Cindy Zhou

T Cell Epitope Mimicry in Antiglomerular Basement Membrane Disease

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.

A Self T Cell Epitope Induces Autoantibody Response: Mechanism for Production of Antibodies to Diverse Glomerular Basement Membrane Antigens

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.

Transient Expression of CC Chemokine TECK in the Ovary during Ovulation: Its Potential Role in Ovulation

Problem:  Chemokine thymus‐expressed chemokine (TECK), which is expressed exclusively in the thymus and small intestine, plays a critical role in T‐cell development. Our previous study revealed its expression in the ovary also. This study investigated its ovarian expression during ovulatory process.

Potential Roles of a Special CD8αα+ Cell Population and CC Chemokine Thymus-Expressed Chemokine in Ovulation Related Inflammation

It is well known that ovulation may be an inflammatory process. However, it remains elusive how immune cells participate in this process. We have identified a novel CD8αα+ population, which resembles tissue dendritic cells, in the theca of antral follicles. We further observed a dramatic influx of the CD8αα+cells into the ovulating follicles. This CD8αα+population was absent in the ovary of estradiol-induced anovulatory C31F1 mice and subfertile athymic nude mice. Expression of a CC chemokine thymus-expressed chemokine (TECK) has previously been found in the ovary; we further demonstrated that TECK attracted CD8αα+cells into the ovary. Anti-TECK Ab, elicited in the female mice by active immunization, depleted the ovarian CD8αα+ cells in vivo. Mice with a high titer of TECK Ab failed to ovulate after superovulation induction. More importantly, the immunized mice had greatly reduced fertility, which was positively correlated with the Ab titers. Ovarian TECK expression was normal in anovulatory C31F1 mice, suggesting that infertility in the immunized mice is due to a block of CD8αα+ cell migration. Finally, the origin of ovarian CD8αα+ cells was explored. Upon being transferred, thymic CD8α+ cells were able to home to the theca of follicles in the recipients. Thus, ovarian CD8αα+ cells, which participate in the ovulation-related inflammation, may originate in the thymus.

IL-33 is required for disposal of unnecessary cells during ovarian atresia through regulation of autophagy and macrophage migration

Physiological processes such as ovarian follicle atresia generate large amounts of unnecessary cells or tissue detritus, which needs to be disposed of rapidly. IL-33 is a member of the IL-1 cytokine gene family. Constitutive expression of IL-33 in a wide range of tissues has hinted at its role beyond immune defense. We have previously reported a close correlation between IL-33 expression patterns and ovarian atresia. In this study, we demonstrated that IL-33 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 life span was shortened to two thirds of that for Il33+/− littermates. IL-33 orchestrated disposal mechanism through regulation of autophagy in degenerating tissues and macrophage migration into the tissues. Our study provides direct evidence supporting an expanded role of IL-33 in tissue integrity and aging through regulating disposal of unnecessary tissues or cells.

Unique Temporal and Spatial Expression Patterns of IL-33 in Ovaries during Ovulation and Estrous Cycle Are Associated with Ovarian Tissue Homeostasis

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. IL-33 is a new member of the IL-1 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 IL-33 was transiently expressed, making it the most significantly upregulated immune gene. During estrous cycle, IL-33 expression levels fluctuated along with numbers of ovarian macrophages and atresia wave. Cells with nuclear form of IL-33 (nIL-33+ 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 nIL-33+ cells showed a tendency similar to that in IL-33 mRNA level during estrous cycle. However, the cell number sharply declined before a rapid increase of macrophages and a surge of atresia. The decline in nIL-33+ cell number was coincident with detection of higher level of the cytokine form of IL-33 by Western blot, suggesting a release of cytokine form of IL-33 before the surge of macrophage migration and atresia. However, IL-33 Ab, either by passive transfer or immunization, showed a limited effect on ovulation or atresia. It raises a possibility of IL-33’s role in tissue homeostasis after ovarian events, instead of a direct involvement in ovarian functions.

Coincident Activation of Th2 T Cells with Onset of the Disease and Differential Expression of GRO-Gamma in Peripheral Blood Leukocytes in Minimal Change Disease

Background: Involvement of Th2 T cells/NFĸB in minimal change disease (MCD) has been postulated. A promising but unconfirmed glomerular permeability factor (GPF) from MCD T cells has been described. We explored whether GPF was the consequence of Th2 cell activation. Methods: Peripheral blood leukocytes (PBL) from 16 MCD patients and 7 normal controls were analyzed and the results were statistically compared. Results: Flow cytometry demonstrated a significant expansion of CD4+ T cell population and dramatically increased CD69+ cells among CD4+ T cells in MCD, suggesting coincident activation of T cells with onset of the disease. RT-PCR on RNA from either freshly isolated PBL or post in vitroactivation showed high-level expression of the Th2 cytokine interleukin-4 in all MCD patients. Importantly, both antibody microarray assay on sera and RT-PCR on mRNA of PBL revealed expression of a CXC chemokine GRO-γ (growth-related oncogene) in all MCD patients as compared with one of 7 controls. Conclusions: Our results reveal an association between onset of MCD and activation of Th2 cells. The GRO family has been implicated in the function of endothelial cells, and its expression is under NFĸB regulation. Thus, GRO-γ is a promising candidate for Th2-associated GPF in MCD.

Identification of a bone marrow-derived CD8αα+ dendritic cell-like population in inflamed autoimmune target tissue with capability of inducing T cell apoptosis.

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.

Blockade of Osteopontin Inhibits Glomerular Fibrosis in a Model of Anti-Glomerular Basement Membrane Glomerulonephritis

Background: In our rat model for anti-GBM GN, severe fibrosis follows glomerular inflammation. A potential role of extracellular matrix protein osteopontin (OPN) in glomerular fibrosis was investigated. Methods: Neutralizing OPN antiserum or control normal serum was injected into the experimental rats at late inflammatory/early fibrotic stage. Glomerular inflammation and fibrosis were determined. Results: OPN antiserum treatment had little effect on glomerular inflammation. However, the antiserum treatment resulted in a significant reduction in number of fibrotic glomeruli (50% of the controls). Histology observation showed that fibrotic tissue in glomeruli of the antiserum treated rats was mild and poorly developed. OPN antiserum treatment resulted in downregulated glomerular expression of collagen 1α1; collagen deposition in the antiserum treated rats reduced to <30% of that for normal serum controls. Conclusion: Neutralization of OPN inhibited progression of fibrosis in vivo when given at early fibrotic stage. Thus, OPN may be a therapeutic target for glomerular fibrosis.

Antibodies to two ZP3 B cell epitopes affect zona pellucida assembly.

Mouse zona pellucida (ZP) proteins are synthesized in developing oocytes and assembled into ZP after their secretion. This study has investigated whether anti-ZP3 antibodies affect ZP assembly. Peptides CP2 and CP3 were used to elicit antibodies to two ZP3 B cell epitopes, ZP3 (335-342) and ZP3 (171-180). Ovulated eggs from mice immunized with a mixture of CP2/CP3 showed an abnormal ZP; importantly, the ZP completely dissolved both in vitro and in vivo 12h after ovulation. Although CP3 immunization resulted also in abnormal ZP, the ZP did not dissociate. Binding of antibodies to the ZP prior to oocyte maturation was requisite, as in vitro incubation of ovulated eggs in combination with the two antibodies failed to induce ZP dissolution. Electron microscopic observation further demonstrated a significant abnormality in ZP structure in CP2/CP3-immunized mice, especially in mature follicles, suggesting that B cell epitopes may be involved in ZP assembly. Though antibody elicited by CP2 has been shown to inhibit fertilization, we now show that antibody induced by CP3 had no effect on fertility. However, immunization with CP3/CP2 resulted in a significantly lower fertility rate than CP2 alone. This suggests that infertility in these mice may be due to an unstable ZP structure. Our model provides a useful tool to study ZP assembly and its structure beyond molecular biology method.