Naohiro Yano

Phenotypic Characterization of Cytokine Expression in Patients With IgA Nephropathy

To identify the cytokines that play a relevant role in the pathogenesis of IgA nephropathy, we analyzed and compared the gene expression of proinflammatory cytokines, immuno-regulatory cytokines, and growth factors in peripheral blood mononuclear cells (PBMC). Expression of IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IFN-γ, TGF-β, TNF-α, and PDGF was examined in 28 patients with IgA nephropathy (IgAN), 20 patients with non-IgA mesangial proliferative glomerulo-nephritis (mesPGN), and 19 healthy controls. Compared with healthy controls, a significant number of IgAN and mesPGN patients showed increased expression of IL-1β, IL-4, IL-10, IL-12, and IFN-γ. The cytokine profile of renal tissue of 10 IgAN and 5 mesPGN biopsies was simultaneously analyzed and compared with that of PBMC. The proinflammatory IL-1α and growth factor PDGF-B were expressed more in renal tissues than in PBMC. Furthermore, the renal profile of IL-α, IFN-γ, and TNF-α expression was associated with the expression of IFN-γ PBMC. The serum level of IFN-γ of IgAN correlated significantly (P = 0.0003) with that of IL-12, suggesting a potential role for cross-stimulation. More importantly, expression of IFN-γ in PBMC and the elevated serum level correlated with the decline in glomerular filtration rate (P = 0.0012) and severity of renal histopathologic grade. To elucidate the role of leukocytes in renal cytokine expression, surface markers of T cells (CD3), monocytes (CD14), natural killer cells (CD16), and B cells (CD19) were also examined in renal tissues. The prominent renal expression of CD3, CD14, and CD16 implicates the leukocytes as the major source of proinflammatory cytokines in IgAN. Collectively, these findings indicate that IFN-γ plays a prominent role in an interactive network of cytokines that contribute to the pathogenesis and progression of IgA nephropathy.

Glomerular FcαR expression and disease activity in IgA nephropathy

In this study, we examined the receptors for the Fc portion of immunoglobulin A (IgA) (Fc alphaR) in the glomeruli as well as circulating polymorphonuclear leukocytes and monocytes at the mRNA level by reverse transcription-polymerase chain reaction (RT-PCR) assay and at the protein level by an immunohistochemistry/flow cytometry technique using a specific anti-Fc alphaR monoclonal antibody (My 43). Glomeruli were isolated from biopsy specimens of renal tissues from IgA nephropathy (IgAN; 20 cases) and non-IgA mesangial proliferative glomerulonephritis (PGN; 13 cases) patients, and from normal renal tissue specimens obtained from kidneys removed because of malignancies (five cases) applying the microdissection method. There was a relative increase in Fc alphaR in the circulating phagocytes from IgAN patients compared with those from PGN and healthy controls. Fc alphaR expression was present in approximately 40% of glomeruli samples from IgAN patients at the message levels. Fc alphaR-positive specimens were also strongly positive for expression of tumor necrosis factor-alpha, interleukin-1, and interleukin-6 mRNA. Specimens from PGN patients and healthy controls did not show any detectable Fc alphaR message. Serum IgA levels and severity of hematuria were significantly higher in patients with positive Fc alphaR expression. A message for Fc alphaR was detected in the tissues that were more damaged histologically. Our data suggest that there is some in vivo induction of glomerular Fc alphaR expression, possibly mediated by a synergistic stimulus from IgA and inflammatory cytokines, and the expressed receptor is likely to be involved in the disease process of IgAN.

Altered production of IgE and IgA induced by IL-4 in peripheral blood mononuclear cells from patients with IgA nephropathy.

In order to elucidate the factors responsible for altered immunoglobulin production in patients with IgA nephropathy (IgAN). the in vitro effects oHL‐4 and intcrferon‐gamma (IFN‐γ) on the synthesis of IgE and IgA by peripheral blood mononuclear cells (PBMC) were studied. Spontaneous IgE and IgA synthesis by PBMC was significantly increased in patients with IgA nephropathy compared with controls. The maximum amounts of IgA and IgE synthesis by PBMC after stimulation with IL‐4 were almost the same both in patients with IgAN and in controls. The enhancement rate of IL‐4‐induccd IgE and IgA synthesis was significantly lower in IgAN than in the controls, suggesting in vivo preactivation of PBMC in IgAN patients. IFN‐γ suppressed IgA and IgE synthesis by PBMC from IgAN patients as well as controls. However, the suppressive effect on IgE synthesis was less prominent in patients with IgAN. These results suggested that altered IL‐4 action might be involved in the development of IgA ncphropathy.

Increase of CD23-Positive Cells in Peripheral Blood from Patients with IgA Nephropathy and Non-lgA Proliferative Glomerulonephritis

CD23 is a surface marker of activated B cells as well as a low-affinity Fc receptor for IgE. In this study, we enumerated CD23-positive peripheral blood lymphocytes and evaluated their clinical significance in patients with IgA nephropathy (IgAN). Twenty-five patients with IgAN and 16 patients with non-IgA proliferative glomerulonephritis (PGN) were studied. Twenty-seven healthy adults served as controls. CD23-bearing cells were enumerated by flow cytometry, and serum IgE levels were measured by latex photometric immunoassay. Significant increases in the number of CD23-positive cells were observed in patients with IgAN (p less than 0.01) and PGN (p less than 0.05) compared with controls. A significant elevation of serum IgE levels was also observed in the patients with IgAN and PGN (p less than 0.05). No positive correlation between the number of CD23-positive cells and serum IgE levels was observed. We also examined the induction of surface CD23 expression on peripheral lymphocytes by interleukin (IL)-2, IL-3, IL-4, IL-5, IL-6, interferon (IFN)-gamma, IFN-alpha, phytohemagglutinin, concanavalin A, pokeweed mitogen, lipopolysaccharide and phorbol myristate acetate. IL-4 was revealed to have a significantly potent effect on the induction of cell surface CD23 compared with other stimulants. It was concluded that many patients with IgAN or PGN show high serum IgE levels and/or high CD23-positive cell counts in their peripheral blood, suggesting that hyperactivation of B cells might be involved in the development of IgAN and non-IgA PGN. It appeared that IL-4 may play a significant role in the etiology of these types of glomerulonephritis.

Immunofluorescence staining of renal biopsy samples in patients with diabetic nephropathy in non-insulin-dependent diabetes mellitus using monoclonal antibody to reduced glycated lysine

This is the first report on immunofluorescence staining of renal biopsy samples in human diabetic nephropathy (DN) using monoclonal antibodies to reduced glycated lysine. In order to detect the localization of glycated lysine in the mesangial matrix and/or the glomerular basement membrane (GBM), we examined immunofluorescence staining using antibodies against reduced glycated lysine in the glomeruli of 16 patients with DN and ten age-matched patients with diffuse mesangial proliferative glomerulonephritis without IgA deposition (DPGN) as controls. In the early stage of DN, immunofluorescence microscopy revealed the presence of intense staining for reduced glycated lysine in the GBM as well as in part of the tubular basement membrane, but not in the mesangial area. In contrast, immunofluorescence microscopy revealed less staining for glycated lysine in the GBM in the advanced stage of DN, and no reaction with any part of the renal tissue in patients with DPGN. It was concluded that detection of reduced glycated lysine in GBM in the early stage of DN might be associated with the initial pathogenesis of this disease.

Value of pathological grading in prediction of renal survival in IgA nephropathy

Summary: In order to clarify the most reliable risk factor to predict renal outcome, 206 patients with IgA nephropathy were studied for mean period of 9.2 years. the histopathological changes of this disease using light microscopy were divided into four grades (grade 1–4). These grades included glomerular, interstitial and vascular lesions. the cumulative rate of kidney survival progressing to end stage renal failure (ESRF) in all patients was 94% at 5 years, 87% at 10 years and 80% at 15 years after renal biopsy. None of the patients in grade 1 reached ESRF. the cumulative rate of kidney survival in grade 2 was 99% at 5 years, 98% at 10 years and 89% at 15 years after renal biopsy. In grade 3, it was 94% at 5 years, 79% at 10 years and 75% at 15 years. In grade 4 it was 53% at 5 years, 33% at 10 years and 22% at 15 years after renal biopsy. Forward stepwise multivariate regression analysis revealed that, in addition to the histopathological findings, three more risk factors were found to influence actuarial renal survival rate. These factors were: (i) the levels of serum creatinine; (ii) the level of serum albumin; and (iii) the amount of proteinuria at the time of renal biopsy. In parallel studies, forward stepwise multivariate regression analysis isolated three risk factors that influenced the progression of the reciprocal of serum creatinine. These factors were: (i) the levels of total protein; (ii) the degree of our pathological grading; and (iii) the amount of proteinuria. It was concluded that our pathological grading was useful as a prognostic parameter because of its simplicity and availability in routine clinical activities.

Altered synthesis of interferon-γ and expression of interferon-γ receptor by peripheral blood mononuclear cells from patients with IgA nephropathy and non-IgA proliferative glomerulonephritis

Previously we reported disease-specific interaction between interferon-γ (IFN-γ) and interleukin-4 (IL-4) in patients with IgA nephropathy (IgAN), suggesting the existence of unusual T cell behavior in this disease. In the present study, we investigated characteristic synthesis of interferon-γ (IFN-γ) and expression of IFN-γ receptor (IFN-γR) in the peripheral blood mononuclear cells (PBMC) from patients with IgAN and other chronic proliferative glomerulonephritis (PGN). Heparinized peripheral blood samples were obtained from 38 patients with chronic mesangial proliferative glomerulonephritis (CGN; including 24 with IgA nephropathy) and 20 healthy controls. PBMC were isolated by gradient centrifugation and fragments were cultured in Iscoves modified Dulbeccos medium (IMDM) supplemented with 10% fetal calf serum (FCS) for 72 hr. IFN-γ concentrations in supernatants were evaluated by the enzyme-linked immunosorbent assay (ELISA). Other parts of PBMC pellets were reacted with anti-human IFN-γR monoclonal antibody and FITC-labeled anti-mouse second antibody for analysis of IFN-γR expression on these cells by FACScan. The remaining PBMC were fractionated into CD4+ T cells, CD8+ T cells, B cells, NK, cells and macrophages using the MACS cell sorting system. The isolated cells were evaluated for IFN-γ or IFN-γR mRNA expression by the semiquantitative RT-PCR method.In vitro IFN-γ synthesis was enhanced in patients with CGN, and NK cells were revealed to be responsible for such enhancement. On the other hand, the expression of IFN-γR on macrophages was suppressed in CGN patients. These results suggest that impairment of regulation of the IFN-γ system might be involved in the development of CGN.

Rheumatoid factors and glomerulonephritis

It is presently unknown whether rheumatoid factors have a pathogenic role in the development of various types of glomerulonephritis with immune deposits. Three isotypes of rheumatoid factors (RFs), which are autoantibodies to IgG, were measured using the solid‐phase fluorescence immunoassay in sera from patients with diffuse proliferative lupus nephritis (DPLN), membranous lupus nephritis (MLN), IgA nephropathy (IgAN) and idiopathic membranous nephropathy (MN). RF activity of immunoglobulins deposited in the glomeruli from these patients was also studied by examining the binding of the FITC‐conjugated human IgG and Fc portion of IgG to the glomeruli of renal biopsy specimens. IgG, IgA and IgM RFs were significantly increased in sera from patients with DPLN. and the increase was significantly lower in patients with MLN. IgAN and MN. Human IgG bound to immunoglobulin on the glomeruli only in DPLN, but not in MLN, IgAN or MN. The Fc portion of IgG was demonstrated to be involved in this reaction. It was suggested that RFs and IgG may play a major role in immune deposits on the glomeruli in DPLN and may be involved in the development of DPLN; however, this is not likely in MLN, IgAN or MN.

Significance of high levels of serum IgA and IgA-class circulating immune complexes (IgA-CIC) in patients with non-insulin-dependent diabetes mellitus

Significance of serum IgA and IgA-class circulating immune complexes (IgA-CIC) elevation in patients with non-insulin-dependent diabetes mellitus (NIDDM) was described. Seventeen patients with NIDDM and 17 patients with diffuse mesangial proliferative glomerulonephritis without deposition of IgA (DPGN) as controls were examined. The levels of serum IgA in patients with NIDDM were significantly higher than those in patients with DPGN (p < or = 0.01). The levels of IgA-CIC in patients with NIDDM were also significantly higher than those in patients with DPGN (p < or = 0.01). Production of IgA derived from B cells and the proportion of IgA bearing B cells in patients with NIDDM were not significantly higher than those in patients with DPGN. Furthermore, the levels of IgA in pharyngeal washings from diabetic patients were not significantly higher than those for DPGN patients. Duration of diabetes, the level of HbA1c, and the presence of hypertension, microalbuminuria, or retinopathy showed no significant correlations with the levels of serum IgA or IgA-CIC in patients with NIDDM. It was postulated that the elevations of serum IgA and IgA-CIC were based on subclinical infection of the mucosa and/or deterioration of IgA clearance in patients with NIDDM.

Interstitial naive and memory T cells in chronic mesangial proliferative glomerulonephritis.

Naohiro Yano, MD, Department of Internal Medicine, Tokai University, Isehara, Kanagawa, 259-11 (Japan) Dear Sir, The degree of cell infiltration in the inter-stitium showed direct correlation with the severity of glomerular damage in typical cases of primary glomerulonephritis [1]. However, the role of interstitial infiltrating cells in the progress of glomerulonephritis has not been clarified. A common finding in previous reports [2, 3] is the dominance of CD4-positive helper/inducer T cells in the interstitium. For further analysis of the phenotypes and the roles of interstitial cells, immunohistochemical staining of CD45RA-positive cells, that is, non-antigen-stimulated ‘naive T cells’ and CD45RO-positive cells, that is, antigen-stimulated ‘memory T cells’ [4, 5] in primary chronic mesangial proliferative glomerulonephritis (CGN) using the avidin-biotin complex peroxidase technique was performed. As shown in table 1, we evaluated renal tissues after separating them into two groups, i.e., mild glomerular change group (grade I or II) and moderate to severe change group (grade III or IV). CD45RO-positive memory T cells were the dominant population of interstitial infiltrating T cells (fig. 1). Memory T cells are known as producers of cytokines [5,6]. In order to evaluate the hypothesis that infiltrating T cells produce cytokines, we attempted immunohistochemical peroxidase-alkaliphosphatase double staining using several anticytokine antibodies, but no clear signals indicating memory T cells produced some cytokines could be obtained. A sophisticated technique such as the in situ hybridization method is required to clarify the role of interstitial memory T cells. Acknowledgement This study was supported by a grant from the Japanese Ministry of Health and Welfare.