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Departments of
*
Microbiology and Molecular Genetics and
Medicine, Division of Rheumatology, School of Medicine, University of California, Los Angeles, CA 90095, and
Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We (7, 8) and others (9) described that the susceptibility to AA of another closely related strain, the Fischer 344 (F344) rat (RT.1lvl), can be modulated significantly by the environmental factors. F344 rats raised under barrier facility (BF) conditions (BF-F344) are susceptible to induction of AA, whereas F344 rats maintained in a conventional facility (CV) (CV-F344) show considerably reduced incidence of AA. However, at this point, the immunologic basis of the reduced incidence and severity of AA in CV-F344 rats has not as yet been defined. In this study, we describe the immunologic mechanism involved in modulation of the susceptibility to AA of CV-F344 rats. Both BF-F344 and CV-F344 rats raise a potent and comparable level of T cell responses to the arthritogenic determinant of Bhsp65 upon challenge with M. tuberculosis. Importantly, we observed that naive unimmunized CV-F344 rats, but not BF-F344 rats, spontaneously raised T cell responses to BCTD described above. Furthermore, adoptive transfer into BF-F344 rats of splenic cells (SPC) of naive CV-F344 rats restimulated in vitro with BCTD can significantly reduce the severity of AA in recipient rats. We attribute the induction of T cell responses to BCTD in CV-F344 rats to the exposure of these rats to environmental agent(s), which presumably display antigenic determinants within homologues of Bhsp65 and/or another Ag that are cross-reactive with BCTD (determinant (molecular) mimicry) (10, 11). Our study provides an explanation of one source of the modulation of an autoimmune disease by the animal’s immediate environment.
In other experimental animal models of autoimmunity, the initiation and/or propagation of autoimmunity has been attributed to the conventional environment; for example, joint and intestinal inflammatory disease in HLA-B27 transgenic rats (12), iodine-induced autoimmune thyroiditis in NOD-H2h4 mice (13), experimental autoimmune thyroiditis in rats (14), hemolytic anemia (15), experimental autoimmune encephalomyelitis (16), and Pristane-induced arthritis (17). In contrast, the current study has revealed that exposure of a susceptible rat strain to the conventional environment rather leads to a significant reduction in the incidence and severity of an autoimmune disease, AA, through spontaneous induction of T cells against regulatory determinants of Bhsp65. These results add a new dimension to the host-microbes relationship in autoimmunity, and are of significance in understanding of the pathogenesis of human autoimmune diseases.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BF-bred F344 rats (RT.1lvl) were purchased from Charles River Laboratories (Wilmington, MA). The CV-F344 rats were derived from breeding BF-F344 rats in the institutional CV (initially at University of California, Los Angeles, CA, and recently at University of Maryland School of Medicine, Baltimore, MD), and then housing the newborns in the same environment. For some experiments, BF-F344 rats were obtained from Harlan Sprague-Dawley (San Diego, CA) (F344/NHsd). The major observations of experiments performed using BF-F344 rats from one source could be reproduced in BF-F344 rats from the other source, and the same was applicable to CV-F344 rats raised under two different conventional vivarium conditions. Male rats, 4–16 wk old, were used in almost all the experiments. A BF system (virus-free or specific-pathogen-free system) uses triple air locks and contains a Hepa filtration device. In addition, the holding cages, bedding, and food are sterilized by autoclaving, and water is filtered, chlorinated, and acidified. In contrast, the conventional animal (rat) facility consists of a single door that leads into the unfiltered housing facility. The caretakers or other workers are not required to take a shower or wear sterile clothes before entering the facility. The rats are housed in cages without filtertops, and the cages and bedding are not autoclaved. Furthermore, rats receive filtered water.
Ags/peptides/mitogens
The peptides containing amino acid sequences of Bhsp65 (18) were either synthesized and purified in the University of California Peptide Core Laboratory as described earlier (19) or obtained from Macromolecular Resources (Fort Collins, CO)/Chiron Mimotopes (San Diego, CA). Hen eggwhite lysozyme (HEL), three-times recrystallized, was purchased from Sigma (St. Louis, MO), and then further purified by chromatography as described (20). Con A was obtained from Sigma.
Induction of AA
Rats were immunized s.c. with 200 µl of M. tuberculosis H37Ra (Difco, Detroit, MI) (10 mg/ml) suspended in IFA (Difco) or in mineral oil (Sigma) (5). Beginning day 7 after immunization, the rats were scored daily for clinical signs of arthritis. The severity of arthritis in each paw was evaluated on the basis of erythema, swelling, and deformity of the joint (21, 22), and graded on a scale of 0 to 4 as follows: 0, no erythema or swelling; 1, slight erythema or swelling of the ankle or wrist; 2, moderate erythema and swelling of the ankle or wrist; 3, moderate erythema and swelling of the entire forepaw or hindpaw; 4, severe erythema and swelling of the entire forepaw or hindpaw (5, 23). The evaluation of the disease score in different groups of rats was performed in a blinded fashion; each day the arthritic scores of rats were recorded on a separate new sheet so that the observer had no information about the scores of the same rats on the preceding days. The sum of the arthritic score of the paws graded yielded the total daily arthritic score for each rat. This information was used to derive the arthritic index (AI) or mean peak arthritic score (PAS). AI indicates the sum of daily scores of individual rats during the entire course of AA, and the mean AI was derived by averaging the AI of the entire group. Mean PAS of a group of rats was derived by finding the average daily score of the group, and then identifying the highest score reached during the course of AA.
Splenic T cell proliferation assay
Rat spleens were removed and a single-cell suspension was prepared (5). Stromal debris was removed, and the cells were washed twice with HBSS (Life Technologies, Rockville, MD). These SPC were cultured in flat-bottom 96-well plates at 6 x 105 cells/well in HL-1 serum-free medium (Ventrex Laboratories, Portland, ME) supplemented with 2 mM L-glutamine, 100 U/ml penicillin G sodium, and 100 µg/ml streptomycin sulfate, with or without Ag (added at different concentrations). Con A or Tuberculin purified protein derivative (PPD) was used as a positive control. A total of 1 µCi of [3H]thymidine (International Chemical and Nuclear, Irvine, CA) was added per well for the last 18 h of a 5-day culture. The cells were then harvested on a Printed Filtermat A glass fiber filter (Wallac, Turku, Finland) using a Micro Cell Harvester (Skatron, Sterling, VA), and the incorporation of radioactivity was assayed by a liquid scintillation LKB 1205 Betaplate counter. The results were expressed as either cpm or stimulation index (S.I. = cpm with Ag/cpm with cells in medium alone). For some repeat experiments, supplemented culture medium was used: HL-1 medium with 1% (v/v) heat-inactivated FCS (Gemini Biological Products, Calabasas, CA) or X-Vivo 10 serum-free medium (BioWhittaker, Walkersville, MD) supplemented with 2% FCS and/or 5 x 10-5 M 2-ME (Sigma). Although generally 6 x 105 SPC were cultured per well, in some assays 5 x 105 cells/well were used.
Lymph node T cell proliferation assay
The draining lymph nodes of rats immunized s.c. with M. tuberculosis were removed and a single-cell suspension prepared (5, 19). Lymph node proliferation assay was performed as described for SPC, except for plating lymph node cells (LNC) at a concentration of 5 x 105 cells/well.
Adoptive transfer experiments
Naive CV-F344 rats (5–16 wk old, male) were used as donors. BF-F344 rats (5–6 wk old, male) were used as recipients or controls. Spleens of CV-F344 rats were removed, a single-cell suspension prepared, and the RBC in the sample were lysed. Thereafter, SPC were washed thoroughly, and cultured in vitro in flasks (2–3 x 106 cells/ml) in RPMI 1640 medium (supplemented with 1% L-glutamine, 1% penicillin-streptomycin, 2.5 x 10-5 M 2-ME, and 5% FCS) in an atmosphere of 5% CO2 and 95% air for 3 days in the presence of an equimolar mixture of five peptides comprising BCTD (peptides 417–431, 441–455, 465–479, 513–527, and 521–535 of Bhsp65) or an irrelevant Ag, HEL. Following restimulation in vitro, SPC were harvested, pooled, and washed with HBSS, live cells were counted and then injected at a concentration of 2 x 108 cells i.v. or i.p. into naive BF-F344 recipients. Within 18 h, the recipient F344 rats and a group of age- and sex-matched naive BF-F344 rats (controls) were challenged with M. tuberculosis s.c. for the purpose of induction of AA. Thereafter, all rats were observed regularly for signs of arthritis, and the severity of the disease was scored as described (5).
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Results |
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F344 rats raised under different environmental conditions
(BF-F344, CV-F344, and BF-F344 rats transferred at the age of 4–5 wk
into, and kept thereafter, in a CV (BF
CV F344)) were tested for
their susceptibility to AA. Cohorts each of BF-F344, BFCV F344, and
CV-F344 rats were injected with M. tuberculosis s.c., and
from day 7 after immunization, were examined regularly for signs of AA.
The results are summarized in Table I
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Strikingly, 20/28 (71.4%) of BF-F344 rats revealed a typical course of
AA in comparison to 16.7% of BFCV F344 rats, and 17.2% of CV-F344
rats. This difference between BF-F344 and each of the remaining two
groups of rats was statistically significant (p
< 0.005). The suppressive effect of the conventional environment on AA
is further highlighted by the differences in the quantitative
parameters including the AI and the PAS of the respective groups (Table I). The differences between BF-F344 vs BFCV F344 as well as those
between BF-F344 vs CV-F344 groups were statistically significant. Thus,
the susceptibility to AA of BF-F344 rats can be modulated significantly
in a conventional environment.
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On the basis of the above results, we reasoned that environmental agents (e.g., gut microbial flora) in the CV might be contributing to the reduced incidence and severity of AA in CV-F344 rats. To determine the relationship between gut microbial load and susceptibility to AA of F344 rats, we studied BF-F344 rats that were transferred (at the age of 4–5 wk) into the conventional environment but maintained thereafter by feeding either regular filtered water (group 1) that was also used for CV-F344 rats, neomycin (2 mg/ml) added to the drinking water (group 2), or acidified water (pH 2–3) (group 3). The objective of feeding neomycin or acidified water was to reduce the existing gut microbial load, and to prevent/reduce further colonization of the gut with bacteria from the environment. All rats received the same food. After 4 wk, all rats were immunized with M. tuberculosis s.c., and then followed for signs of arthritis. Interestingly, F344 rats in group 1 showed a significant reduction in the incidence of AA; only 2 of 12 (16.6%) rats developed arthritis. On the contrary, 14 of 14 (100%) F344 rats in groups 2 and 3 combined maintained their susceptibility to AA, and developed arthritis. This difference in relative susceptibility to AA (16.6 vs 100%) was statistically significant (p < 0.005). These results suggest that there is down-modulation of the disease process (AA) in rats with comparatively higher microbial load.
CV-F344 rats are not deficient in mounting responses to the potentially arthritogenic determinant of Bhsp65
AA-susceptible Lewis rats develop potent responses to peptide
177–191 of Bhsp65 (p177–191) (which contains the minimal
arthritogenic determinant 180–188 for the Lewis rat and is
cross-reactive with it) upon challenge with M. tuberculosis
(5). We tested whether AA-susceptible BF-F344 and
relatively less susceptible CV-F344 rats differ significantly in their
ability to respond to p177–191 after immunization with M.
tuberculosis. The results shown in Fig. 1 demonstrate that both BF-F344 and
CV-F344 rats have strong and comparable levels of proliferative T cell
responses to p177–191. Thus, the reduction in the incidence of AA of
CV-F344 rats cannot be attributed to a lack of response to the
potentially arthritogenic determinant of Bhsp65. In this regard, a more
likely explanation is the induction of Bhsp65-reactive T cells,
potentially capable of modulating the course of AA, in CV-F344 rats in
the conventional environment.
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The results of our previous study suggest that T cell responses to
BCTD (represented by peptides 417–431, 441–455, 465–479, 513–527,
and 521–535 of Bhsp65) are involved in natural remission from AA in
the Lewis rat (5). Based on these results, we reasoned
that the reduced incidence and severity of AA in CV-F344 rats might be
due to inadvertent induction of T cell responses to BCTD, possibly
following their exposure to environmental agents, such as bacteria, in
the CV. Because hsp are highly conserved, the environmental agent(s)
might possess a homologue of Bhsp65, which could fortuitously prime T
cell responses to BCTD through determinant mimicry. To examine the
above proposition, we tested SPC of three groups of naive unimmunized
F344 rats (BF-F344, BFCV F344, and CV-F344 rats, none of which had
been exposed to M. tuberculosis in any form) in a
proliferation assay using peptides comprising BCTD and other regions of
Bhsp65. The results are given in Fig. 2.
Clearly, CV-F344 rats raised relatively much higher proliferative
responses to BCTD and to peptides comprising two of the amino-terminal
determinants of Bhsp65 (namely, 1–15 and 13–27) compared with BF-F344
rats. Of different Bhsp65 peptides, the lowest responses were observed
with peptides 33–48, 121–135, and 177–191 of Bhsp65 in CV-F344. The
difference in the response of BF-F344 and CV-F344 rats to three of the
BCTD peptides (417–431, 513–527, and 521–535) was statistically
significant (p < 0.05–0.025). Interestingly,
there was a gradual increase in the level of proliferative response to
Bhsp65 peptides in the three groups of rats (BF-F344, BFCV F344, and
CV-F344, in increasing order of response) in direct relation to the
duration, and thereby, extent of exposure of these rats to the
conventional environment. Importantly, CV-F344 and BFCV F344 but not
BF-F344 show reduction in the incidence and severity of AA (Table I
).
According to the results of our previous study, the amino-terminal
determinants of Bhsp65 did not play any role in attenuation of AA under
the conditions of the test (5). In this regard, BCTD
appear to be the most relevant candidate in modulating the course of AA
in CV-F344 and BFCV F344 rats. Considering that the proliferative T
cell responses shown in Fig. 2 are of naive unimmunized rats, the cut
off level to determine a positive response was set at a S.I. of 1.5 to
account for relatively lower responses of naive rats (compared with
rats traditionally immunized with Ag in adjuvant, whose responses are
expectedly much higher). In a recently reported study, the change in
the pattern/trend of proliferative T cell response of patients with
multiple sclerosis was similarly deemed to be of physiologic
significance despite the relatively low levels of S.I. (24, 25).
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To determine the specificity of T cell response to Bhsp65 peptides
of CV-F344 rats, and to rule out a generalized hyporesponsiveness of T
cells of BF-F344 rats, we have tested the SPC of these two groups of
rats in a proliferation assay using Con A as a mitogen and HEL as an
irrelevant Ag. The results of a representative experiment are shown in
Fig. 3. Similar results were obtained in
repeat experiments (data not shown). Collectively, these results
demonstrate that T cells of both groups of rats raised comparable level
of proliferative responses to Con A. Therefore, T cells of naive
BF-F344 rats are not deficient in their ability to proliferate in
comparison to that of naive CV-F344 rats. Also evident from the results
of Fig. 3 is the issue of specificity of spontaneously induced T cell
response of CV-F344 rats to Bhsp65 peptides (shown above in Fig. 2); T
cells of neither naive CV-F344 nor BF-F344 rats gave any proliferative
recall response to HEL. Thus, emergence of the
Bhsp65/BCTD-specific T cell responses in naive CV-F344 rats is
attributable to exposure of these rats to the conventional
environment.
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To further define the immunologic
basis of relatively reduced susceptibility to AA of CV-F344 rats, we
determined whether splenic T cells (SPC) of CV-F344 rats could
down-modulate the course of AA in naive BF-F344 rats. For this purpose,
SPC of naive CV-F344 rats were cultured for 3 days in vitro with an
equimolar pool of five peptides comprising BCTD (peptides
417–431, 441–455, 465–479, 513–527, and 521–535 of Bhsp65), and
then injected i.p. into naive BF-F344 rats (experimental group). Age-
and sex-matched naive BF-F344 rats, which had either not received any
SPC at all or received HEL-restimulated SPC, served as controls. All
experimental and control rats were immunized with M.
tuberculosis s.c. to induce AA. The results of a representative
experiment are shown in Fig. 4, and in
another format, in Table II (experiment no. 1). Also included in Table II are the results of several repeat adoptive transfer experiments.
Collectively, these results demonstrate that transfer into BF-F344 rats
of BCTD-restimulated SPC of CV-F344 rats led to a significant reduction
in the severity of subsequent AA in the recipients, whereas the control
rats revealed the usual course of AA. Collectively, the incidence of AA
with a typical disease course in the three groups of rats (Table II)
was as follows: BF-F344, (26/27 (96.3%)); BCTD group, (4/24 (16.7%));
and HEL group, (13/13 (100%)). These results underscore the extent of
down-modulation of AA effected by adoptive transfer into naive BF-F344
rats of BCTD-restimulated SPC of CV-F344 rats in comparison to
recipients of HEL-restimulated SPC or no cells at all. In most
experiments, the incidence as well as overall course of disease in
BF-F344 and HEL group of rats was comparable. In one experiment (no. 3,
Table II), apparently there was a slight suppression of disease
activity in HEL group; however, the difference between the two groups
was not statistically significant. These results show that the presence
of potentially BCTD-reactive T cells in the donor CV-F344 SPC
restimulated in vitro with an irrelevant Ag (HEL) was not sufficient
for effecting reduction in the severity of subsequent AA in recipients
to a significant level unlike that in experimental groups given
BCTD-restimulated SPC. These results suggest that re-activation and
expansion of BCTD-reactive T cells above a certain threshold was
essential for modulating the severity of AA. We have described above
adoptive transfer experiments using whole SPC. Such an approach has
successfully been used in a recent study in animal model of type I
diabetes (26).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CV F344 rats are most likely
to be involved in modulating the course of AA in these rats. This
proposition is further validated by the results showing that T cells of
naive BF-F344 and CV-F344 rats give a comparable level of proliferative
response to Con A but no recall response at all to an irrelevant Ag
like HEL; and adoptive transfer of BCTD-restimulated but not
HEL-restimulated SPC derived from naive CV-F344 rats can lead to
reduced severity of AA in recipient BF-F344 rats. The approach using
whole SPC in adoptive transfer experiments in this study is similar to
that used in a recently reported study in the murine model of
autoimmune diabetes (26). Despite the relatively low S.I.
of T cell response to BCTD, the trend revealed by the pattern of
response of CV-F344/BFCV F344 rats compared with BF-F344 rats
underscores a phenomenon of physiologic significance. Such
interpretation of results based on low S.I. values has also been
emphasized by other investigators studying T cell response of multiple
sclerosis patients (24, 25). Thus, the results of this
study provide an immunologic explanation underlying the reduced
incidence and severity of AA observed in CV-F344 rats: priming of
BCTD-reactive T cells. We suggest that this T cell priming is owing to
molecular mimicry (10, 11, 27, 28, 29) between
mycobacterial hsp65 and its homologues within environmental
microbial agents, e.g., gut microbial flora. Alternatively, because of
the degeneracy of TCRs (28, 29, 30, 31, 32), fortuitous priming might
also occur by microbial proteins other than hsp65 (33).
These T cells could then be restimulated by peptides comprising BCTD
when tested for proliferative response in vitro or in adoptive transfer
experiments. In another study, induction of, rather than protection
from experimental autoimmune encephalomyelitis in transgenic mice
expressing a TCR specific for myelin basic protein (16)
was proposed to be due to molecular mimicry between myelin basic
protein and a yet undefined environmental Ag. A similar mechanism was
proposed to explain the influence of gut microbial flora in the
induction of autoimmune thyroiditis in rats (14).
Recently, it has been reported that hsp60 cross-reactive T cells
mediate autoimmune pathology of the small intestine (29).
In contrast to the above-mentioned studies (11, 12, 13, 14, 15, 16), in
this study, we attribute reduced incidence and severity of, rather than
induction of, AA to determinant mimicry.
Although suppressive effect of the conventional environment on the
incidence of diabetes or arthritis in animal models has also been
observed by other investigators (9, 34, 35, 36, 37), the
mechanisms underlying this down-modulation of autoimmunity have yet to
be defined. In this study, we have experimentally documented through
Ag-specific T cell proliferation and adoptive transfer experiments at
least one of the immunological bases of the suppressive effect of
environmental microbial flora on the course of AA. Our results suggest
that exposure to conventional environment leads to priming and
expansion of T cells against regulatory determinants within Bhsp65 as
opposed to "tolerance induction" to disease-inducing determinant(s)
(36). The mechanism by which BCTD bring about these
effects is currently under investigation. However, because the nature
of microbial flora in different conventional facilities might be
different, it is likely that T cell responses to certain Bhsp65
determinant regions (6, 38, 39) other than, or in addition
to, BCTD might be crucial in inducing suppressive effect on AA in F344
rats. In addition, microbial agents can also influence susceptibility
to autoimmunity by mechanisms other than determinant mimicry, e.g.,
bystander activation of potentially autoreactive T cells (34, 40), and by the action of superantigen (41).
Interestingly, it has been shown that infection with Mycoplasma
pulmonis can modulate the course of AA in Lewis rats
(42). An additional factor that might contribute to the
reduced incidence and severity of AA in CV-F344 rats might be the level
of activity of the hypothalamic-pituitary-adrenal axis
(43). However, in one study, no significant difference in
the levels of plasma corticosterone in response to IL-1
between
germ-free F344 and CV-F344 rats was observed (37). The
outcome of the influence of the above factors on AA is further
compounded by the genetic susceptibility/resistance to the disease
(44).
The results of this study using F344 rats has important implications for the susceptibility to autoimmunity of heterogeneous human populations living under different environmental conditions. It is conceivable that exposure to various agents (such as bacteria and viruses) is one of the instrumental causes in modulating the incidence, severity, or even the type of autoimmune process acquired by an individual (45, 46, 47, 48, 49, 50, 51). Likewise, this type of adventitious microbial stimulation occurring throughout the life of an individual could play an important role in establishing expanded repertoires of pathogenic/regulatory memory T cells (52, 53, 54). These factors, for example may contribute to the lack of concordance of susceptibility to certain autoimmune diseases between siblings, including identical human twins.
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Acknowledgments |
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Footnotes |
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2 Current address: University of Maryland School of Medicine, Baltimore, MD 21201.
3 Address correspondence and reprint requests to Dr. Kamal D. Moudgil, Department of Microbiology and Immunology, University of Maryland School of Medicine, BRB 13-019, 655 West Baltimore Street, Baltimore, MD, 21201-1509.
4 Current address: La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121.
5 Abbreviations used in this paper: AA, adjuvant arthritis; Bhsp65, the 65-kDa mycobacterial heat-shock protein; BCTD, Bhsp65 C-terminal determinants; BF, barrier facility; CV, conventional facility; HEL, hen eggwhite lysozyme; SPC, spleen cells; F344, Fischer 344; BF-F344, F344 rats raised in a BF; CV-F344, F344 rats maintained in a CV; AI, arthritic index; PAS, peak arthritic score; S.I., stimulation index; BFCV F344, BF-F344 rats transferred at the age of 4–5 wk into, and kept thereafter, in a CV.
Received for publication July 8, 2000. Accepted for publication January 9, 2001.
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References |
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cells. Science 284:1183.This article has been cited by other articles:
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-
-
) rats were tested in a
proliferation assay. The assay was performed as described in
Materials and Methods. The response of SPC was tested
using 0.5 µg/ml Con A as a mitogen and HEL as an irrelevant Ag. The
results are expressed as a S.I. Similar results were obtained in repeat
experiments (data not shown).