Jean Wu

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.

Hypoxia-induced autophagic response is associated with aggressive phenotype and elevated incidence of metastasis in orthotopic immunocompetent murine models of head and neck squamous cell carcinomas (HNSCC)

Hypoxia confers resistance to chemoradiation therapy and promotes metastasis in head and neck squamous cell carcinomas (HNSCC). We investigated the effects of hypoxia in tumor phenotype using immunocompetent murine HNSCC models. Balb/c mice were injected intraorally with murine squamous cell carcinoma cells LY-2 and B4B8. Intratumoral hypoxia fraction was evaluated by the immunohistochemical detection of hypoxic probe pimonidazole and carbonic anhydrase IX (CAIX). Tumor cell apoptosis and autophagy in hypoxic areas of these tumors were examined immunohistochemically. Hypoxia-induced apoptotic and autophagic responses in vitro were examined by treating LY2 cells with CoCl(2). B4B8 tumors exhibited a non-aggressive phenotype characterized by its slow growth rate and the lack of metastatic spread. LY2 tumors demonstrated an aggressive phenotype characterized by rapid growth rate with regional and distant metastasis. Intratumoral hypoxia fraction in B4B8 tumors was significantly lower than in LY2 tumors. The hypoxic areas in B4B8 tumors exhibited increased apoptosis rate than that of LY2 tumors. In contrast, the hypoxic areas in LY2 tumors revealed autophagy. The induction of hypoxia in vitro elicited autophagy and not apoptosis in LY2 cells. The induction of autophagy coupled with blockage of apoptosis in hypoxic areas promotes tumor cell survival and confers aggressive phenotype in immunocompetent murine HNSCC models.

Repression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but not its receptors during oral cancer progression

BackgroundTRAIL plays an important role in host immunosurveillance against tumor progression, as it induces apoptosis of tumor cells but not normal cells, and thus has great therapeutic potential for cancer treatment. TRAIL binds to two cell-death-inducing (DR4 and DR5) and two decoy (DcR1, and DcR2) receptors. Here, we compare the expression levels of TRAIL and its receptors in normal oral mucosa (NOM), oral premalignancies (OPM), and primary and metastatic oral squamous cell carcinomas (OSCC) in order to characterize the changes in their expression patterns during OSCC initiation and progression.MethodsDNA microarray, immunoblotting and immunohistochemical analyses were used to examine the expression levels of TRAIL and its receptors in oral epithelial cell lines and in archival tissues of NOM, OPM, primary and metastatic OSCC. Apoptotic rates of tumor cells and tumor-infiltrating lymphocytes (TIL) in OSCC specimens were determined by cleaved caspase 3 immunohistochemistry.ResultsNormal oral epithelia constitutively expressed TRAIL, but expression was progressively lost in OPM and OSCC. Reduction in DcR2 expression levels was noted frequently in OPM and OSCC compared to respective patient-matched uninvolved oral mucosa. OSCC frequently expressed DR4, DR5 and DcR1 but less frequently DcR2. Expression levels of DR4, DR5 and DcR1 receptors were not significantly altered in OPM, primary OSCC and metastatic OSCC compared to patient-matched normal oral mucosa. Expression of proapoptotic TRAIL-receptors DR4 and DR5 in OSCC seemed to depend, at least in part, on whether or not these receptors were expressed in their parental oral epithelia. High DR5 expression in primary OSCC correlated significantly with larger tumor size. There was no significant association between TRAIL-R expression and OSSC histology grade, nodal status or apoptosis rates of tumor cells and TIL.ConclusionLoss of TRAIL expression is an early event during oral carcinogenesis and may be involved in dysregulation of apoptosis and contribute to the molecular carcinogenesis of OSCC. Differential expressions of TRAIL receptors in OSCC do not appear to play a crucial role in their apoptotic rate or metastatic progression.

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.