Blockade of TGF-beta signaling in T cells prevents the development of experimental glomerulonephritis

Anti-glomerular basement membrane (GBM) Ab-induced glomerulonephritis (GN) at late stage is thought to be mediated by T cells. However, signaling pathways of T cells that are involved in the development of anti-GBM Ab-induced GN are unclear. We have recently established transgenic mice expressing Smad7, an inhibitor of TGF-beta signaling, in mature T cells, where signaling by TGF-beta was blocked specifically in T cells. In this study, we showed that anti-GBM Ab-induced GN was suppressed in several measures in the transgenic mice including the severity of glomerular changes, proteinuria, renal function, and CD4 T cell infiltration into the glomeruli without down-regulation of CD62 ligand (CD62L) (L-selectin) expression on CD4 T cells. Furthermore, treatment with the soluble fusion protein of CD62L and IgG enhanced anti-GBM Ab-induced GN. These findings indicated that blockade of TGF-beta signaling in T cells prevented the development of anti-GBM Ab-induced GN. Because CD62L on T cells appears to be inhibitory for the development of anti-GBM Ab-induced GN, persistent expression of CD62L on CD4 T cells may explain, at least in part, the suppression of anti-GBM Ab-induced GN in the transgenic mice. Our findings suggest that the development of anti-GBM Ab-induced GN requires TGF-beta/Smad signaling in T cells.     

Recruitment of CTL activity by tumor-specific antibody-mediated targeting of single-chain class I MHC-peptide complexes

Acute and lethal ileitis can be elicited in certain strains of inbred mice after oral infection with the intracellular protozoan parasite Toxoplasma gondii. The development of this inflammatory process is dependent upon the induction of a robust Th1 response, including overproduction of IFN-gamma, TNF-alpha, and NO, as has been reported in other experimental models of human inflammatory bowel disease. In this study we have investigated the role of CD4(+) T cells from the lamina propria (LP) in the early inflammatory events after T. gondii infection using isolated and primary cultured intestinal cells from infected mice and immortalized mouse mIC(cl2) intestinal epithelial cells. Primed LP CD4(+) T cells isolated from parasite-infected mice produce substantial quantities of both IFN-gamma and TNF-alpha. IFN-gamma- and TNF-alpha-producing LP CD4(+) T cells synergize with infected mIC(cl2) and enhance the production of several inflammatory chemokines including macrophage-inflammatory protein-2, monocyte chemoattractant protein-1, monocyte chemoattractant protein-3, macrophage-inflammatory protein-1alphabeta, and IFN-gamma-inducible protein-10. Furthermore, primed LP CD4(+) T cells cocultured with infected mIC(cl2) inhibited replication of the parasite in the intestinal epithelial cells. Thus, LP CD4(+) T cells can interact with parasite-infected intestinal epithelial cells and alter the expression of several proinflammatory products that have been associated with the development of intestinal inflammation. The interaction between these two components of the gut mucosal compartment (CD4(+) T cells and enterocytes) may play a role in the immunopathogenesis of this pathogen-driven experimental inflammatory bowel disease model.     

Failure of CD25+ T cells from lupus-prone mice to suppress lupus glomerulonephritis and sialoadenitis

The development of organ-specific autoimmune diseases in mice thymectomized on day 3 of life (d3tx mice) can be prevented by transferring CD4(+)CD25(+) T cells from syngeneic, normal adult mice. Using a d3tx model, we asked whether CD4(+)CD25(+) T cell deficiency contributes to glomerulonephritis (GN) in lupus-prone mice. New Zealand Mixed 2328 (NZM2328) mice spontaneously develop autoantibodies to dsDNA and female-dominant, fatal GN. After d3tx, both male and female NZM2328 mice developed 1) accelerated dsDNA autoantibody response, 2) early onset and severe proliferative GN with massive mesangial immune complexes, and 3) autoimmune disease of the thyroid, lacrimal gland, and salivary gland. The d3tx male mice also developed autoimmune prostatitis. The transfer of CD25(+) cells from 6-wk-old asymptomatic NZM2328 donors effectively suppressed dsDNA autoantibody and the development of autoimmune diseases, with the exception of proliferative lupus GN and sialoadenitis. This finding indicates that NZM2328 lupus mice have a selective deficiency in T cells that regulates the development of lupus GN and sialoadenitis. After d3tx, the proliferative GN of female mice progressed to fatal GN, but largely regressed in the male, thereby revealing a checkpoint in lupus GN progression that depends on gender.     

Chemokine receptor expressions and responsiveness of cord blood T cells

Chemokines and their receptors play a critical role in the selective attraction of various subsets of leukocytes. We examined the chemokine receptor expressions and responsiveness of cord blood (CB) T cells. Flow-cytometric analysis revealed that peripheral blood (PB) T cells expressed CCR-1, CCR-2, CCR-5, CCR-6, CXC chemokine receptor-3 (CXCR-3), and CXCR-4, while CB T cells expressed only CXCR-4 on their surface. Chemotactic migratory response of CB T cells to macrophage-inflammatory protein (MIP)-1alpha, monocyte chemoattractant protein-1, RANTES, MIP-3alpha, monokine induced by IFN-gamma, and IFN-gamma-inducible protein-10 was significantly impaired compared with those of PB T cells. In contrast, the ability of CB T cells to migrate to MIP-3beta, 6Ckine, and stromal cell-derived factor-1alpha was greater than that of PB T cells, and these events were correlated with the expression levels of CCR-7 and CXCR-4, respectively. Engagement of CD3 and CD28 specifically up-regulated CXCR-3 expression and chemotaxis to monokine induced by IFN-gamma and IFN-gamma-inducible protein-10, whereas this stimulation down-regulated CCR-7 expression and chemotaxis to MIP-3beta and 6Ckine in PB T cells, but not in CB T cells. These results suggest that PB T cells and CB T cells exhibit distinct chemokine responsiveness via different chemokine receptor repertoire.     

Early local generation of C5a initiates the elicitation of contact sensitivity by leading to early T cell recruitment

We have shown previously that an early complement C5-dependent cascade is required to recruit T cells to elicit 24-h contact sensitivity (CS) responses. In this paper, we have characterized molecular events of this early required cascade by biochemically analyzing extracts of mouse ears undergoing elicitation of CS. Chemotactic activity was found after local Ag challenge, in CS ear extracts early (by 1 h), in CS ear extracts late (through 24 h), in previously immunized mice, but not in ears of vehicle-immunized or non-immune-challenged mice. The early chemotactic activity at 2 h was likely caused by C5a, because it was neutralized in vitro by anti-C5a Ab, was inactive on C5aR-deficient (C5aR-/-) macrophages, and was absent in C5-deficient mice. The activity was present in T cell-deficient mice, but elaboration was Ag-specific. This T cell-independent, Ag-specific elaboration of C5a early in CS ear responses likely led to T cell recruitment, because subsequent local IFN-gamma mRNA and protein expression, as markers of T cell arrival and activation, began by 4 h after Ag challenge. In contrast to early C5a chemotactic activity, late chemotactic activity 24 h after Ag challenge was unaffected by anti-C5, was active on C5aR-/- macrophages, was T cell-dependent, and by ELISA appeared largely due to chemokines (macrophage-inflammatory protein-1alpha and -1beta, IFN-gamma-inducible protein-10, and monocyte chemoattractant protein-1). Importantly, early generation of C5a was required for T cell recruitment because C5aR-/- mice had absent 24-h CS. Taken together, these findings indicate an important linkage of C5a as a component of early activated innate immunity that is required for later elicitation of acquired T cell immunity, probably by facilitating the initial recruitment of T cells into the Ag-challenged local site in CS responses.     

FcR-bearing myeloid cells are responsible for triggering murine lupus nephritis

Lupus glomerulonephritis is initiated by deposition of IgG-containing immune complexes in renal glomeruli. FcR engagement by immune complexes (IC) is crucial to disease development as uncoupling this pathway in FcRgamma(-/-) abrogates inflammatory responses in (NZB x NZW)F1 mice. To define the roles of FcR-bearing hemopoietic cells and of kidney resident mesangial cells in pathogenesis, (NZB x NZW)F1 bone marrow chimeras were generated. Nephritis developed in (NZB x NZW)F1 mice expressing activating FcRs in hemopoietic cells. Conversely, recipients of FcRgamma(-/-) bone marrow were protected from disease development despite persistent expression of FcRgamma in mesangial cell populations. Thus, activating FcRs on circulating hemopoietic cells, rather than on mesangial cells, are required for IC-mediated pathogenesis in (NZB x NZW)F1. Transgenic FcRgamma(-/-) mice expressing FcRgamma limited to the CD11b+ monocyte/macrophage compartment developed glomerulonephritis in the anti-glomerular basement disease model, whereas nontransgenic FcRgamma(-/-) mice were completely protected. Thus, direct activation of circulating FcR-bearing myeloid cells, including monocytes/macrophages, by glomerular IC deposits is sufficient to initiate inflammatory responses.     

An IL-12-independent role for CD40-CD154 in mediating effector responses: studies in cell-mediated glomerulonephritis and dermal delayed-type hypersensitivity

Crescentic glomerulonephritis (GN) results from IL-12-driven Th1-directed cell-mediated responses (akin to delayed-type hypersensitivity (DTH)) directed against glomerular Ags. CD40-CD154 interactions are critical for IL-12 production and Th1 polarization of immune responses. Crescentic anti-glomerular basement membrane GN was induced in C57BL/6 (wild-type (WT)) mice (sensitized to sheep globulin) by planting this Ag (as sheep anti-mouse glomerular basement membrane globulin) in their glomeruli. Crescentic GN did not develop in CD40(-/-) mice due to significantly reduced nephritogenic Th1 responses. IL-12 was administered to CD40(-/-) mice with GN to dissect interactions between IL-12 and CD40 in inducing nephritogenic immunity and injury. Administration of IL-12 to CD40(-/-) mice restored Th cell IFN-gamma production, and up-regulated intrarenal chemokines and glomerular T cell and macrophage accumulation compared with WT control mice. Despite this, renal macrophages were not activated and renal injury and dermal DTH were not restored. Thus, CD40-directed IL-12 drives Th1 generation and effector cell recruitment but CD40 is required for activation. To test this hypothesis, activated OT-II OVA-specific CD4(+) cells and OVA(323-339)-loaded nonresponsive APCs were transferred into footpads of WT, CD40(-/-), and macrophage-depleted WT mice. WT mice developed significant DTH compared with CD40(-/-) and macrophage-depleted WT mice. This study demonstrated that CD40-induced IL-12 is required for generation of systemic Th1 immunity to nephritogenic Ags, and that IL-12 enhances Th1 effector cell recruitment to peripheral sites of Ag presentation via generation of local chemokines. Effector cell activation, renal DTH-like injury, and dermal DTH require direct Th1 CD154/macrophage CD40 interactions.     

Chemokine amplification in mesangial cells.

Mesangial cells are specialized cells of the renal glomerulus that share some properties of vascular smooth muscle cells and macrophages. They are implicated in the pathogenesis of many forms of nephritis. The murine CXC-chemokines macrophage inflammatory protein-2 (MIP-2) and KC induce migration of mouse mesangial cells. Mesangial cells also exhibit a unique chemokine feedback mechanism. Treatment with nanomolar concentrations of MIP-2 or KC markedly up-regulates monocyte chemoattractant protein-1 and RANTES expression in mesangial cells. Autoinduction of MIP-2 and KC mRNA was also noted. Low levels of MIP-1alpha, MIP-1beta, and IFN-gamma-inducible protein-10 were induced following treatment with higher doses of MIP-2 or KC. These effects are specific to mesangial cells, as MIP-2 or KC treatment of renal cortical epithelial cells or peritoneal macrophages failed to induce chemokine production. This cascade of chemokine interactions may contribute to renal infiltration and leukocyte activation. The abilities of MIP-2 or KC to stimulate their own synthesis may also contribute to the maintenance and chronic course of glomerular inflammation. The mesangial cell receptor for MIP-2 and/or KC is unknown but is not CXC-chemokine receptor-2.     

Role for nephritogenic T cells in lupus glomerulonephritis: progression to renal failure is accompanied by T cell activation and expansion in regional lymph nodes

Autoreactive T cells are critical in the initiation and maintenance of autoantibody responses that are a hallmark of systemic lupus erythematosus. However, the direct contribution of T cells in end-organ disease like lupus glomerulonephritis (GN) is poorly understood. In this study, we investigated the role of T cells in progression of lupus GN in NZM2328 mice, a murine model of spontaneous systemic lupus erythematosus. At 26 wk of age, NZM2328 female mice showed glomerular immune complex deposits and acute proliferative GN. This was associated with up-regulation of MHC class II and the detection of T cells and CD11c(+) dendritic cells in the glomeruli. The regional lymph nodes (LN) showed preferential activation of T cells and an oligoclonal T cell response with skewed expansion of certain Vbeta families. This suggests an Ag-driven response occurring in the regional LN of nephritic mice during acute GN. In contrast, male NZM2328 mice developed glomerular immune complexes and acute GN, but rarely progressed to fatal chronic GN. Significantly, male kidneys at 40 wk of age did not have detectable dendritic cells and T cells in the glomeruli. Thus, glomerular immune complex deposition initiates an immune response against renal Ags in the regional LN, leading to T cell recruitment into the kidney during acute proliferative GN. This T cell activation and infiltration are influenced by gender-dependent end-organ factors and may determine the progression of acute GN to chronic GN and renal failure.     

Experimental autoimmune encephalomyelitis on the SJL mouse: effect of gamma delta T cell depletion on chemokine and chemokine receptor expression in the central nervous system

Experimental autoimmune encephalomyelitis (EAE) is a demyelinating disease of the central nervous system (CNS) that is a model for multiple sclerosis. Previously, we showed that depletion of gamma delta T cells significantly reduced clinical and pathological signs of disease, which was associated with reduced expression of IL-1 beta, IL-6, TNF-alpha, and lymphotoxin at disease onset and a more persistent reduction in IFN-gamma. In this study, we analyzed the effect of gamma delta T cell depletion on chemokine and chemokine receptor expression. In the CNS of control EAE mice, mRNAs for RANTES, eotaxin, macrophage-inflammatory protein (MIP)-1 alpha, MIP-1 beta, MIP-2, inducible protein-10, and monocyte chemoattractant protein-1 were detected at disease onset, increased as disease progressed, and fell as clinical signs improved. In gamma delta T cell-depleted animals, all of the chemokine mRNAs were reduced at disease onset; but at the height of disease, expression was variable and showed no differences from control animals. mRNA levels then fell in parallel with control EAE mice. ELISA data confirmed reduced expression of MIP-1 alpha and monocyte chemoattractant protein-1 at disease onset in gamma delta T cell-depleted mice, and total T cell numbers were also reduced. In normal CNS mRNAs for CCR1, CCR3, and CCR5 were observed, and these were elevated in EAE animals. mRNAs for CCR2 were also detected in the CNS of affected mice. Depletion of gamma delta T cells reduced expression of CCR1 and CCR5 at disease onset only. We conclude that gamma delta T cells contribute to the development of EAE by promoting an inflammatory environment that serves to accelerate the inflammatory process in the CNS.     

Exuberant inflammation in nicotinamide adenine dinucleotide phosphate-oxidase-deficient mice after allogeneic marrow transplantation

We have shown that NO and superoxide (O-*2)contribute to donor T cell-dependent lung dysfunction after bone marrow transplantation (BMT) in mice. We hypothesized that inhibiting superoxide production during inducible NO synthase induction would suppress oxidative/nitrative stress and result in less severe lung injury. Irradiated mice lacking the phagocytic NADPH-oxidase (phox(-/-)), a contributor to superoxide generation, were conditioned with cyclophosphamide and given donor bone marrow in the presence or absence of inflammation-inducing allogeneic spleen T cells. On day 7 after allogeneic BMT, survival, weight loss, and indices of lung injury between phox(-/-) and wild-type mice were not different. However, the majority of macrophages/monocytes from phox(-/-) mice given donor T cells produced fewer oxidants and contained less nitrotyrosine than cells obtained from T cell-recipient wild-type mice. Importantly, suppressed oxidative stress was associated with marked infiltration of the lungs with inflammatory cells and was accompanied by increased bronchoalveolar lavage fluid levels of the chemoattractants monocyte chemoattractant protein-1 and macrophage-inflammatory protein-1alpha and impaired clearance of recombinant mouse macrophage-inflammatory protein-1beta from the circulation. Furthermore, cultured macrophages/monocytes from NADPH-deficient mice produced 3-fold more TNF-alpha compared with equal number of cells from NADPH-sufficient mice. The high NO production was not modified during NADPH-oxidase deficiency. We conclude that phox(-/-) mice exhibit enhanced pulmonary influx of inflammatory cells after BMT. Although NO may contribute to increased production of TNF-alpha in phox(-/-) mice, the data suggest that NADPH-oxidase-derived oxidants have a role in limiting inflammation and preventing lung cellular infiltration after allogeneic transplantation.     

Hydrogen Peroxide–Inducible Clone-5 Regulates Mesangial Cell Proliferation in Proliferative Glomerulonephritis in Mice

Hydrogen peroxide-inducible clone-5 (Hic-5) is a transforming growth factor (TGF)-β1-inducible focal adhesion protein. We previously demonstrated that Hic-5 was localized in mesangial cells and its expression was associated with glomerular cell proliferation and matrix expansion in human and rat glomerulonephritis (GN). In the present study, we first assessed the role of Hic-5 in mesangioproliferative GN by injecting Habu venom into heminephrectomized wild type (Hic-5+/+) and Hic-5-deficient (Hic-5-/-) mice. Hic-5+/+ GN mice exhibited glomerular cell proliferation on day 7. Surprisingly, glomerular cell number and Ki-67-positive cells in Hic-5-/- GN mice were significantly greater than those in Hic-5+/+ GN mice on day 7, although the number of glomerular apoptotic cells and the expression of growth factors (platelet-derived growth factor-BB and TGF-β1) and their receptors were similarly increased in both Hic-5+/+ and Hic-5-/- GN mice. In culture experiments, proliferation assays showed that platelet-derived growth factor-BB and TGF-β1 enhanced the proliferation of Hic-5-/- mesangial cells compared with Hic-5+/+ mesangial cells. In addition, mitogenic regulation by Hic-5 was associated with altered and coordinated expression of cell cycle-related proteins including cyclin D1 and p21. The present results suggest that Hic-5 might regulate mesangial cell proliferation in proliferative GN in mice. In conclusion, modulation of Hic-5 expression might have a potential to prevent mesangial cell proliferation in the acute mitogenic phase of glomerulonephritis.     

Intraallograft chemokine RNA and protein during rejection of MHC-matched/multiple minor histocompatibility-disparate skin grafts

Chemokines direct leukocyte recruitment into sites of tissue inflammation and may facilitate recruitment of leukocytes into allografts following transplantation. Although the expression of chemokines during rejection of MHC-disparate allografts has been examined, chemokine expression in MHC-matched/multiple minor histocompatibility Ag-disparate allografts has not been tested. The intraallograft RNA expression of several C-X-C and C-C chemokines was tested during rejection of full thickness skin grafts from B10. D2 donors on control Ig-, anti-CD4 mAb-, and anti-CD8 mAb-treated BALB/c recipients. In all recipients, two patterns of intragraft chemokine expression were observed during rejection of these grafts: 1) macrophage-inflammatory protein-1alpha, macrophage-inflammatory protein-1beta, GRO-alpha (KC), JE, and IFN-gamma-inducible protein (IP-10) were expressed at equivalent levels in allo- and isografts for 2-4 days posttransplant and then returned to low or undetectable levels; and 2) IP-10 and monokine induced by IFN-gamma (Mig) were expressed in the allografts 3 days before rejection was completed, suggesting a possible role in recruiting primed T cells into the allograft. Three days before completion of rejection, intraallograft IP-10 protein was restricted to the epidermis, whereas Mig was located in the lower dermis and associated with the intense infiltration of mononuclear cells. Treatment of B10.D2 recipients with rabbit antiserum to Mig, but not to IP-10, delayed rejection of the allografts 3-4 days. The results suggest that Mig mediates optimal recruitment of T cells into MHC-matched/multiple minor histocompatibility Ag-disparate allografts during rejection.     

T cell chemokine receptor expression in aging

Changes in chemokine receptor expression are important in determining T cell migration and the subsequent immune response. To better understand the contribution of the chemokine system in immune senescence we determined the effect of aging on CD4(+) T cell chemokine receptor function using microarray, RNase protection assays, Western blot, and in vitro chemokine transmigration assays. Freshly isolated CD4(+) cells from aged (20-22 mo) mice were found to express a higher level of CCR1, 2, 4, 5, 6, and 8 and CXCR2-5, and a lower level of CCR7 and 9 than those from young (3-4 mo) animals. Caloric restriction partially or completely restored the aging effects on CCR1, 7, and 8 and CXCR2, 4, and 5. The aging-associated differences in chemokine receptor expression cannot be adequately explained by the age-associated shift in the naive/memory or Th1/Th2 profile. CD4(+) cells from aged animals have increased chemotactic response to stromal cell-derived factor-1 and macrophage-inflammatory protein-1alpha, suggesting that the observed chemokine receptor changes have important functional consequences. We propose that the aging-associated changes in T cell chemokine receptor expression may contribute to the different clinical outcome in T cell chemokine receptor-dependent diseases in the elderly.     

Single-cell analyses reveal two defects in peptide-specific activation of naive T cells from aged mice

Confocal fluorescent microscopy was used to study redistribution of membrane-associated proteins in naive T cells from young and old mice from a transgenic stock whose T cells express a TCR specific for a peptide derived from pigeon cytochrome C. About 50% of the T cells from young mice that formed conjugates with peptide-pulsed APC were found to form complexes, at the site of binding to the APC, containing CD3epsilon, linker for activation of T cells (LAT), and Zap-70 in a central area and c-Cbl, p95(vav), Grb-2, PLC gamma, Fyn, and Lck distributed more uniformly across the interface area. Two-color staining showed that those cells that were able to relocalize c-Cbl, LAT, CD3epsilon, or PLC gamma typically relocalized all four of these components of the activation complex. About 75% of conjugates that rearranged LAT, c-Cbl, or PLC gamma also exhibited cytoplasmic NF-AT migration to the T cell nucleus. Aging had two effects. First, it led to a diminution of approximately 2-fold in the proportion of T cell/APC conjugates that could relocalize any of the nine tested proteins to the immune synapse. Second, aging diminished by approximately 2-fold the frequency of cytoplasmic NF-AT migration among cells that could generate immune synapses containing LAT, c-Cbl, or PLC gamma. Thus naive CD4 T cells from old mice exhibit at least two separable defects in the earliest stages of activation induced by peptide/MHC complexes.     

Localization in situ of type VI collagen protein and its mRNA in mesangial proliferative glomerulonephritis using renal biopsy sections

 Extracellular matrix accumulation is crucial in the pathogenesis of glomerulosclerosis in mesangial proliferative glomerulonephritis (GN). In an attempt to explore the distribution of type VI collagen and its synthesizing cells in normal and diseased glomeruli, we investigated mRNA and protein expression of type VI collagen in renal biopsy sections, histologically diagnosed as mesangial proliferative GN. Five renal biopsies from patients diagnosed as having minor glomerular abnormalities and one surgical renal tissue were also simultaneously examined as controls. Immunohistochemical studies revealed type VI collagen immunostaining in the mesangium and glomerular basement membrane of the control glomeruli. Compared to the control, increased deposition of type VI collagen was noted in the mesangial proliferative and sclerotic lesions in GN. To identify the cells responsible for the synthesis of type VI collagen mRNA, renal sections were hybridized in situ with digoxigenin-labeled antisense oligo-DNA probe complementary to a part of α1 (VI) mRNA. Occasionally intraglomerular cells hybridized with digoxigenin-labeled antisense pro α1 (VI) oligo-DNA in control glomeruli. An increased number of intraglomerular cells (mostly epithelial cells) were, however, positive for α1 (VI) mRNA expression in GN sections. The present study documents the distribution of type VI collagen in the normal glomeruli and provides further evidence of accelerated synthesis of this collagen in mesangial proliferative GN. Accepted: 21 July 1998