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,
*
Veterans Affairs Medical Center, Memphis, TN 38104; and Departments of
Medicine,
Pathology, and
Pediatrics, University of Tennessee Health Sciences Center, Memphis, TN 38163
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells express the Tg, these mice can be rendered
completely tolerant to the induction of arthritis by i.v.
administration of 200 µg of CII. As early as 24 h after CII
administration, CII-specific T cells demonstrated a decreased ability
to proliferate in response to the CII immunodominant peptide and
phenotypically altered the expression of L-selectin to
CD62Llow and of phagocytic glycoprotein-1 to
CD44high, expression levels consistent with the phenotype
of memory T cells. In addition, they up-regulated the expression of the
activation markers CD71 and CD69. Functionally, following tolerogenic
stimulation, the CII-specific T cells produced similar levels of IL-2
in comparison to controls when challenged with CII peptide, however, by
48 h after exposure to tolerogen, IL-2 production dropped and was
replaced by high levels of IL-10 and IL-4. Based on their production of
Th2 cytokines, these data suggest that T regulatory cells expressing
activation and memory markers are induced by the tolerogen and may
exert their influence via cytokines to protect the animals from the
induction of arthritis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Here, we describe the development of a transgenic (Tg)3 mouse that expresses a TCR specific for type II collagen (CII) in the context of I-Aq, a haplotype susceptible to the induction of collagen-induced arthritis (CIA; Ref. 18). These mice express the CII-specific TCR on >90% of the CD4+ T cells and, despite the fact that they are capable of recognizing murine CII (mCII), they are not deleted in the thymus. We have analyzed the function of these T cells with the goal of determining their role in mediating autoimmune arthritis and using the Tg TCR for the study of the early events in tolerance induction. This model has the advantage that the tolerance in vivo is directly linked to prevention of a disease process, the ultimate goal in the development of therapeutic tolerance. Our results indicate that, despite an overwhelming capacity to establish an autoimmune response and elicit disease, these cells can be tolerized and that the tolerance appears to be mediated by an active mechanism involving regulatory control. Thus, this Tg model will serve as an excellent vehicle for defining the molecular mechanisms of tolerance induction in T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TCR transgenes were established in (C57BL/6 x C3H)F1 mice and backcrossed onto DBA/1LacJ mice (The Jackson Laboratory, Bar Harbor, ME). Mice used in these experiments were from backcross generations N7 to N9 and were heterozygous for the TCR transgenes.
Generation of TCR Tg mice
TCR constructs encoding a CII-specific receptor were made by
RT-PCR amplification of V and V gene
segments expressed by an I-Aq-restricted CII
(260–267)-specific T cell hybridoma, qCII85.33 (19), and
subcloned into TCR-2B4 and TCR-3A9 expression vectors as
described by Sakaguchi et al. (20). The endogenous
VJ and VDJ segments of these
expression vectors were removed by digestion with
XhoI/NotI and ClaI/NotI,
respectively. The 5' and 3' ends of V11.1-J17
(TCRAV11S1J17) cDNA and the V8.3-Db1-J1.4
(TCRBV8S3D1J1S4) cDNA were modified by PCR to include these
restrictions sites for insertion into their respective expression
vectors and to incorporate intronic splicer donor and acceptor sites
for recombination to the genomic leader domains and the C and C
domains (20). Tg mice were established by coinjection of
linearized constructs (SalI and ClaI for TCR
and PvuI and SalI for TCR) into the pronuclei
of F1(C57BL/6 x C3H) eggs. Resulting
progeny were screened by PCR amplification of genomic DNA using PCR
primers specific for the rearranged V11.1-J17 and the
V8.3-D1-J1.4 gene segments. PCR-positive mice were
subsequently backcrossed to DBA/1LacJ mice and offspring were screened
for the presence of the transgenes by PCR and by immunofluorescence
using the V8.3-specific Ab, 1B3.3. No Ab is available for detecting
the V11.1 genetic allele used by this TCR.
Antibodies
The Abs used for flow cytometry were: PerCP-conjugated
anti-CD4 (clone RM4-5), PE-conjugated anti-CD8 (clone 53-6.7),
APC-conjugated anti-CD44 (clone 1 M7), PE-conjugated anti-CD62L
(clone Mel-14), PE-conjugated anti-CD69 (clone H1.2F3),
PE-conjugated anti-CD71 (clone C2), and FITC-conjugated
anti-V8.3 (clone 1B3.3). All Abs were purchased from BD
PharMingen (San Diego, CA) and used according to the
manufacturer’s recommendations.
Flow cytometry
Spleens were minced in cold RPMI and RBC were lysed with Gey’s solution. Splenocytes were stained with specific Abs in PBS at 4°C for 30 min. Flow cytometry was performed on a FACSCaliber (BD Biosciences, Mountain View, CA) and analyzed using CellQuest version 3.3 (BD Biosciences, Mountain View, CA).
Collagen preparation
Native bovine CII was solubilized from articular cartilage and native mCII was solubilized from the sternal cartilage of young mice. CII was extracted by limited proteolysis with pepsin and purified by repeated differential salt precipitation as described (21, 22).
Immunizations and arthritis induction
Six- to 8-wk-old mice were immunized with CII for the induction of arthritis. CII was dissolved in 10 mM cold acetic acid by stirring overnight at 4°C and emulsified at a 1:1 (v/v) ratio with CFA (Life Technologies, Gaithersburg, MD), as previously described (23). Mice were immunized s.c. at the base of the tail with 100 µg of CII. Each paw was evaluated and scored for the degree of inflammation on a scale of 0–4 (18, 22). The average severity per arthritic mouse was calculated by adding the score of all four paws of the arthritic animals and dividing by the number of arthritic mice.
Tolerization induction
For the study of early events following administration of a tolerogen in the TCR Tg mice, a single dose of 200 µg of tolerogen was administered i.v. via the sinus at the retro-orbital plexus. Ags were dissolved at 2 mg/ml in PBS. For the experiments in which arthritis incidence was measured, animals were challenged with bovine CII (see Immunizations and arthritis induction) 1 wk following i.v. administration of Ag.
Peptide synthesis
Peptides were synthesized by F-moc chemistry using an Applied Biosystems automated peptide synthesizer (model 430; Foster City, CA) and were purified by HPLC.
Proliferation assays
Spleen cells were cultured in 96-well plates at 4.5 x 105/well in 300 µl of HL-1 medium supplemented with 50 µM of 2-ME and 0.1% BSA (fraction V, IgG free, low endotoxin; Sigma-Aldrich, St. Louis, MO) at 37°C, 5% humidified CO2 for 4 days. Eighteen hours before the termination of the cultures, 1 µCi of [3H]thymidine (New England Nuclear, Boston, MA) was added to each well. Cells were harvested onto glass fiber filters and counted on a Matrix 96 direct ionization beta counter (Packard Instrument, Meriden CT). Results were confirmed by replicate experiments and all data are expressed as dpm. Some proliferation assays included the addition of human rIL-2 (PeproTech, Rocky Hill, NJ).
T cell hybridomas and Ag presentation assays
T cell hybridomas were established by polyethylene glycol
(Boehringer Mannheim, Indianapolis, IN)-induced fusion of lymph node
cells with TCR --
BW5147 thymoma cells (24, 25). Lymph node cells were
obtained from DBA/1LacJ mice immunized 10 days previously with
bCII/CFA. The recovered T cells were cultured with bovine 1(II) for
4 days, followed by IL-2 for 3 days before fusion. Resulting hybridomas
were screened for their ability to recognize bovine 1(II) chains and
the CII (256–273) peptide presented by I-Aq. Ag
presentation experiments were performed in 96-well microtiter plates in
a total volume of 0.3 ml containing either 105
M12.Aq cells (19) or 4 x
105 syngeneic spleen cells as APC and
105 T hybridoma cells. Cell cultures were
maintained at 37°C in 5% humidified CO2 for
20–24 h, after which 2-fold serial dilutions were made for
determination of IL-2 titers using the IL-2-addicted cell line HT-2
(26, 27). HT-2 cell viability was assessed by cleavage of
MTT and quantitation of OD650 (28, 29). IL-2
titers were quantified by the reciprocal of the highest 2-fold serial
dilution maintaining HT-2 cell viability >2-fold over control
cultures. Results are presented as units of IL-2 per milliliter of
undiluted supernatant as described by Kappler et al.
(30).
ELISA
Ab titers specific for mCII were determined using a solid-phase ELISA as previously described (31). Briefly, microtiter plates were coated with 500 ng of mCII at 4°C overnight. After extensive washing with 0.15 M saline, 0.05% Tween 20, dilutions of sera ranging from 1/4,000 to 1/24,000 in 2% normal goat sera were added to each well and incubated overnight at 4°C. After washing with saline and Tween 20, a goat anti-mouse Ig (1/5,000) was added for 2 h. The plates were then washed and developed by the addition of o-phenyldiamine (Sigma-Aldrich). After stopping the reaction with 2.5 N H2SO4, the degree of color development was measured at 490 nm with the background OD650 subtracted. The quantity of specific Ab was measured for each animal and data are expressed as mean relative units of activity based on a standard anti-CII serum.
Cytokine analysis
Cytokine analyses were performed using the R&D Systems
Quantikine M ELISA (Minneapolis, MN) according to the manufacturer’s
protocol. Splenocytes were harvested from tolerized animals at the
times indicated and were cultured in HL-1 with 33 µg/well of CII
(245–270) peptide as described for Proliferation assays
using 2-ml cultures of 2 x 106 cells/well
in 24-well plates. Cultures were incubated according to the conditions
listed below. Supernatants from each culture were collected at 24
h for IL-2 measurement, 48 h for IFN-
and IL-4 measurement, and
72 h for IL-10 measurement. These time points have been found to
be optimal for the detection of these cytokines. Culture supernatants
were stored frozen at -20°C until analyzed by ELISA. Cytokine
quantities were calculated based on a standard curve generated by
SoftMaxPro version 2.1 software on a SpectroMax (Molecular Devices,
Sunnyvale, CA) using recombinant standards supplied by the
manufacturer. Results are based on the mean of two individual wells of
each supernatant and the data are representative of at least two
individual experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To study the role of CII (260–267)-specific T cells in the
development of CII autoimmune arthritis and the mechanism by which
these T cells are rendered tolerant, a TCR Tg mouse was developed. cDNA
encoding a CII (260–267)-specific TCR was cloned by RT-PCR from an
I-Aq-restricted T cell hybridoma (qCII85.33; Fig. 1
A) that recognizes both
bovine CII (immunogen) and mCII (autoantigen). This TCR uses
V11.1-J17 gene segments (TCRAV11S1J17) and
V8.3-D1-J1.4 gene segments
(TCRV8S3D1J1S4; Fig. 1B), similar to other CII
(260–267)-specific I-Aq-restricted TCR
previously described (32, 33). The V and V cDNA were
cloned into the TCR and transgenesis vectors described by
Sakaguchi et al. (20). Following coinjection, two founders
were identified by PCR and immunofluorescence and backcrossed to
DBA/1LacJ mice. Initially, the TCR Tg expression could not be detected
in the founders and first generation offspring
(H-2q/b). This appeared to be the result of
negative selection by the I-Ab molecule, as
expression was easily detectable in the H-2q/q
second generation backcrosses (N2) but still remained undetectable in
the H-2q/b littermates. As shown in Fig. 1C, the V8.3 transgene was expressed by
90% of the CD4+ T cell population of the
spleen and >95% of the CD4+ T cells in the
lymph nodes, which is significantly higher than the 8%
CD4+ cells that express the endogenous
V8.3+ in the non-Tg littermates. Similarly,
>90% of the + T cells
expressed the Tg TCR (data not shown). In the thymus,
CD4+/V8.3+ cells predominated
while the percent of CD4+/CD8+ and
CD4+ T cells were unchanged, although a decrease
in the percent of CD8+ T cells was evident.
Similar data for TCR Tg expression were obtained with a second founder,
27 (see Fig. 3) with the exception of the Tg being expressed by only
25% of the T cell population (data not shown).
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The TCR Tg is fully functional as measured by the
ability of the T cells to proliferate specifically in response to
peptide presentation by I-Aq and by their ability
to mediate the development of autoimmune arthritis. When naive Tg T
cells were stimulated in vitro with either native CII or a CII peptide
containing the immunodominant determinant, CII (260–267), the cells
proliferated vigorously (Fig. 2). No
response was observed to irrelevant Ags, nor when using T cells from
non-Tg littermates. When the Tg mice were immunized with an emulsion
containing CII and CFA, they developed a strong T cell-dependent Ab
response to both the immunogen and the autoantigen, mCII (Table I), and developed a severe accelerated
autoimmune arthritis (Fig. 3
). The Tg
mice developed arthritis as early as 11 days after immunization and
generally reached their maximum incidence and severity by day 35. In
comparison, non-Tg littermates first developed arthritis around day 20
and did not reach their maximum incidence until around day 50, a rate
similar to that observed with normal DBA/1 mice (34).
Offspring from both Tg founders exhibited accelerated kinetics of
response, but those from founder 24 appeared to have a more severe form
of disease as compared with the negative littermates and those derived
from founder 27 (Fig. 3A). As might be predicted from the
data in Fig. 3, the anti-CII Ab levels (Table I) were higher in the
Tg mice at day 19, but by day 34 when the disease was equally well
established in both groups, the Ab levels were high in both Tg and
negative littermate groups. We have not observed spontaneous arthritis
in Tg mice up to 9 mo of age, despite the overwhelming numbers of
CII-specific T cells.
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Based on the overwhelming T cell response and accelerated
arthritis development following CII immunization, we were initially
concerned that it may be difficult to tolerize these mice and prevent
the development of CIA. However, as shown in Fig. 4B, i.v. administration of a
single dose of 200 µg of CII was capable of significantly reducing
the incidence of arthritis in the Tg mice. Tg mice tolerized with OVA
developed arthritis at an expected accelerated rate, reaching 100% by
day 24, whereas only two of the CII tolerized mice developed arthritis
by day 42. The severity of the disease exhibited by the two
CII-tolerized mice that did develop arthritis was indistinguishable
from that exhibited by control (OVA-tolerized) animals (Fig. 4A). When the protocol was changed to a regimen of i.v.
administration of three consecutive daily doses of 200 µg of CII or
OVA (a total of 600 µg), disease was completely prevented in all 10
of the animals tolerized with CII, whereas all 10 of the OVA-tolerized
animals had disease by day 13 (Fig. 4D). This protection was
afforded for at least 8 wk after the challenge with CII + CFA.
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To determine how CII-specific T cell function had been altered
following exposure to the CII tolerogen, TCR Tg mice were treated i.v.
with a tolerizing dose of CII, and T cell function, phenotype, and
cytokine production were analyzed. As early as 24 h after a single
dose of 200 µg of Ag, a clear effect on T cell proliferation could be
observed. T cells from CII tolerized mice had a greatly reduced
capacity to be stimulated to proliferate by CII peptide in comparison
to Tg T cells from OVA-treated mice (Fig. 5). At 48 h after exposure to
tolerogen, these cells were consistently refractory to stimulation by
peptide concentrations ranging from 130 nM to 130 µM. However, by
72 h after tolerization, some proliferative capacity was restored,
although the quantity of peptide required to stimulate the
CII-tolerized T cells was >2 logs higher than that required to
stimulate the Tg T cells from the OVA-tolerized mice. This suppressed
proliferative response could still be observed at 7 days post CII
treatment (data not shown).
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To determine whether the lymphocytes that were refractory to
stimulation by peptide were exhibiting signs of anergy, we assayed the
ability of CII-tolerized lymphocytes to proliferate when various
concentrations of exogenous rIL-2 were added to cultures containing a
concentration of CII (245–270) peptide known to optimally stimulate
the TCR Tg lymphocytes. The data in Fig. 6 demonstrate that the addition of
exogenous IL-2 can restore the ability of CII-tolerized lymphocytes to
proliferate in response to CII (245–270) peptide, but only at very
high concentrations of IL-2. Concentrations exceeding 100 U/ml were
required to bring the level of proliferation of the CII-tolerized
lymphocytes up to that of the control (OVA-tolerized) lymphocytes. The
addition of exogenous IL-2 to cultures containing lymphocytes from
OVA-tolerized animals had little effect on these highly stimulated
lymphocytes.
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In addition to a reduced proliferative capacity of the Tg T cells
from i.v.-tolerized mice, significant phenotypic changes also occurred
shortly after CII tolerization. Following tolerization as described
above, spleen cells were recovered and subjected to four-color
immunofluorescent analysis focusing on activation and memory marker
expression by the
CD4+/V8.3+ Tg T cells.
These analyses revealed that as early as 24–48 h post treatment, two
cell surface markers associated with the activation state of the cell,
CD69 and CD71, were significantly elevated in the splenocytes from
CII-tolerized animals as compared with those from OVA or PBS
(control)-tolerized mice (Fig. 7). CD69
was expressed by 62% of the CII-treated Tg T cells in comparison with
only 11% on the Tg T cells from the OVA-treated control mice. CD71
expression was also induced on the majority of the Tg T cells from
CII-treated mice, while only 5% of the T cells from the control mice
expressed this marker. In addition to activation markers, changes in
expression of two cell surface proteins associated with the memory
phenotypes of CD44high and
CD62low were also observed. CD44 expression
levels shifted from the majority of the cells expressing
CD44low (OVA-and PBS-treated groups) to the vast
majority of the CII-tolerized T cells expressing
CD44high. Thus, using this model, changes in T
cell proliferation and phenotype were detected very early after the
tolerogenic administration of CII. These changes suggest that the
CII-tolerized T cells enter a period of activation before the loss of
ability to proliferate in response to challenge with the CII
immunodominant peptide and that they also acquire a memory
phenotype.
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To determine whether CII-specific T cell function had also been
altered shortly after i.v. administration of CII, spleen cells were
recovered 24, 48, and 72 h after treatment with CII or OVA and
tested for their ability to produce IL-2, IL-4, and IL-10 and IFN-.
When the CII-or OVA-tolerized lymphocyte cultures were examined without
stimulation of any kind, we determined that the levels of cytokines
secreted were below our level of detection. However, following an
overnight stimulation with the CII peptide, CII-specific T cells from
CII-treated mice were found to produce similar amounts of IL-2 as
OVA-treated mice, but by 48 h after tolerization the IL-2 response
was significantly reduced, achieving less than half the response of
those from the control mice (Fig. 8C). In contrast to the IL-2
response, the production of the Th2 cytokines IL-4 and IL-10 by the
CII-specific T cells was enhanced (Fig. 8, A and
B). By 48 h after tolerance induction, when the IL-2
production had dropped significantly, the CII-specific T cells from the
CII-treated mice produced high levels of IL-10 and IL-4, whereas the T
cells from the OVA-treated mice produced very little of either these
Th2 cytokines. In contrast, IFN- production was elevated in cultures
containing spleen cells from animals treated with CII (Fig. 8D). In the absence of stimulation with the CII peptide, no
cytokines could be detected (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Th1 and Th2 cell types are defined by the spectrum of cytokines that
they produce, with Th1 cells producing IFN-, but not IL-4, and Th2
cells producing IL-4 and IL-10, but not IFN- (41). Our
data suggest that based on their changing cytokine profiles by 48–72 h
after tolerization with CII, the Ag-specific T cells have acquired a
Th2 phenotype. It has been hypothesized that several factors can
influence the fate of the precommitted Th0 cell during its primary
encounter with Ag, driving it toward the expression of a Th1 or Th2
phenotype (42). These factors include the strength of the
signal received through the TCR (43), specific
costimulatory signals (37), and the cytokine milieu in
which the cells encounter their Ag (44, 45). Some of the
studies pertaining to differentiation via specific costimulatory
signals include the concept of B7 binding with high affinity to CTLA-4
instead of to CD28, thereby down-regulating T cell activation
(46). Using CTLA-4 knockout mice, Oosterwegel et al.
(47) were able to determine that the B7-CD28/CTLA-4
pathway plays a critical role in determining the phenotype of the Th0
cell. They found that CD28 promotes Th2 differentiation while CTLA-4
limits Th2 differentiation. Although, we have not yet tested the
ability of the CTLA-4 Ig fusion protein to modulate the induction of
tolerance in this TCR Tg model, it has been used successfully to
prevent CIA disease in DBA/1 mice (48) and rats
(49). An understanding of the role of negative
costimulation such as that supplied through CTLA-4 would be important
to understanding the signals driving the differentiation of
CII-specific T cells toward the Th2 pathway as we have observed.
It has also been suggested that the cytokine milieu present when the
Ag-specific cell encounters its Ag can influence its future
differentiation. For instance, autoimmune T cells which encounter IL-12
when they are activated with Ag are likely to differentiate toward the
pathogenic Th1 phenotype (50), whereas those that
encounter Ag in the presence of IL-4 may differentiate into
suppressive IL-4- and IL-10-secreting Th2 cells (51).
The characteristics of these specific cytokine environments have a
large influence over the series of events leading to the induction of
disease in the CIA model and also have important implications for its
prevention through tolerance. Studies using the CIA model have
suggested that the Mycobacterium, the key component in CFA,
stimulates the dendritic cells found in the intradermal site of
injection to produce IL-12 (50, 52). It is this IL-12 and
a combination of IFN--inducing factor and IFN-
(53, 54, 55) that are suspected to drive the differentiation
of the precommitted CII-specific CD4+ T cell
toward the Th1 phenotype so critical for the precipitation of the
inflammatory response that defines the disease state. However, the
converse situation is not as well understood. The initial source of
IL-4 that is suspected to be important to furthering the
differentiation of Ag-specific T cells toward the Th2 pathway has not
yet been determined (56). Le Gros et al. (57)
suggest that the initial production of IL-4 by naive murine T cells is
dependent on the in situ secretion of IL-2 . It is possible that an
initial spike in IL-2 secretion such as we have observed at 24 h
posttolerance may supply the environment necessary for the
differentiation of the nascent Th2 response toward the production of
enough IL-4 to initiate a cascade of Th2 development. The high levels
of IL-4 we detected at 48 h post-CII treatment suggest that our
cultures were, in fact, becoming biased in the Th2 direction. Although
IFN- is traditionally classified as a Th1 cytokine, there is some
evidence to suggest that IFN- is often elevated in cultures of
lymphocytes from tolerized mice and that its presence is required for
the induction of tolerance (58, 59). This would support
our observations of the elevated Th2 cytokines IL-4 and IL-10 and the
concurrent presence of elevated levels of IFN-. In addition, IFN-
has been shown to be a negative regulator in several autoimmune disease
models (60, 61, 62, 63, 64).
As mentioned previously, because the distinctions between the categories described for mechanisms hypothesized to be at work in immune tolerance are not as clear as was once believed, it becomes important to clearly define the functional and phenotypic attributes of the Ag-specific cells during tolerance induction. Analysis of the early events in the tolerance of our CII TCR Tg mouse model suggest that several of these mechanisms may be functioning to some degree. The proliferative responses suggest the appearance of anergy in the cultures of lymphocytes from CII-tolerized TCR Tg animals, yet the T cells express activation markers. However, our cytokine analyses indicate the emergence of Tr cells that exhibit a Th2 phenotype. Taken together, these data support the notion by Lanoue et al. (17) that functionally anergic cells may be early stages of Tr cells that will eventually exert their influence through immune deviation.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David D. Brand, Veterans Affairs Medical Center, 1030 Jefferson Avenue, Memphis, TN 38104. E-mail address: dbrand{at}utmem.edu
3 Abbreviations used in this paper: Tg, transgenic; CII, type II collagen; CIA, collagen-induced arthritis; mCII, murine CII; Tr, T regulatory cells.
Received for publication March 5, 2001. Accepted for publication October 22, 2001.
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References |
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-chains. Biochemistry 10:1652.[Medline]
domain of a TCR prevents the development of collagen-induced arthritis. J. Immunol. 155:4504.[Abstract]
production by T cells. Nature 378:88.[Medline]
production and activates IRAK and NF
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2 subunit expression in developing T helper 1 (Th1) and Th2 cells. J. Exp. Med. 185:817. plays a critical role in induced cell death of effector T cell: a possible third mechanism of self-tolerance. J. Exp. Med. 172:1735.. J. Exp. Med. 186:71. plays a critical down-regulatory role in the induction and effector phase of myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis. J. Immunol. 157:3223.[Abstract]
is critical to the control of murine autoimmune encephalomyelitis and regulates both in the periphery and in the target tissue: a possible role for nitric oxide. J. Immunol. 163:5278. protects C57BL/6 mice from chronic-progressive experimental autoimmune encephalomyelitis by increasing apoptosis of central nervous system-infiltrating lymphocytes. J. Immunol. 167:1821. receptor-deficient mice. J. Immunol. 158:5507.[Abstract]
receptors. J. Immunol. 158:5501.[Abstract]This article has been cited by other articles:
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
)
and 24 V
). All animals were challenged
with a dose of 100 µg of CII emulsified in CFA for the induction of
disease. Mice were scored for arthritis incidence and severity as
described in Materials and Methods.
), but IFN-