Tetsuya Ootaka

The role of macrophages in diabetic glomerulosclerosis

To elucidate the role of macrophages in diabetic glomerulosclerosis (DGS), an immunohistologic study was performed using monoclonal antibodies to common leukocyte antigen (DAKO-LC), T cells (T3), B cells (CD22), and macrophages (MAC 387, Leu-M5, and EBM-11). Kidney biopsy specimens were obtained from 28 patients with non-insulin-dependent diabetes mellitus. Cells were identified by a three-layer immunoperoxidase technique applied to cold ethanol-fixed, paraffin-embedded sections and quantitated as the number of cells per glomerular cross-sections and number of cells per square millimeter of glomerulus. The severity of the diffuse lesions in each glomerulus was graded semiquantitatively. The average grades for all the glomeruli were calculated and registered as an index of DGS for a biopsy specimen. There was no relationship between the index of DGS and the number of T or B cells. However, the number of macrophages and common leukocyte-positive cells increased significantly in the moderate stage of glomerulosclerosis compared with the mild or advanced stage. The results suggest that macrophages may transiently infiltrate during the moderate stage of diffuse DGS, contributing to irreversible structural damage.

Contribution of cellular infiltration to the progression of IgA nephropathy: A longitudinal, immunocytochemical study on repeated renal biopsy specimens

Summary: A retrospective immunocytochemical study was performed on repeated renal biopsy specimens from 47 patients with IgA nephropathy, 23 of whom received steroid therapy after the initial biopsy. Immune cells in renal tissues were detected by the immunoperoxidase method using monoclonal antibodies against common leukocyte antigens, T cells, B cells and monocytes/macrophages.

Intercellular adhesion molecule- 1/leukocyte function associated antigen-1-mediated and complement receptor type 4—mediated infiltration and activation of glomerular immune cells in immunoglobulin a nephropathy

Glomerular expression of intercellular adhesion molecule-1 (ICAM1) (CD54) and membrane cofactor protein (MCP; CD46) and positive infiltrating cells in leukocyte function associated antigen-1 (LFA1)alpha (CD11a) and C3bi receptors (CR3/CD11b, CR4/CD11c) were examined by the indirect immunoperoxidase method on 43 sets of repeated renal biopsy specimens from patients with immunoglobulin A nephropathy. Twenty-four-hour urine protein at the time of renal biopsy was also evaluated. Glomerular infiltration of LFA1alpha+ cells was significantly correlated with glomerular expression of ICAM1 (r = 0.494, P < 0.0001). Glomerular complement receptor type 4 (CR4)+ cells were significantly correlated with glomerular expression of MCP (r = 0.405, P < 0.0001). The glomerular expressions of ICAM1 and MCP were significantly correlated with each other (r = 0.700, P < 0.00001). The glomerular infiltrations of LFA1alpha+ and CR4+ cells were highly correlated with each other (r = 0.884, P < 0.00001), and both cell types were significantly correlated with urine protein (respectively, r = 0.426 and 0.478, P < 0.001 and 0.0001). When the change in these parameters between the time of the initial and follow-up biopsies was evaluated, there was a significant correlation between the change in glomerular expression of ICAM1 (DeltaICAM1) and MCP (DeltaMCP) as well as between the change in glomerular infiltration of LFA1alpha+ cells (DeltaLFA1alpha+) and CR4+ cells (DeltaCR4+). Both DeltaLFA1alpha+ and DeltaCR4+ were significantly correlated with the change in urine protein. These findings suggest that ICAM1/LFA1 interaction and MCP-mediated C3bi/C3biR interaction cooperate and participate in persistent glomerular infiltration of immune cells in immunoglobulin A nephropathy, and that these LFA1alpha+ and C3biR+ cells contribute to the induction of proteinuria.

The involvement of advanced glycation endproducts (AGEs) in renal injury of diabetic glomerulosclerosis: association with phenotypic change in renal cells and infiltration of immune cells.

AbstractBackground. Glycation of proteins is regarded as one of the major causes of the progression of diabetic nephropathy (DN) and of the phenotypic changes in glomerular mesangial cells (MC) and the cellular infiltration that occurs in MC from DN. It thus is very important to study the interrelationships and interaction between these causes. Methods. The localization of advanced glycation endproducts (AGEs), cytoskeletal proteins, and immune cell infiltration was evaluated by immunohistochemical study in patients with DN. Results. NΕ-(carboxymethyl)lysine (CML), Α-smooth muscle actin (SMA), and low-molecular-weight caldesmon (L-CLD) were localized in the mesangial area in DN. The intensity of mesangial SMA and L-CLD expression was significantly correlated with the index of diabetic glomerulosclerosis (IDGS), while the expression of pentosidine was correlated with the IDGS and with 24-h urinary protein. Pentosidine was localized in glomerular basement membrane (GBM) only in DN and had a significant correlation with the mesangial expression of SMA and L-CLD. Pyrraline deposition on the tubular basement membrane, and the expression of SMA and L-CLD, and the infiltration of immune cells were observed in interstitial areas in DN. The intensity of L-CLD expression had a close relationship with pyrraline deposition and immune cell infiltration. The expression of SMA and L-CLD in interstitial areas was significantly correlated with the percent interstitial volume. Conclusions. AGEs are involved in renal injury in DN, and their effect is, at least in part, exercised via phenotypic changes in intrinsic renal cells and by the infiltration of immune cells.

Glomerulointerstitial interaction of adhesion molecules in IgA nephropathy and membranoproliferative glomerulonephritis.

The expression of two adhesion molecules, ICAM1 (CD54) and ICAM3 (CD50), infiltrating cells positive for their ligand, LFA1 (CD11a), and the markers of total leukocytes (CD45), T cells (CD3), granulocytes/monocytes (CD15), and macrophages (CD68) in renal interstitium were examined by an indirect immunoperoxidase method. The study was longitudinally performed on repeat renal biopsy specimens from 69 patients with two different proliferative glomerulonephritides: 43 with IgA nephropathy (IgAN) and 26 with membranoproliferative glomerulonephritis (MPGN). Interstitial ICAM1 (iICAM1) was mainly expressed on endothelium of peritubular venules and sometimes on tubular epithelium, and interstitial ICAM3 (iICAM3) on infiltrating immune cells. In IgAN, iICAM1 was significantly correlated with glomerular infiltration of LFA1+ cells (gLFA1) and CD68+ cells (gCD68) (r = 0.478/0.500; P < 0.0001) as well as CD3+ cells (gCD3) (r = 0.402; P < 0.002). In MPGN, iICAM1 was significantly correlated only with gCD68 (r = 0.382; P < 0.05). In both diseases, iICAM1 and iICAM3 were significantly correlated with interstitial infiltration of LFA1+ cells (iLFA1) and CD68+ cells (iCD68) (r = 0.616 to 0.815; P < 0.0001) and with interstitial infiltration of CD3+ cells (iCD3) (r = 0.474 to 0.816; P < 0.01). The iICAM3 was also significantly correlated with interstitial CD45+ cells (iCD45) (r = 0.672 in IgAN and 0.769 in MPGN; P < 0.00001). Interstitial infiltration of these immune cells was significantly correlated with the histologic parameters indicating renal injury, such as the index of glomerular lesion and the percent interstitial volume (r = 0.410 to 692; P < 0.05). Longitudinal analysis revealed that the parameters described above showed corresponding change with each other at the follow-up biopsy. These findings suggest that the glomeruler infiltration of T cells and macrophages influences the ICAM1/ICAM3 expression of the interstitial cells, especially In IgAN, and that ICAM1/LFA1 and ICAM3/LFA1 interactions contribute to the persistent infiltration of the interstitium by immune cells in both diseases.

Analysis of leukocyte surface antigens on ethanol-fixed paraffin-embedded tissue material

Ethanol-fixed paraffin-embedded specimens of human tissues were studied whether the surface antigens of leukocytes in these tissues can be stained and analyzed. Three-layer indirect immunoperoxidase staining was performed on the ethanol-fixed paraffin-embedded sections by the use of several monoclonal antibodies for whole human leukocytes (Dako LC), B cells (Dako CD-22, 4KB5, and L26; Leu 14), T cells and their subsets (Dako UCHL-1, T1, T3, T4 and T8; Leu 4, 3a and 2a) and monocyte/macrophage lineage (Dako macrophage, Leu M1, M3 and M5). The results were compared with those on fresh-frozen sections. No essential differences were obtained between the paraffin-embedded and the fresh frozen sections stained by the following antibodies; Dako LC for whole human leukocytes; Dako UCHL-1, T3 and Leu 4 for T cells; Dako CD22, 4KB5, L26 and Leu 14 for B cells; Dako macrophage, Leu M1 and M5 for monocyte/macrophage lineage. On the other hand, the subsets of T cells could only be detected on the fresh-frozen sections. The results of the leukocyte analysis on the paraffin-embedded specimens of several renal diseases were very similar to those reported by other investigators on fresh-frozen sections or PLP-fixed materials. Thus, by the use of appropriate monoclonal antibodies, the ethanol-fixed paraffin-embedded material can be used for leukocyte analysis except for the definition of T cell subsets.

Participation of CR1 (CD35), CR3 (CD11b/CD18) and CR4 (CD11c/CD18) in membranoproliferative glomerulonephritis type I

Intraglomerular expression of complement receptors (CR) was investigated chronologically in 22 repeatedly biopsied patients with membranoproliferative glomerulonephritis (MPGN) type I by indirect immunoperoxidase staining using MoAbs. Patients were divided into two groups based on whether intraglomerular C3c deposition was decreased at the second biopsy (2nd Bx) (group A, n= 12), or not (group B, n= 10). At the first biopsy (1st Bx), the severity of glomerular injury and the degree of glomerular C3c deposition were compatible between the two groups. Four patterns of CR1 (CD35) expression on podocytes were recognized: normal; generally decreased; focally/ segmentally lost; and completely lost. The numbers of CR3 (CD11b/CD18)‐ and CR4 (CD11c/ CD18)‐positive cells per glomerular cross‐section were counted. At the 1st Bx, no significant difference was found in the number of CR3+ or CR4+ cells between the two groups. At the 2nd Bx. the numbers of both the CR3+ and CR4+ cells were significantly decreased only in group A (P <0·01). The numbers of CR3+ and CR4+ cells were significantly higher in cases with moderate or marked C3c deposits than in those with no or mild C3c deposits. The intensity of CR1 expression in group B was less than that in group A at both the 1st and 2nd Bx (1 st, P <0·05; 2nd, P < 0·01), and chronological improvement of CR1 expression was observed only in group A. The severity of glomerular injury was increased only in group B (P <0·01), and was associated with persistent massive proteinuria and hypocomplementaemia. Our results suggest that, in cases with an adverse outcome, a more severe defect of CR1 initially exists and the expression of CR1 is not recoverable chronologically. This irreversible decrease or loss of CR1 may partly contribute to the continuous C3c deposition and intraglomerular infiltration of CR3+ and CR4+ cells.

Significance of Leukocyte Infiltration in Membranous Nephropathy with Segmental Glomerulosclerosis

Aim: To clarify the role of leukocyte subsets in the development of glomerulosclerosis (GS) and interstitial lesions which may contribute to the prognosis of membranous nephropathy (MN), we investigated infiltrating cells in both glomeruli and interstitium in biopsy specimens. Methods: Forty-one cases of MN were divided into two groups: MN with segmental GS (MN+GS; n = 21) and MN without GS (MN–GS; n = 20). There was no significant difference between both groups regarding clinical data at the time of the renal biopsy. The cells were analyzed with a three-layer indirect immunoperoxidase method using monoclonal antibodies against leukocyte common antigen, T cells, B cells, and monocytes/macrophages (Mo/Mφ). Results: Renal function tended to deteriorate during the final follow-up period in group MN+GS, but not in group MN–GS. The number of glomerular and interstitial leukocytes in group MN+GS were significantly higher than those in group MN–GS. Leukocytes were mostly Mo/Mφ in the glomerulus, while T cells and Mo/Mφ were predominant in the interstitium. In group MN+GS, there was a significant correlation in number of Mφ (CD68+) between glomeruli and interstitium, but not in group MN–GS. Conclusion: The results suggest that Mo/Mφ may play an important role in the development of segmental GS and interstitial lesions in MN which may be responsible for the incurability and poor prognosis.

Chemically Modified Ribozyme to V Gene Inhibits Anti-DNA Production and the Formation of Immune Deposits Caused by Lupus Lymphocytes

A variety of autoantibodies is responsible for the tissue injury in autoimmune diseases. We have demonstrated that the human anti-DNA Ab O-81, of which Ids are commonly detected in renal glomeruli of active lupus nephritis, uses the V3-7 gene. We tried to develop a new therapy for lupus nephritis by using chemically modified ribozymes to specifically inhibit the expression of the mRNA of Ig V gene. The transfection of hammerhead ribozyme or the addition of chemically modified ribozyme against the flanking region of V3-7 caused a potent and selective inhibition of anti-DNA production in V3-7-using B cell clones, but not in irrelevant V gene-using clones in vitro. Chemically modified ribozyme was long-acting and resistant to RNase, and nonspecific cytotoxicity of the ribozyme was negligible. To know the efficacy of the ribozyme in vivo, we used a model of immune complex nephritis in SCID mice in which 5 × 106 PBLs from patients with active lupus nephritis (lupus PBL) were transferred twice. The injection of lupus PBL in combination with chemically modified ribozyme to increase resistance to RNase significantly reduced anti-DNA Ab levels in blood and decreased levels of urinary protein in the immune deposit models. Immunofluorescence study also revealed a marked decrease in IgG deposits at renal glomeruli in the ribozyme-treated group. These results indicate an efficacy of chemically modified ribozyme therapy for autoantibody-mediated immune diseases.

Intercellular adhesion molecule-1, intercellular adhesion molecule-3, and leukocyte integrins in leukocyte accumulation in membranoproliferative glomerulonephritis type i☆☆☆

Marked intraglomerular infiltration of leukocytes is observed in membranoproliferative glomerulonephritis (MPGN). We recently demonstrated that this leukocyte infiltration develops partly through macrophage-1 (Mac-1)-positive cells and glomerular C3c deposits (Clin Exp Immunol 100:269-276, 1995). To further investigate the mediation of adhesion molecules in the leukocyte accumulation, we immunohistochemically examined the expression of intraglomerular leukocyte integrins and their ligands as well as surface markers for granulocytes/monocytes (CD15) and macrophages (CD68) in 26 patients with MPGN type I who had undergone repeated biopsies. These patients were divided into two groups. Group A included the patients who showed both normo-complementemia and urinary protein excretion less than 1 g/d at the follow-up biopsy (recovery group: n = 14). Group B (persistent group: n = 12) included the patients other than those in group A. At the initial biopsy, there was no difference in the degree of glomerular C3c deposition, glomerular intercellular adhesion molecule (ICAM)-1 expression, or the numbers of cells bearing leukocyte function-associated antigen-1 (LFA-1), Mac-1, and ICAM-3 between the two groups. At the follow-up biopsy, the degree of glomerular C3c deposition, and the numbers of cells bearing LFA-1, Mac-1, and ICAM-3, were significantly decreased only in group A (P < 0.01, P < 0.001, P < 0.001, and P < 0.01, respectively). No chronological change in ICAM-1 expression was observed in either group. Group B showed a chronological increase in the severity of glomerular injury and serum creatinine level, associated with persistent heavy proteinuria. Neither LFA-1- nor Mac-1-positive cells were positively correlated with ICAM-1 expression. Most of Mac-1-positive cells were CD15-positive cells (granulocytes/monocytes), and a considerable number of Mac-1-positive cells concurrently expressed ICAM-3. In contrast, most LFA-1-positive cells were considered to be CD68-positive cells (macrophages). The number of cells bearing LFA-1 was positively correlated with that of cells bearing ICAM-3 (P < 0.00001). These results suggest that the glomerular leukocytes, infiltrating through Mac-1/complement interaction, express ICAM-3 by themselves, and that LFA-1/ICAM-3 interaction might participate in the glomerular aggregation of leukocytes in MPGN type I. In this study, we could not conclude that LFA-1/ICAM-1 or Mac-1/ICAM-1 interaction was involved in the leukocyte accumulation in this disease.