Clin. exp. Immunol. (1991) 84, 256-262
ADONIS
0009910491001380
Localization of 20-kD homologous restriction factor (HRF20) in diseased human glomeruli. An immunofluorescence study H. TAMAI*, S. MATSUO*, A. FUKATSU*, K. NISHIKAWA*, N. SAKAMOTO*, K. YOSHIOKAt, N. OKADA$ & H. OKADA§ *Third Department of Internal Medicine, Nagoya University School of Medicine, tDepartment of Paediatrics, Kinki University School of Medicine, IDepartment of Microbiology, Fukuoka University School of Medicine and §Department of Molecular Biology, Nagoya City University School of Medicine, Nagoya, Japan
(Acceptedfor publication 21 November 1990)
SUMMARY The 20-kD homologous restriction factor (HRF20), which is identical to CD59, is a membraneassociated protein which inhibits the reaction of C9 to form membrane attack complex (MAC) of homologous complements. In various human glomerular diseases deposition of complement components is frequently seen and MAC is reported to associate with immune deposits. Using a specific monoclonal antibody, F5, against H RF20, we attempted to study the localization of HRF20 in human glomerulonephritides and to compare the localization of HRF20 with those of immune deposits and MAC. The frozen sections of kidney specimens were fixed in acetone at room temperature before staining. In normal kidneys and kidney specimens from the patients with minimal change nephrotic syndrome, membranous nephropathy, and IgA nephropathy, HRF20 was strongly localized in the peritubular capillaries and along Bowman's capsules. A weaker but well-defined staining was obtained in the mesangial area and faint staining was seen along the glomerular capillary walls. In contrast, glomerular capillary walls were rather strongly stained in the cases with diffuse lupus nephritis which had subendothelial dense deposits. These data suggest that HRF20 (CD59) is present in the human glomeruli and its expression is enhanced under certain conditions such as lupus nephritis.
Keywords complement membrane attack complex HRF20 CD59 glomerulonephritis
1985; Adler et al., 1984; Perkinson et al., 1985; Cybulsky, Quigg & Salant, 1986) and in immunohistochemical analysis on kidney specimens of human glomerulonephritis (Biesecker, Katz & Koffler, 1981; Hinglais et al., 1986; Falk et al., 1987; Rauterberg et al., 1987). It has been known that cells are rather resistant to lysis caused by homologous complements even if the complement system is activated (Okada, Tanaka & Okada, 1983), and now a family of membrane factors which are anchored by phosphatidylinositol and inhibit the cell lysis by homologous complements have been reported (Schonermark et al., 1986; Zalman, Wood & Miller-Eberhard, 1986; Sugita, Nakano & Tomita, 1988; Davies et al., 1989; Holguin et al., 1989; Okada et al., 1989a, 1989b). Among these, HRF20 has been purified from the membrane of human erythrocyte (Harada et al., 1990) and a mouse monoclonal antibody, IF5, against HRF20 has been generated (Okada et al., 1989a). IF5 inhibits the action of HRF20 specifically and makes the erythrocyte prone to homologous complement attack (Okada et al., 1989a). Recently it was shown that HRF20 was identical to CD59 (Davies et al., 1989; Okada et al., 1989c) and present in the human glomeruli,
INTRODUCTION The complement system plays various roles in the modulation of inflammatory reactions. Although various components or products in each step of the complement cascade have unique biological properties, one of the multiple functions is the formation of membrane attack complex (MAC) on the plasma membrane of the target cells, which forms a channel in the plasma membrane (Mayer et al., 1981; Biesecker, 1983). Nonlethal effects of MAC on the nucleated cells such as production of reactive oxygen metabolites and arachidonic acid metabolites are thought to be associated with the intracellular increase of CA2 + (Morgan & Campbell, 1985) and to be involved in promoting the inflammatory reaction (Morgan, 1989). The role of MAC in the pathogenesis of glomerular injury and of proteinuria has been suggested in the studies on animal models of glomerulonephritis (Koffler et al., 1983; Groggel et al., 1983, Correspondence: Sejichi Matsuo, MD, The Third Department of Internal Medicine, Nagoya University School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, 466 Japan.
256
HRF20 in human glomerulonephritis possibly on the glomerular endothelium, epithelium and on the mesangial cells (Nose et al., 1990), suggesting that HRF20 (CD59) might have a protective role against immune injury of the glomeruli because the glomerulus is a target structure of immune complex deposition. In an attempt to evaluate the role of HRF20 (CD59) in the pathogenesis of human glomerulonephritides, we investigated the localization of HRF20 (CD59) in various types of immune complex-mediated glomerular injuries by immunofluorescence (IF) technique. MATERIALS AND METHODS Kidney specimens Specimens from normal human kidneys and pathologic human kidneys were studied. Normal human kidney specimens were prepared from the normal portion of the nephrectomized kidneys of three patients who had renal tumours. These specimens were confirmed as histologically normal. Pathologic kidney specimens were obtained by the percutaneous renal biopsy from the patients with various renal diseases. These included nine cases of minimal change nephrotic syndrome (MCNS), 31 cases of IgA nephropathy (IgAN), 18 cases of idiopathic membranous nephropathy (MN), and nine cases of diffuse lupus nephritis (DLN). Specimens diagnosed as lupus nephritis were obtained from patients who fulfilled the American Rheumatism Association 1982 revised criteria for systemic lupus erythematosus (SLE) (Tan et al., 1982), and the categorization of lupus nephritis was based on WHO morphologic classification of lupus nephritis (Churg & Sobin, 1982). These specimens were placed in OCT compound (Miles, Elkhart, IN) and were snap-frozen in liquid nitrogen. Frozen tissues were cut by a cryostat and 2-pm thick sections were obtained.
Immunofluorescence staining In the preliminary experiments, we tested several fixatives including 4% paraformaldehyde, periodate lysine paraformaldehyde fixative (PLP), 10% buffered formaline, glutaraldehyde and acetone. Frozen sections were fixed in these fixatives for 10 min either at room temperature or at 4°C. The resolution and reproducibility were superior when frozen tissues were fixed in acetone at room temperature. Therefore this fixation method was employed in the following experiments. For the detection of human IgG, IgM, IgA, Clq, C3, C4 and fibrinogen the frozen sections were incubated with FITC-labelled goat antibodies against these antigens (Behringwerke, Marburg, Germany). For the staining of HRF20, Terminal complement complexes (TCCs), and leucocyte common antigen (LCA) the sections fixed with acetone at room temperature were immersed in 0-01 M phosphate-buffered saline (PBS) containing 1% normal rabbit serum for 20 min to avoid non-specific binding of the subsequent antibodies, especially through the Fc receptors of the glomerular and infiltrating cells. After washing with PBS, the sections were incubated at room temperature for 20 min with an optimal concentration of the mouse monoclonal anti-HRF20 antibody (1 F5), with a mouse monoclonal antibody against neoantigen of polymerized C9 (anti-TCCs antibody), kindly provided by Dr A. F. Michael (University of Minnesota, Minneapolis), or with a mouse monoclonal antibody against human leucocyte common antigen (CD45) (Dako Corporation, Carpinteria, CA). The specificity and properties of I F5 and the
257
anti-TCC antibody have been previously reported (Falk et al., 1983; Okada et al., 1989a). In all cases, control tissue sections were incubated with corresponding amount of an irrelevant mouse monoclonal antibody of IgGl class which had no reactivity with human renal tissues. The sections were washed three times with PBS and they were incubated with FITClabelled rabbit anti-mouse IgG antibodies (Zymed, San Francisco, CA) absorbed with normal human serum. After the final wash in PBS, all the sections were mounted with medium containing p-phenylenediamine (Platt & Michael, 1983) and examined with an Olympus BH-2 epifluorescence microscope.
Evaluation of the immunofluorescence findings The intensity of the fluorescence was graded without the knowledge of the diagnosis. Intensity of immunofluorescence staining (IF grades) were assigned as described in Results. The procedure described above was done within 24 h after renal biopsy or nephrectomy. In order to diagnose the pathologic kidney, all biopsy specimens were examined by light and electron microscopy as well as by immunofluorescence. Evaluation of clinical activity in diseased kidney The clinical data of the patients at the time of renal biopsy were obtained, including the concentration of serum creatinine, total protein, complement level (C3, C4 and CH50) and daily urine protein secretion. Relationship between each clinical parameter and the intensity of IF5 staining was analysed statistically in each disease group, using Student's t-test. RESULTS
Localization of HRF20 in normal kidneys In normal human kidneys (Fig. 1), the localization of HRF20 was most strongly seen in the peritubular capillaries. Endothelial aspects of other arteries and veins were similarly stained. In contrast, brush borders of renal tubular cells were only faintly stained by I F5. In glomeruli, Bowman's capsules demonstrated moderate reactivity with 1 F5, and the intensity was a little weaker than that of the peritubular capillaries. Mesangial area was clearly stained by 1 F5 but the glomerular capillary wall was less prominently stained in a finely granular pattern (Fig. 1, inset). Since the peritubular capillaries were most strongly stained, we assigned the staining intensity of the peritubular capillaries in normal human kidneys as 4+ and the intensity of immunofluorescence staining was graded from 0 to 4 + accordingly. According to this grading, normal kidneys exhibited 2 + or 3 + of staining for HRF20 in Bowman's capsules, 2 + in the mesangial area, 1 + in the glomerular capillary wall, and + in the proximal tubules. Localization of HRF20 in diseased kidneys In two-thirds of the kidneys from patients with MCNS, the localization and the intensity of HRF20 was not different from those seen in the normal kidneys (Fig. 2a). In the remaining kidneys the staining intensity for HRF20 in the glomerular capillary wall was slightly increased. In about half of the patients with MN or IgAN, intensity of the staining for HRF20 in the glomerular capillary wall was similar to that seen in the normal subjects (I +), whereas the remaining cases showed increased reactivity with I F5 (mainly 2+) in the glomerular
258
H. Tamai et al.
Fig. 1. Localization of HRF20 in the normal human kidney visualized by indirect immunofluorescence. M, mesangial area; BC, Bowman's capsule; CW, capillary wall; PTC, peritubular capillary. Magnification x 300, inset x 750.
Fig. 2. Localization of HRF20 (a, b, c) and leukocyte common antigen positive cells (d) in diseased human kidneys. a, minimal change nephrotic syndrome; b, membranous nephropathy; c, IgA nephropathy; d, diffuse lupus nephritis. Magnification x 300.
capillary wall. Staining for HRF20 in the mesangial area was comparable to that seen in normal subjects. The representative immunofluorescence pictures of MN and IgAN are shown in Fig. 2b and 2c. In contrast to IgAN and MN, the staining intensity for HRF20 in all the DLN cases was
increased impressively (3 + to 4 + in most cases) in the glomerular capillary wall. It was shown that the endothelial aspect of the glomerular capillary wall was more strongly stained than the epithelial side. The staining in the mesangial area in DLN patients was similar to that seen in normal subjects.
259
HRF20 in human glomerulonephritis
Fig. 3. Localization of HRF20 in the same glomerulus of Fig. 2d, diffuse lupus nephritis. CL, capillary lumen; BC, Bowman's capsule; M, mesangial area. Magnification x 300, inset x 750.
Table 1. Summary of staining for HRF20 in the glomerular capillary wall in diseased human kidneys
Number of patients IF grade (mean+s.e.m.)
Normal
MCNS
IgAN
MN
DLN
3 100+0000
9 144+0242
31 145+0102
18 172+0211
9 311+0261
*P
ADONIS
0009910491001380
Localization of 20-kD homologous restriction factor (HRF20) in diseased human glomeruli. An immunofluorescence study H. TAMAI*, S. MATSUO*, A. FUKATSU*, K. NISHIKAWA*, N. SAKAMOTO*, K. YOSHIOKAt, N. OKADA$ & H. OKADA§ *Third Department of Internal Medicine, Nagoya University School of Medicine, tDepartment of Paediatrics, Kinki University School of Medicine, IDepartment of Microbiology, Fukuoka University School of Medicine and §Department of Molecular Biology, Nagoya City University School of Medicine, Nagoya, Japan
(Acceptedfor publication 21 November 1990)
SUMMARY The 20-kD homologous restriction factor (HRF20), which is identical to CD59, is a membraneassociated protein which inhibits the reaction of C9 to form membrane attack complex (MAC) of homologous complements. In various human glomerular diseases deposition of complement components is frequently seen and MAC is reported to associate with immune deposits. Using a specific monoclonal antibody, F5, against H RF20, we attempted to study the localization of HRF20 in human glomerulonephritides and to compare the localization of HRF20 with those of immune deposits and MAC. The frozen sections of kidney specimens were fixed in acetone at room temperature before staining. In normal kidneys and kidney specimens from the patients with minimal change nephrotic syndrome, membranous nephropathy, and IgA nephropathy, HRF20 was strongly localized in the peritubular capillaries and along Bowman's capsules. A weaker but well-defined staining was obtained in the mesangial area and faint staining was seen along the glomerular capillary walls. In contrast, glomerular capillary walls were rather strongly stained in the cases with diffuse lupus nephritis which had subendothelial dense deposits. These data suggest that HRF20 (CD59) is present in the human glomeruli and its expression is enhanced under certain conditions such as lupus nephritis.
Keywords complement membrane attack complex HRF20 CD59 glomerulonephritis
1985; Adler et al., 1984; Perkinson et al., 1985; Cybulsky, Quigg & Salant, 1986) and in immunohistochemical analysis on kidney specimens of human glomerulonephritis (Biesecker, Katz & Koffler, 1981; Hinglais et al., 1986; Falk et al., 1987; Rauterberg et al., 1987). It has been known that cells are rather resistant to lysis caused by homologous complements even if the complement system is activated (Okada, Tanaka & Okada, 1983), and now a family of membrane factors which are anchored by phosphatidylinositol and inhibit the cell lysis by homologous complements have been reported (Schonermark et al., 1986; Zalman, Wood & Miller-Eberhard, 1986; Sugita, Nakano & Tomita, 1988; Davies et al., 1989; Holguin et al., 1989; Okada et al., 1989a, 1989b). Among these, HRF20 has been purified from the membrane of human erythrocyte (Harada et al., 1990) and a mouse monoclonal antibody, IF5, against HRF20 has been generated (Okada et al., 1989a). IF5 inhibits the action of HRF20 specifically and makes the erythrocyte prone to homologous complement attack (Okada et al., 1989a). Recently it was shown that HRF20 was identical to CD59 (Davies et al., 1989; Okada et al., 1989c) and present in the human glomeruli,
INTRODUCTION The complement system plays various roles in the modulation of inflammatory reactions. Although various components or products in each step of the complement cascade have unique biological properties, one of the multiple functions is the formation of membrane attack complex (MAC) on the plasma membrane of the target cells, which forms a channel in the plasma membrane (Mayer et al., 1981; Biesecker, 1983). Nonlethal effects of MAC on the nucleated cells such as production of reactive oxygen metabolites and arachidonic acid metabolites are thought to be associated with the intracellular increase of CA2 + (Morgan & Campbell, 1985) and to be involved in promoting the inflammatory reaction (Morgan, 1989). The role of MAC in the pathogenesis of glomerular injury and of proteinuria has been suggested in the studies on animal models of glomerulonephritis (Koffler et al., 1983; Groggel et al., 1983, Correspondence: Sejichi Matsuo, MD, The Third Department of Internal Medicine, Nagoya University School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, 466 Japan.
256
HRF20 in human glomerulonephritis possibly on the glomerular endothelium, epithelium and on the mesangial cells (Nose et al., 1990), suggesting that HRF20 (CD59) might have a protective role against immune injury of the glomeruli because the glomerulus is a target structure of immune complex deposition. In an attempt to evaluate the role of HRF20 (CD59) in the pathogenesis of human glomerulonephritides, we investigated the localization of HRF20 (CD59) in various types of immune complex-mediated glomerular injuries by immunofluorescence (IF) technique. MATERIALS AND METHODS Kidney specimens Specimens from normal human kidneys and pathologic human kidneys were studied. Normal human kidney specimens were prepared from the normal portion of the nephrectomized kidneys of three patients who had renal tumours. These specimens were confirmed as histologically normal. Pathologic kidney specimens were obtained by the percutaneous renal biopsy from the patients with various renal diseases. These included nine cases of minimal change nephrotic syndrome (MCNS), 31 cases of IgA nephropathy (IgAN), 18 cases of idiopathic membranous nephropathy (MN), and nine cases of diffuse lupus nephritis (DLN). Specimens diagnosed as lupus nephritis were obtained from patients who fulfilled the American Rheumatism Association 1982 revised criteria for systemic lupus erythematosus (SLE) (Tan et al., 1982), and the categorization of lupus nephritis was based on WHO morphologic classification of lupus nephritis (Churg & Sobin, 1982). These specimens were placed in OCT compound (Miles, Elkhart, IN) and were snap-frozen in liquid nitrogen. Frozen tissues were cut by a cryostat and 2-pm thick sections were obtained.
Immunofluorescence staining In the preliminary experiments, we tested several fixatives including 4% paraformaldehyde, periodate lysine paraformaldehyde fixative (PLP), 10% buffered formaline, glutaraldehyde and acetone. Frozen sections were fixed in these fixatives for 10 min either at room temperature or at 4°C. The resolution and reproducibility were superior when frozen tissues were fixed in acetone at room temperature. Therefore this fixation method was employed in the following experiments. For the detection of human IgG, IgM, IgA, Clq, C3, C4 and fibrinogen the frozen sections were incubated with FITC-labelled goat antibodies against these antigens (Behringwerke, Marburg, Germany). For the staining of HRF20, Terminal complement complexes (TCCs), and leucocyte common antigen (LCA) the sections fixed with acetone at room temperature were immersed in 0-01 M phosphate-buffered saline (PBS) containing 1% normal rabbit serum for 20 min to avoid non-specific binding of the subsequent antibodies, especially through the Fc receptors of the glomerular and infiltrating cells. After washing with PBS, the sections were incubated at room temperature for 20 min with an optimal concentration of the mouse monoclonal anti-HRF20 antibody (1 F5), with a mouse monoclonal antibody against neoantigen of polymerized C9 (anti-TCCs antibody), kindly provided by Dr A. F. Michael (University of Minnesota, Minneapolis), or with a mouse monoclonal antibody against human leucocyte common antigen (CD45) (Dako Corporation, Carpinteria, CA). The specificity and properties of I F5 and the
257
anti-TCC antibody have been previously reported (Falk et al., 1983; Okada et al., 1989a). In all cases, control tissue sections were incubated with corresponding amount of an irrelevant mouse monoclonal antibody of IgGl class which had no reactivity with human renal tissues. The sections were washed three times with PBS and they were incubated with FITClabelled rabbit anti-mouse IgG antibodies (Zymed, San Francisco, CA) absorbed with normal human serum. After the final wash in PBS, all the sections were mounted with medium containing p-phenylenediamine (Platt & Michael, 1983) and examined with an Olympus BH-2 epifluorescence microscope.
Evaluation of the immunofluorescence findings The intensity of the fluorescence was graded without the knowledge of the diagnosis. Intensity of immunofluorescence staining (IF grades) were assigned as described in Results. The procedure described above was done within 24 h after renal biopsy or nephrectomy. In order to diagnose the pathologic kidney, all biopsy specimens were examined by light and electron microscopy as well as by immunofluorescence. Evaluation of clinical activity in diseased kidney The clinical data of the patients at the time of renal biopsy were obtained, including the concentration of serum creatinine, total protein, complement level (C3, C4 and CH50) and daily urine protein secretion. Relationship between each clinical parameter and the intensity of IF5 staining was analysed statistically in each disease group, using Student's t-test. RESULTS
Localization of HRF20 in normal kidneys In normal human kidneys (Fig. 1), the localization of HRF20 was most strongly seen in the peritubular capillaries. Endothelial aspects of other arteries and veins were similarly stained. In contrast, brush borders of renal tubular cells were only faintly stained by I F5. In glomeruli, Bowman's capsules demonstrated moderate reactivity with 1 F5, and the intensity was a little weaker than that of the peritubular capillaries. Mesangial area was clearly stained by 1 F5 but the glomerular capillary wall was less prominently stained in a finely granular pattern (Fig. 1, inset). Since the peritubular capillaries were most strongly stained, we assigned the staining intensity of the peritubular capillaries in normal human kidneys as 4+ and the intensity of immunofluorescence staining was graded from 0 to 4 + accordingly. According to this grading, normal kidneys exhibited 2 + or 3 + of staining for HRF20 in Bowman's capsules, 2 + in the mesangial area, 1 + in the glomerular capillary wall, and + in the proximal tubules. Localization of HRF20 in diseased kidneys In two-thirds of the kidneys from patients with MCNS, the localization and the intensity of HRF20 was not different from those seen in the normal kidneys (Fig. 2a). In the remaining kidneys the staining intensity for HRF20 in the glomerular capillary wall was slightly increased. In about half of the patients with MN or IgAN, intensity of the staining for HRF20 in the glomerular capillary wall was similar to that seen in the normal subjects (I +), whereas the remaining cases showed increased reactivity with I F5 (mainly 2+) in the glomerular
258
H. Tamai et al.
Fig. 1. Localization of HRF20 in the normal human kidney visualized by indirect immunofluorescence. M, mesangial area; BC, Bowman's capsule; CW, capillary wall; PTC, peritubular capillary. Magnification x 300, inset x 750.
Fig. 2. Localization of HRF20 (a, b, c) and leukocyte common antigen positive cells (d) in diseased human kidneys. a, minimal change nephrotic syndrome; b, membranous nephropathy; c, IgA nephropathy; d, diffuse lupus nephritis. Magnification x 300.
capillary wall. Staining for HRF20 in the mesangial area was comparable to that seen in normal subjects. The representative immunofluorescence pictures of MN and IgAN are shown in Fig. 2b and 2c. In contrast to IgAN and MN, the staining intensity for HRF20 in all the DLN cases was
increased impressively (3 + to 4 + in most cases) in the glomerular capillary wall. It was shown that the endothelial aspect of the glomerular capillary wall was more strongly stained than the epithelial side. The staining in the mesangial area in DLN patients was similar to that seen in normal subjects.
259
HRF20 in human glomerulonephritis
Fig. 3. Localization of HRF20 in the same glomerulus of Fig. 2d, diffuse lupus nephritis. CL, capillary lumen; BC, Bowman's capsule; M, mesangial area. Magnification x 300, inset x 750.
Table 1. Summary of staining for HRF20 in the glomerular capillary wall in diseased human kidneys
Number of patients IF grade (mean+s.e.m.)
Normal
MCNS
IgAN
MN
DLN
3 100+0000
9 144+0242
31 145+0102
18 172+0211
9 311+0261
*P
