RESUMEN
It has been previously shown that the cytokine interleukin 33 is required for two processes, i.e., autophagic digestion of granulosa cells and recruitment of macrophages into atretic follicles, for full disposal of atretic follicles. Now, this study shows that activation of interleukin 33-suppression of tumorigenicity 2-Nuclear Factor ĸB (NFκB) axis in granulosa in early atretic follicles may regulate those two events. Injection of human chorionic gonadotropin has been shown to induce a transient peak of interleukin 33 expression with synchronized atresia. In this model, interleukin 33-independent expression of suppression of tumorigenicity 2 in granulosa cells was detected in early atretic follicles before macrophage invasion. The activation of NFκB pathway in ovaries was further demonstrated in vivo in Tg mice with luciferase-reporter for NFκB activation; the activation was microscopically localized to granulosa cells in early atretic follicles. Importantly, antibody blockage of interleukin 33 or interleukin 33 Knock-out (KO) (Il33-/-) not only inhibited NFκB activity in ovaries, but it also altered expression of two key genes, i.e., reduction in proinflammatory interleukin6 (IL6) expression, and a surge of potential autophagy-inhibitory mammalian target of rapamycin (mTOR) expression in atretic follicles. By contrast, apoptosis and other genes, such as interleukin1ß (IL1ß) were not affected. In conclusion, in parallel to apoptosis, atresia signals also trigger activation of the interleukin 33-suppression of tumorigenicity 2-NFκB pathway in granulosa, which leads to (1) down-regulated expression of mTOR that is a negative regulator of autophagy and (2) up-regulated expression of proinflammatory IL6.
Asunto(s)
Atresia Folicular , Células de la Granulosa , Interleucina-33 , FN-kappa B , Folículo Ovárico , Femenino , Animales , Células de la Granulosa/metabolismo , Células de la Granulosa/efectos de los fármacos , Ratones , FN-kappa B/metabolismo , Atresia Folicular/metabolismo , Folículo Ovárico/metabolismo , Interleucina-33/metabolismo , Interleucina-33/genética , Transducción de Señal , Ratones Noqueados , Autofagia/fisiologíaRESUMEN
Late-onset Alzheimer's disease (LOAD) is the most common age-related dementia, and its etiology remains unclear. Recent studies have linked abnormal neuronal aging to LOAD. Neurons are non-proliferative, and thus, majority of aged neurons must be rejuvenated through repairing or eliminating damaged molecules to regain their healthy status and functionalities. We discovered a surge of oxidative stress in neurons at middle age in mice. A rapid upregulation of neuronal rejuvenation is vital, while astrocyte-expressed interleukin33 (IL33), an IL1-like cytokine, is critical for this process. Thus, IL33-deficiency cripples the neuronal rejuvenation mechanisms, such as repairing DNA double strand breaks, eliminating damaged molecules by autophagy or by glymphatic drainage. IL33-deficient mice develop tau deposition and age-related dementia following a path similar to LOAD. We hypothesize that any interferences on IL33-initiated rejuvenation process for aged neurons after middle life is a potential risk for LOAD development.
RESUMEN
Defective aquaporin4 (AQP4)-mediated glymphatic drainage has been linked to tauopathy and amyloid plaque in Alzheimer's disease. We now show that brain interleukin33 (IL33) is required for regulation of AQP4 expression in astrocytes, especially those at neuron-facing membrane domain (n-AQP4). First, IL33-deficient (Il33-/-) mice showed a loss of n-AQP4 after middle age, which coincided with a rapid accumulation of abnormal tau in neurons and a reduction in drainage of abnormal tau to peripheral tissues. Second, injection of recombinant IL33 induced robust expression of AQP4 at perivascular endfoot (p-AQP4) of astrocytes, but not n-AQP4, in Il33-/- brains. Although the increased p-AQP4 greatly accelerated drainage of intracerebroventricularly injected peptides, it did not substantially accelerate drainage of abnormal tau. These results suggest that p-AQP4 drives overall convective flow toward perivenous space, i.e., glymphatics, whereas n-AQP4 may generate an aqueous flow away from neurons to remove neuronal wastes, e.g., abnormal tau. We have previously shown the role of brain IL33 in DNA repair and autophagy in neurons with oxidative stress. Now, we show that IL33 deficiency also impairs glymphatic drainage. Defects in those mechanisms together may lead to chronic neurodegeneration and tauopathy at old age in IL33-deficient mice.
Asunto(s)
Enfermedad de Alzheimer , Tauopatías , Animales , Acuaporina 4/genética , Acuaporina 4/metabolismo , Astrocitos/metabolismo , Encéfalo/metabolismo , Interleucina-33 , Ratones , Placa Amiloide , Proteínas tauRESUMEN
Inter-molecular epitope spreading during autoimmune pathogenesis leads to generation of new pathogenic epitopes on other autoantigens beyond the original one. It raises an important question as whether autoimmunity extends beyond the target tissues if new epitopes are on the molecules shared with other tissues. This study is aimed addressing this question in a rat anti-glomerular basement membrane (GBM) glomerulonephritis model induced by a T cell epitope of glomerulus-specific collagen4α3. We have demonstrated inter-molecular B cell epitope spreading. Four novel epitopes were first identified by screening a phage display random peptide library against autoantibodies isolated from the GBM of immunized rats. All four epitopes were derived from GBM proteins with three from laminins and one from collagen4α4. Three out of four synthetic peptides were nephritogenic. Importantly, two peptides from lamininα1 and lamininß1, respectively, induced severe inflammation in glomeruli but not in the interstitial tissues, despite the presence of more abundant laminins in the tubular basement membranes. Our study suggests that surrounding tissues may display a lower or altered susceptibility to autoimmune inflammation. Thus, preventing extension of autoimmune inflammation beyond the original target tissue.
Asunto(s)
Autoinmunidad , Epítopos/inmunología , Glomerulonefritis/inmunología , Animales , Autoanticuerpos/inmunología , Femenino , Inmunización , Laminina/metabolismo , Ratas , Linfocitos T/inmunologíaRESUMEN
Alzheimer's Disease or other dementias are characterized by the accumulation of abnormal tau and amyloid ß peptides in brains. Therefore, abnormal tau and amyloid peptides in peripheral tissues or blood have been explored as diagnostic biomarkers. On the other hand, recent studies have revealed glymphatics a special drainage system for brain's wastes. We aimed to investigate whether effectiveness of glymphatic system affects the quantity of abnormal tau in the peripheral tissues. We have previously shown that aged IL33 KO (Il33 -/-) mice develop Alzheimer's like disease. Despite a large quantity of abnormal tau in brains, Il33 -/- mice showed a much lower amount of abnormal tau drained to the peripheral tissues kidneys than in wild type mice. Our further study showed that it was caused by defective glymphatic drainage since Il33 KO impaired glymphatics. Thus, it is necessary to identify biomarkers, which can evaluate efficiency of glymphatic drainage. Simultaneous measurement of these biomarkers and abnormal tau in peripheral tissues or blood may be critical for accurate diagnosis of Alzheimer's disease.
RESUMEN
Discovery of immune tolerance mechanisms, which inhibit pre-existing autoimmune inflammation, may provide us with new strategies for treating autoimmune diseases. We have identified a CD8αα+MHC class II+ cell with professional APC capacity during our investigation on spontaneous recovery from autoimmune glomerulonephritis in a rat model. This cell actively invades inflamed target tissue and further terminates an ongoing autoimmune inflammation by selective killing of effector autoreactive T cells. In this study, we show that this cell used a cytotoxic machinery of Ly49s+ NK cells in killing of target T cells. Thus, this CD8αα+MHC class II+ cell was a dually functional Ag-presenting NK-like (AP-NK) cell. Following its coupling with target T cells through Ag presentation, killing stimulatory receptor Ly49s6 and coreceptor CD8αα on this cell used rat nonclassic MHC class I C/E16 on the target T cells as a ligand to initiate killing. Thus, activated effector T cells with elevated expression of rat nonclassic MHC class I C/E16 were highly susceptible to the killing by the CD8αα+ AP-NK cell. Granule cytolytic perforin/granzyme C from this cell subsequently mediated cytotoxicity. Thus, inhibition of granzyme C effectively attenuated the killing. As it can recognize and eliminate effector autoreactive T cells in the inflamed target tissue, the CD8αα+ AP-NK cell not only represents a new type of immune cell involved in immune tolerance, but it also is a potential candidate for developing a cell-based therapy for pre-existing autoimmune diseases.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Enfermedades Autoinmunes/inmunología , Antígenos CD8/inmunología , Glomerulonefritis/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Células Asesinas Naturales/inmunología , Animales , Células Presentadoras de Antígenos/patología , Enfermedades Autoinmunes/patología , Femenino , Glomerulonefritis/patología , Granzimas/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Inflamación/inmunología , Inflamación/patología , Células Asesinas Naturales/patología , Ratas , Ratas Endogámicas Lew , Ratas Endogámicas WKYRESUMEN
BACKGROUND: Inflammation is the hallmark of nephrotoxic nephritis. Stanniocalcin-1 (STC1), a pro-survival factor, inhibits macrophages, stabilizes endothelial barrier function, and diminishes trans-endothelial migration of leukocytes; consistently, transgenic (Tg) overexpression of STC1 protects from nephrotoxic nephritis. Herein, we sought to determine the phenotype of nephrotoxic nephritis after conditional and kidney-specific knockdown of STC1. METHODS: We used Tg mice that, express either STC1 shRNA (70% knockdown of STC1 within 4d) or scrambled shRNA (control) upon delivery of Cre-expressing plasmid to the kidney using ultrasound microbubble technique. Sheep anti-mouse GBM antibody was administered 4d after shRNA activation; and mice were euthanized 10 days later for analysis. RESULTS: Serum creatinine, proteinuria, albuminuria and urine output were similar 10 days after anti-GBM delivery in both groups; however, anti-GBM antibody delivery to mice with kidney-specific knockdown of STC1 produced severe nephrotoxic nephritis, characterized by severe tubular necrosis, glomerular hyalinosis/necrosis and massive cast formation, while control mice manifested mild tubular injury and crescentic glomerulonephritis. Surprisingly, the expression of cytokines/chemokines and infiltration with T-cells and macrophages were also diminished in STC1 knockdown kidneys. Staining for sheep anti-mouse GBM antibody, deposition of mouse C3 and IgG in the kidney, and antibody response to sheep IgG were equal. CONCLUSIONS: nephrotoxic nephritis after kidney-specific knockdown of STC1 is characterized by severe tubular and glomerular necrosis, possibly due to loss of STC1-mediated pro-survival factors, and we attribute the paucity of inflammation to diminished release of cytokines/chemokines/growth factors from the necrotic epithelium.
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Glicoproteínas/metabolismo , Riñón/metabolismo , Nefritis/etiología , Actinas/metabolismo , Albúminas/análisis , Animales , Anticuerpos/administración & dosificación , Anticuerpos/inmunología , Línea Celular , Creatinina/sangre , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Glicoproteínas/antagonistas & inhibidores , Glicoproteínas/genética , Peróxido de Hidrógeno/toxicidad , Inmunoglobulina G/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Riñón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Nefritis/metabolismo , Nefritis/patología , Fenotipo , Índice de Severidad de la EnfermedadRESUMEN
BACKGROUND: Many types of glomerulonephritis (GN) undergo tandem connected phases: inflammation and fibrosis. Fibrosis in human GNs leads to irreversible end-stage disease. This study investigated how these 2 phases were controlled. METHODS: Using a rat anti-glomerular basement membrane GN model, we established bone marrow (BM) chimeras between GN-resistant Lewis (LEW) and GN-susceptible Wistar Kyoto (WKY) rats. Glomerular inflammation and fibrosis were compared between chimeras. RESULTS: LEW's BM to WKY chimeras with or without co-transfer of host WKY's T cells were GN-resistant. On the other hand, WKY's BM to LEW (LEW(WKY)) chimeras developed glomerular inflammation and albuminuria upon immunization. Quantitative analysis showed that the number and composition of inflammatory cells in glomeruli of immunized LEW(WKY) chimeras were similar to those in immunized WKY rats at their inflammatory peak. Thus, glomerular inflammation was controlled by BM-derived non-T cell populations. However, unlike WKY rats, LEW(WKY) rats did not develop fibrosis until the end of experiments (84 days) in spite of persistent inflammation and albuminuria. CONCLUSION: Inflammation alone was not sufficient to trigger fibrosis, suggesting a critical role of glomerular cells in the fibrotic process. As LEW(WKY) chimera allows us to separate glomerular inflammation from fibrosis, this model provides a useful tool to study how fibrosis is initiated following inflammation.
Asunto(s)
Enfermedad por Anticuerpos Antimembrana Basal Glomerular/inmunología , Enfermedad por Anticuerpos Antimembrana Basal Glomerular/patología , Glomérulos Renales/patología , Células Mieloides/inmunología , Linfocitos T/inmunología , Albuminuria/inmunología , Animales , Nitrógeno de la Urea Sanguínea , Trasplante de Médula Ósea , Quimera , Colágeno Tipo IV/inmunología , Creatinina/sangre , Epítopos de Linfocito T/inmunología , Femenino , Fibrosis , Células Mieloides/trasplante , Fragmentos de Péptidos/inmunología , Ratas , Ratas Endogámicas Lew , Ratas Endogámicas WKY , Linfocitos T/trasplante , Factores de TiempoRESUMEN
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.
Asunto(s)
Fertilidad/inmunología , Atresia Folicular/inmunología , Interleucinas/inmunología , Folículo Ovárico/inmunología , Animales , Autofagia , Senescencia Celular/inmunología , Femenino , Atresia Folicular/genética , Eliminación de Gen , Regulación de la Expresión Génica , Interleucina-33 , Interleucinas/deficiencia , Interleucinas/genética , Lipofuscina/inmunología , Macrófagos/inmunología , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Folículo Ovárico/patología , Transducción de Señal , Factores de TiempoRESUMEN
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.
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Ciclo Estral/inmunología , Regulación de la Expresión Génica/inmunología , Homeostasis/inmunología , Interleucinas/inmunología , Proteínas Nucleares/inmunología , Ovario/inmunología , Ovulación/inmunología , Animales , Femenino , Atresia Folicular/inmunología , Interleucina-33 , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/inmunologíaRESUMEN
In an anti-glomerular basement membrane (GBM) glomerulonephritis (GN) model, GN-resistant Lewis rats naturally recover from early glomerular inflammation. Here we investigated recovery mechanisms for development of a potential immunotherapy for autoimmune GN. Our previous studies suggested that glomeruli-infiltrating leukocytes with a phenotype of CD8αα+CD11c+MHC-II+CD3- (GIL CD8αα+ cells) were responsible for recovery through induction of T-cell apoptosis. Now, we identified peripheral blood CD8αα+CD11c+MHC-II+CD3- cells (PBMC CD8αα+CD3- cells), which shared 9 markers with GIL CD8αα+ cells. Upon incubation, PBMC CD8αα+CD3- cells displayed a morphology resembling that of dendritic cells. Similar to GIL CD8αα+ cells, PBMC CD8αα+CD3- cells were capable of inducing T-cell apoptosis in vitro. Hence, PBMC CD8αα+CD3- cells were likely the precursor of GIL CD8αα+ cells. We next tested their potential in vivo function. PBMC CD8αα+CD3- cells were able to infiltrate inflamed but not normal glomeruli. Isolated PBMC CD8αα+CD3- cells of Lewis rats were transferred into GN-prone Wistar-Kyoto rats at early inflammatory stage (days 17-25). When examined at day 45, both histopathology and blood urea nitrogen/serum creatinine level showed significantly attenuated GN in 80% of cell recipient Wistar-Kyoto rats. Separate experiments verified infiltration of transferred Lewis PBMC CD8αα+CD3- into the glomeruli, accompanied with apoptotic CD4+ T cells in the glomeruli of the recipient Wistar-Kyoto rats. Thus, PBMC CD8αα+CD3- cells of Lewis rats were able to terminate ongoing autoimmune inflammation in the glomeruli.
Asunto(s)
Traslado Adoptivo , Autoinmunidad , Antígeno CD11c/sangre , Complejo CD3/sangre , Antígenos CD8/sangre , Linfocitos T CD8-positivos/trasplante , Glomerulonefritis/prevención & control , Antígenos de Histocompatibilidad Clase II/sangre , Glomérulos Renales/inmunología , Animales , Apoptosis , Biomarcadores/sangre , Nitrógeno de la Urea Sanguínea , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/inmunología , Células Cultivadas , Quimiotaxis de Leucocito , Técnicas de Cocultivo , Creatinina/sangre , Citotoxicidad Inmunológica , Modelos Animales de Enfermedad , Femenino , Adyuvante de Freund , Glomerulonefritis/sangre , Glomerulonefritis/inducido químicamente , Glomerulonefritis/inmunología , Glomerulonefritis/patología , Glomérulos Renales/patología , Técnicas de Cultivo de Órganos , Péptidos , Ratas Endogámicas Lew , Ratas Endogámicas WKY , Factores de TiempoRESUMEN
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/IEâ»F4/80â» was also frequently observed. A preliminary age-dependent study showed dramatic increases in IA/IE⺠macrophages and IA/IE⺠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.
Asunto(s)
Ovario/citología , Fagocitos/citología , Maduración Sexual , Animales , Biomarcadores/metabolismo , Antígeno CD11b/metabolismo , Linaje de la Célula , Transdiferenciación Celular , Células Cultivadas , Cuerpo Lúteo/citología , Cuerpo Lúteo/crecimiento & desarrollo , Cuerpo Lúteo/metabolismo , Células Dendríticas/citología , Células Dendríticas/metabolismo , Femenino , Citometría de Flujo , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Microscopía Fluorescente , Folículo Ovárico/citología , Folículo Ovárico/crecimiento & desarrollo , Folículo Ovárico/metabolismo , Ovario/crecimiento & desarrollo , Ovario/metabolismo , Fagocitos/metabolismo , FagocitosisRESUMEN
BACKGROUND/AIMS: In an antiglomerular basement membrane glomerulonephritis (GN) model, GN-resistant Lewis (LEW) rats naturally recover from early glomerular inflammation (days 21-23). We have previously identified a glomeruli-infiltrating CD8α(+)CD11(high)MHC II(+) cell (GIL CD8α(+) cell) in GN-prone Wistar Kyoto (WKY) rats, which terminates glomerular inflammation through inducing T cell apoptosis prior to glomerular fibrosis at days 35-40. We investigated if GIL CD8α(+) cells were also associated with the recovery in LEW rats. METHODS: GIL CD8α(+) cells in LEW rats were characterized; their infiltration was observed in connection with T cell apoptosis in glomeruli. RESULTS: An influx of GIL CD8α(+) cells into inflamed glomeruli was confirmed in the immunized LEW rats at days 17-22, which was much earlier than days 28-35 in WKY rats. Notably, LEW rats had a GIL CD8α(+)CD11(high) subpopulation after day 17, while WKY rats lacked this population until after day 30. Analyses further revealed a large number of clustered apoptotic CD4(+) or CD3(+) T cells in the glomeruli during recovery (day 23) in LEW rats, as compared to day 35 (transition to fibrosis) in WKY rats. Thus, infiltration of GIL CD8α(+) cells coincided with decline of glomerular inflammation and T cell apoptosis during recovery in LEW rats. Isolated GIL CD8α(+) cells were able to infiltrate glomeruli in both WKY and LEW rats at day 20. CONCLUSION: Our data revealed a strong association between GIL CD8a+ cells and recovery from early glomerular inflammation. It raises a possibility of involvement of GIL CD8a+ cells in the recovery.
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Autoanticuerpos/inmunología , Glomerulonefritis/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Glomérulos Renales/inmunología , Animales , Apoptosis , Antígeno CD11c/biosíntesis , Antígenos CD8/biosíntesis , Femenino , Citometría de Flujo/métodos , Inflamación , Glomérulos Renales/metabolismo , Microscopía Confocal/métodos , Ratas , Ratas Endogámicas Lew , Ratas Endogámicas WKY , Linfocitos T/citología , Factores de TiempoRESUMEN
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.
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Enfermedad por Anticuerpos Antimembrana Basal Glomerular/metabolismo , Enfermedad por Anticuerpos Antimembrana Basal Glomerular/patología , Fibrosis/patología , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Osteopontina/antagonistas & inhibidores , Osteopontina/sangre , Animales , Anticuerpos/administración & dosificación , Femenino , Citometría de Flujo , Modelos Animales , Ratas , Ratas Endogámicas WKYRESUMEN
BACKGROUND: Glomerular fibrosis is the common end result of glomerulonephritis (GN) regardless of etiology. In our rat model for anti-glomerular basement membrane GN, severe fibrosis follows glomerular inflammation. We investigated the association between expression of extracellular matrix (ECM) proteins and progression of glomerular fibrosis. METHODS: Expression of ECM genes in glomeruli was determined at RNA and protein levels. Immunofluorescence was applied to identify cell sources for the molecules. RESULTS: DNA microarray for ECM genes, quantitative RT-PCR and Western blot revealed significant upregulation of osteopontin (OPN), a multifunctional molecule, in the glomeruli only after onset of glomerular fibrosis. Two-dimensional electrophoresis showed that the expressed OPN was in three major isoforms. Immunofluorescence showed that fibrotic tissues in glomeruli accumulated massive deposits of extracellular OPN. Both in vivo and in vitro experiments showed that a novel population of multinucleated α-smooth muscle actin(+)CD90(-) myofibroblast-like cells, which surrounded fibrotic tissue, was the main source of OPN during progression of fibrosis. Since senescence-associated ß-galactosidase activity was detected in those cells both in vitro and in vivo, these cells probably were terminally differentiated senescent myofibroblasts. CONCLUSION: OPN has been implicated in fibrosis in several organs. Our results suggest potential roles of OPN and its main source, the senescent myofibroblasts, in glomerular fibrosis.
Asunto(s)
Enfermedad por Anticuerpos Antimembrana Basal Glomerular/metabolismo , Enfermedad por Anticuerpos Antimembrana Basal Glomerular/patología , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Osteopontina/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Fibrosis , Expresión Génica , Osteopontina/genética , Ratas , Ratas Endogámicas WKY , Regulación hacia Arriba/genéticaRESUMEN
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.
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Células de la Médula Ósea/inmunología , Antígenos CD8/análisis , Células Dendríticas/inmunología , Inflamación/inmunología , Linfocitos T/inmunología , Animales , Apoptosis/inmunología , Antígenos CD11/aislamiento & purificación , Antígenos CD8/aislamiento & purificación , Muerte Celular , Línea Celular , Supervivencia Celular , Femenino , Glomérulos Renales/inmunología , Linfocitos/inmunología , Ratas , Ratas Wistar , Linfocitos T/citologíaRESUMEN
We have previously shown that stanniocalcin-1 (STC1) inhibits the transendothelial migration of macrophages and T cells, suppresses superoxide generation in macrophages, and attenuates macrophage responses to chemoattractants. To study the effects of STC1 on inflammation, in this study we induced a macrophage- and T-cell-mediated model of anti-glomerular basement membrane disease in STC1 transgenic mice, which display elevated serum STC1 levels and preferentially express STC1 in both endothelial cells and macrophages. We examined the following parameters both at baseline and after anti-glomerular basement membrane antibody treatment: blood pressure; C(3a) levels; urine output; proteinuria; blood urea nitrogen; and kidney C(3) deposition, fibrosis, histological changes, cytokine expression, and number of T cells and macrophages. Compared with wild-type mice, after anti-glomerular basement membrane treatment STC1 transgenic mice exhibited: i) diminished infiltration of inflammatory macrophages in the glomeruli; ii) marked reduction in crescent formation and sclerotic glomeruli; iii) decreased interstitial fibrosis; iv) preservation of kidney function and lower blood pressure; v) diminished C(3) deposition in the glomeruli; and vi) reduced expression of macrophage inhibitory protein-2 and transforming growth factor-beta2 in the kidney. Compared with baseline, wild-type mice, but not STC1 transgenic mice, had higher proteinuria and a marked reduction in urine output. STC1 had minimal effects, however, on both T-cell number in the glomeruli and interstitium and on cytokine expression characteristic of either TH1 or TH2 activation. These data suggest that STC1 is a potent anti-inflammatory and renal protective protein.
Asunto(s)
Enfermedad por Anticuerpos Antimembrana Basal Glomerular/inmunología , Glicoproteínas/inmunología , Mediadores de Inflamación/inmunología , Animales , Enfermedad por Anticuerpos Antimembrana Basal Glomerular/patología , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Western Blotting , Citocinas/biosíntesis , Citocinas/inmunología , Modelos Animales de Enfermedad , Membrana Basal Glomerular/inmunología , Membrana Basal Glomerular/patología , Glicoproteínas/genética , Inmunohistoquímica , Inflamación/inmunología , Inflamación/patología , Pruebas de Función Renal , Ratones , Ratones TransgénicosRESUMEN
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.
Asunto(s)
Antígenos CD8/biosíntesis , Quimiocinas CC/fisiología , Mediadores de Inflamación/fisiología , Ovario/inmunología , Ovario/patología , Ovulación/inmunología , Timo/inmunología , Traslado Adoptivo , Animales , Antígenos CD8/fisiología , Inhibición de Migración Celular/inmunología , Quimiocinas CC/inmunología , Femenino , Infertilidad Femenina/inmunología , Mediadores de Inflamación/metabolismo , Macrófagos/citología , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Desnudos , Folículo Ovárico/citología , Folículo Ovárico/inmunología , Folículo Ovárico/metabolismo , Ovario/metabolismo , Timo/citología , Timo/metabolismo , Timo/trasplanteRESUMEN
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.
Asunto(s)
Proteínas del Huevo/inmunología , Epítopos de Linfocito B/inmunología , Glicoproteínas de Membrana/inmunología , Oocitos/metabolismo , Receptores de Superficie Celular/inmunología , Zona Pelúcida/metabolismo , Animales , Proteínas del Huevo/metabolismo , Epítopos de Linfocito B/metabolismo , Femenino , Fertilización/inmunología , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica de Transmisión , Oocitos/citología , Oocitos/inmunología , Oocitos/ultraestructura , Receptores de Superficie Celular/metabolismo , Zona Pelúcida/inmunología , Zona Pelúcida/ultraestructura , Glicoproteínas de la Zona PelúcidaRESUMEN
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 through immunization with the nephritogenic T cell epitope pCol(28-40) from Col4alpha3NC1 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 to the 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.