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*
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104;
University of North Carolina School of Medicine, Chapel Hill, NC 27599;
Department of Surgery, University of Wisconsin Medical School, Madison, WI 53706;
§
Department of Comparative Medicine, University of Tennessee, Knoxville, TN 37901; and
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Department of Medicine, University of Colorado Health Sciences Center, Denver, CO 80262
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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50% smaller
LNs compared with sex-matched GF-NI controls. Autoantibody formation
was present in both groups. Histologic analysis of the kidneys revealed
that GF-AF mice had much lower levels of nephritis, while
immunofluorescence analysis demonstrated no difference in Ig deposits
but did reveal a paucity of C3 deposition in the kidneys of GF-AF mice. These data do not support a role for infectious agents in the induction of lymphoproliferation and B cell autoimmunity in MRL-lpr mice. Furthermore, they suggest that autoantibodies do not originate from B cells that were initially committed to exogenous Ags. They do suggest a possible contributory role for dietary exposure in the extent of lymphoproliferation and development of nephritis in this strain.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Some previous studies have addressed the role of environmental stimuli, including the germfree (GF)4 state, in several models of autoimmunity in mice. Organ-specific autoimmunity was unaffected by GF conditions, as seen in the susceptibility to postgastrectomy autoimmune gastritis in BALB/c mice (4). The transfer of myelin basic protein-specific TCR transgenic mice from a conventional to an specific pathogen-free (SPF) facility resulted in the suppression of the spontaneous development of experimental autoimmune encephalomyelitis (5). Previous work noted that New Zealand black (NZB) mice maintained under GF conditions had lower levels of serum IgG, although they had higher levels of antinuclear Abs (6, 7). Also noted were a lower incidence and later onset of moderate to severe renal disease (7). In contrast, transferring nonobese diabetic (NOD) mice from SPF to GF conditions resulted in a marked increase in the incidence of diabetes (8). These findings suggest that some features of systemic autoimmunity may be dependent on microbiota. They do not, however, address the possible role of dietary Ags, including bacterial byproducts.
In normal mouse strains, GF environment has a quantitative effect on the immune system. GF mice have smaller lymphoid tissue and reduced serum Ig levels. The GF state does not, however, abrogate their ability to respond to specific antigenic stimuli, although primary responses are often less vigorous than those of conventionally housed mice (9, 10).
GF mice have never encountered live microorganisms, but they are routinely exposed to nonviable microbes, primarily in their diet (10). Alternatively, GF mice can be made "Ag-free" (AF), if they are fed an ultrafiltered, low m.w., chemically defined liquid diet and housed on ashless filter-paper bedding. Such GF-AF animals are as close to being immunologically shielded from nonself antigenic exposure as is currently possible (11, 12). The diet itself contains no potentially antigenic macromolecules, while ultrafiltration further guarantees the absence of intermediate m.w. Ags of bacterial origin, such as endotoxin, or viral contamination (10). The AF diet was originally developed for rigorous determination of nutritional requirements in GF mice and has been extensively characterized for its nutritional adequacy over several generations (11, 13, 14, 15).
Studies of the immunological parameters of GF-AF mice demonstrate a profound depression in levels of circulating IgG and IgA (with nearly normal IgM levels), low numbers of circulating white blood cells and reduced number of splenic Ig-secreting cells (10, 15, 16). In spite of these findings, GF-AF mice do have normal numbers of Ag-specific IgM-secreting cells, and the diversity of the IgM repertoire is conserved as measured by a quantitative immunoblot assay (16, 17). Primary responses to T cell-dependent Ag immunization actually produce higher numbers of Ag-specific IgG-secreting cells in GF-AF mice than in conventional controls (18). T cell-independent responses and affinity maturation following T cell-dependent immunizations are also comparable to what is seen in conventionally housed mice (19, 20). Analysis of LPS-induced B cell hybridomas demonstrated similar patterns of VH gene family usage with a high frequency of reactivity to either self or bacterial Ags in both neonatal SPF and adult GF-AF mice, but not in adult SPF mice (21, 22). While GF-AF mice have lower numbers of activated T cells than do SPF mice, T cell-effector function, as measured in a variety of ways, is normal, as is the TCR Vß repertoire measured by flow cytometry analysis (19, 23, 24, 25). All this work suggests that background Ig synthesis is mostly driven by foreign antigenic exposure, but the basic ontogeny of the immune system is autochthonous.
In the present work, we investigated the role of environmental stimuli on the development of autoimmunity in MRL-lpr mice. These mice have the faslpr mutation, which affects the expression of Fas, a cell surface receptor member of the TNFR gene family, which is involved in the induction of apoptosis. They spontaneously develop an autoimmune syndrome characterized by elevated levels of Ig, multiple autoantibodies, nephritis, and vasculitis, in association with massive lymphoproliferation involving all lymphocyte subsets and dominated by an aberrant CD4-CD8- (double negative (DN)) T cell subset. MRL/MpJ mice, without the faslpr gene, also spontaneously develop autoimmunity, although at a later stage in life, so the MRL background clearly plays an important genetic role (26). The general reproducibility of the MRL-lpr syndrome from colony to colony over more than 20 years suggests that the basic disease is not dependent on any particular infectious agent. This and the wide range of autoimmune features seen in these mice make them suitable candidates to examine the relative roles of environment vs genetics in the development of autoimmunity.
We planned our experiments to test both the role of live microbiota and the role of other environmental Ags. In the first experiment, MRL-lpr mice raised in a GF environment were compared with conventional mice. In the second experiment, GF MRL-lpr mice were fed an AF diet or an autoclaved natural ingredient (NI) diet. Our results show no role for microbiota and a selective effect of dietary Ags in the development of the MRL-lpr autoimmune syndrome.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MRL/MpJ-Faslpr (MRL-lpr) mice were originally
obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained
under conventional conditions at the University of North Carolina
School of Medicine (Chapel Hill, NC) animal facility. Pregnant dams
were sent as founders to the Gnotobiotic Division of the Medical School
Animal Care at the University of Wisconsin-Madison (Madison, WI). A
group of mice were rederived into GF housing via cesarean section from
these pregnant dams and maintained under these conditions from then on
(Table I
). A second group of mice, the
control group for experiment 1, was delivered in a conventional
facility and maintained there for the duration of the experiment. Mice
were fed a standard NI Purina brand mouse chow (no. 5010) and purified
water ad libitum (all autoclaved for the GF mice). GF mice were
periodically tested and determined to be free of parasites and aerobic
and anaerobic microbial growth. Sentinel mice were also routinely
tested and found to be negative for viral serologies. In experiment 2,
a group of GF dams were placed on a chemically defined, low m.w., water
soluble, ultrafiltered AF diet within 10 days of delivery and
throughout nursing. The GF-AF offspring were maintained on this AF diet
following weaning. The water soluble and lipophilic components of the
diet were presented to the mice separately. The preparation and
composition of the aqueous diet was as previously described (solution A
in 27). The lipophilic component of the diet, consisting of the
fat-soluble vitamins dissolved in chemically synthesized triglycerides,
has been previously described (14). The diet did not contain
polysorbates to avoid the known immunological effects and
hepatotoxicity associated with such diets (11, 28). The techniques for
sterilization of diets and equipment and for monitoring for microbial
contamination was as described (14, 27). We are grateful to Fred
Dickeson and Julian Pleasants (American Biogenetic Sciences, South
Bend, IN) for their assistance with the ultrafiltration of the aqueous
portion of the diet through a membrane with a m.w. cut-off of 10,000
Da. The GF-AF mice were maintained on shredded ashless filter-paper
bedding. The paper bedding is consumed by the mice and is considered
important for normal intestinal function in GF-AF mice (29). BALB/c
mice used were obtained from the GF breeding colony maintained at the
University of Wisconsin-Madison since 1973 and were housed under
identical conditions as the MRL-lpr mice described above.
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Preparation of cells
Single-cell suspensions of all LN and spleens were made by passing them through cell strainers (Becton Dickinson, Franklin Lakes, NJ) in cold medium (RPMI 1640 with 15 mM HEPES, 5% FCS (HyClone, Logan, UT), 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Gaithersburg, MD)). RBC were lysed with NH4Cl, and cells were washed twice before counting by an automated cell counter (Coulter, Hialeah, FL).
Immunofluorescence staining and flow cytometry
In 96-well microtiter plates, 1.5 x 106 cells/well were stained in cold media containing 3% FCS and 0.1% NaN3. In general, first and second step reagents were incubated with cells on ice for 30 min. Cell surface immunofluorescence analysis of LN and spleen cells was performed by two- and three-color flow cytometry analysis with size gating on the lymphocyte population. At least 104 events were collected for each sample on a FACScan (Becton Dickinson Immunocytometry Systems, San Jose, CA) with Cytomation (Fort Collins, CO) or CellQuest (Becton Dickinson Immunocytometry Systems) data acquisition and software.
Reagents for immunofluorescence staining and flow cytometry
Reagents used for immunofluorescence staining included:
anti-IgM (Bet-2-BNHS), anti-CD45R/B220-FITC (RA3-6B2;
PharMingen, San Diego, CA), anti-CD4 (172.4 overgrown supernatant;
RM4-5-PE; PharMingen), anti-CD8 (31 M overgrown supernatant;
53-6.72-PE; PharMingen), anti-Thy1.2 (MmT1 overgrown supernatant;
53-2-1-BNHS; PharMingen), SAv-Cy-Chrome (PharMingen), SAv-R-PE
(Southern Biotechnology Associates, Birmingham, AL), and anti-rat
light chain (MAR-18.5) (31). Two-color immunofluorescence was used
to analyze TCR Vß and TCR 
/
expression separately in the
CD4+, CD8+, CD4-CD8-
(analyzed by costaining with both fluoresceinated anti-CD4 plus CD8
Abs) T cell populations, as previously described (32).
Quantification of Ig and autoantibody levels by ELISA
In experiment 1, serum samples were assayed in duplicate for total IgG, IgG2a, and IgM levels, as well as the following autoantibodies: IgG anti-chicken chromatin, anti-ssDNA, anti-P, and anti-Sm; and IgG3 rheumatoid factor (RF) anti-IgG2aa activity (clone P1.17 (mouse IgG2a) as target and clone 2E.6 F(ab')2-BNHS (rat IgG1 anti-mouse IgG3) as the secondary reagent), as previously described (33, 34, 35). In experiment 2, IgG anti-dsDNA and IgM RF anti-IgG2aa were performed in addition (35, 36). Results for both male and female sera for each group were pooled, as no significant differences were noted between them. The Crithidia assay for detecting anti-dsDNA Ab was performed as previously described (37). Additional sera from non-SPF MRL-lpr mice and nonautoimmune B6.C20 mice were also assayed in experiment 1 for comparison. In Experiment 9, sera from BALB/c mice housed under identical conditions as the MRL-lpr mice were assayed as nonautoimmune controls. Some results are reported in equivalent dilution factors (EDF) of standardized reference MRL/lpr sera, as previously defined by the formula: EDF = (dilution of standard reference sera, which gives the equivalent OD of the test serum) x 106 (38).
Kidney histopathology (light microscopy and immunofluorescence)
The presence and severity of nephritis was determined on hematoxylin-eosin light and immunofluorescence microscopy as previously described (37, 39). In experiment 1, a blinded observer (Jennette) evaluated and graded the overall severity by light microscopy, while IgG, IgM, IgA, and C3 deposits were individually scored by immunofluorescence microscopy. In experiment 2, a blinded observer (Madaio) evaluated and scored independently the severity of glomerular, interstitial, and vascular lesions by light microscopy. Similarly, the presence of glomerular, tubular basement membrane and vascular deposits of IgG and C3 by immunofluorescence microscopy was judged independently. Multiple sections at a minimum of two different levels were observed. Each section typically involved evaluation of >50 glomeruli, >25 blood vessels, and the interstitium contained within two to three longitudinal sections of kidney.
Statistics
Student’s t test was used to determine the statistical significance of differences between most groups. Fisher’s exact test was used for comparisons of Crithidia assay anti-dsDNA titers. Differences were considered significant at p < 0.05. The significance level for the results of the ELISAs was adjusted using the Bonferroni correction for multiple comparisons of means to a p < 0.0063 (0.05 ÷ 8 = 0.0063, where 8 equals the number of assays performed). Error bars in graphs represent the 95% confidence intervals (CI). The differences in kidney histological and C3 and IgG deposition scores were analyzed using the two-sample Wilcoxon rank-sum (Mann-Whitney) test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the role of environmental stimulus on the development
of spontaneous autoimmune disease in MRL-lpr mice, we
performed two separate experiments with MRL-lpr mice (Table I
). In experiment 1, we wanted to establish the role of live,
nonpathogenic stimuli. We housed one group of mice under conventional
conditions, while a second group was reared in a GF state. In
experiment 2, we wanted to isolate the mice from environmental
antigenic stimuli as much as possible. For this experiment, two groups
of GF mice were established. One group was fed a conventional,
autoclaved NI (GF-NI) diet, while the second group was maintained on a
chemically defined, low m.w., water soluble, ultrafiltered AF (GF-AF)
diet.
At 5 mo of age, experiment I mice, reared in a GF environment, had body
weights no different from those of conventional controls (Fig. 1A). In experiment 2, same sex
comparisons demonstrated that differences in total body weight (TBW)
were only present between GF-AF and GF-NI males at 4 mo of age (33 g vs
38 g, p < 0.01) (Fig. 2A). These differences may be
due in part to differences in LN weights and bowel weight and content
(GF-AF mice have a reduction in cecal size, as a percentage of body
weight, from 10% to 3% compared with GF-NI mice and have very little
luminal fecal content (12)). Of note, 7/8 female, but only 1/9 male,
GF-AF mice had ear lesions typical of MRL-lpr mice, while
none of the GF-NI diet-fed mice had ear lesions at the time of
sacrifice. None of the mice demonstrated clinical signs of inflammatory
arthritis at the time of sacrifice.
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Analyses of the LN and spleens collected at sacrifice are
presented in Figs. 1 and 2. In experiment 1, the GF state did not
affect the weight or total cell counts of either LN or spleens (Fig. 1, A and B), as both groups demonstrated the usual
degree of lymphoproliferation seen in MRL-lpr mice, well in
excess of the levels seen in normal C57BL/6 mice (31). LN T cell subset
analysis likewise did not reveal any differences (Fig. 1B).
There was also no significant difference in the percentage of /
DN T cells between the groups (conventional, 1.6% vs GF, 2.5%;
p > 0.05). Fig. 3 shows
that T cell subset TCR Vß-chain usage, as determined by flow
cytometric analysis of LN cells, was indistinguishable between the
groups of mice and included the selective expansion of
Vß8.3+ DN T cells (32).
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, A and
B). Cell surface marker expression analysis revealed that
all mice demonstrated the characteristic expansion of lymphocyte
subsets seen in MRL-lpr mice (Fig. 2B). No
significant differences were found in the percentage of
CD4+ T cells, CD8+ T cells, or B cells
(B220+/sIgM+) in same sex comparisons. GF-AF
mice tended to have lower absolute numbers of cells. There were small
but significant differences in the percentage of DN T cells (data not
shown) with larger differences in the total number of DN T cells (GF-AF
vs GF-NI; female, 2123 vs 3360 and male, 821 vs 2014 x
10-6 cells; both comparisons p < 0.01). Autoantibody production in GF and GF-AF MRL-lpr mice
All sera collected from mice at 3 mo of age and at the time of
sacrifice were analyzed for levels of Ig isotypes and autoantibody
specificities. At 3 mo of age, conventional and GF MRL-lpr
mice in experiment 1 had roughly equivalent levels of all
autoantibodies tested, although GF mice made higher levels of both
total IgG and IgG2a (data not shown). At the time of sacrifice (5 mo),
GF and conventional MRL-lpr mice, again, had similar levels
of autoantibodies (Fig. 4). Conventional
mice appeared to have higher levels of IgM and anti-ssDNA
autoantibodies (p = 0.05 and 0.04,
respectively, NS using the Bonferroni correction for multiple
comparisons), but GF mice showed a trend toward higher titers in others
(total IgG2a and anti-Sm). Anti-dsDNA was tested by the Crithidia
assay when sufficient serum was available. Eight of nine 5-mo sera in
the GF group (89%) had titers of at least 1/160, while only 6/14 mice
in the conventional group (43%) showed a similar level of reactivity
(p < 0.01).
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, and data not shown). GF-AF
MRL-lpr mice, compared with GF-NI MRL-lpr mice,
however, trended toward lower titers of most serologies tested, a
pattern also repeated in the BALB/c mice. While GF-AF
MRL-lpr mice produced significantly lower titers of total
IgG, anti-ssDNA, IgM, and IgG3 RF anti-IgG2aa (Fig. 5, A, D, E, and G), they
produced comparable titers of anti-Sm, IgM RF
anti-IgG2aa, anti-chromatin, and anti-dsDNA
(Fig. 5, B, C, F, and H) at
4 mo of age. Serologies at 3 mo of age, before the monocontamination,
demonstrated similar patterns (data not shown). It is of note
that even in those assays in which GF-AF MRL-lpr mice
produced lower average titers, there was significant overlap in the
level of the individual titers between both groups of
MRL-lpr mice.
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Nephritis involving the glomeruli, interstitia and blood vessels
with deposition of Ig and complement is a hallmark of the autoimmune
syndrome of MRL-lpr mice. As seen in Fig. 6, A–C, GF MRL-lpr
mice developed both light and immunofluorescence findings that were
indistinguishable from those seen in conventional controls. (The
patterns of IgM and IgA deposition were similar to those of IgG, data
not shown). Both groups also demonstrated similar levels of proteinuria
at the time of sacrifice (Fig. 7A).
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D; p < 0.05; similar
results were seen for vascular and interstitial changes, data not
shown). When the kidneys were analyzed by immunofluorescence, extensive
and indistinguishable amounts of glomerular IgG deposits were present
in both groups (Fig. 6E; p > 0.05; results
for vascular and interstitial deposits were comparable, data not
shown). Staining of five to six representative kidneys from each group
for IgG2a and IgG3 subclass deposition suggested a switch away from
these subclasses in the GF-AF group. Analysis of C3 deposition,
however, revealed that it was minimal in the kidneys of GF-AF mice. By
contrast, there where extensive deposits found in the control GF-NI
mice (Fig. 6F; p < 0.05). GF-AF mice from
experiment 2 also demonstrated much less proteinuria than NI mice (Fig. 7B). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The possibility that the changes resulting from the AF diet were due primarily to nutritional inadequacy is unlikely, although we cannot rule out that the particular constituents of the diet may have played a role. The AF diet was originally developed for rigorous determination of nutritional requirements and has been characterized as the most studied dietary model (14). AF diet-fed mice are usually smaller at weaning but as adults achieve weights comparable to those of NI diet-fed mice, in spite of their higher resting O2 consumption (40). Previous experiments in which diets deficient in any number of components led to the amelioration of autoimmune features, including nephritis, in MRL-lpr and NZB/W F1 mice were always associated with concomitant significantly lower body weights (41, 42). Additional work in MRL-lpr females, however, showed that a nutritionally adequate diet low in fat, none of it saturated, compared with isocaloric, higher fat diets, can decrease the incidence and severity of proteinuria, autoantibodies, and mortality without significantly affecting body weights (43). Other forms of dietary interventions, such as caloric restriction, fatty acid supplementation, or restriction of tyrosine and phenylalanine, are known to have some beneficial effects on the progression of nephritis in MRL-lpr or (NZB x NZW)F1 (NZB/W F1) mice (42, 44, 45, 46, 47, 48, 49, 50). The significant difference in the fat composition of our AF diet could thus account for the observed decrease in the renal pathology of AF mice (51). Similarly, NZB/W F1 mice fed a diet in which the protein source was replaced with synthetic amino acids (as in our AF diet) had delayed onset of their autoimmune disease, without abnormalities of body weight (50). In our experiments, the decreases in certain parameters of autoimmunity in the GF-AF group were probably not due to malnutrition, as the mice displayed no obvious differences in appearance or behavior and little difference in weight compared with their GF-NI controls. However, it is impossible to differentiate between a modest role for dietary Ags vs a "chemical" effect of the synthetic diet’s constituents.
One of the most striking features of the faslpr phenotype is the massive, nonmalignant lymphoproliferation that can be found in all strains of mice deficient in Fas or Fas ligand (FasL) expression. These expanded lymphocyte populations, particularly the aberrant DN T cells, may play a role in the development of the autoimmune features of the syndrome. Previous work by us and others has suggested that the DN T cells derive mostly from the postthymic CD8+ T cell population (31, 32, 52). This led to the suggestion that DN T cells arise because defects in the Fas-FasL signaling pathway prevent normal deletion of T cells that have encountered Ag in the periphery. The development of substantial numbers of DN T cells in our GF-AF mice supports the contention that the lymphoproliferation, including the DN T cells, does not predominantly arise from reactivity to foreign Ags, but rather is largely driven by recognition of self.
Our results offer some insight into the origin of autoantibodies in
MRL-lpr mice. The GF-AF MRL-lpr mice produced
elevated titers of all autoantibodies tested, although certain
specificities were decreased compared with GF-NI MRL-lpr
mice. Thus, dietary Ags play, at most, a modifying role on the
autoimmune process. Significantly lower levels of serum IgG were found
in all GF-AF mice and were not due to synthetic defects brought on by
protein-calorie malnutrition as seen in other experimental models (53, 54). As previously mentioned, while background levels of serum IgG are
extremely low in GF-AF mice, as we found in our BALB/c controls (Fig. 5), GF-AF mice are capable of mounting high titer, specific Ab
responses following Ag exposure. In addition, adult GF mice transferred
from an NI diet to an AF diet demonstrate a dramatic fall of serum IgG
levels after 3–4 mo (10). These findings imply that baseline IgG
levels are predominantly driven by continued foreign antigenic stimuli.
In contrast, then, the significant titers of autoantibodies produced by
GF-AF MRL-lpr mice and the relatively high levels of their
serum IgG must be driven by specific Ags, which can only be autologous.
This is further supported by experiments that indicate that the
production of autoantibodies in MRL-lpr mice is cognate and
follows the pattern of an Ag-driven response focused on the identified
autoantigens, i.e., dsDNA, Ig, and Sm (55, 56, 57, 58, 59). Thus, the
autoantibodies characteristic of MRL-lpr mice do not
originate from B cells that were initially committed to exogenous Ags,
and their ongoing production must be driven by continued exposure to
autoantigen. What remains undetermined is the nature and the site of
the original self-antigenic stimuli. It is apparent, however, that
foreign Ags neither initiate nor exacerbate (by epitope spreading, for
example) the endogenous propensity to make autoantibodies.
Nonautoimmune prone strains of mice have been shown to develop
spontaneously certain RF specificities, mostly IgA and IgM
anti-IgG2a, that can vary depending on environmental factors (60).
For example, DBA/2 mice housed under SPF conditions made RF, while
animals kept under GF conditions in the same colony did not. Our
present work showed parallel results, since GF-NI BALB/c mice made
significant titers of IgM RF anti-IgG2aa (Fig. 5C), while the GF-AF BALB/c mice did not. On the other hand,
the spontaneously autoimmune MRL-lpr mice made high titers
of RF, both IgM and IgG3, whether they were housed under conventional,
GF, or GF-AF conditions (Figs. 4 and 5). Therefore, this autoantibody
specificity can be driven by environmental stimuli in some situations,
but can also be independent of foreign Ags.
It was striking to find that dietary Ags, but not microbiota, played a major role in the development of nephritis in MRL-lpr mice. In the GF-AF group, paucity of C3 deposition in the setting of large amounts of IgG may point to an explanation of this observation. The development of nephritis may depend on the absorption of dietary Ags that participate as cofactors in the binding of complement by renal immune complexes. Alternatively, a switch in the pattern of Ig subclass deposited in the kidneys may account for the observed differences in the amount of complement deposited and the subsequent development of nephritis. Peng et al. (61) also reported a disassociation between renal Ig deposition and the development of nephritis. A change in the C3 binding capacity of IgG deposited in kidney lesions by GF-AF MRL-lpr mice is possible in their model as well as ours. It is also possible that the absence of complement deposition is due to decreased serum levels due to the AF diet. This seems unlikely as decreased levels of serum complement in both humans and experimental animals have only been reported in the setting of severe protein-calorie malnutrition and inanition (62).
B cells are necessary for the development of glomerulonephritis in MRL-lpr mice, but their exact roles are not known (63, 64). Previous work in both MRL-lpr and NZB/W F1 mice has suggested that anti-retroviral gp70 Ag-Ab complexes (gp70 complexes) are important in the pathogenesis of renal disease (47, 65). As a low-calorie diet is able to decrease the level of gp70 complexes, it is possible that the decreased incidence of nephritis in our AF mice may be due to a decrease in gp70 complexes (66). This concern is less likely as Datta et al. (67) first demonstrated that the expression of serum gp70 did not correlate with the levels circulating immune complexes nor the presence of nephritis in the progeny of NZB and SWR crosses. Andrews et al. (68) later demonstrated a lack of a direct relationship between serum gp70 complexes levels and the development of nephritis in MRL-lpr mice. Hence, nephritis, indistinguishable in severity and age of onset, can be found in mice with either high and low titers of gp70 or gp70 Ag-Ab complexes. Another autoantibody, IgG3 RF anti-IgG2aa, has been implicated in the development of various pathological manifestations, including glomerulonephritis, seen in MRL-lpr mice (35). It is possible that the lower levels of this autoantibody in GF-AF mice may in part explain the decreased severity of glomerulonephritis.
This work supports the view that the specific immunoregulatory mechanisms involved in the induction of tolerance to certain autoantigens, like those targeted in systemic lupus erythematosus (SLE), fail in MRL-lpr mice due to genetic features in T and B cells that are largely independent of foreign antigenic stimuli. The striking similarity between the profile of autoantibodies produced in MRL-lpr mice and humans with SLE suggests that the immunoregulatory mechanisms that fail in humans with SLE may also be primarily genetic.
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Acknowledgments |
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Footnotes |
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2 This work was presented at the American Academy of Allergy, Asthma, and Immunology/The American Association of Immunologists/Clinical Immunology Society Joint Meeting, San Francisco, February 1997.
3 Address correspondence and reprint requests to Dr. Robert A. Eisenberg, Division of Rheumatology, Department of Medicine, University of Pennsylvania, 504 Maloney, 3600 Spruce Street, Philadelphia, PA 19104-4283. E-mail address:
4 Abbreviations used in this paper: GF, germfree; AF, Ag-free; NI, natural ingredient; SPF, specific pathogen-free; DN, double negative; TBW, total body weight; LN, lymph node; CI, confidence intervals; RF, rheumatoid factor; NZB, New Zealand black; NZW, New Zealand white.
Received for publication November 17, 1998. Accepted for publication March 4, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß T cells. J. Immunol. 156:4041.[Abstract]
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T. Catterall, D. Stockwell, V. Marshall, A. Strasser, and J. Allison Autoimmune kidney disease and lymphadenopathy in NODlpr mice are not modified by deficiency in tumor necrosis factor receptor 1 or {beta}2-microglobulin Int. Immunol., June 1, 2003; 15(6): 679 - 690. [Abstract] [Full Text] [PDF]
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 P. L. Cohen Autoimmunity and Lymphoproliferation: Two Genes are Worse than One Mol. Interv., November 1, 2002; 2(7): 427 - 430. [Abstract] [Full Text]
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 D. W. van Bekkum Experimental basis of hematopoietic stem cell transplantation for treatment of autoimmune diseases J. Leukoc. Biol., October 1, 2002; 72(4): 609 - 620. [Abstract] [Full Text] [PDF]
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M. Lockshin Sex ratio and rheumatic disease: excerpts from an Institute of Medicine report Lupus, October 1, 2002; 11(10): 662 - 666. [Abstract] [PDF]
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 O.M. Z. Howard, H. F. Dong, D. Yang, N. Raben, K. Nagaraju, A. Rosen, L. Casciola-Rosen, M. Hartlein, M. Kron, D. Yang, et al. Histidyl-tRNA Synthetase and Asparaginyl-tRNA Synthetase, Autoantigens in Myositis, Activate Chemokine Receptors on T Lymphocytes and Immature Dendritic Cells J. Exp. Med., September 16, 2002; 196(6): 781 - 791. [Abstract] [Full Text] [PDF]
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T. Miwa, M. A. Maldonado, L. Zhou, X. Sun, H. Y. Luo, D. Cai, V. P. Werth, M. P. Madaio, R. A. Eisenberg, and W.-C. Song Deletion of Decay-Accelerating Factor (CD55) Exacerbates Autoimmune Disease Development in MRL/lpr Mice Am. J. Pathol., September 1, 2002; 161(3): 1077 - 1086. [Abstract] [Full Text] [PDF]
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P. L. Cohen, R. Caricchio, V. Abraham, T. D. Camenisch, J. C. Jennette, R. A.S. Roubey, H. S. Earp, G. Matsushima, and E. A. Reap Delayed Apoptotic Cell Clearance and Lupus-like Autoimmunity in Mice Lacking the c-mer Membrane Tyrosine Kinase J. Exp. Med., July 1, 2002; 196(1): 135 - 140. [Abstract] [Full Text] [PDF]
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L. A. Trimble, K. A. Prince, G. A. Pestano, J. Daley, and H. Cantor Fas-Dependent Elimination of Nonselected CD8 Cells and lpr Disease J. Immunol., May 15, 2002; 168(10): 4960 - 4967. [Abstract] [Full Text] [PDF]
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 M. D. Lockshin Genome and Hormones: Gender Differences in Physiology: Invited Review: Sex ratio and rheumatic disease J Appl Physiol, November 1, 2001; 91(5): 2366 - 2373. [Abstract] [Full Text] [PDF]
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J. H. Freed, A. Marrs, J. VanderWall, P. L. Cohen, and R. A. Eisenberg MHC Class II-Bound Self Peptides from Autoimmune MRL/lpr Mice Reveal Potential T Cell Epitopes for Autoantibody Production in Murine Systemic Lupus Erythematosus J. Immunol., May 1, 2000; 164(9): 4697 - 4705. [Abstract] [Full Text] [PDF]
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