Potential roles of a special CD8 alpha alpha+ 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 CD8alpha alpha(+) population, which resembles tissue dendritic cells, in the theca of antral follicles. We further observed a dramatic influx of the CD8alpha alpha(+) cells into the ovulating follicles. This CD8alpha alpha(+) population was absent in the ovary of estradiol-induced anovulatory C31F(1) 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 CD8alpha alpha(+) cells into the ovary. Anti-TECK Ab, elicited in the female mice by active immunization, depleted the ovarian CD8alpha alpha(+) 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 C31F(1) mice, suggesting that infertility in the immunized mice is due to a block of CD8alpha alpha(+) cell migration. Finally, the origin of ovarian CD8alpha alpha(+) cells was explored. Upon being transferred, thymic CD8alpha(+) cells were able to home to the theca of follicles in the recipients. Thus, ovarian CD8alpha alpha(+) cells, which participate in the ovulation-related inflammation, may originate in the thymus.

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.

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/NFkappaB 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-gamma (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 NFkappaB regulation. Thus, GRO-gamma is a promising candidate for Th2-associated GPF in MCD.

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 4alpha3 NC1 (Col4alpha3NC1) 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 Col4alpha3NC1 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.

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. METHOD OF STUDY: Super-ovulation was induced in young female CD1 mice by equine chorionic gonadotropin (eCG) and human chorionic gonadotropic (hCG). Ovarian TECK expression during ovulation was determined by: (1) reverse transcriptase-polymerase chain reaction (RT-PCR) at mRNA level, (2) Western blot and immunohistology at the protein level, and (3) leukocyte infiltration assay at the bioactive level. RESULTS: A transient, high-level expression of TECK in murine ovaries at the mRNA level during hCG-induced ovulation was detected. Sequencing of directly cloned PCR product confirmed the ovarian expression of TECK. The peak expression of TECK was observed at 10-12 hr post-hCG injection; real-time PCR revealed an 800-fold increase during its expression peak over 0 hr. The expressed ovarian TECK protein was readily detectable by Western blot. Immunohistochemistry localized TECK expression to the ovarian interstitial tissue surrounding, or in the theca layer of the mature follicles undergoing ovulatory process. Expression of TECK receptor, the CC chemokine receptor (CCR9) was also detected in the ovulating ovaries. Using in vitro leukocyte infiltration assay, we first demonstrated that ovaries undergoing the ovulatory process were able to selectively chemoattract mononuclear cells. Importantly, neutralization of TECK by the antibody resulted in a 85% reduction in the chemotactic activities of the ovaries. CONCLUSION: This study suggested that ovarian expression of TECK is under a tight hormonal regulation, and expressed TECK may be responsible for recruitment of mononuclear cells into the ovary to participate in the ovulatory process.

Ovarian expression of chemokines and their receptors.

Recent studies suggest involvement of the immune system, including leukocytes and cytokines/chemokines, in various ovarian functions such as ovulation. Using the RT-PCR method, we examined expression of various chemokines and their receptors in normal mouse ovaries. Among seventeen examined chemokines (17 CC types and two CXC types), expressions of CC types MCP-1 and RANTES, and CXC type IP-10 were detected at high levels, while most CC types expressed at variable or low levels. Only five chemokines were not detected in the ovary. We next examined expression of chemokine receptors. CCR1 and CCR2, which are the receptors for MCP-1 and RANTES, were also expressed at constitutively high levels while others were not detectable. We further showed that a significant part of expression of both detected chemokines and receptors originated from peripheral blood leukocytes (PBL) circulating in the ovary. However, ovarian tissue was the major contributor of expression. Constitutive expression of several chemokines and their receptors suggests frequent migrations/movements of leukocytes in the ovary, which may be involved in ovarian functions other than ovulation.

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 pCol(28-40) of noncollagen domain 1 of collagen type IV alpha3 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 pCol(28-40); 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 pCol(28-40). 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.

T-cell epitope of alpha3 chain of type IV collagen induces severe glomerulonephritis.

BACKGROUND: Anti-glomerular basement membrane (GBM) glomerulonephritis is among the earliest recognized human autoimmune diseases. However, the etiology of anti-GBM glomerulonephritis remains unclear. We have previously shown that CD4+ T cells, specific to alpha3 NC1 of type IV collagen (Col4alpha3NC1), were able to induce anti-GBM glomerulonephritis in Wistar-Kyoto (WKY) rats. In the present study, we continued to map the nephritogenic T cell epitope in Col4alpha3NC1. METHODS: Synthetic peptides, which covered Col4alpha3NC1, were used as immunogens to induce glomerulonephritis in WKY rats. T-cell and B-cell responses to the peptides in the animals were analyzed. RESULTS: One potent nephritogenic T-cell epitope, pCol(28-40) (SQTTANPSCPEGT), was identified. A single immunization with pCol(28-40) induced extremely severe glomerulonephritis in all 23 rats. Renal pathology revealed nearly 100% of glomeruli with crescentic lesions or tuft necrosis in 21 animals. pCol(28-40) elicited a T-cell response to the peptide; T cells isolated from rats immunized with recombinant Col4alpha3NC1 reacted with pCol(28-40). pCol(28-40) elicited a peptide specific antibody response, which did not react with polypeptide Col4alpha3NC1 or native GBM. An 11-mer peptide, pCol(a30-40) (Ac-TTANPSCPEGT), was further mapped to be the core of the T-cell epitope in pCol(28-40). As expected, immunization with pCol(a30-40) induced severe glomerulonephritis in 10 out of 19 rats. CONCLUSION: Our study not only demonstrated that a single T-cell epitope of Col4alpha3NC1 is sufficient to induce severe glomerulonephritis, but also provides a unique model for studying T-cell-mediated mechanisms in anti-GBM glomerulonephritis pathogenesis.

Migration of T cells from nearby inflammatory foci into antibody bound tissue: a relay of T cell and antibody actions in targeting native autoantigen.

We have reported that binding of antibody to native autoantigen is prerequisite for T cells to target the native antigen in murine autoimmune ovarian disease model (AOD). As a result, ovarian follicles, with target antigen ZP3 (Zona Pellucida 3), are destroyed. In this study, AOD was induced by co-transfer of ZP3-specific CD4(+)T cells and ZP3 antibody. ZP3 CD4(+)T cells, labeled with CFSE, were found to target macrophages in degenerated follicles to form inflammatory foci, which were composed of mainly endogenous CD4(+)T cells (85%). Only endogenous T cells in the foci, with increased CD69(+)expression, further migrated into antibody bound follicles. No F4/80 or MHC II(+)cells were found to co-migrate with the T cells or in follicles. Co-transfer of ZP3 T cell and antibody also induced (1) a transient PMN influx at early stage and (2) a dramatic increase in IL-1 beta expression coincident with the migration in the ovary. These results suggest that ZP3 antibody binding, only in the presence of ZP3 T cells, may cause an inflammatory change in follicles, which attract endogenous T cells from nearby inflammation. Thus, through a relay between T cell and antibody mediated mechanisms, native autoantigen is targeted and destroyed. This mechanism may explain the requirement of antibody in several T cell mediated autoimmune diseases.

CD4(+) T cells specific to a glomerular basement membrane antigen mediate glomerulonephritis.

Ab-mediated mechanisms have been considered the major causes of glomerulonephritis (GN). However, recent studies suggest that T cells may be more important in mediating GN. To investigate the effects of antigen-specific CD4(+) T cells, we generated Th1 cell lines specific for this antigen from rats that had been immunized with a recombinant form of the glomerular basement membrane (GBM) antigen, Col4alpha3NC1. Upon the transfer of in vitro-activated T cell lines to pertussis toxin-primed, naive syngeneic rats, the recipients developed severe proteinuria/albuminuria, which plateaued after approximately 35 days. Although no IgG binding to GBM or C3 deposition could be detected by immunofluorescence, five out of eleven rats exhibited severe GN, as judged by the formation of characteristic crescent-shaped lesions in the glomeruli, whereas the others exhibited modest GN. Thus Col4alpha3NC1-specific T cells directly initiated glomerular injury in the recipients. One notable difference from GN induced by active immunization was a T cell infiltration in the renal interstitium, which affected some tubules. We therefore injected fluorescence-labeled Col4alpha3NC1-specific into naive rats, and we found that they were enriched 4.5-fold in the kidney cortex relative to nonspecific control T cells 24 hours later. Many of the T cells were located in the Bowman's space and had a flattened shape, suggesting that the primary target for the T cells was in or adjacent to the Bowman's capsule.