The anti-glomerular basement membrane (GBM) Ab has been regarded as a prototypical example of pathogenic autoantibodies. However, the mechanism for elicitation of this Ab remains unknown. In the present paper, we report that the Ab to diverse GBM Ags was induced by a single nephritogenic T cell epitope in a rat model. The T cell epitope pCol28–40 of noncollagen domain 1 of collagen type IV α3 chain not only uniformly induced severe glomerulonephritis but also elicited anti-GBM Ab in 76% of the immunized rats after prominent glomerular injury. Furthermore, we demonstrated that the anti-GBM Ab was not related to the peptidic B cell epitope nested in pCol28–40; that is, 1) elimination of the B cell epitope, either by substitution of the critical residues of the B cell epitope or by truncation, failed to abrogate anti-GBM Ab production, and 2) the anti-GBM Ab, eluted from the diseased kidneys, reacted only with native GBM, but not with pCol28–40. Confocal microscopy and immunoprecipitation further demonstrated that the eluted anti-GBM Ab recognized conformational B cell epitope(s) of multiple native GBM proteins. We conclude that autoantibody response to diverse native GBM Ags was induced by a single nephritogenic T cell epitope. Thus, anti-GBM Ab may actually be a consequence of T cell-mediated glomerulonephritis.

Antiglomerular basement membrane (GBM) disease or Goodpasture’s syndrome is among the earliest recognized human autoimmune diseases. Although collagen 4α3 NC1 (Col4α3NC1) has been identified as the responsible autoantigen, it remains unknown how autoimmunity to this autoantigen is provoked. We have demonstrated in our rat model that a single nephritogenic T cell epitope pCol28–40 of Col4α3NC1 induces glomerulonephritis. We hypothesized that microbial peptides that mimic this T cell epitope could induce the disease. Based on the critical residue motif (xxtTxNPsxx) of pCol28–40, seven peptides derived from human infection-related microbes were chosen through GenBank search and synthesized. All peptides showed cross-reactivity with pCol28–40-specific T cells at various levels. Only four peptides induced transient proteinuria and minor glomerular injury. However, the other three peptides induced severe proteinuria and modest to severe glomerulonephritis in 16–25% of the immunized rats. Unexpectedly, the most nephritogenic peptide, pCB, derived from Clostridium botulinum, also induced modest (25%) to severe (25%) pulmonary hemorrhage, another important feature of anti-GBM disease; this was not correlated with the severity of glomerulonephritis. This finding suggests that subtle variations in T cell epitope specificity may lead to different clinical manifestations of anti-GBM disease. In summary, our study raises the possibility that a single T cell epitope mimicry by microbial Ag may be sufficient to induce the anti-GBM disease.

DCs play critical roles in promotion of autoimmunity or immune tolerance as potent APCs. In our anti-GBM GN model, WKY rats develop severe T cell-mediated glomerular inflammation followed by fibrosis. A DC-like cell population (CD8αα(+)CD11c(+)MHC-II(+)ED1(-)) was identified in the inflamed glomeruli. Chimera experiments demonstrated that the CD8αα(+) cells were derived from BM. The CD8αα(+) cells infiltrated glomeruli at a late stage (Days 28-35), coincident with a rapid decline in glomerular inflammation before fibrosis. The CD8αα(+) cells isolated from inflamed glomeruli were able to migrate rapidly from the bloodstream into inflamed glomeruli but not into normal glomeruli, suggesting that the migration was triggered by local inflammation. Despite high-level expression of surface and cellular MHC class II molecules, in vitro experiments showed that this CD8αα(+) DC-like cell induced apoptosis but not proliferation in antigen-specific CD4(+) T cells from T cell lines or freshly isolated from lymph nodes; they were not able to do so in the absence of antigens, suggesting induction of apoptosis was antigen-specific. Furthermore, apoptotic T cells were detected in a large number in the glomeruli at Day 32, coincident with the infiltration of the cells into glomeruli, suggesting that the cells may also induce T cell apoptosis in vivo. A potential role of this CD8αα(+) DC-like population in peripheral immune tolerance and/or termination of autoimmune inflammation was discussed.

Linear binding of IgG to the glomerular basement membrane (GBM) is the hallmark of anti-GBM glomerulonephritis (GN).However, the precise mechanism by which diverse autoantibodies to GBM are induced in GN has not been determined.

Ovaries are among the most active organs. Frequently occurring events such as ovulation and ovarian atresia are accompanied with tissue destruction and repairing. Critical roles of immune cells or molecules in those events have been well recognized. Interleukin 33 (IL33) is a new member of IL1 cytokine gene family. Recent studies suggest its roles beyond immune responses. We systemically examined its expression in ovaries for its potential roles in ovarian functions. During ovulation, a high level of IL33 was transiently expressed, making it the most significantly up-regulated immune genes. During estrous cycle, IL33 expression levels fluctuated along with numbers of ovarian macrophages and atresia wave. Cells with nuclear form of IL33 (nIL33+ cells) were mostly endothelial cells of veins, either in the inner layer of theca of ovulating follicles during ovulation, or surrounding follicles during estrous cycle. Changes in number of nIL33+ cells showed a tendency similar to that in IL33 mRNA level during estrous cycle. However, the cell number sharply dropped before a rapid increase of macrophages and surge of atresia. The drop in nIL33+ cell number was coincident with detection of higher level of the cytokine form of IL33 by western blot, suggesting a release of cytokine form of IL33 before the surge of macrophage migration and atresia. However, IL33 Ab, either by passive transfer or immunization, showed a limited effect on ovulation or atresia. It raises a possibility of IL33’s role in tissue homeostasis following ovarian events, instead of a direct involvement in ovarian functions.

Physiological processes such as ovarian follicle atresia generate large amounts of unnecessary cells or tissue detritus, which needs to be disposed of rapidly. Interleukin33 (IL33) is a member of the IL1 cytokine gene family. Consecutive expression of IL33 in a wide range of tissues has hinted at its role beyond immune defense. We have previously reported a close correlation between IL33 expression patterns and ovarian atresia. Here, we demonstrated that IL33 is required for disposal of degenerative tissue during ovarian atresia using Il33−/− mice. Deletion of the Il33 gene impaired normal disposal of atretic follicles, resulting in massive accumulations of tissue wastes abundant with aging-related catabolic wastes such as lipofuscin. Accumulation of tissue wastes in Il33−/− mice, in turn, accelerated ovarian aging and functional decline. Thus, their reproductive lifespan was shortened to 2/3 of that for Il33+/− littermates. IL33 orchestrated disposal mechanism through regulation of autophagy in degenerating tissues and macrophage migration into the tissues. Our study provided direct evidence supporting an expanded role of IL33 in tissue integrity and aging through regulating disposal of unnecessary tissues or cells.

Different susceptibility to anti-GBM glomerulonephritis (GN) among animal strains has been reported. Using our rat model for T cell-mediated anti-GBM GN, this study initiated an investigation on the mechanism related with GN-susceptibility. Anti-GBM GN was induced either though immunization with the nephritogenic T cell epitope pCol(28-40) from Col4α3NC1 or through the transfer of specific T cells. WKY rats were highly susceptible to GN while immuno-compatible LEW rats were GN-resistant. GN-resistance in LEW rats was not associated immune response to pCol (28-40). First, both strains mounted a Th1 T cell response to pCol(28-40) with identical specificities; transfer of T cells from LEW to WKY rats induced glomerular injury. Second, co-transfer of antibody from WKY to LEW failed to induce GN. Time-course studies revealed that LEW rats did develop T cell-mediated inflammation in glomeruli at early stages similar to WKY rats, as evidenced by histopathology, proteinuria, CD4 + T cell infiltration in glomeruli, and glomerular expression of inflammatory molecules. However, glomerular inflammation in LEW rats was transient followed by a full recovery. Thus, GN-resistance in LEW rats was due to its ability to contain early T cell-mediated autoimmune glomerular damage. Our model may reveal a potential tolerance mechanism after autoimmune tissue damage has been initiated.

Ovarian macrophages, which play critical roles in various ovarian events, are probably derived from multiple lineages. Thus, a systemic classification of their subsets is a necessary first step for determination of their functions. Utilizing antibodies to five phagocyte markers, i.e. IA/IE (major histocompatibility complex class II), F4/80, CD11b (Mac-1), CD11c, and CD68, this study investigated subsets of ovarian phagocytes in mice. Three-color immunofluorescence and flow cytometry, together with morphological observation on isolated ovarian cells, demonstrated complicated phenotypes of ovarian phagocytes. Four macrophage and one dendritic cell subset, in addition to many minor phagocyte subsets, were identified. A dendritic cell-like population with a unique phenotype of CD11c high IA/IE K F4/80 K was also frequently observed. A preliminary age-dependent study showed dramatic increases in IA/IE C macrophages and IA/IE C dendritic cells after puberty. Furthermore, immunofluorescences on ovarian sections showed that each subset displayed a distinct tissue distribution pattern. The pattern for each subset may hint to their role in an ovarian function. In addition, partial isolation of ovarian macrophage subset using CD11b antibodies was attempted. Establishment of this isolation method may have provided us a tool for more precise investigation of each subset's functions at the cellular and molecular levels.