Nephrol Dial Transplant (1991) 6: 917-922 © 1991 European Dialysis and Transplant Association-European Renal Association
Nephrology Dialysis Transplantation
Original Article
D. Seron, J. S. Cameron and D. O. Haskard Renal Unit and Department of Rheumatology, United Medical and Dental Schools of Guy's and St. Thomas's Hospitals, Guy's Campus, London
Abstract. We have conducted an immunocytochemical analysis to investigate the presence of the recently described vascular cell adhesion molecule-1 (VCAM-1) in human kidney, using the anti-VCAM-1 monoclonal antibody 1.4C3. In normal control tissue VCAM-1 was present on some (but not all) parietal epithelial cells lining Bowman's capsule. Forty-nine of fifty clinical biopsy specimens were characterised by the additional presence of VCAM-1 on proximal tubular cells. This was most marked in biopsies of patients with interstitial nephritis or systemic vasculitis with crescentic nephritis, but was also observed in biopsies with minimal change, IgA or lupus nephropathy, or from patients with diabetic nephropathy, amyloid, or gout. Proximal tubule VCAM-1 correlated significantly with the number of transferrin-receptor-positive leukocytes (r = 0.607,/? < 0.0001) in the interstitium, but not with expression of HLA-DR by tubular cells. Surprisingly, VCAM-1 was not observed on vascular endothelial cells in these biopsies, even in the presence of a marked infiltrate; this contrasts with other tissues (e.g. skin and synovium). The presence of VCAM-1 on tubular cells in the inflamed kidney indicates the potential for these cells to interact with mononuclear cells, either as accessory cells or as cytotoxic targets. The unexpected absence of VCAM-1 in renal vascular endothelial cells suggests local differences in the endothelial cells of this organ.
Key words: VCAM-1 Monoclonal antibodies; Glomerulonephritis; Renal tubular cells; MHC class II antigens; Transferrin receptor
Introduction
There is increasing recongnition that the migration of leukocytes into and within tissues is regulated by interactions between surface adhesion molecules on infiltrating and resident cells [1]. Vascular cell adhesion molecule-1 (VCAM-1) (also known as INCAM-10) is a member of the immunoglobulin supergene family that was first cloned using COS cells transfected with cDNA derived from cytokine-activated umbilical vein endothelial cells [2]. On endothelial cells the molecule is minimally expressed in resting cells but is induced by activation with interleukin-1, tumour necrosis factor, lipopolysaccharide (LPS) or interleukin-4 [3-5]. The leukocyte receptor for VCAM-1 has been shown to be the pi integrin VLA-4 (CD49d/CD29), which is expressed on both lymphocytes and monocytes [6]. Although it is clear that the expression of VCAM-1 is not limited to endothelial cells [7], at present little information exists about its cellular distribution in different organs and different pathological states. In this study we have used an anti-VCAM-1 monoclonal antibody (m.Ab) [4] to investigate the presence of this molecule in the normal and diseased kidney. We found Correspondence and offprint requests to: Professor J. S. Cameron, Clinical Science Laboratories, 17th Floor, Guy's Tower, Guy's that the m.Ab stained pariethal epithelial cells in Bowman's capsule and a variable proportion of proximal Hospital, London SE1 9RT, UK.
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Expression of VCAM-1 in the Normal and Diseased Kidney
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tubule cells. There was little evidence for vascular staining in the kidney in the specimens examined.
Table 2. Monoclonal antibodies moAb
CD Specificity designation
2D1
CD45
UCHT1 UCHT4
CD3 CD8
Source
Materials and Methods Patients and Biopsy Material Kidney biopsies were taken for diagnostic purposes using a standard Trucut needle. A total of 50 biopsies were classified according to pathological diagnosis in seven groups (Table 1).
Disease
n 2B6C
Minimal-change nephropathy Non-immune chronic renal disease Gout Diabetes mellitus Amyloid IgA-associated nephropathy Membranous nephropathy Lupus nephritis WHO Class II WHO Class III WHO Class IV WHO Class V NSAID-induced1 acute interstitial nephritis Vasculitis with crescentic nephritis Total ' NSAID, non-steroidal anti-inflammatory drug.
HLA-DR CD71
ELAM-1
Dr P. Beverly Dr P. Beverly Dr P. Beverly Serotec Dako Dako Dr D. Haskard Bradsure Biologicals, Loughborough, UK Dr D. Haskard
4 6 2 2 2 13 8 7 1 1 3 2 5 7 50
normal rabbit serum for 10 min. Each m.Ab was then applied at an appropriate dilution and incubated for 90 min in a damp chamber. The sections were then washed in phosphate-buffered saline (PBS) and incubated with rabbit anti-mouse peroxidase conjugate (Dakopatts, Copenhagen) (1:40) for 30 min. The final reaction was developed by incubating the sections in PBS containing diaminobenzidine (0.5 mg/ml) and 0.3% hydrogen peroxide for 1-2 min. Sections were washed, counterstained with Mayer's haemalum, dehydrated and mounted in DPX (Raymond Lamb, London). In the case of m.Ab Ber-T9, reactivity was enhanced with sodium gold chloride as previously described [10]. Control sections were submitted to the same technique except for the omission of the primary m.Ab.
Four biopsies obtained from donor kidneys at the time of transplantation served as control material. Two cores were taken at each biopsy, with one processed for routine histology and the other embedded in OCT, snap frozen and stored at — 70°C until required. Microscopy and Cell Counting
Quantification was performed in randomly selected fields by a single observer (DS) in the absence of clinical The m.Ab used in the study are shown in Table 2. m.Ab or histological information. Cells and positively stained 1.4C3 was first characterised as reacting with a cytokine- tubular cross-sections were counted at a magnification inducible antigen on umbilical vein endothelial cells [4] of x 400 using a graticule in the eyepiece of a Leitz West Germany) measurand has subsequently been shown to react specifically Orthplan microscope (Leitz, 2 an area of 0.058 mm . Between six and ten fields ing with VCAM-1 [8]. were assessed depending upon the size of the biopsy. Biopsies that measured less than six fields were discarded. Cells were scored as positive only if they Immunoperoxidase Technique displayed a distinct membrane staining. Tubular crossFrozen sections (5-6 \im) were cut on a Bright's cryostat, sections were considered to be positive if at least one air dried and fixed in acetone for 10 min. Sections were epithelial cell in a tubular cross-section displayed a clear stained with primary m.Ab, employing an indirect cytoplasmic staining. The results were expressed as immunoperoxidase method previously described [9]. number of cells per mm2 and number of positive tubular Briefly, slides were incubated with a 1:5 dilution of cross-sections per mm2.
Monoclonal Antibodies
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Table 1. Renal biopsies studied
FMC32 DK22 Ber-T9 1.4C3 PAL-E
Leukocyte common antigen T cells Suppressor/cytotoxic T cells Monocytes/macrophages HLA-DR positive cells Transferrin receptor VCAM-1 Vascular endothelial cells
VCAM-1 in the Kidney
Statistical Analysis Results are given as the mean ± 1 standard deviation. Student's t test was used to compare means between two groups and ANOVA to compare more than two groups. Individual comparisons were made with Fisher's method combining p values. Multiple linear regression analysis was employed to study the relationship between quantitiative variables. Ap value of < 0.05 was considered significant.
1
Results
Fig. 1. VCAM-1 positive proximal tubular cross-sections in a case of membranous nephropathy stained with 1.4C3 MAb (xlO7).
Despite the fact that the number of cases included in each group was low, the number of VCAM-1-positive tubular cross sections in vasculitis and NSAID was significantly greater when compared to the number of VCAM-1-positive tubular cross-sections with minimalchange disease, membranous nephropathy, and IgA nephropathy (p < 0.05). There did not appear to be a relationship between the intensity of proximal tubular staining and the proportion of positive tubular cells. When all cases were considered together, the mean number of tubules expressing VCAM-1 (53 ± 35 tubules/mm2) was significantly higher than the mean number of tubules expressing HLA-DR antigens (p = 0.0001). Only eight biopsies showed activated leukocytes infiltrating the interstitium, as indicated by the presence of transferrin-receptor-positive cells. In these biopsies the mean number of activated cells was low (31 ± 18 cells/mm2. Transferrin-receptor-positive cells were seen
Table 3. Staining with monoclonal antibodies' Diagnosis
Minimal change Non-immune IgA Membranous Lupus NSAID Vasculitis
+ve Tubular cross-sections/mm2
Cells/mm2
1.4C3 VCAM-1
DK22 HLA-DR
2D1 All leukocytes
4
34±14
13±9
6 13 8 7 5 7
47±17 38±28 43±24 45±27 78±37 97±42
Cases
14±24 18+43 12±20 14±22 60+67 19±30 53 ±35 +ve tubular cross-sections/mm2
UCHT1 T-Cells
UCHT4 CD8+ve
177±95
70+75
16±8
86±31
5±10
236±66 449±270 335+113 258±75 858±470 592+348
86±57 250±256 122±71 172±107 447±252 317±237
18±10 87±59 48±42 76±64 172+185 106+59
119+39 322 ±154 184±104 126±91 574±321 286±129
0 0 1±3 0 12±22 21 ±24
FMC32 monocytes
Ber-T9 TfR
1 Monoclonal antibody staining was quantified as described in Materials and Methods. Values for moAb 1.4C3 and DK22 are positive tubular cross-sections/mm2±lSD.
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Analysis of four biopsies of renal tissue taken from donor kidneys at the time of transplantation showed m.Ab 1.4C3 to stain some, but not all, parietal epithelial cells lining Bowman's capsule. It did not stain visceral epithelium or endothelium. Very few proximal tubular cross-sections (0-6 tubules/mm2) were positively stained in these control biopsies. The 50 clinical specimens were all, apart from a single case of IgA nephropathy, characterised by the additional cytoplasmic staining of variable numbers of proximal tubular cells with the anti-VCAM-1 antibody 1.4C3 (Fig. 1) ranging up to 173 tubules/mm2 (Table 3). The mean number of positive tubular cross-sections was greater in vasculitis, but fewer VCAM-1 positive tubules were seen in minimal-change disease, IgA nephropathy, membranous nephropathy, lupus nephropathy, and in the group of non-immune mediated kidney diseases (p = 0.0024). In this group, the two cases of diabetic glomerulopathy showed a mean of 67 tubules/mm2, the two cases of amyloidosis a mean of 38 tubules/mm2, and the two cases of gouty nephropathy a mean of 36 tubules/mm2.
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in four of seven cases of vasculitis, two of five cases of interstitial nephritis due to NSAID, one of eight cases of membranous nephropathy, and one of four cases of minimal-change disease. When the biopsies were divided into two groups depending upon the presence or absence of transferrin-receptor-positive cells in the interstitium, the number of VCAM-1-positive tubular cross-sections was significantly higher (p = 0.0001) in cases displaying this activation marker (104 ± 34 tubules/mm2) compared to cases with no transferrinreceptor-positive cells (43 ± 26 tubules/mm2) (Table 4). In contrast, there was no difference to the number of HLA-DR-positive tubules between transferrinreceptor-positive and -negative biopsies.
Transferrin receptor moAb a
Positive
Negative
1.4C3 DK22
104±34 33+48 729+402 390+246 154+153 401+275
43 ±26 17±32 361+244 182±186 64±58 220+164
2D1
UCHT1 UCHT4 FMC32
Significance (P)