm on a Titer-Tek Multiskan plate reader (Flow
Laboratories, Irvine, U.K.). For IL-12 analysis, the secondary Ab, HRP-
labeled anti-p40 POD-5C3, was detected by incubation with substrate
buffer prepared as described for determination of plasma Ab titers. Cyto-
kine concentrations in the cell cultures were determined from the standard
curve (regression coefficient, r 5 0.970 or better). Comparisons between
groups were made using Student’s t test.
Results
Electron microscopy
Analysis of vesicle preparations extruded through 800 nm (Fig.
1a), 400 nm (Fig. 1b), 200 nm (Fig. 1c), and 100 nm (Fig. 1d) pore
size polycarbonate membranes was performed by electron micros-
copy. The micrographs demonstrate that vesicle characteristics are
retained following extrusion and also that extrusion through suc-
cessively smaller pore size membranes results in correspondingly
smaller vesicles. Mean vesicle diameters were estimated from the
micrographs as being approximately 560 6 60 nm (Fig. 1a) and
100 6 10 nm (Fig. 1d). Photon correlation spectroscopy indicated
that the mean size of preparation B was 225 6 25 nm, that of
preparation C was 155 6 10 nm, and that of nonextruded vesicles
was 3100 6 660 nm in diameter.
4002 Th1 AND Th2 RESPONSES ARE DETERMINED BY LIPID VESICLE SIZE
FIGURE 1. Freeze fracture electron mi-
crographs of vesicle suspensions extruded
through 800-nm (a), 400-nm (b), 200-nm
(c), and 100-nm (d) pore diameter polycar-
bonate membranes. The micrographs dem-
onstrate that vesicle suspensions retain
vesicle characteristics after extrusion and
also that extrusion through successively
smaller pore size membranes results in cor-
respondingly smaller vesicles. All panels
are of the same magnification; the bar in C
represents 400 nm.
Effect of vesicle size on Th1/Th2-type responses
Reducing the size of the vesicle preparations did not signifi-
cantly affect their overall adjuvant activity compared with that
in nonextruded control vesicles as assessed by OVA-specific
IgG titers (data not shown). Similarly, vesicle size had no sig-
nificant effect on the production of OVA-specific IgG1, the ves-
icle preparations again inducing significantly higher IgG1 titers
in mice than those detected following inoculation with OVA
alone (Fig. 2A). In contrast, the production of OVA-specific
IgG2a was very much a function of vesicle size (Fig. 2B). While
no difference could be observed in IgG2a production in groups
treated with 3100-, 560-, or 225-nm vesicles, significantly re-
duced titers of IgG2a were observed when OVA was prepared
in NISV extruded through smaller pore size membranes (560
nm . 100 nm or 155 nm, p , 0.025; 225 nm . 100 nm or 155
nm, p , 0.025).
IFN-g and IL-5 production in Con A- and OVA-stimulated
lymph node cells isolated from mice 10 to 14 days after admin-
istration of Ag alone or prepared in different sizes of vesicle
preparations was also compared (Fig. 2, C–E). Ag-stimulated
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production of IFN-g increased with increasing vesicle size,
such that only vesicles with mean diameters of 560 nm ( p ,
0.025) and 225 nm ( p , 0.01), but not 155 nm, stimulated
significantly higher levels of IFN-g compared with inoculation
of Ag alone. Furthermore, both 560- and 225-nm vesicle prep-
arations produced significantly higher concentrations of IFN-g
than inoculation with 155-nm vesicles (Fig. 2D; p , 0.05 and
p , 0.01, respectively). Similar results were obtained when Con
A was employed as the in vitro stimulus (Fig. 2F). In contrast,
IL-5 production by lymph node cells appeared to be preferen-
tially stimulated by immunization with smaller vesicles, with
155-nm diameter vesicles producing significantly more IL-5
than 225-nm diameter vesicles ( p , 0.025) or OVA alone ( p ,
0.05) following in vitro stimulation with Con A (Fig. 2E). Lev-
els of this cytokine in Ag-stimulated cultures were not signif-
icantly greater than background levels (Fig. 2C). Thus, increas-
ing vesicle size results in a switch from predominantly Th2-
associated IL-5 production to Th1-associated IFN-g production,
with the switch in response occurring between 155- and 225-nm
vesicle preparations.
The Journal of Immunology 4003

FIGURE 2. Analysis of Th1- and

Th2-type responses induced by Ag en-
trapped in extruded (100 –560 nm) vesi-
cles. A, Although vesicle size had no sig-
nificant effect on production of OVA-
specific IgG1, the production of OVA-
specific IgG2a was a function of vesicle
size. B, While no difference could be ob-
served in IgG2a production in groups
treated with 560- or 225-nm vesicles,
significantly reduced titers of IgG2a
were observed when OVA was prepared
in vesicles extruded through smaller pore
size membranes (560 nm . 155 nm or
100 nm, p , 0.025; 225 nm . 155 nm or
100 nm, p , 0.025). While all the vesi-
cle formulations could enhance OVA-
specific IgG1 titers, IgG2a titers were
only enhanced in the animals adminis-
tered vesicles with mean diameters of
225 nm (p 5 0.01) and 560 nm (p 5

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0.01). Data shown are representative of
two experiments; results are expressed as
the mean reciprocal end-point dilution 6
SEM (n 5 5). C, Although Ag-stimu-
lated cultures produced very low levels
of IL-5 (#60 pg/ml), levels of IFN-g
were readily detectable. D, Thus, inocu-
lation with both 560- and 225-nm vesicle
preparations induced significantly higher
IFN-g production by lymph node cells
than inoculation with either OVA alone
or 155-nm vesicle preparations (p ,
0.05 and p , 0.01, respectively). E, In
contrast, lymph node cells from mice in-
oculated with Ag prepared in 155-nm
vesicles produced significantly higher
IL-5 production following mitogen stim-
ulation than cells from mice inoculated
with either 225-nm diameter vesicles
(p , 0.025) or OVA alone (p , 0.05). F,
Lymph node cultures from mice inocu-
lated with 560- and 225-nm vesicles also
produced significant levels of IFN-g af-
ter mitogen stimulation (p , 0.05). Data
shown are representative of two experi-
ments; results are expressed as the mean
cytokine concentration 6 SEM (n 5 5).

Role of vesicle size in determining uptake by B cells
and macrophages
Reducing vesicle size from 225 to 155 nm did not affect the ability
of J.774 macrophages or B cells to internalize lipid vesicles (Fig.
3). While B220-positive spleen cells did not accumulate either size
of lipid vesicle compared with the pinocytic marker, FITC-dextran
(Fig. 3A), J774 macrophages avidly injested both sizes of lipid
vesicle tested (Fig. 3B). The data shown are representative of three
experiments, and in each experiment the number of positive J.774
macrophages was in excess of 90%.
Role of the protein/lipid ratio in determining Th1/Th2 responses
Because reducing the size of lipid vesicles will effectively reduce
the amount of protein delivered to APCs per vesicle, we examined
how Th1/Th2 responses were affected by this parameter, known as
the protein/lipid ratio, in vesicles with constant size (Fig. 4). Vary-
ing the protein/lipid ratio above and below normally employed
levels (typically 1:30) had little effect on titers of OVA-specific
IgG1 or IgG2a in mice inoculated twice with each preparation. In
fact, as demonstrated in Figure 4, altering this parameter over a
50-fold range did not significantly affect the IgG1/IgG2a response
in any fashion and certainly not in a manner similar to reducing
vesicle size.
Effect of vesicle size on in vitro macrophage cytokine production
The ability of Ag-containing vesicle preparations to initiate macro-
phage cytokine production was compared with that of LPS from Sal-
monella abortus equi. Significant IL-1b production could be induced
by each of preparations tested compared with cells incubated with
medium alone (Fig. 5A; p , 0.01). However, cells incubated with
560-nm vesicles produced less IL-1b than cells treated with either
155-nm vesicles ( p , 0.01) or LPS ( p , 0.02). The levels of IL-1b
induced by the 155-nm vesicles was not significantly different from
those induced following incubation with LPS.
4004 Th1 AND Th2 RESPONSES ARE DETERMINED BY LIPID VESICLE SIZE
FIGURE 3. Analysis of the ability of B220-positive B cells and J.774
macrophages to accumulate different sizes of vesicles. A, Uptake of FITC-
labeled dextran (m.w., 20,000) by splenic B cells was inhibited equally by
preparation of FITC-dextran in vesicles with mean diameters of 155 and
225 nm. B, Uptake of 1,19-dioctadecyl-3,3,39,39-tetramethylindocarbocya-
nine perchlorate-labeled vesicles by J.774 macrophages was similar re-
gardless of the vesicle diameter. The data shown are expressed as the mean
fluorescence intensity as determined by flow cytometry and are represen-
tative of two or three separate experiments.
Although 560-nm vesicles did induce IL-12 production, no de-
tectable IL-12 production was observed following incubation of
cells with smaller, 155-nm diameter vesicles (Fig. 5B). The levels
of IL-12 detected following incubation with 560-nm vesicles were
significantly greater than those detected in cultures of untreated
cells ( p , 0.01) or cells treated with 155-nm vesicles ( p , 0.01)
or LPS ( p , 0.01). As previously described (26), treatment of
resident peritoneal macrophages with LPS did not induce detect-
able levels of IL-12 p70.
The role of IL-12 in lipid vesicle determined Th1/Th2 responses
in vivo
To evaluate the in vivo significance of the in vitro-driven IL-12
response, we compared cytokine production by spleen cells of IL-
12-deficient BALB/c mice with those of wild-type control mice
(Fig. 6A). In agreement with the data presented above, spleen cells
from wild-type mice inoculated with OVA entrapped in 560-nm
vesicles produced significantly higher concentrations of IFN-g
than cells from mice immunized with OVA prepared in 155-nm
vesicles following in vitro stimulation with Ag ( p 5 0.01). In
contrast, although cells from the IL-12-deficient mice exhibited
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FIGURE 4. Effect of altering the protein/lipid ratio on the induction
of Th1/Th2 responses as determined by IgG1/IgG2a analysis. A, Alter-
ing the protein/lipid ratio from that normally employed (typically 1:30)
produced no significant variation in the level of Ag-specific IgG1 de-
tected 2 wk after secondary inoculations. B, Similarly, no significant
alteration in IgG2a titer could be observed by varying the protein/lipid
ratio in these studies. Results are expressed as the mean reciprocal
end-point dilution 6 SEM (n 5 5).
significant proliferation (Fig. 6B), they only produced low levels of
IFN-g regardless of whether they were from mice immunized with
OVA entrapped in 560- or 155-nm vesicles (Fig. 6A).
Discussion
Inoculation of BALB/c mice with OVA entrapped in lipid vesicles
of different sizes alters the subsequently induced immune response
in a qualitative fashion. As has been described previously (14) and
as characterized here by electron microscopy, the effect of extru-
sion of lipid vesicles through polycarbonate membranes was to
reduce the diameter of the lipid vesicles in a manner proportional
to the pore diameter of the membrane. When vesicles containing
OVA were extruded through successively smaller sized pore mem-
branes and inoculated s.c. into BALB/c mice, a size-dependent and
significant reduction in the Ag-specific IgG2a Ab titer was ob-
served. In contrast, IgG1 and IgG titers were unaffected by the
effects of extrusion of vesicles through reducing pore size poly-
carbonate membranes. While production of the IgG2a subclass of
Ab by murine B cells is associated with Th1-type cytokine pro-
duction (27, 28), IgG1 Abs, on the other hand, are associated with
The Journal of Immunology
FIGURE 5. Comparison of IL-1b and IL-12 production by peritoneal
macrophages. A, Significant IL-1b production could be induced by each of
preparations tested in comparison with cells incubated with medium alone
(p , 0.01). However, cells incubated with 560-nm vesicles produced less
IL-1b than cells treated with either 155-nm vesicles (p , 0.01) or LPS
(p , 0.01). The levels of IL-1b induced by the 155-nm vesicles were not
significantly different from those induced following incubation with LPS.
B, The levels of IL-12 detected following incubation with 560-nm vesicles
were significantly greater than those detected in cultures of untreated cells
(p , 0.01) or cells treated with either 155-nm vesicles (p , 0.01) or LPS
(p , 0.01). The results shown are representative of three experiments. Data
are expressed as the mean cytokine concentration 6 SEM (n 5 5).
Th2-type cells (27, 28). Therefore, these results indicate that as
vesicle size is reduced, the adjuvant effect produced by the vesicles
switches from inducing a mixed Th1/Th2-type response charac-
terized by B cell IgG1 and IgG2a production to the production of
a Th2-dominated response in the absence of IgG2a. The critical
factor in this switch appears to be the change in particle size that
lies between mean diameters of 155 and 225 nm.
To further study this phenomenon, we have analyzed cytokine
production by draining lymph node cells isolated from BALB/c
mice 10 days after footpad inoculation with differently sized ves-
icles containing OVA. The production of the Th1-associated cy-
tokine, IFN-g (2, 29), was only elevated in the lymph node cells of
mice inoculated with vesicles with a mean diameter of 225 nm or
greater. Conversely, elevated levels of the Th2-associated cyto-
kine, IL-5 (2, 29), could only be detected in mitogen-stimulated
cultures of lymph nodes taken from mice following inoculation
with smaller vesicles with a mean diameter of 155 nm or less. In
agreement with the Ab data presented above, this switch in re-
sponse appears to lie in between the mean vesicle diameters of 155
and 225 nm. However, the cytokine data also indicate that rather
4005
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FIGURE 6. Effect of vesicle size on IFN-g production and proliferation
by spleen cells from IL-12-deficient mice. A, Wild-type BALB/c or IL-12-
deficient mice were immunized with OVA entrapped in 560- or 155-nm
vesicles as described in Materials and Methods. Spleen cells were cultured
with OVA (1000 mg/ml), and concentrations of IFN-g in the culture su-
pernatants were compared. Spleen cells from wild-type mice inoculated
with 560-nm vesicles produced more IFN-g than those from BALB/c mice
immunized with 155-nm vesicles (p # 0.05). However, spleen cells from
IL-122/2 mice inoculated with 560-nm vesicles produced similar levels of
IFN-g as spleen cells from IL-122/2 mice inoculated with 155-nm vesi-
cles. Levels of IFN-g produced by spleen cells from 560-nm inoculated
IL-122/2 mice were also significantly lower than those in similarly immu-
nized BALB/c control mice (p # 0.01). Results are expressed as the mean
cytokine concentration 6 SEM (n 5 5). B, In contrast, T cell proliferation
was unaffected by IL-12 deficiency or by the size of vesicle in which the
Ag was entrapped. Results are expressed as the mean stimulation index 6
SEM (n 5 5).
than a mixed Th1/Th2 response, vesicles with a diameter $225 nm
induce a predominantly Th1-type response. Thus, the Th1 and Th2
responses induced by large and small vesicles, respectively, are
more polarized than indicated by the Ab data. While we could not
detect IL-4 (,50 pg/ml) in Ag-stimulated spleen cells from
BALB/c mice immunized with either large or small vesicles (data
not shown) this may merely reflect the sensitivity of the assay.
However, we and others have demonstrated that Th2-type re-
sponses can still be generated in the absence of IL-4 (30 –33) or its
receptor.4
Previous studies have indicated that, unlike administration of
soluble Ag alone, which induces neither a Th1- or a Th2-biased
4
M. Welte, B. Ledermann, A. Dorfmuller, and F. Brombacher. Submitted for publi-
cation.
4006 Th1 AND Th2 RESPONSES ARE DETERMINED BY LIPID VESICLE SIZE
response, high m.w. glutaraldehyde-polymerized OVA induces a
strong Th1-like response in vivo (21). This change in response was
associated with the increased ability of polymerized OVA to in-
duce IFN-g production and decreased uptake of the immunogen by
B cells (34). While the physical size of the polymerized OVA
particles was not determined and the effect of the change in com-
position of the formulation by the addition of glutaraldehyde could
not be assessed (34), the data presented in our present report dem-
onstrate that size is an important factor in altering the Th1 or Th2
balance of an immune response. Furthermore, the present study
indicates that the critical cut-off in the ability to stimulate Th1-like
as opposed to Th2-like responses lies between 155 and 225 nm.
Intriguingly, this figure also corresponds to the lower limit of the
macrophage phagocytic response (35, 36).
The ability to phagocytose large particles is one of the features
that distinguishes macrophages from other APCs (37, 38), al-
though some capacity for ingestion of particulate Ags has been
demonstrated for dendritic cell progenitors in vitro (39, 40). In
contrast, other APCs, such as B lymphocytes, cannot phagocytose
(37, 38) and must presumably ingest Ag by pinocytic mechanisms,
which effectively means they cannot internalize particles greater
than approximately 150 nm (35, 36). A number of studies in vivo
and in vitro demonstrate an essential role for phagocytic cells in
both Th1 cell expansion (6, 7, 10, 41– 44) and the processing of
exogenous Ag via the endogenous pathway to stimulate CD81 T
cell expansion (36, 45– 47). Similarly, B cells have been impli-
cated in preferentially stimulating Th2 cell expansion (42, 48, 49).
In vitro studies have previously demonstrated that entrapment of
Ag within liposomes, which requires phagocytic ingestion, totally
inhibits the ability of B cells to present that Ag (37, 38). Therefore,
it is possible that preparation of Ag in vesicles of different sizes
alters the distribution of Ag among the APC populations. The cru-
cial factor in this arrangement would then be whether the size of
the particle made it more likely to be phagocytosed or pinocytosed.
However, in vivo localization of horseradish peroxidase-labeled
vesicles up to 24 h after administration demonstrated similar pat-
terns of distribution for both large (560 nm) and small (155 nm)
vesicles (data not shown). Furthermore, the ability of B2201
splenic B cells to acquire FITC-dextran, a marker of fluid phase
endocytosis (23), could be totally inhibited by preparation of
FITC-dextran in any size of vesicle. Alternatively, larger vesicles
may be more avidly phagocytosed by macrophages than smaller
vesicles; however, our experiments with J.774 macrophages dem-
onstrate that this is clearly not the case. These observations suggest
that the mechanism by which different sizes of vesicles induce Th1
or Th2 responses does not involve differential distribution of the
vesicles among APC populations, and therefore that the ultimate
destination for vesicles in vivo is most likely the macrophage.
A number of studies have suggested that the density of T cell
epitopes on APCs can influence the Th1/Th2 bias of the develop-
ing T cell response (50, 51). It is possible, therefore, that the vary-
ing abilities of different sizes of vesicle to deliver protein to APCs
may affect this parameter. To explore this possibility we prepared
vesicles of constant size with different amounts of entrapped Ag.
By manipulating the ratio of protein to lipid we demonstrated that
across a large range of ratios the relative Th1/Th2 response was
not affected. We would conclude from these data that the ability of
different sizes of lipid vesicle to influence the Th1/Th2 response
while not being mediated through variations in the amount of Ag
per vesicle, is more likely to be influenced by the direct effects of
vesicles on macrophages.
To determine the potential role of macrophages in the mecha-
nism of Th1 or Th2 induction by vesicles we analyzed the abilities
of different sizes of vesicles to induce the production of the T cell
stimulatory cytokines IL-1b (52) and IL-12 (53) by macrophages
in vitro. Our results indicate that while small vesicles induce high
levels of IL-1b in macrophages, they cannot induce IL-12 produc-
tion. In contrast, when macrophages were treated with large ves-
icles, both IL-12 and IL-1b were produced, although IL-1b levels
were significantly lower than those observed for small vesicles. In
agreement with this result, we have previously demonstrated the
ability of large nonextruded vesicles administered i.p. to potentiate
splenocyte IL-12 production upon in vitro restimulation (12). As
macrophage-derived IL-12 is known to be an essential factor for
the development of Th1 responses (53–56), these results would
suggest a mechanism for the preferential generation of Th1 re-
sponses by large vesicles via induction of macrophage IL-12. The
importance of IL-12 in the Th1 response induced by large vesicles
was further confirmed in vivo in studies using IL-12 (p40)-defi-
cient mice. These results demonstrated that the elevated production
of IFN-g observed in vitro with spleen cells from BALB/c mice
inoculated with OVA prepared in large vesicles (560 nm) was
absent in IL-12-deficient mice.
Downloaded from http://www.jimmunol.org/ by guest on December 7, 2019
The endocytic pathways through which large and small vesicles
are most likely to be sequestered may determine the differential
responses of the macrophage to these stimuli. As large vesicles
have a mean diameter above the lower limit of the phagocytic
response (150 nm), they are likely to act as a phagocytic stimulus
to macrophages. This would not be the case with smaller vesicles,
which have a mean diameter below this limit. In this context, it is
relevant that the ability of chitin particles to induce the production
of IL-12 by spleen cells has recently been shown to be ablated by
incubation with cytochalasin D (25), an inhibitor of phagocytosis
(57). Collectively, these observations indicate a relationship be-
tween phagocytosis and the production of IL-12 and, conse-
quently, the induction of Th1-type responses in vivo.
In conclusion, the study described here indicates that the same
adjuvant can be physically manipulated to preferentially stimulate
either a Th1- or a Th2-type response to an Ag. This capacity is a
function of the size of the adjuvant particle, and our further anal-
yses indicate a central role for macrophages in distinguishing these
stimuli and influencing the generation of Ag-specific Th1- or
Th2-type responses by the respective secretion or nonsecretion
of IL-12.
Acknowledgments
We thank Margaret Mullin, Joan McColl, and June Irvine for their tech-
nical assistance.
References
1. Parish, C. R., and F. Y. Liew. 1972. Immune response to chemically modified
flagellin. III. Enhanced cell mediated immunity during high and low zone anti-
body tolerance to flagellin. J. Exp. Med. 135:298.
2. Mosmann, T. R., and R. L. Coffman. 1989. Th1 and Th2 cells: different patterns
of lymphokine secretion lead to different functional properties. Annu. Rev. Im-
munol. 7:145.
3. Liew, F. Y., and C. A. O’Donnell. 1993. Immunology of leishmaniasis. Adv.
Parasitol. 32:161.
4. Grencis, R. K., K. J. Else, A. J. Bancroft, and D. A. P. Bundy. 1993. Trichuris
update ’93. Parasitol. Today 9:309.
5. Rocken, M., J. H. Saurat, and C. Hauser. 1992. A common precursor for CD41
T cells producing IL-2 or IL-4. J. Immunol. 148:1031.
6. Boom, W. H., D. Liano, and A. K. Abbas. 1988. Heterogeneity of helper/inducer
T lymphocytes. II. Effects of interleukin 4- and interleukin 2-producing T cell
clones on resting B lymphocytes. J. Exp. Med. 167:1352.
7. Brewer, J. M., J. Richmond, and J. Alexander. 1994. The demonstration of an
essential role for macrophages in the in vivo generation of IgG2a antibodies. Clin.
Exp. Immunol. 97:164.
8. Weaver, C. T., C. M. Hawrylowicz, and E. R. Unanue. 1988. T helper cell subsets
require the expression of distinct costimulatory signals by antigen presenting
cells. Proc. Natl. Acad. Sci. USA 85:8181.
9. Gajewski, T. F., and F. W. Fitch. 1988. Anti-proliferative effect of IFN-g in
immune regulation. I. IFN-g inhibits proliferation of Th2 but not Th1 murine
helper T lymphocyte clones. J. Immunol. 140:4245.
The Journal of Immunology
10. Macatonia, S. E., C.-S. Hsieh, K. M. Murphy, and A. O’Garra. 1993. Dendritic
cells and macrophages are required for Th1 development of CD41 T cells from
ab TCR transgenic mice: IL-12 substitution for macrophages to stimulate IFN-g
production is IFN-g dependent. Int. Immunol. 5:1119.
11. Brewer, J. M., and J. Alexander. 1992. The adjuvant activity of non-ionic sur-
factant vesicles (niosomes) on the BALB/c humoral response to bovine serum
albumin. Immunology 75:570.
12. Brewer, J. M., C. W. Roberts, M. Conacher, J. McColl, B. A. Blaney, and
J. Alexander. 1996. An adjuvant formulation which preferentially induces Th1
cytokine and CD81 cytotoxic responses is associated with up-regulation of IL-12
and suppression of IL-10 production. Vaccine Res. 5:77.
13. Roberts, C. W., J. M. Brewer, and J. Alexander. 1994. Congenital toxoplasmosis
in the BALB/c mouse: prevention of vertical disease transmission and foetal
death by vaccination. Vaccine 12:1389.
14. Nayar, R., M. J. Hope, and P. R. Cullis. 1989. Generation of large unilamellar
vesicles from long chain saturated phosphatidylcholines by extrusion techniques.
Biochim. Biophys. Acta 986:200.
15. Brewer, J. M., C. W. Roberts, W. H. Stimson, and J. Alexander. 1995. Accurate
determination of adjuvant-associated protein or peptide by ninhydrin assay. Vac-
cine 13:1441.
16. Kelso, A., P. Groves, A. B. Troutt, and M. H. Pech. 1994. Rapid establishment
of a stable IL-4/IFN-g production profile in the antigen-specific CD41 T cell
response to protein immunization. Int. Immunol. 6:1515.
17. Magram, J., S. E. Connaughton, R. R. Warrier, C. M. Carvajal, D. Y. Wu,
J. Ferrante, C. Stewart, U. Sarmiento, D. A. Faherty, and M. K. Gately. 1996.
IL-12-deficient mice are defective in IFN-g production and type 1 cytokine re-
sponses. Immunity 4:471.
18. Johnson, D. A., J. W. Gautsch, J. R. Sportsman, and J. H. Elder. 1984. Improved
technique utilising non fat dry milk for analysis of proteins and nucleic acids
transferred to nitrocellulose. Gene Anal. Tech. 1:3.
19. Boyle, W. 1968. An extension of the 51-Cr release assay for the estimation of
mouse cytotoxins. Transplantation 6:761.
20. Maloy, K. J., A. M. Donachie, and A. M. Mowat. 1995. Induction of Th1 and Th2
CD41 T cell responses by oral or parenteral immunization with ISCOMS. Eur.
J. Immunol. 25:2835.
21. Yang, X., R. S. Gieni, T. R. Mosmann, and K. T. HayGlass. 1993. Chemically
modified antigen preferentially elicits induction of Th1-like cytokine synthesis
patterns in vivo. J. Exp. Med. 178:349.
22. Burstein, H. J., C. M. Shea, and A. K. Abbas. 1992. Aqueous antigens induce in
vivo tolerance selectively in IL-2- and IFN-g- producing (Th1) cells. J. Immunol.
148:3687.
23. Wang, E., J. Michl, L. M. Pfeffer, S. C. Silverstein, and I. Tamm. 1984. Interferon
suppresses pinocytosis but stimulates phagocytosis in mouse peritoneal macro-
phages: related changes in cytoskeletal organization. J. Cell Biol. 98:1328.
24. Claassen, E. 1992. Post-formation fluorescent labelling of liposomal membranes:
in vivo detection, localisation and kinetics. J. Immunol. Methods 147:231.
25. Shibata, Y., W. J. Metzger, and Q. N. Myrvik. 1997. Chitin particle-induced
cell-mediated immunity is inhibited by soluble mannan: mannose receptor-me-
diated phagocytosis initiates IL- 12 production. J. Immunol. 159:2462.
26. Chensue, S. W., J. H. Ruth, K. Warmington, P. Lincoln, and S. L. Kunkel. 1995.
In vivo regulation of macrophage IL-12 production during type 1 and type 2
cytokine-mediated granuloma formation. J. Immunol. 155:3546.
27. Snapper, C. M., and J. J. Mond. 1993. Towards a comprehensive view of im-
munoglobulin class switching. Immunol. Today 14:15.
28. Snapper, C. M., and W. E. Paul. 1987. Interferong and B cell stimulatory factor-1
reciprocally regulate immunoglobulin isotype control. Science 239:944.
29. Mosmann, T. R., H. Cherwinski, M. W. Bond, M. A. Giedlin, and R. L. Coffman.
1986. Two types of murine helper T cell clone. I. Definition according to profiles
of lymphokine activities and secreted proteins. J. Immunol. 136:2348.
30. Noben-Trauth, N., P. Kropf, and I. Muller. 1996. Susceptibility to Leishmania
major infection in interleukin-4 deficient mice. Science 271:987.
31. Morawetz, R. A., L. Gabriele, L. V. Rizzo, N. Nobentrauth, R. Kuhn,
K. Rajewsky, W. Muller, T. M. Doherty, F. Finkelman, R. L. Coffman, and
H. C. Morse. 1996. Interleukin (IL)-4 independent immunoglobulin class switch
to immunoglobulin (Ig)E in the mouse. J. Exp. Med. 184:1651.
32. Brewer, J. M., M. Conacher, A. Satoskar, H. Bluethmann, and J. Alexander.
1996. In interleukin-4 deficient mice, alum not only generates T helper-1 re-
sponses equivalent to Freund’s complete adjuvant, but continues to induce T
helper-2 cytokine production. Eur. J. Immunol. 26:2062.
33. von der Weid, T., M. Kopf, G. Kohler, and J. Langhorne. 1994. The immune
response to Plasmodium chabaudi malaria in interleukin-4-deficient mice. Eur.
J. Immunol. 24:2285.
34. HayGlass, K. T., and W. P. Stefura. 1991. Antigen specific inhibition of ongoing
murine IgE responses. II. Inhibition of IgE responses induced by treatment with
4007
glutaraldehyde modified allergens is paralleled by reciprocal increases in IgG2a
synthesis. J. Immunol. 147:2455.
35. Rabinovitch, M. 1995. Professional and non-professional phagocytosis: an intro-
duction. Trends Cell Biol. 5:85.
36. Harding, C. V., and R. Song. 1994. Phagocytic processing of exogenous partic-
ulate antigens by macrophages for presentation by class I MHC molecules. J. Im-
munol. 153:4925.
37. Harding, C. V., D. S. Collins, O. Kanagawa, and E. R. Unanue. 1991. Liposome
encapsulated antigens engender lysosomal processing for class II presentation
and cytosolic processing for class I presentation. J. Immunol. 147:2860.
38. DalMonte, P. R., and F. C. Szoka. 1989. Effect of liposome encapsulation on
antigen presentation in vitro: comparison of presentation by peritoneal macro-
phages and B cell tumors. J. Immunol. 142:1437.
39. Reis e Sousa, C., P. D. Stahl, and J. M. Austyn. 1993. Phagocytosis of antigens
by Langerhans cells in vitro. J. Exp. Med. 178:509.
40. Inaba, K., M. Inaba, M. Naito, and R. M. Steinman. 1993. Dendritic cell pro-
genitors phagocytose particulates, including Bacillus Calmette-Guerin organisms,
and sensitize mice to mycobacterial antigens in vivo. J. Exp. Med. 178:479.
41. Schmitz, J., and A. Radbruch. 1992. Distinct antigen presenting cell derived
signals induce Th cell proliferation and expression of effector cytokines. Int.
Immunol. 4:43.
42. Gajewski, T. F., M. Pinnas, T. Wong, and F. W. Fitch. 1991. Murine Th1 and Th2
clones proliferate optimally in response to distinct antigen presenting cell popu-
lations. J. Immunol. 146:1750.
43. Bogen, S. A., D. S. Weinberg, and A. K. Abbas. 1991. Histological analysis of
Downloaded from http://www.jimmunol.org/ by guest on December 7, 2019
T lymphocyte activation in reactive lymph nodes. J. Immunol. 147:1537.
44. Bogen, S. A., I. Fogelman, and A. K. Abbas. 1993. Analysis of IL-2, IL-4 and
IFN-g producing cells in situ during immune responses to protein antigens. J. Im-
munol. 150:4197.
45. Reis e Sousa, C., and R. N. Germain. 1995. Major histocompatibility complex
class-I presentation of peptides derived from soluble exogenous antigen by a
subset of cells engaged in phagocytosis. J. Exp. Med. 182:841.
46. Kovacsovics-Bankowski, M., K. Clark, B. Benacerraf, and K. L. Rock. 1993.
Efficient major histocompatibility complex class I presentation of exogenous an-
tigen upon phagocytosis by macrophages. Proc. Natl. Acad. Sci. USA 90:4942.
47. Zhou, F., B. T. Rouse, and L. Huang. 1992. Induction of cytotoxic T lymphocytes
in vivo with protein antigen entrapped in membranous vehicles. J. Immunol.
149:1599.
48. Saoudi, A., S. Simmonds, I. Huitinga, and D. Mason. 1995. Prevention of ex-
perimental allergic encephalomyelitis in rats by targeting autoantigen to cells:
evidence that the protective mechanism depends on changes in the cytokine re-
sponse and migratory properties of the autoantigen-specific T-cells. J. Exp. Med.
182:335.
49. McKnight, A. J., C. M. Shea, and G. R. Weins. 1994. Differentiation of Vb8.21
CD41 T cells induced by a superantigen: roles of antigen presenting cells and
cytokines. Immunology 81:513.
50. Constant, S., C. Pfeiffer, A. Woodard, T. Pasqualini, and K. Bottomly. 1995.
Extent of T cell receptor ligation can determine the functional differentiation of
naive CD41 T cells. J. Exp. Med. 182:1591.
51. Hosken, N. A., K. Shibuya, A. W. Heath, K. M. Murphy, and A. O’Garra. 1995.
The effect of antigen dose on CD41 T helper cell phenotype development in a T
cell receptor-ab-transgenic model. J. Exp. Med. 182:1579.
52. Lichtman, A. H., J. Chin, J. A. Schmidt, and A. K. Abbas. 1988. Role of inter-
leukin 1 in the activation of T lymphocytes. Proc. Natl. Acad. Sci. USA 85:9699.
53. D’Andrea, A., M. Rengaraju, N. M. Valiante, J. Chehimi, M. Kubin, M. Aste,
S. H. Chan, M. Kobayashi, D. Young, E. Nickbarg, R. Chizzonite, S. F. Wolf, and
G. Trinchieri. 1992. Production of natural killer cell stimulatory factor (Interleu-
kin 12) by peripheral blood mononuclear cells. J. Exp. Med. 176:1387.
54. Germann, T., M. K. Gately, D. S. Schoenhaut, M. Lohoff, F. Mattner, S. Fischer,
S.-C. Jin, E. Schmitt, and E. Rude. 1993. Interleukin-12/T cell stimulating factor,
a cytokine with multiple effects on T helper type 1 (Th1) but not on Th2 cells.
Eur. J. Immunol. 23:1762.
55. Morris, S. C., K. B. Madden, J. J. Adamovicz, W. C. Gause, B. R. Hubbard,
M. K. Gately, and F. D. Finkelman. 1994. Effects of IL-12 on in vivo cytokine
gene expression and Ig isotype selection. J. Immunol. 152:1047.
56. Schmitt, E., P. Hoehn, C. Huels, S. Goedert, N. Palm, E. Rude, and T. Germann.
1994. T helper type 1 development of naive CD41 T cells requires the coordinate
action of interleukin-12 and interferon-g and is inhibited by transforming growth
factor-b. Eur. J. Immunol. 24:793.
57. Allison, A. C., P. Davies, and S. De Petris. 1971. Role of contractile microfila-
ments in macrophage movement and endocytosis. Nature 232:153.