NEFROLOGÍA. Vol. XXI. Número 3. 2001 ORIGINALES Quantitative analysis of the interstitial mast cells in idiopathic mesangiocapillary glomerulonephritis type I M. Danilewicz y M. Wa growska-Danilewicz ^ / Department of Pathology (Morphometry Division) Medical University of Lódz, Poland. ´ SUMMARY Fourteen renal biopsy specimens from patients with mesangiocapillary glomerulonephritis type I (MCGN-I) for whom both light and electron microscopy as well as immunofluorescence microscopy and full clinical data were available were examined quantitatively. As a control 10 biopsy specimens of kidneys removed because of trauma were used. Morphometric investigations were performed by means of a computer image analysis system to evaluate whether mast cells have a role in tubuloinsterstitial fibrosis in MCGN-I and to examine the relationship between mast cells and intertitial -smooth muscle actin (-SMA) expression as well as interstitial infiltrates. The morphometric study revealed that the mean values of the interstitial tryptase positive cells, expresion of -SMA, interstitial volume, CD 68+, CD 45RB+, CD 43+ and CD 20+ cells were significantly increased in MCGN-I patients in comparison with control group. In MCGN-I group there were significant positive correlations between interstitial tryptase positive cells and interstitial expression of -SMA, interstitial volume, serum creatinine as well as CD 43+ and CD 68+ cells. The correlations between interstitial tryptase positive cells and CD 45+, as well as CD 20+ cells did not reach statistical significance. In conclusion, our findings demonstrate that mast cells are one of the constitutive cell types in the interstitium in MCGN-I. Additionally, significant positive correlations between interstitial mast cell count and relative interstitial volume as well as serum creatinine concentration suggest the role of these cells in the development of interstitial fibrosis which may contribute to the renal deterioration in patients with MCGN-I. Key words: Mast cells. Mesangiocapillary glomerulonephritis type I. Morphometry. Recibido: 4-VIII-2000. En versión definitiva: 4-III-2001. Aceptado: 8-III-2001. Correspondencia: Marian Danilewicz M.D., Ph.D. ul. Zamenhofa 5 m. 4 90-431 Lódz, Poland / ´ E-mail: hobo@psk2.am.lodz.pl 253 M. DANILEWICZ y M. WAGROWSKA-DANILEWICZ ANÁLISIS CUANTITATIVO DE LOS MASTOCITOS INTERSTICIALES EN LA GLOMERULONEFRITIS MESANGIOCAPILAR IDIOPÁTICA TIPO I RESUMEN Se examinaron 14 biopsias renales de enfermos con glomerulonefritis mesangiocapilar tipo I (GNMC-I) por microscopía óptica, electrónica e inmunofluorescencia, así como sus datos clínicos y analíticos. Como control se emplearon 10 muestras de nefrectomías por traumatismos renales. Se efectuaron investigaciones morfométricas mediante un sistema de imágenes computarizadas con el fin de valorar si los mastocitos juegan algún papel en la fibrosis intersticial de la GNMC-I y examinar la relación entre dichas células, los infiltrados intersticiales y la actina de músculo liso (x-SMA). El estudio morfométrico reveló que los valores medios de las células intersticiales triptasa positivas, la expresión de x-SMA, el volumen intersticial, y las células CD 68+, CD 45RB+, CD 43+ y CD 20+ estaban significativamente aumentadas en comparación con el grupo control. En el grupo de GNMC-I existía una correlación significativa positiva entre las células intersticiales triptasa + y a la expresión intersticial de x-SMA, el volumen intersticial, la creatinina sérica y las células CD 43+ y CD 68+. Las correlaciones entre las células intersticiales triptasa positivas y las células CD 45+ y CD 20+ no alcanzaron significación estadística. En conclusión, nuestros hallazgos demuestran que los mastocitos son uno de los tipos de células que forman el intersticio de las GNMC-I. Además, el hallazgo de unas correlaciones positivas significativas entre el recuento de células intersticiales y el volumen intersticial relativo y los niveles séricos de creatinina sugieren el papel de estas células en el desarrollo de fibrosis intersticial pudiendo contribuir al deterioro de la función renal en estos enfermos. Palabras clave: Mastocitos. Glomerulonefritis mesangiocapilar tipo I. Morfometría. INTRODUCTION Mesangiocapillary glomerulonephritis type I (MCGN-I) is a well-defined histopathologic entity that, although it may be found in a variety of clinical settings 1, 2, is usually idiopathic 3. This disease is characterised morphologically by the presence of granular, subendothelian electron-dense material presumed to be immune deposit 4. Although morphometric investigations confirmed prominent tubulointerstitial fibrosis in this glomerulopathy 5 the fibrogenic mechanisms operating locally in the kidney are not well understood ar the present time 6. A great number of cytokines and growth factors produced by the interstitial leukocyte subpopulations are probably involved in tubulointerstitial fibrosis 7. Recent evidence also suggest a role for mast cells in the pathogenesis of renal scarring in various glomerulopathies 8-11. Mast cells contain inflammatory mediators such as histamine, prostaglandin, leukotriens, plate254 let activating factors and basic fibroblast growth factor 10-12. Although mast cells are well known to cause type I hypersensitivity 13 and play a role in chronic inflammatory diseases such as scleroderma, rheumatoid arthritis and inflammatory bowel diseases 8-14, the role of these cells in tubulointerstitial damage in glomerulonephritis is not fully understood 11. The involvement of mast cells in the process of renal interstitial fibrosis has not been investigated, because these cells are not easily detected by routine histochemical staining 8. Recently, it is suggested that immunostaining with anti-tryptase monoclonal antibody detects almonst all the mast cells in the renal interstitium 10, 15, 16. Therefore, the present study was undertaken to ascertain whether mast cells have a role in tubulointerstitial fibrosis in MCGN-I and to examine the relationship between mast cells and interstitial smooth muscle actin (-SMA) expression as well as interstitial infiltrates. MAST CELLS IN GLOMERULONEPHRITIS PATIENTS AND METHODS Patients Fourteen renal biopsy specimens from patients with MCGN-I were examined by percutaneous renal biopsy. Most of our patients were young adults: the mean age was 36.8 ± 8.4 and the male to female ratio was 6:8. The mean duration of the disease prior to biopsy was 6.5 month. Morphological diagnosis of MCGN-I was established independently by two experienced nephropathologists and based on light microscopy, immunofluorescence and electron microscopy. As a control 10 biopsy specimens of the kidneys removed because of trauma were used (the male to female ratio was 7:3, the mean age was 38.1 ± 7.2). None of the persons from whom renal tissue originated were known to have had previous or actual renal disease. Before the quantitative examination was carried out, all control specimens were histologically examined by a nephropathologist and found to be normal renal tissue. Light microscopy Tissue specimens were embedded in paraffin, sections cut precisely at 4 µm, and stained by hematoxylin and eosin, periodic acid-Shiff (PAS)-alcian blue, trichrome light green (Masson), and by silver impregnation (Jones). Thickness of each section was controlled according to the method described by Weibel 17. Immunofluorescence microscopy Tissue was snap frozen, sectioned at 5 µm and fixed in 95% alcohol for 10 min. Sections incubated with FITC-antisera (Hoechst) to human IgG, IgA, IgM and complement (C3) were viewed on Carl Zeiss (Jena) NU-2 microscope, using and HBO 200 lamp and proper filters. Electron microscopy Tissue was fisex in glutaraldehyde, post-fixed in 1% osmium tetroxide, embedded in epon and sectioned on a LKB ultratome. Sections were stained by lead citrate and uranyl acetate, and viewed in a JEM 100B electron microscope. Immunohistochemistry The preparation and staining of tissue sections for immunohistochemistry (an indirect StreptABComplex/HRP technique) was carried out as follows: paraffin-embedded tissues, after deparaffinization and rehydratation were reacted for 5 minutes with 3% hydrogen peroxide in distilled water. If necessary, pre-treatment with Trypsin (DAKO) for 15 minutes at 37 °C was performed (mouse monoclonal antihuman mast cell tryptase antibody and monoclonal antibody anti-CD68). After blocking by normal rabbit serum (DAKO) during 20 minutes (dilution 1:5), the sections were incubated with the following solutions, each followed by TBS washing: ­ appropriately diluted mouse antihuman monoclonal antibodies (in a moist chamber for 30 minutes at room temperature), ­ biotinylated rabbit anti-mouse immunoglobulin (DAKO) diluted 1:600 in TBS for 20 minutes, and ­ StreptABComplex/HRP (DAKO) for 30 minutes prepared according to the instruction. The final reaction was achieved by incubating the sections with 3.3'-diaminobenzidine (DAB tablets DAKO) 0.5 mg/ml Tris-HCL buffer, pH 7.6, containing 0.02% hydrogen peroxide, for 10 minutes. After washing, sections were counter-stained with hematoxylin and coversliped. The monoclonal antibodies (DAKO) employed, their specific reactivities and dilutions are listed in table I. Table I. Monoclonal antibodies employed and their specificity Monoclonal antibody Anti-tryptase (AA-1) -SMA CD 45RB CD 43+ CD 20+ CD 68+ Specificity Mast cells Myofibroblasts, smooth muscles All leucocytes Pan - T cells Pan - B cells Monocytes/macrophages Source Dako Dako Dako Dako Dako Dako Dilution 1: 1: 1: 1: 1: 1: 100 150 100 100 100 100 255 M. DANILEWICZ y M. WAGROWSKA-DANILEWICZ Tissue control for immunostaining For each MoAb and for each sample a positive control was processed (for mast cells paraffin-embedded sections of nasal polyp where mast cells were found with toluidine blue staining, paraffin-embedded sections of surgically removed lymph node for interstitial leukocyte infiltrates and vascular smooth muscle cells of the renal biopsy specimens for myofibroblasts). Moreover, the following negative controls were used: 1) omission of primary antibody, 2) incubation with appropriately diluted mouse IgG (DAKO) as a first layer. The samples were prepared by the same method as described above. Specificity of labelling was shown by lack of staining in these samples. Morphometry Histological morphometry was performed by means of image analysis system consisting of a IBMcompatible computer equipped with an optical mouse, Indeo Fast card (frame grabber, true-colour, real-time), produced by Indeo (Taiwan), and colour TV camera Panasonic (Japan) linked to a Carl Zeiss Jenaval microscope (Germany). This system was programmed (program MultiScan 8.08, produced by Computer Scanning Systems, Poland) to calculate: ­ the number of objects (semiautomatic function) ­ the surface area of a structure using stereological net (with regulated number of points). The immunophenotype of mast cells and leukocyte interstitial infiltration was determined by counting all positive cells for each monoclonal antibody (semiautomatic function) in a sequence of ten consecutive computer images of 400 x high power fields - 0.0047 mm2 each. Mast cells were scored positive when displayed cytoplasmic granules stained positively with anti-tryptase monoclonal antibody. The only adjustments of field were made to avoid glomeruli and large vessels. The results were expressed as a mean number of immunopositive cells per mm2. Interstitial myofibroblasts were identified by their morphology and positive staining with anti--SMA. The interstitial immunoperoxidase staining for -SMA was measured using point counting method, which is an adaptation of the principles of Weibel 17. The point spacing being 16 µm. Total number of the points of a net was 169, and total area was 36,864 sq.µm. Under the net described above, 8-10 randomly selected adjacent fields of the renal cortex were investigated. Glomeruli and large blood vessels were neglected. The percentage of -SMA positive 256 staining was an expression of the number of points overlying -SMA positive areas as a percentage of the total points counted. The same method was used to estimate interstitial volume in sections stained with Masson trichrome. The percentage interstitial volume was an expression of the number of points overlying renal cortical interstitium as a percentage of the total points counted. Statistical methods Differences between groups were tested using unpaired Student's t-test preceded by evaluation of normality and Levene's test. The Mann-Whitney U test was used where appropriate. Correlation coefficients were calculated using Spearman's method. Results were considered statistically significant if p < 0.05. RESULTS At the time of renal biopsy, a high percentage of patients with MCGN-I showed nephrotic syndrome or heavy proteinuria. Clinical renal impairment (serum creatinine greater than 1.5 mg/100 ml) was noted in 5 patients. Elevated blood pressure was observed in 9 MCGN-I cases. Haematuria accompanied proteiuria in 11 patients. The morphometric data of the interstitial tryptase positive cells (figs. 1 y 2), interstitial expression of -SMA, interstitial fibrosis and interstitial infiltrates appear from table II. The mean values of the interstitial tryptase positive cells, expression of -SMA, interstitial volume, CD 68+, CD 45RB+, CD 43+ and Fig. 1.--Immunostaining with ant-tryptase monoclonal antibody of a control case. Mast cell between tubules in periglomerular area (x 400). MAST CELLS IN GLOMERULONEPHRITIS as interstitial infiltrates are shown in table III. In MCGN-I group there were significant positive correlations between interstitial tryptase positive cells and interstitial expression of -SMA, interstitial volume, serum creatinine as well as CD 43+ and CD 68+ cells (respectively: r = 0.77, p < 0.002, r = 0.71, p < 0.005, r = 0,69, p < 0.01, r = 0.6, p < 0.03, r = 0,67, p < 0.01). The correlations between interstitial tryptase positive cells and CD 45+, as well as CD 20+ cells were positive, but they have not reached statistical significance. No significant correlation was noted between interstitial mast cells and proteinuria. Similarly, correlations between all these parameters in controls were weak and not significant. Fig. 2.--Immunostaining with ant-tryptase monoclonal antibody of in a patient with MCGN-I. Numerous polygonal in shape mast cells can be seen (x 400). DISCUSSION It was well documented that irrespective of the nature of primary disease the decline in renal function correlates better with the degree of tubulointerstitial injury (as measured by the relative interstitial volume) than with the glomerular changes 18-21. The idea that mast cells are important to the pathogenesis of renal interstitial fibrosis was recently confirmed in CD 20+ cells were in MCGN-I patients significantly increased in comparison with control group. The correlations between interstitial tryptase positive cells and interstitial expression of -SMA, interstitial volume, serum creatinine, proteinuria as well Table II. Morphometric data of interstitial mast cells, expression of -SMA and a interstitial infiltrates in MCGN-I group and controls Number of immunopositive cells per 1 mm2 Number of cases Controls [n = 10] MCGN-I [n = 14] P value -SMA (%) 0.52 ± 0.26 16.38 ± 12.37 < 0.001 Interstitial volume (%) 10.08 ± 1.25 23.22 ± 9.67 < 0.001 Tryptase+ 1.24 ± 0.86 (0.76%) 19.65 ± 12.2 (2.88%) < 0.001 CD 68+ CD 45RB+ CD 43+ CD 20+ 33.96 ± 17.91 83.43 ± 20.91 42.35 ± 17.05 1.31 ± 1.52 (20.92%) (51.41%) (26.1%) (0.81%) 98.81 ± 39.28 311.57 ± 126.29 232.93 ± 135.1 19.75 ± 8.81 (14.47%) (45.64%) (34.12%) (2.89%) < 0.001 < 0.001 < 0.001 < 0.001 Data are expressed as mean ± standard deviation. Data in parentheses are percentages of total immunopositive cells. Table III. The correlations between tryptase positive cells and interstitial expression of -SMA, interstitial volume as well as interstitial infiltrates in MCGN-I and controls Correlation between Interstitial Interstitial Interstitial Interstitial Interstitial Interstitial Interstitial interstitial tryptase tryptase tryptase tryptase tryptase tryptase tryptase tryptase positive positive positive positive positive positive positive positive cells cells cells cells cells cells cells cells and and and and and and and and interstitial expression of -SMA interstitial volume CD 68+ cells CD 45RB+ cells CD 43+ cells CD 20+ cells serum creatinine proteinuria r r r r r r r r = = = = = = = = MCGN-I 0.77, 0.71, 0.67, 0.45, 0.6, 0.01, 0.69, 0.06, p p p p p p p p < < < = < = < = 0.002 0.005 0.01 0.1 (NS) 0.03 0.69 (NS) 0.01 0.65 (NS) r r r r r r r = = = = = = = Controls 0.77, 0.25, 0.11, 0.10, 0.07, 0.44, 0.22, p p p p p p p = = = = = = = 0.8 0.4 0.7 0.7 0.8 0.2 0.4 (NS) (NS) (NS) (NS) (NS) (NS) (NS) 257 M. DANILEWICZ y M. WAGROWSKA-DANILEWICZ IgA-nephropathy 10, in membranous nephropathy, diabetic nephropathy, focal segmental glomerular sclerosis and lupus nephritis 8. To out knowledge, however, no data have documented quantitatively interstitial staining for mast cells in representative group of MCGN-I. Our morphometric study showed that average number of interstitial tryptase positive mast cells was in MCGN-I patients significantly increased as compared with control group. Similar results were obtained by Hiromura y cols. 8, in membranoproliferative glomerulonephritis, however, in this study only 5 patients were investigated. Moreover, no information was included if these cases were by electron microscopy identified as MCGN-I subgroup. We observed tryptase positive cells in the interstitium between tubules, around glomeruli and blood vessels. No mast cells were found in the medullary interstitium and in glomeruli. Similar distribution of mast cells was reported in IgA nephropathy 10. Contrary to our results in proliferative glomerulopathies some mast cells were detected in the medullary interstitium 8. Similarly like in IgA nephropathy 10 in the present study the average number of mast cells was lower than the average number of T lymphocytes, but in contrast to these authors, the mean number of mast cells was relatively small in comparison with the number of interstitial monocytes/macrophages. It mus be noted, however, that in cited study the difference between these cells was not significant. Whereas our results showed that mast cells were one of the constitutive interstitial cell types in MCGN-I, a major finding in this study was the demonstration that there were significant positive correlations between the number of interstitial mast cells and interstitial volume as well as between interstitial mast cells and serum creatinine concentration. Although these findings are consistent with earlier suggestions that mast cells might play a role in the development of interstitial fibrosis and, in the process, may contribute to the renal deterioration 8, 9, we agree with point of view of Ehara and Shigematsu 10 that at the present time is not possible decide whether or not mast cell increased as the result of fibrosis. The precise mechanisms of renal interstitial fibrosis and the role of mast cells in this process are not clear 11. Mast cells have been shown to produce cytokines and growth factors that may contribute fo fibrosis 22, 23, whereas heparin and tryptase may enhance fibroblasts migration and proliferation 24, 25. Moreover, fibroblasts and mast cells are known to influence each other 12, 26 and is suggested that mast cell granule synthesis is also dependent on fibroblasts 27. Fibroblasts secrete mast cells growth factors, 258 for example c-kit ligand 26. Recently, Ehara and Shigematsu showed using double-labelled immunohistochemistry that some tryptase-positive mast cells had basic fibroblast growth factor in their cytoplasm 10. On the other hand, we found positive correlations between mast cells and interstitial T lymphocytes as well as monocytes/macrophages. Similar results were obtained by Toth y cols., in rapidly progressive glomerulonephritis 11. Also in study of Hiromura y cols., interstitial mast cells correlated well with the degree of tubulointerstitial leukocyte infiltration 8. The role of leukocyte infiltrates in tubulointerstitial damage in human MCGN-I is well documented 28, but the relationship between interstitial mast cells and leukocytes is less clear 27. In skin with scleroderma mast cell proliferation is considered to involve IL-3 and IL-4 stimulation by T lymphocytes 29. It is noteworthy, that only tryptase positive mast cells have been suspected to be dependent on the T lymphocyte 30. Furthermore, we observed significant positive correlation betwee the mast cell count and interstitial expression of -SMA. This result appears to be consistent with data of Toth y cols., who revealed similar relationship in rapidly progressive glomerulonephritis 11. Although it has been confirmed that interstitial -SMA positive cells play a role in the development of interstitial fibrosis in MCGN-I, as they may act as active, extracellular matrix producing cells 31, 32, the relationship between mast cells and interstitial myofibroblasts remains to be shown. In conclusion, our findings demonstrate that mast cells are one of the constitutive cell types in the interstitium of MCGN-I. 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