Local macrophage proliferation in the pathogenesis of glomerular crescent formation in rat anti-glomerular basement membrane (GBM) glomerulonephritis

Glomerular crescent formation is a feature of aggressive forms of glomerulonephritis. The conventional view of crescent formation within Bowman's space involves proliferation of parietal epithelial cells and the recruitment of blood monocytes. However, the potential role of local macrophage proliferation in this process has not been investigated. The current study examines macrophage proliferation within Bowman's space on the basis of expression of the proliferating cell nuclear antigen (PCNA) in a rat model of crescentic glomerulonephritis (accelerated anti-GBM disease). ED1⁺ macrophages accounted for 42% of cells within early cellular crescents, and 38% of these crescent macrophages were proliferating on the basis of PCNA expression. Macrophages became the dominant cell population in advanced cellular and fibrocellular crescents (64–71%), and there was a significant increase in the level of macrophage proliferation, with 62% and 67% of ED1⁺ macrophages expressing the PCNA, respectively. This high level of macrophage proliferation was confirmed by incorporation of bromo-deoxyuridine and the presence of mitotic figures within crescents. Indeed, macrophages accounted for 73% of all proliferating cells within advanced and fibrocellular crescents. Macrophage proliferation within Bowman's space was a local event, as shown by a lack of proliferating monocytes in the circulation, the presence of mitotic figures within crescents and a reciprocal relationship between the numbers of ED1⁺PCNA⁺ cells within Bowman's space compared with that in the capillary tuft during the progression from early to advanced and fibrocellular crescents. In conclusion, this study has changed the conventional view of the pathogenesis of crescent formation in glomerulonephritis with the demonstration of substantial local macrophage proliferation within Bowman's space. It is proposed that local proliferation is a major mechanism of macrophage accumulation within crescents and plays an important role in the progression of epithelial-dominated early cellular crescents to macrophage-dominated advanced and fibrocellular cellular crescents.     

Fc receptor-mediated accumulation of macrophages in crescentic glomerulonephritis induced by anti-glomerular basement membrane antibody administration in WKY rats

Anti-glomerular basement membrane (GBM) glomerulonephritis induced in WKY rats is characterized by glomerular accumulation of CD8(+) T cells and monocytes/macrophages, followed by crescent formation. The mechanism of leukocyte accumulation after antibody binding to GBM is still unclear. To unveil an involvement of Fcgamma receptors (FcgammaR) in leukocytes recruitment we examined the expression of FcgammaR in glomeruli and the effects of the administration of F(ab')(2) fragment of anti-GBM antibody or FcgammaR blocking on the initiation and progression of this model. A gradual increase of FcgammaR mRNA expression in glomeruli during the time course of disease suggested their significance in the development of glomerulonephritis. Glomerular lesions and proteinuria were induced only in rats injected with intact IgG of anti-GBM antibody, but not with the F(ab')(2) fragment. In vivo blocking of FcgammaR by administering heat-aggregated IgG led to the decrease of mRNA expression for all types of FcgammaR (types 1, 2 and 3) and a significant amelioration of glomerulonephritis manifestations. By flow cytometry and immunohistochemistry FcgammaR2-expressing cells in glomeruli were identified as macrophages, but not CD8(+) T cells. The expression of FcgammaR1 and 3 was significantly decreased, and that of FcgammaR2 became undetectable in CD8(+) T cell-depleted rats. Thus, CD8(+) T cells may stimulate FcgammaR expression on macrophages, contributing to their glomerular accumulation and injury. These studies provide direct evidence for a crucial involvement of IgG Fc-FcgammaR interaction in glomerular recruitment of macrophages and following induction of anti-GBM glomerulonephritis in WKY rats.     

Effects of CTLA4-Fc on glomerular injury in humorally-mediated glomerulonephritis in BALB/c mice

The effect of cytotoxic T-lymphocyte-associated molecule 4-immunoglobulin fusion protein (CTLA4-Fc) on humorally-mediated glomerulonephritis was studied in accelerated anti-glomerular basement membrane (anti-GBM) glomerulonephritis induced in BALB/c mice. This strain of mice develops antibody and complement dependent glomerulonephritis under this protocol. Sensitized BALB/c mice developed high levels of circulating autologous antibody titres, intense glomerular deposition of mouse immunoglobulin and complement, significant proteinuria, renal impairment, significant glomerular necrosis and a minor component of crescent formation 10 days after challenge with a nephritogenic antigen (sheep anti-GBM globulin). Early treatment during the primary immune response, or continuous treatment throughout the disease with CTLA4-Fc, significantly suppressed mouse anti-sheep globulin antibody titres in serum, and immunoglobulin and complement deposition in glomeruli. The degree of glomerular necrosis was improved and proteinuria was reduced, particularly in the earlier stages of disease. Late treatment by CTLA4-Fc starting one day after challenge with sheep anti-mouse GBM did not affect antibody production and did not attenuate glomerulonephritis. The low level of crescent formation found in BALB/c mice developing glomerulonephritis was not prevented by the administration of CTLA4-Fc. These results demonstrate that CTLA4-Fc is of benefit in this model of glomerulonephritis by its capacity to attenuate antibody production, without affecting the minor degree of cell-mediated glomerular injury.     

MHC class I pathway is not required for the development of crescentic glomerulonephritis in mice

MHC II and CD4+ T cells are required for anti-glomerular basement membrane (GBM) globulin-initiated crescentic glomerulonephritis (GN) in mice, but the role of MHC I and CD8+ T cells is unclear. The cytolytic function of CD8+ T cells requires recognition of peptide antigens presented on MHC I. CD8+ T cells can also perform helper functions via cytokine production. The contribution of MHC I to crescentic GN was investigated using TAP-1 gene knock out (TAP-1-/-) mice, which have deficient MHC I antigen presentation. Heterozygous TAP-1 mice have normal MHC I expression and developed GN with crescents in 42 +/- 4% of glomeruli (normal 0%), proteinuria (9.1 +/- 1.6 mg/20 h, normal 1.5 +/- 0.3 mg/20 h) and impaired renal function (creatinine clearance 110 +/- 8 microl/min, normal 193 +/- 10 microl/min) following administration of sheep anti-mouse GBM globulin. TAP-1-/- mice, which have extremely low MHC I expression and reduced CD8+ T cells, developed similar GN with 39 +/- 3% crescents, proteinuria (12.7 +/- 4.3 mg/20 h) and impaired renal function (creatinine clearance 123 +/- 20 microl/min). In vivo antibody-induced CD8 depletion did not attenuate crescent formation or protect renal function in C57Bl/6 mice developing GN, although significant reduction in proteinuria (5.3 +/- 1.2 mg/20 h, P = 0. 012) and glomerular recruitment of CD4+ T cells and macrophages were observed compared with control treated mice with GN. These data demonstrate that MHC I is not required for development of crescentic GN in mice. The MHC I-independent contribution of CD8+ T cells to proteinuria and inflammatory cell recruitment suggests that they may serve a 'helper' rather than cytolytic role in this disease.     

Novel molecular mechanisms of dendritic cell-induced T cell activation

In this study we have re-examined the molecular mechanisms involved in activation of T cells by dendritic cells (DC). Human peripheral blood DC (PBDC) were derived by 2 h adhesion followed by 7 day culture in a combination of granulocyte macrophage colony stimulating factor and IL-4, and depletion of residual T and B cells. These PBDC were used to induce autologous T cell proliferation in a CD3-dependent response, and antibodies against CD11a/18 and CD86 were used as control inhibitors of accessory function. Antibodies against five of the cell surface molecules that we have recently identified on the surface of DC, CD13, CD87, CD98, CD147 and CD148, and an antibody which recognizes a molecule that has not as yet been identified, all inhibited the CD3-induced T cell proliferation. These findings were observed not only when antibodies were present throughout the culture, but also when they were prepulsed on to the surface of the DC, suggesting the inhibition was mediated via the antigen-presenting cells rather than the T cell. The same set of antibodies also inhibited an allospecific mixed lymphocyte reaction, confirming that the inhibitory effect was not dependent on the use of a CD3 antibody as the stimulating agent. All the antibodies of known specificity inhibited both CD4 and CD8 T cells equally. Unlike CD87, CD98 and CD147 antibodies, which inhibited activation of both CD45RA (naive) T cells and CD45RO (memory) T cells, CD13 and CD148 appeared to be involved in activation of naive cells only. The molecules identified in this study have not previously been demonstrated to play a role as accessory molecules on DC, the cells that are pivotal for immune induction. Therefore they may provide new potential targets for modulation of the immune response at the APC level.     

Role of mast cells in experimental anti-glomerular basement membrane glomerulonephritis

Recently, divergent reports on the role of mast cells (MC) in different glomerular diseases have brought our attention to their role in an accelerated model of anti-glomerular basement membrane (GBM) glomerulonephritis (GN). Genetically MC-deficient Kit(W)/Kit(W-v) mice, MC-reconstituted Kit(W)/Kit(W-v) mice and Kit+/+ control mice were subjected to anti-GBM GN. Kit(+/+) mice developed moderate proteinuria and glomerular damage following the induction of anti-GBM nephritis. In contrast, proteinuria and glomerular damage were dramatically increased in MC-deficient Kit(W)/Kit(W-v) mice. MC-reconstituted Kit(W)/Kit(W-v) mice showed proteinuria and glomerular damage comparable to Kit+/+ mice. A significant increase in infiltrating T cells and macrophages was detected in MC-deficient Kit(W)/Kit(W-v) mice as compared to Kit+/+ control mice and MC-reconstituted Kit(W)/Kit(W-v) mice. Accordingly, we observed an increase of TGF-beta1 mRNA in kidneys from Kit(W)/Kit(W-v) mice. Interestingly, we did not detect MC in the kidney using either Giemsa staining or RT-real-time PCR, but MC were found in the regional lymph nodes. Finally, mortality of Kit(W)/Kit(W-v) mice was significantly increased after the induction of anti-GBM GN due to uremia. Our report provides the first direct evidence that MC are protective in anti-GBM GN, possibly by modulating the influx of effector T cells and macrophages to inflammatory sites in the kidney.     

Anti-neutrophil cytoplasmic (ANCA) and anti-glomerular basement membrane (GBM) autoantibodies in necrotizing and crescentic glomerulonephritis

Anti-neutrophil cytoplasmic autoantibodies (ANCA) and anti-glomerular basement membrane (GBM) necrotizing and crescentic glomerulonephritis are aggressive and destructive glomerular diseases that are associated with and probably caused by circulating ANCA and anti-GBM antibodies. These necrotizing lesions are manifested by acute nephritis and deteriorating kidney function often accompanied by distinctive clinical features of systemic disease. Prompt diagnosis requires clinical acumen that allows for the prompt institution of therapy aimed at removing circulating autoantibodies and quelling the inflammatory process. Continuing exploration of the etiology and pathogenesis of these aggressive inflammatory diseases have gradually uncovered new paradigms for the cause of and more specific therapy for these particular glomerular disorders and for autoimmune glomerular diseases in general.     

T细胞疫苗免疫前后外周血CD4~ 和CD8~ T细胞变化的分析

目的 观察T细胞疫苗免疫前后外周血CD4~+和CD8~+T细胞的变化情况,探讨T细胞疫苗诱导特异性免疫耐受的作用及其机理。方法 制备针对Wistar大鼠的SD大鼠T细胞疫苗,用制备好的T细胞疫苗去免疫正常的SD大鼠,同时设特异性抗原对照组和空白对照组。于免疫前和免疫后规定时点分别进行单向混合淋巴细胞反应(MTT法),于相同时点通过流式细胞分析对外周血CD4~+和CD8~+T细胞进行检测。结果 在T细胞疫苗组,平均OD值免疫后比免疫前显著降低(P<0.01)CD4/CD8比值免疫后比免疫前显著降低(P<0.05);在特异性抗原对照组,免疫后的OD值显著高于免疫前(P<0.01),同时CD4/CD8比值于免疫后显著增高(P<0.05);空白对照组各时点各指标比较无显著差异(P>0.05)。结论T细胞疫苗可以诱导同种抗原特异性免疫耐受,CD4~+反应性T细胞克隆与CD8~+抗独特型T细胞克隆相对比例优势的转换可能在T细胞疫苗诱导的免疫耐受形成中发挥关键作用。     

Implication of the complement system in the induction of anti-glomerular basement membrane glomerulonephritis in WKY rats

In WKY rats, administration of a small dose of anti-glomerular basement membrane antibody induces severe crescentic glomerulonephritis, which is characterized by infiltration of CD8-positive lymphocytes and monocytes/macrophages into the glomeruli with crescent formation. In this study, the involvement of the complement system was examined in the induction of this model. To deplete complement, cobra venom factor (CVF) was used. By a single injection with CVF at 24 h prior to the induction of this model, plasma C3 level from days 0-2 was less than 10% compared with pre-injection of CVF. Complement was almost depleted in the early phase of this model. No significant differences were observed in proteinuria and the frequency of glomeruli associated with the extracapillary crescentic lesions between the CVF-treated group and the control group. In addition, the number of monocytes/macrophages and CD8-positive lymphocyte infiltration into the glomeruli showed no significant differences between both groups. These results indicate that the possibility of complement system involvement is considered low in the induction of this model.     

Protection against anti-glomerular basement membrane (GBM)-mediated nephritis in C3- and C4-deficient mice

Mice rendered completely deficient of the complement components C3 or C4 were used to determine the influence of complement activation in the heterologous phase of the anti-GBM disease model. In wild-type animals the disease is characterized by a neutrophil infiltrate, capillary thrombosis, proteinuria and C3 and C4 deposited within the glomerulus. The early infiltration of neutrophils into the glomeruli is greater in wild-type mice (2.8 ± 0.3) compared with C3-deficient (1.4 ± 0.2) and C4-deficient (1.2 ± 0.003) mice. Deficiency also protects against the subsequent development of proteinuria (2.99 ± 1.11 mg/24 h, 0.059 mg/24 h and 0.327 ± 0.14 mg/24 h in wild-type, C3-deficient and C4-deficient mice, respectively) and decreases glomerular capillary thrombosis in both C3- and C4-deficient mice. The degree of protection is greater in the C3-deficient than the C4-deficient animals, suggesting both classical and alternative pathway involvement. These studies support a critical role for complement in the development of anti-GBM disease. However, the protective effect of complement deficiency can be broken if the dose of nephritogenic antibody is increased.     

Immunohistochemical characterization of glomerular inflammatory cells and expression of adhesion molecules in anti-glomerular basement membrane (anti-GBM) glomerulonephritis induced in WKY rats with monoclonal anti-GBM antibodies of different subclasses

According to previous report, adhesion of CD8-positive cells and macrophages to glomerular endotherial cells through the lymphocyte function-associated antigen-1 (LFA-1)/intercellular adhesion molecule-1 (ICAM-1) pathway is crucial for the initiation and subsequent progression of anti-glomerular basement membrane (anti-GBM) antibody-induced glomerulonephritis (anti-GBM nephritis) in WKY rats. In the present study glomerular inflammatory cell infiltration and LFA-1/ICAM-1 expression were examined in anti-GBM nephritis induced in WKY rats with monoclonal anti-GBM antibodies of different subclasses: IgG1, IgG2a, and IgG2b. The IgG2a and IgG2b subclasses induced significant proteinuria from day 3 as compared with the IgG1 subclass. Glomerular infiltration of macrophages and CD8-positive cells after administration of IgG2a and IgG2b subclass antibodies was significantly elevated compared to that for the IgG1 subclass. The intensity of glomerular ICAM-1 immunostaining by the IgG2a and IgG2b subclass antibodies tended to be stronger than that by the IgG1 subclass. Glomerular LFA-1-positive cell infiltration by the IgG2a and IgG2b subclasses was significantly higher than that of the IgG1 subclass. These results demonstrate that monoclonal antibodies belonging to the IgG2a and IgG2b subclasses strongly induce glomerular infiltration of inflammatory cells and expression of adhesion molecules in rat anti-GBM nephritis.     

The role of T cells in the mediation of glomerular injury in Heymann's nephritis in the rat.

Heymann's nephritis (HN), a rat model of the membranous glomerulonephritis in man, is thought to be mediated by auto-Ig with subsequent activation of C. Whether T cell mechanisms are involved in the mediation of HN, apart from CD4+ cells providing help for auto-Ig production, was examined by treatment with mAb specific for T cell subsets for 6 weeks after immunization to induce HN. Anti-CD4 mAb therapy totally prevented proteinuria, in that at 6, 8, and 12 week treated rats had less than 15 mg/day of protein compared to controls that all had greater than 260 mg/day. Ig and C deposition in the glomerulus was significantly less and auto-Ig titers in serum were partially suppressed by anti-CD4 therapy. Anti-CD8 mAb therapy markedly reduced proteinuria at all time points, for example at 6 weeks there was 51 +/- 40 mg/day compared to 183 +/- 120 mg/day (P = 0.0003), but had no effect on auto-Ig titers or on Ig and C deposition in the glomerulus. A non-specific effect of high dose mouse mAb therapy was excluded by the findings that a mAb that did not bind to rat cells had no effect on the induction of HN and that serum C was not depleted in any of the mAb treated animals. A role for T effector mechanisms was further supported by the finding that therapy with mAb to T cell receptor alpha/beta chain or with cyclosporine also markedly delayed the onset of proteinuria. Examination of renal biopsies showed a T cell infiltrate in glomeruli and the interstitium of the untreated HN controls that was not present in MRC Ox35 or MRC Ox8 treated groups. This infiltrate included CD4+ and CD8+ T cells and macrophages. These results suggest induction of proteinuria in HN was totally dependent upon CD4+ T cells, and that CD4+ and CD8+ cells may have a direct role in the mediation of glomerular dysfunction in HN.     

Anti-glomerular basement membrane (GBM) glomerulonephritis in the mouse: development of disease and cell proliferation.

In a mouse model of anti-glomerular basement membrane (GBM) glomerulonephritis, associated with the nephrotic syndrome, a wide range of morphological and proliferative responses was seen in the renal corpuscle, at 6 days. The severity of the damage could be assessed by measuring the average daily weight gain between days 0 and 3 (DWG) of the animal. Those animals with a high DWG showed capsular proliferation, whereas animals with a low DWG showed predominantly tuft cell proliferation. Capsular cell birth rate increased with DWG whilst tuft cell birth rate was negatively related. A computer simulation suggests that the results are compatible with the induction of successive but overlapping waves of tuft and capsular cell proliferation.     

Lower numbers of circulating natural killer T (NK T) cells in individuals with human T lymphotropic virus type 1 (HTLV‐1) associated neurological disease

Human T lymphotropic virus type 1 (HTLV‐1) infects 10–20 million people worldwide. The majority of infected individuals are asymptomatic; however, approximately 3% develop the debilitating neurological disease HTLV‐1‐associated myelopathy/tropical spastic paraparesis (HAM/TSP). There is also currently no cure, vaccine or effective therapy for HTLV‐1 infection, and the mechanisms for progression to HAM/TSP remain unclear. NK T cells are an immunoregulatory T cell subset whose frequencies and effector functions are associated critically with immunity against infectious diseases. We hypothesized that NK T cells are associated with HAM/TSP progression. We measured NK T cell frequencies and absolute numbers in individuals with HAM/TSP infection from two cohorts on two continents: São Paulo, Brazil and San Francisco, CA, USA, and found significantly lower levels when compared with healthy subjects and/or asymptomatic carriers. Also, the circulating NK T cell compartment in HAM/TSP subjects is comprised of significantly more CD4⁺ and fewer CD8⁺ cells than healthy controls. These findings suggest that lower numbers of circulating NK T cells and enrichment of the CD4⁺ NK T subset are associated with HTLV‐1 disease progression.     

Presence of glomerular basement membrane (GBM) antibodies in HIV− patients with Pneumocystis carinii pneumonia

Following the unexpected finding of antibodies to GBM in a patient with Pneumocystis carinii pneumonia in the absence of kidney abnormalities, the presence of anti-GBM antibodies was analysed in 14 patients with pulmonary P. carinii infection who did not have clinical evidence of autoimmune glomerulonephritis. Patients were divided into three groups: HIV⁻ with P. carinii pneumonia (n = 4), HIV⁺ with P. carinii pneumonia (n = 5) and HIV⁻ carriers of P. carinii without pneumonia (n = 5). As control groups, HIV⁻ patients with community-acquired non-P. carinii pneumonia (n = 6) and healthy individuals (n = 16) were included. Anti-GBM antibodies, studied with a quantitative enzyme immunoassay (EIA) for anti-α3 chain of collagen IV antibodies, were detected in three out of the four HIV⁻ patients with P. carinii pneumonia, but not in any individuals of the other categories. These results suggest that P. carinii alveolar injury or the host response to the organism could affect the basal membrane Goodpasture antigen or a similar antigen, and induces anti-GBM antibody production in HIV⁻ patients, and support the hypothesis that, at least in some cases, Goodpasture's syndrome could be triggered by an alveolar lesion induced by a P. carinii pneumonia.     

Tubulointerstitial nephritis and glomerulonephritis in Brown-Norway rats immunized with heterologous glomerular basement membrane.

Tubulointerstitial nephritis and glomerulonephritis were produced in Brown-Norway rats (BN) by a single immunization with 2 mg of lyophilized bovine glomerular basement membrane. Tubulointerstitial nephritis was evident before glomerulonephritis. Antibody first bound to tubular basement membranes (TBM), and then the renal cortex was infiltrated with inflammatory cells. The TBM was split, and many renal tubules, especially proximal tubules, were destroyed. Approximately 14 days after the beginning of the tubular phase, antibody was observed to be bound to glomerular basement membranes (GBM) in linear fashion. There was epithelial and mesangial cell proliferation, splitting and reduplication of GBM, crescent formation, and glomerular scarring and atrophy.     

Relative contributions of chemo-attractant and terminal components of complement to anti-glomerular basement membrane (GBM) glomerulonephritis.

The relative contributions of chemo-attractant and terminal components of complement to heterologous phase glomerular injury was studied in anti-GBM glomerulonephritis in rabbits. Normal rabbits (complement intact) were given anti-GBM antibody at a dose which resulted in 140 micrograms specific kidney-fixed antibody per gram of renal cortex, and developed significant proteinuria (1910 +/- 327 mg/24 h; control 18.2 +/- 6.1 mg/24 h; P less than 0.01). Leucocyte depletion significantly reduced but did not abolish proteinuria (574 +/- 186 mg/24 h, P less than 0.05). Complement depletion of neutrophil-depleted rabbits resulted in a further significant reduction in proteinuria 50.1 +/- 12.2 mg/24 h, P less than 0.05; versus neutrophil-depleted, complement-intact rabbits), indicating that both neutrophil accumulation and complement activation independent of neutrophils contribute to injury in this model. Rabbits congenitally deficient in the sixth component of complement (C6D) developed similar levels of proteinuria (2099 +/- 796 mg/24 h) to normal rabbits given an identical dose of antibody. However, after leucocyte depletion, C6D rabbits developed significantly less proteinuria (135 +/- 56 mg/24 h) than did leucocyte-depleted, complement-intact rabbits (P less than 0.05). These studies show that terminal complement components are not necessary for the full expression of acute anti-GBM antibody-initiated injury in leucocyte-intact rabbits. However, in the absence of leucocytes, C6 and the terminal complement components are apparently responsible for the majority of the complement-dependent glomerular injury.     

Role of L-selectin in the development of autoimmune diabetes in non-obese diabetic mice

Autoimmune diabetes is characterized by an early mononuclear infiltration of pancreatic islets and later selective autoimmune destruction of insulin-producing beta cells. Lymphocyte homing receptors have been considered candidate targets to prevent autoimmune diabetes. L-selectin (CD62L) is an adhesion molecule highly expressed in naive T and B cells. It has been reported that blocking L-selectin in vivo with a specific antibody (Mel-14) partially impairs insulitis and diabetes in autoimmune diabetes-prone non-obese diabetic (NOD) mice. In the present study we aimed to elucidate whether genetic blockade of leukocyte homing into peripheral lymph nodes would prevent the development of diabetes. We backcrossed L-selectin-deficient mice onto the NOD genetic background. Surprisingly NOD/L-selectin-deficient mice exhibited unaltered islet mononuclear infiltration, timing of diabetes onset and cumulative incidence of spontaneous diabetes when compared to L-selectin-sufficient animals. CD4, CD8 T cells and B cells were present in islet infiltrates from 9-week-old L-selectin-sufficient and -deficient littermates. Moreover, total splenocytes from wild-type, heterozygous or NOD/L-selectin-deficient donor mice showed similar capability to adoptively transfer diabetes into NOD/SCID recipients. On the other hand, homing of activated, cloned insulin-specific autoaggressive CD8 T cells (TGNFC8 clone) is not affected in NOD/L-selectin-deficient recipients. We conclude that L-selectin plays a small role in the homing of autoreactive lymphocytes to regional (pancreatic) lymph nodes in NOD mice.