Jason Borillo

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 Bowmans space and had a flattened shape, suggesting that the primary target for the T cells was in or adjacent to the Bowmans capsule.

Glomerulonephritis Induced by Recombinant Collagen IVα3 Chain Noncollagen Domain 1 Is Not Associated with Glomerular Basement Membrane Antibody: A Potential T Cell-Mediated Mechanism

Glomerulonephritis is believed to result commonly from Ab-mediated glomerular injury. However, Ab-associated mechanisms alone cannot explain many cases of human glomerulonephritis. We developed a rat model of human anti-glomerular basement membrane (GBM) disease to investigate T cell and Ab response, and their associations with the disease. A single immunization of highly denatured recombinant mouse collagen IVα3 chain noncollagen domain 1 (rCol4α3NC1) induced severe glomerulonephritis in 100% of Wistar Kyoto rats, 33% of which died of this disease around day 35 postimmunization. The renal pathology demonstrated widespread glomerular damage and a mononuclear cell infiltration within the interstitial tissue. T cells from immunized rats responded not only to rCol4α3NC1, but also to isolated rat GBM. Sera Abs to rCol4α3NC1 were detectable in 100% of the rats, but only 20% of the rats had low levels of Ab to isolated rat GBM by Western blot, and none by immunofluorescence. Furthermore, IgG/M binding to or C3 deposition on endogenous GBM in immunized rats were not detected in most of the experimental rats, and showed no statistical correlation with disease severity. Additionally, no electronic dense deposition in the glomeruli was detected in all rats. Those data revealed a disassociation between the disease and anti-GBM Ab. T cell-mediated mechanisms, which are currently under our investigation, may be responsible for the glomerular disease.

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.

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.

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.

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.