Interleukin-15 and interleukin-12 have an additive effect on the release of vascular permeability factor by peripheral blood mononuclear cells in normals and in patients with nephrotic syndrome.

BACKGROUND: The vascular permeability factor (VPF) is a lymphokine that has been shown to play a role in minimal-change nephrotic syndrome (MCNS). A better understanding of the mechanisms that upregulate VPF release is of basic importance to control the immune system in nephrotic syndrome (NS). Interleukin (IL)-15 is a key inducer of differentiation of uncommitted T helper cells, which regulates cellular immunity. The cytokine IL-15 appears to mimic the stimulatory activity of IL-2 on T cells. PATIENTS AND METHODS: In the present report, we studied the ability of IL-15, alone or in combination with IL-12, to influence the release of VPF by peripheral blood mononuclear cells (PBMC) from nephrotic patients. We have analyzed the release of VPF by concanavalin-A- (Con A) stimulated PBMC in normals, 16 patients with MCNS and 16 patients with IgA nephropathy (IgAN). RESULTS: In both patient groups 50% had a proteinuria below 0.8 g/day. We demonstrate that nephrotic, but not non-nephrotic patients (both MCNS and IgAN), exhibit a high VPF release, which can be stimulated further by IL-15 + IL-12. To determine the specificity of the stimulatory effect, neutralizing anti-IL-15 and anti-IL-12 antibodies were preincubated with IL- 15 and IL-12 prior to the addition of responder cells, respectively. The antibodies completely inhibited the effects of IL-15 and IL-12. CONCLUSION: These results indicate that IL-15 plus IL-12 acted additively to augment VPF release. These biological interactions between IL-15 and IL-12 may be important in the pathophysiology of VPF in vitro.     

Interleukin-4 cooperates with interleukin-10 to inhibit vascular permeability factor release by peripheral blood mononuclear cells from patients with minimal-change nephrotic syndrome

Increased production of a vascular permeability factor (VPF) from peripheral blood mononuclear cells (PBMC) in patients with minimal-change nephrotic syndrome (MCNS) has been reported. Interleukin-4 (IL-4) and interleukin-10 (IL-10), both produced by T-helper type-2 cells, are cytokines with the capacity to downregulate proinflammatory responses. To gain insight into the immunoregulatory properties of these cytokines, we analyzed the effects of recombinant human IL-4 and IL-10 on VPF release in MCNS patients. In the present study we show that the regulatory cytokines IL-4 and IL-10 are potent inhibitors of the VPF activity of concanavalin A-activated MCNS PBMC. Each cytokine was found to suppress VPF release in a dose-dependent manner. Moreover, when used at suboptimal concentrations, a combination of the two cytokines resulted in enhanced suppression of VPF release. Neutralization of endogenously produced IL-4 and IL-10 by both anti-IL-4 and anti-IL-10 antibodies resulted in an increased release of VPF. These data demonstrate that IL-4 acts in concert with IL-10 to inhibit VPF release and suggest that they are effective biologic regulators of the VPF responses in vitro.     

Transforming growth factor-beta1 inhibits vascular permeability factor release by T cells in normal subjects and in patients with minimal-change nephrotic syndrome

BACKGROUND/AIM: A lymphokine, the vascular permeability factor (VPF), which increases vascular permeability, has been characterized in minimal-change nephrotic syndrome (MCNS). Transforming growth factor-beta (TGF-beta) is an immunosuppressive cytokine that inhibits proliferation, cytokine production, and cytotoxic activity of T cells and natural killer cells. We, therefore, investigated the effects of TGF-beta1 on the release of VPF by peripheral blood T cells from MCNS patients. The aim of our study was to determine the in vitro immunosuppressive capacity of TGF-beta1 in patients with MCNS. METHODS: To further test the effect of TGF-beta1 on concanavalin A (Con A)-induced VPF release, normal and MCNS T cells were stimulated with 5 microg/ml of Con A in the presence or absence of TGF-beta1, and the culture supernatants were tested for the presence of VPF by the method of Lagrue et al. The disease controls included 16 patients with IgA nephropathy. RESULTS: Significantly increased spontaneous and Con A-stimulated secretion of VPF was detected in T-cell cultures of MCNS patients with the nephrotic syndrome as compared with those of normal controls. Addition of TGF-beta1 to these cultures inhibited the release of VPF in a dose-dependent manner. The effect of TGF-beta1 on the release of VPF was specific, since a reversion was obtained with a neutralizing monoclonal antibody to human TGF-beta1. CONCLUSION: Together, our data demonstrate that TGF-beta1 antagonizes the ability of T cells to release VPF, and suggest a role of TGF-beta1 in the pathophysiology of VPF in vitro.     

Augmented interleukin-18 production by peripheral blood monocytes in patients with minimal-change nephrotic syndrome

BACKGROUND/AIM: The etiology of minimal-change nephrotic syndrome (MCNS) is poorly understood. It has been proposed that cell-mediated immunity and T-cell activation are key features of this glomerular disease. Interleukin (IL)-18, a novel interferon-gamma-stimulating factor, may act as an important effector molecule involved in various immune responses. To our knowledge, very little is known about the involvement of IL-18 in NCNS. The aim here was to define further the involvement of IL-18 in MCNS. METHODS: To understand the role of this cytokine, in vitro IL-18 levels were analyzed by a sensitive enzyme-linked immunosorbent assay (ELISA) method in 16 patients with MCNS who were either in a stable or active condition. The disease controls included 16 patients with IgA nephropathy (IgAN). The IL-18 levels were compared with values in healthy controls. RESULTS: Significantly increased spontaneous and lipopolysaccharide (LPS)-stimulated production of IL-18 was detected in peripheral blood monocyte (PBM) cultures of MCNS patients with the nephrotic syndrome (NS) as compared with those of normal controls. Moreover, when individual MCNS patients were followed through their clinical illness, IL-18 levels were increased during the active phase and normalized as the patients went into remission. The amounts of IL-18 are significantly correlated with the levels of vascular permeability factor (VPF) in MCNS patients. CONCLUSIONS: Thus, in MCNS patients, the level of IL-18 was increased and this increase was related to the activity of this disease. The data provide circumstantial evidence for a role of IL-18 in MCNS.     

Studies of glomerular permeability factor (GPF) in focal segmental glomerular sclerosis and the relationship between GPF and vascular permeability factor (VPF)

BACKGROUND: We previously demonstrated that the supernatants of cultured concanavalin-A (con-A) stimulated peripheral blood mononuclear cells (PBMC) from patients with minimal change nephrotic syndrome (MCNS) increased the urinary protein excretion in injected rats and suggested that PBMC released a factor, which we called glomerular permeability factor (GPF), changes in the glomerular permeability and thus resulted in proteinuria in MCNS. MATERIAL AND METHODS: In this study we investigated the GPF activity in focal segmental glomerular sclerosis (FGS) and other conditions of chronic glomerulonephritis (CGN), and also the relationship between GPF and vascular permeability factor (VPF). In experiment 1 the supernatants of the cultured con-A stimulated PBMC from patients with 10 FGS, 5 other CGN and 10 controls were tested regarding their ability to produce GPE The GPF activity was defined as positive when the 8-hour urinary protein excretion after the injection of the supernatant in Sprague-Dawley rats exceeded the mean value plus 2 standard deviations (M + 2 SD) of that before injection. RESULTS: Three out of 10 FGS patients and 1 membranous nephropathy patient out of the 5 other CGN patients were positive for GPF activity. In experiment 2 the relationship between GPF and VPF was analyzed using culture supernatants of PBMC from 10 nephrotic MCNS patients and 15 controls. The VPF activity was measured following the method developed by Ovary [1975]. All 7 cases that were positive for GPF activity were simultaneously positive for VPF activity. On the other hand, 16 cases that were positive for VPF activity were not always positive for GPF activity (7 cases were positive and 9 were negative for VPF activity). CONCLUSION: Experiments 1 and 2 thus suggested that GPF was not active in MCNS alone, but also in other CGN conditions and it was therefore not considered to be the same factor/substance(s) as VPF.     

Elevated vascular endothelial growth factor levels in the urine of patients with minimal-change nephrotic syndrome

BACKGROUND: Vascular endothelial growth factor (VEGF) is a selective endothelial mitogen and vascular permeability factor (VPF), that is mainly produced by activated monocytes/macrophages and T cells. To our knowledge, very little is known about the involvement of VEGF in minimal-change nephrotic syndrome (MCNS). The aim here was to define further the involvement of VEGF in MCNS. PATIENTS AND METHODS: Urine samples were obtained from 20 MCNS patients. The disease controls included 20 patients with IgA nephropathy (IgAN). The samples were assayed for VEGF protein by a sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared with normal controls, markedly increased urinary levels of VEGF were detected in both MCNS and IgAN patients with the nephrotic syndrome (NS). The urinary VEGF (uVEGF) levels correlated with the degree ofproteinuria in MCNS and IgAN patients. Moreover, when individual MCNS patients were followed through their clinical illness, uVEGF levels were increased during the active phase and decreased as the patients went into remission. Our main concern is to distinguish between two possibilities: Increases in uVEGF excretion might indeed relate to specific glomerular pathology and thus have a pathophysiological role. Alternatively, uVEGF may be derived from the circulation and as such may be nothing more than an assay for proteinuria. In fact, given the strict correlation between uVEGF excretion and the amount of proteinuria, the second possibility appears quite conceivable. CONCLUSION: Therefore, this may be a coincidental finding which has no bearing on the pathophysiology of MCNS.     

Studies of vascular permeability factor derived from T lymphocytes and inhibitory effect of plasma on its production in minimal change nephrotic syndrome

Peripheral T lymphocytes from patients with minimal change nephrotic syndrome (MCNS) and controls were treated for their ability to produce vascular permeability factors (VPF) without concanavalin A stimulation. In vitro cultures of T lymphocytes from active MCNS produced VPF in the supernatant, whereas T lymphocytes from inactive MCNS or normal subjects did not. Furthermore, the plasma from patients with active MCNS markedly inhibited VPF production when compared with plasma taken from inactive MCNS or fetal calf serum alone. However, the plasma from MCNS in neither the active nor the inactive stage had any direct blocking effect on VPF activity. These results seem to suggest that the plasma from patients with MCNS in the active stage inhibits VPF production, but does not neutralize T lymphocytes derived VPF activity.     

Increased urinary excretion of interleukin-17 in nephrotic patients

BACKGROUND/AIM: Interleukin (IL)-17 is a newly discovered cytokine that is secreted by activated memory CD4+ T cells and modulated the early stage of immune response. To elucidate the pathophysiology of minimal-change nephrotic syndrome (MCNS), we focused on IL-17, which is one of the key factors in regulating an inflammatory response, and thus determined the daily excretion of IL-17 in urine. METHODS: For this purpose, excretion levels of IL-17 were measured in the urine of patients with MCNS during relapse and remission using a highly sensitive sandwich enzyme-linked immunosorbent assay. The data obtained were compared with levels of daily urinary excretion of IL-17 in patients with IgA nephropathy (IgAN). A group of healthy subjects served as control. In both experimental groups urine levels of IL-17 excretion were plotted against their daily urinary protein excretion. RESULTS: We demonstrated increased levels of IL-17 excretion in the urine of patients with MCNS and IgAN as compared to the non-nephrotic and healthy controls. In MCNS the daily urinary IL-17 (uIL-17) excretion was increased and returned to baseline with remission of the nephrotic syndrome (NS). We also demonstrated a positive correlation between urinary protein excretion and daily uIL-17 excretion. CONCLUSION: Taken together, these data indicate that uIL-17 excretion is increased during the NS, suggesting the possibility that daily uIL-17 excretion may reflect the disease activity of NS.     

Elevated interleukin-18 levels in the urine of nephrotic patients

BACKGROUND/AIM: The etiology of minimal-change nephritic syndrome (MCNS) is obscure. It has been speculated that T cells play a role in the pathogenesis of MCNS. Interleukin (IL)-18, a novel immunoregulatory cytokine with potent inferon-gamma-inducing activities, may play an important role in T-helper type 1-mediated immune responses. To examine further the possible role of IL-18 in nephrotic syndrome (NS), in the present study we measured IL-18 levels in the urine in different clinical stages of MCNS. The aim of this study was to investigate the involvement of IL-18 in MCNS. METHODS: Urine samples were obtained from 20 MCNS patients. The disease controls included 20 patients with IgA nephropathy. The samples were assayed for IL-18 protein by a sandwich enzyme-linked immunosorbent assay. RESULTS: Compared with normal controls, significantly increased urinary levels of IL-18 were detected in MCNS patients with the NS. The urinary IL-18 (uIL-18) levels correlated with the degree of proteinuria in MCNS patients. Moreover, when individual MCNS patients were followed through their clinical illness, uIL-18 levels were increased during the active phase and decreased as the patients went into remission. CONCLUSIONS: These results indicate that uIL- 18 is detectable in a subgroup of patients with active NS and correlates to their disease activity in patients with MCNS. Our findings support the notion that IL-18 may play a role in the pathophysiology of NS.     

Interleukin-17 stimulates the release of pro-inflammatory cytokines by blood monocytes in patients with IgA nephropathy

OBJECTIVE: Interleukin-17 (IL-17) is a newly discovered cytokine implicated in the regulation of inflammation. The present study was designed to explore whether IL-17 is involved in the immunoregulatory response in IgA nephropathy (IgAN). MATERIAL AND METHODS: We examined the in vitro effect of recombinant human IL-17 (rhIL-17) on pro-inflammatory cytokine release by peripheral blood monocytes (PBM) from patients with IgAN. Measurement of IL-1beta and tumor necrosis factor-alpha (TNF-alpha) was performed by means of a sandwich enzyme-linked immunosorbent assay. RESULTS: IL-1beta and TNF-alpha release were upregulated by rhIL-17 in a dose- and time-dependent fashion. Treatment of PBM from patients with IgAN with lipopolysaccharide and rhIL-17 resulted in significant activation and release of IL-1beta and TNF-alpha protein. Levels of IL-1beta and TNF-alpha were significantly higher in IgAN patients with the nephrotic syndrome (NS) than in patients without NS or in healthy subjects. We also provide information indicating that there is excessive production of IL-17 in IgAN patients. When IL-17-activated PBM were incubated in the presence of IL-10, IL-10 exerted a significant suppressive effect on IL-1beta and TNF-alpha release in vitro. CONCLUSION: IL-17 can stimulate the release of pro-inflammatory cytokines from PBM in patients with IgAN.     

Increased production of macrophage migration inhibitory factor by T cells in patients with IgA nephropathy.

BACKGROUND/AIM: Several studies have demonstrated an upregulation of macrophage migration inhibitory factor (MIF) synthesis in experimental glomerulonephritis. To our best knowledge, no investigation of MIF production by T cells from patients with IgA nephropathy (IgAN) has been reported so far. MIF is one of the immunoregulatory cytokines involved in T-cell activation and delayed-type hypersensitivity. In this study, we examined MIF production of T cells from patients with IgAN to investigate the contribution of the cells to elevated serum MIF content and to its pathologic characteristics. METHODS: We measured MIF production by T cells from the peripheral blood of 20 healthy controls and 20 patients with IgAN before and approximately 10 days after the beginning of steroid therapy. The disease controls included 20 patients with minimal-change nephrotic syndrome (MCNS) before therapy. MIF concentrations in the supernatants of T-cell cultures were measured with a specific enzyme-linked immunosorbent assay (ELISA). We also investigated the relationship between MIF levels and disease activity. RESULTS: The mean level for MIF in patients with IgAN was significantly elevated compared with that of healthy controls and also higher than that of patients with MCNS. When T cells were stimulated by concanavalin A, MIF production by T cells of patients with IgAN was more enhanced than in control subjects or patients with MCNS. We also investigated the relationship between MIF levels and pathological features in IgAN patients. Our findings reveal that MIF levels correlated with the grade of glomerular crescent formation and immunofluorescent C3 deposits in the glomeruli. Moreover, MIF overproduction was significantly related to the acute exacerbation stage of IgAN patients. Elevated MIF levels during the acute phase or exacerbations were found to be decreased during spontaneous or steroid therapy-induced remission in all IgAN patients examined. CONCLUSIONS: We provide data indicating that a T-cell population isolated by negative selection from peripheral blood mononuclear cells shows increased in vitro production of MIF in IgAN, and a reduction in that production when patients are treated with corticosteroids. In this paper, we describe the hypothetical role of MIF in the pathophysiology of IgAN.     

Inhibition of vascular permeability factor production by ciclosporin in minimal change nephrotic syndrome.

In order to ascertain whether ciclosporin (Cs) has an inhibitory effect on the vascular permeability factor (VPF) production, various quantities of Cs were added to T lymphocyte cultures from 8 children with minimal change nephrotic syndrome (MCNS) and the VPF activity of culture supernatants was assayed. As a result of the addition of Cs, a dose-dependent inhibition of VPF production was seen. VPF production was inhibited by a level of between 100 and 250 ng/ml of Cs in vitro. The reduction of proteinuria by Cs in MCNS might be due to the inhibition of VPF production.     

Interleukin-18 and interleukin-12 synergize to stimulate the production of vascular permeability factor by T lymphocytes in normal subjects and in patients with minimal-change nephrotic syndrome

BACKGROUND/AIM: In a previous study, we reported that interleukin (IL)-12 could upregulate the production of vascular permeability factor (VPF) derived from activated human peripheral blood mononuclear cells. Since IL-18, a novel immunoregulatory cytokine with potent interferon-gamma inducing activities, has been shown to be a strong cofactor for T helper type 1 cell development, we tested the hypothesis that IL-18 in combination with IL-12 can act synergistically to modulate the production of VPF. METHODS: For this purpose, T cells were isolated from heparinized venous blood, stimulated with concanavalin A, and incubated in the presence of IL-18 or IL-12, and the production of VPF was determined by the method of Lagrue. RESULTS: There was a significant increase in VPF production from concanavalin A-stimulated T cells following incubation with IL-18 or IL-12. More importantly, the combination of the cytokines was found to give a potent synergistic stimulation of VPF by concanavalin A-activated T cells from normal subjects. To determine the specificity of the stimulatory effect, neutralizing anti-IL-18 and anti-IL-12 antibodies were preincubated with IL- 18 and IL-12, respectively, prior to the addition of responder cells. The antibodies completely inhibited the effects of IL-18 and IL-12. Thus, these data show that IL-18 can synergize with IL-12 to selectively increase the production of VPF from T cells. The present study further demonstrates that IL-18 and IL-12 are in fact acting in synergy in patients with minimal-change nephrotic syndrome. CONCLUSIONS: Taken together, our results indicate that both IL-18 and IL-12 contribute to the VPF production in vitro and suggest that they play key roles in the complexity of cytokine regulation in the pathophysiology of VPF.     

Interleukin-12 and interferon-γ production in childhood idiopathic nephrotic syndrome

Cellular immune disturbances, and T lymphocyte function in particular, have been previously implicated in idiopathic nephrotic syndrome (INS) of childhood. There are different patterns of cytokine expression in various forms of glomerulonephritis, which suggests that local production of these peptides plays an important role in the pathogenesis and progression of glomerulonephritis. To investigate T-cell and monocyte/macrophage cytokine production in INS, interleukin-12 (IL-12) and interferon-γ (IFN-γ) production by peripheral blood mononuclear cells (PBMC) of 11 children with steroid-sensitive nephrotic syndrome (SSNS), 9 with focal segmental glomerulosclerosis (FSGS), and 17 healthy controls was determined. Children with SSNS were studied in relapse, during corticosteroid treatment, and in stable remission, off corticosteroid treatment. IL-12 was not detected in serum, urine, and in supernatants of unstimulated PBMC. IL-12 production by concanavalin A (Con A)-stimulated PBMC of children with SSNS and FSGS was not different from controls. IFN-γ production by Con A-stimulated PBMC was decreased in children with relapsing SSNS, both in relapse and and during corticosteroid treatment. However, in stable remission it was similar to controls. Markedly decreased IFN-γ production (P<0.001) was observed by pokeweed mitogen-stimulated PBMC of relapsing SSNS patients and moderately decreased production by PBMC of FSGS patients. This study has established a decreased production of IFN-γ by PBMC of relapsing SSNS and FSGS patients, but does not allow differentiation between these two different conditions. IL-12 did not have a pathogenic role in either SSNS or FSGS. Received May 24, 1997; received in revised form March 19, 1998; accepted March 23, 1998     

Natural killer activity and antibody-dependent cell-mediated cytotoxicity in primary glomerular diseases

This study was performed to investigate the role of cell-mediated immunity in primary glomerular diseases (PGD), by estimating natural killer (NK) activity from peripheral blood mononuclear cells (PBMC) assayed against K562 cells, and antibody-dependent cell-mediated cytotoxicity (ADCC) activity from PBMC assayed against chicken red blood cells. The proportion of PBMC was examined by flow cytometry using the monoclonal antibodies OKT and Leu series. NK activity in patients with nephrotics of minimal change nephrotic syndrome (MCNS), membranous glomerulonephritis (MGN) and membranoproliferative glomerulonephritis (MPGN) was significantly lower compared to those of the normal controls. In addition, ADCC activity in patients with nephrotics of MPGN was significantly lower compared to the normal controls. The sera from patients with diminished NK activity inhibited NK activity of PBMC from healthy individuals. Furthermore, OKM1 positive lymphocytes were significantly higher in the MCNS, MGN and MPGN patients and Leu7 positive lymphocytes were significantly higher in the MPGN patients. These data suggest that several mechanisms contribute to the defects of NK and ADCC activities in patients with PGD, and the defects in NK and killer cells are common in patients with nephrotics of MCNS, MGN and MPGN, and may be important in the pathogenesis of PGD.     

Effect of mizoribine on IL-6 release by peripheral blood mononuclear cells

BACKGROUND/AIM: A novel immunosuppressant, mizoribine (MZB), has recently been reported to be effective in the treatment of IgA nephropathy (IgAN), although its mechanism of action remains unknown. This study was conducted to investigate whether the efficacy of MZB on IgAN is exerted by suppression of interleukin-6 (IL-6) release. METHODS: Peripheral blood mononuclear cells (PBMC) were collected from 4 children with IgAN (median age 13.0 years) and 4 control children (median age 5.2 years). PBMC were cultured with medium alone or medium with lipopolysaccharide (LPS), and then incubated with LPS and MZB. Culture supernatants were assayed for lL-6. RESULTS: IL-6 release was increased by LPS in all subjects. Although the median value was higher in IgAN patients (median increase in IL-6 release 1,298.1%) than in controls (median 489.2%), statistical significance was not reached (p > 0.05). 10 mg/ml of MZB suppressed the release of IL-6 in both IgAN patients (median decrease in IL-6 release 39.3%) and controls (median 43.2%), with statistical significance (p < 0.05 and p < 0.01, respectively). CONCLUSION: This study suggests that MZB could suppress IL-6 release in vitro and thus may exert its efficacy on IgAN.     

Tumor necrosis factor-α production from mononuclear cells in nephrotic syndrome

Tumor necrosis factor-alpha (TNF-alpha) levels in supernatant fluid from cultured peripheral blood mononuclear cells (PBMC) were measured by ELISA in 54 children with active non-inherited forms of primary nephrotic syndrome (PNS), 10 nephrotics in remission, and 10 healthy controls. Children with active PNS included 21 patients with steroid-sensitive (SS) minimal change nephrotic syndrome (MCNS), 5 patients with steroid-resistant (SR) MCNS, 11 with SR focal segmental glomerulosclerosis (FSGS), 6 patients with SS diffuse mesangial proliferation (DMP), 5 patients with SR DMP, and 6 patients with mesangiocapillary glomerulonephritis (MCGN). Patients with active PNS had elevated TNF-alpha production compared with controls. Remission was associated with normalization of TNF-alpha production. There was a positive correlation between TNF-alpha production and the degree of proteinuria ( r=0.34, P=0.013), mesangial hypercellularity ( r=0.42, P=0.028), and glomerulosclerosis ( r=0.46, P=0.001). By using ROC curve, TNF-alpha production greater or equal to a cut-off point of 50 pg/ml could be used to predict resistance to steroid therapy (predictability 93.2%). By discriminate analysis, TNF-alpha production could be used to discriminate between patients with SR MCNS, SR FSGS, and SR DMP (predictability 100%). In conclusion, TNF-alpha from cultured PBMC might be involved in the pathogenesis of proteinuria as well as the pathological changes that occur in non-inherited forms of PNS. TNF-alpha levels in PBMC culture could be used to predict the pathological type of PNS and the response of these patients to steroid therapy.