Ingrid Lantz,â Emijke Dim&y,â Erik Larsson,b Bengt Fellstriimâ and Keiko Funaa. âLudwig Institute for Cancer Research, Biomedical Center, Uppsala,. bDepartme ...
Transplant
Immunology
1996; 4: 209-214
Increased immunoreactivity of transforming growth factor-p in human kidney transplants Ingrid Lantz,” Emijke Dim&y,” Erik Larsson,b Bengt Fellstriim” and Keiko Funaa “Ludwig Institute for Cancer Research, Biomedical Center, Uppsala, bDepartme nt of Pathology and ‘Department of Internal Medicine, University Hospital, Uppsala Received 7 February 1996; accepted 22 March 1996
Abstract: Transforming growth factor-p (TGF-p) has been known to be involved in the pathogenesis of various kidney diseases. TGF-p is also a potent immunosuppressor that has been shown to be induced after allogeneic transplantation. We have studied the distribution of immunoreactive TGF-P proteins in different compartments of 21 allogeneic transplanted kidneys that had been rejected through acute (eight interstitial or six vascular) and chronic (seven vascular) processes. This distribution was compared with that in seven non-rejected transplanted and five non-transplanted kidneys with intact morphology. There were no obvious differences between the three groups of rejected grafts and the transplanted non-rejected group for the expression of TGP-Ps. A major difference was seen between transplanted kidneys, which exhibited clearly positive TGF-P and LTBPl (latent TGF-P binding protein) immunoreactivities, and the non-transplanted kidneys. The non-transplanted kidneys showed only very weak or no immunoreactivity for these proteins. The morphologically intact non-rejected grafts showed a significantly increased immunoreactivity compared with the non-transplanted kidneys. When the transplanted kidneys were classified into two groups (i.e. with or without diabetes mellitus) and compared with regard to the expression of all TGF-Ss, no difference was found. Thus, transplantation was the most important predictor for expression of TGF-Ss and LTBPl, and the largest expression increase in the allografts occurred in the inter&mm, followed by the glomeruli and blood vessels. Tubuli and lymphocyte aggregates stained only faintly. The results imply that TGF-P is induced rapidly after kidney transplantation. This induction can suppress immunoreactivation, but concomitantly promotes changes such as arteriosclerosis and fibrosis associated with rejection.
Introduction Allograft rejection is still a major problem in renal transplantation despite a dramatic improvement in survival by introduction of cyclosporine A (CyA) and other immunosuppressive drugs.’ This therapeutic possibility is still limited to chronic rejection due to transplantation-induced arteriosclerosis and interstitial fibrosis.’ Various cytokines have been shown to be responsible for both acute and chronic rejection through activation of lymphocytes and vascular cells.2*3 These include Address for correspondence: Keiko Funa, Ludwig Institute for Cancer Research, Biomedical Center, Box 595, S-751 24 Uppsala, Sweden. 0 Arnold 1996
interleukins mainly produced by lymphocytes, as well as growth factors derived of macrophages, stromal and epithelial cells, all promoting a cascade of immunological and inflammatory responses. 4*5Some of them, however, are capable of inhibiting cell proliferation and suppressing immunoactivation. Interferon-y, IL-10 (inter-let&in 10) and TGF-P (transforming growth factor-s) belong to these cytokines.4*6 One therapeutic possibility is to administer or to modify the generation of these cytokines, thus preventing the activation of immune and inflammatory cells.7 One of these moelcules, TGF-I3, has recently been defined as an important modulator in organ transplantation, but has also shown to be involved in the pathogenesis of various renal diseases.8*9
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The molecules of the TGF-B family are multifunctional peptides acting on many different cell typeslo Three different isoforms, TGF-Bl, TGF-B2, and TGF-B3, have so far been found in humans. The active form of TGF-Bl is a dimeric molecule of 25 kDa, which is synthesized and secreted in two different latent forms. A small latent complex is composed of mature TGF-B which is non-covalently bound to the N-terminal remnant of the TGF-B precursors, denoted latency-associated peptide (LAP). In a large latent complex, LAP is covalently bound to an additional protein, the latent TGF-B binding protein (LTBPl).” All three human isoforms of TGF-B are produced as latent complexes. ” The mature TGF-Bs can bind to their receptors after proteolytical dissociation from the latent complex. TGF-B inhibits the growth of most cell types, including lymphocytes, and suppresses their functions.13*14 However, it stimulates the production of extracellular matrix and inhibits enzymes that degrade the matrix.‘5-17 Furthermore, TGF-B can stimulate fibrosis of various tissues.L0,18,19Therefore, TGF-B can act both beneficially and deleteriously in the inflammatory process after kidney transplantation.
Objectives The aim of the present study was to characterize the pattern and intensity of the expression of the TGF-B isoforms in transplanted kidneys by imrnunohistochemical techniques. Various types of rejected allografts and functionally intact transplants as well as specimens from histologically normal non-transplanted human kidney were compared.
Materials and methods Renal allograft biopsies Biopsy specimens obtained from 28 renal transplant recipients were investigated. The patients were on triple immunosuppressive therapy with CyA. Seven patients (3/7 with diabetes mellitus) had a well-functioning stable allograft 6-24 months after the transplantation with normal morphology. Fourteen patients displayed a clinical picture of acute rejection, six of them showing an acute vascular process (3/6 with diabetes mellitus) and the remaining cases histopathologically had a cellular interstitial rejection (3/8 with diabetes mellitus). Seven patients had a clinical picture of slowly progressive deteriation of graft function and morphological signs compatible with chronic rejection (l/7 with diabetes mellitus). Five normal kidney tissue samples (O/5 with diabetes mellitus) were examined for comparison and obtained from donor kidneys that were not used for transplantation or from kidneys removed because of small kidney tumours. Antibodies A polyclonal rabbit antiserum raised against human plateletderived LTBPI, Ab39, was prepared as described elsewhere.” The affinity-purified rabbit polyclonal antibodies, Ab96, Ab94 and Ab95, directed against synthetic peptides in the respective LAP portions of the TGF-Bl, TGF-B2 and TGF-B3 precursors were used as described previously.‘* hnmunohistochemistry The kidneybiopsies were cryosectioned (7 p,rn thickness), airdried and fixed in 100% acetone for 8 min, then stored at Transplant Immunology 1996; 4: 209-214
-20°C until used. Endogenous peroxidase activity was quenched by 0.3% hydrogen peroxide in methanol. To prevent non-specific reactions between endogenous biotin and the detection system, avidin (0.1%) followed by biotin (0.01%) was applied to the sections. Prior to addition of the primary antibody, non-specific binding was blocked with phosphate buffered saline (PBS) containing 20% normal goat serum (NGS) and 10% bovine serum albumin (BSA). The sections were incubated overnight with the primary antibodies in a humidified chamber at 4°C and then with a biotinylated goat anti-rabbit Ig antibody for 45 min at room temperature, which was coupled with an ABC Elite complex (Vectastain). The immunocomplexes were visualized by addition of 3,3’-diaminobenzidine tetrahydrochloride (DAB) as a chromogen and 0.006% hydrogen peroxide as a substrate. The sections were counterstained with haematoxylin. The slides were washed between all incubations with PBS for 3 x 5 min. The specificity of the antibodies was demonstrated by blocking the immunoreaction by preincubation with the excess synthetic peptides that were used for the immunization. Non-specific binding of the secondary antibody or the detection complex to the sections was excluded by replacement of the primary antibody by an irrelevant antibody or 0.1% BSA. The assessment of growth factor expresison was done semiquantitatively. A staining-intensity scale of - to +++ (-, negative staining; +, intense staining on majority of the cells) was applied to estimate the level of immunoreactivity and thus the expression levels of the various molecules. All sections were examined in duplicate and evaluated by two of the authors independently. Statistical analysis Statistical calculations were carried out using the SuperAnovaTM program. Factors contributing to a dependent variable (immunohistochemical results) were analysed by analysis of variance. Independent variables included transplantation status (yes/no) and a diagnosis of diabetes mellitus (yes/no). This analysis of variance model was tested separately in three different areas (blood vessels, interstitium, glomeruli), always predicting the expression of each TGF-B isoform or LTBPl. When focusing on diabetes mellitus, no distinction was made between areas and proteins. The results were considered significant at a probability level of < 0.05.
Results The distribution of immunoreactive TGF-B proteins was evaluated in the different compartments of the kidney, particularly in the tubuli, the glomeruli, the interstitia and the blood vessels. The means of evaluated staining intensity were calculated separately in each group and for every compartment, and are summarized in Table 1. Normal non-transplanted kidneys The tubuli of the non-transplanted kidneys showed no immunoreactivity for the LTBPl and TGF-Bs, whereas the glomeruli stained either negative or very weakly positive (Figure IA, C, E, G). In the interstitium there was a weak expression of LTBPl (Figure lA), TGF-Bl (Fig. 1C) and TGF-B3 (Figure lG), but not of TGF-B2 (Figure 1E). The TGF-Bl antibody stained all compartments weakly. The most intense immunoreactivity in the non-transplanted kidney was detected for LTBPl and TGF-B2 in the blood vessels, while the TGF-B3
Transforming
Table 1 Average
expression of TGF-p isoforms in human transplanted and non-transplanted kidneys as determined
Diagnosis Untransplanted
Transplanted
kidney
‘normal’ allograft
Chronic vascular
Acute vascular
Acute interstitial
+tt,
growth factor-p in human kidney transplants
rejection
rejection
rejection
strong expression;
-H, moderate
by immunohistochemistry
n
TGF-f!
Blood vessels
Interstitium
Glomerali
Tubuli
5
LTBPl TGF-Pl TGF-P2 TGF-P3
* + ++ +
+ -
-
-
I
LTBPl TGF-PI TGF-p2 TGF-P3
++ + +++ ++t
* tt + i-t+
-H + +Ii+k
_
-
7
LTBPl TGF-Pl TGF-f32 TGF-P3
-H-t + i+k +++
i-k ++ ++ +++
t+ + +i i-l-k
_ _ _ -
6
LTBPl TGF-Pl TGF-f32 TGF-P3
+H i-k ++ -H
+++ ++ + ++
+ + + +
-
LTBPl TGF-fll TGPp2 TGPP3
+t tt -I+ -H-
+t + + +-I+
i-k + + +
-
+, low expression;
-, very weak or no expression.
8
expression;
immunostaining was weak throughout the tissue and seen only in the internal elastic lamina of the blood vessels. Transplanted kidneys There were no obvious differences in the expression of TGF@s among the three groups of rejected grafts and the transplanted non-rejected group. A major difference was seen between transplanted kidneys, which exhibited clearly positive TGF-P immunoreactivity, and the non-transplanted kidneys. Thus, the staining in the non-transplanted tissue was very weak and the morphologically intact non-rejected grafts showed an increased immunoreactivity compared to the non-transplanted kidneys. When all materials were classified into two groups as for the presence of diabetes mellitus and compared with the expression of all TGF-Ps, including LTBPl, a higher expression was found in kidneys from the diabetic patients (F = 5.92; P = 0.016). However, none of the five non-transplanted kidneys were from patients with diabetes mellitus, and this significant difference disappeared when non-transplanted kidneys were excluded from the material. In the transplants the immunoreactivity was generally stronger than that in the non-transplanted tissue. However, the tubuli cells in the transplanted kidneys lacked expression of the TGF-Ps (Figure lB, D, F, H). Lymphocyte aggregates, occasionally found in the transplanted kidneys, also stained mostly negative (Figure lB, F). In the allografts the expression of LTBPl was moderately increased in all three compartments compared to the non-transplanted kidney (Figure lA, B). The LTBPl staining was moderately strong in the blood vessels and less strong in the interstitium, whereas the glomendi was mostly negative (Figure 1B). When compared with the nontransplanted kidneys (Figure lA), a significant difference was seen in the interstitium of the transplanted tissue (F= 13.63; P = 0.001) and in the glomeruli (F = 17.55; P = 0.0004), but not in the blood vessels (F = 1.04; NS). Transplant Immunology 1996; 4: 209-214
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Transplantation was the most important predictor for expression of TGF-P 1, and the largest increase in the allografts occurred in the interstitium (F = 14.03; P = 0.0009). The intensity of the immunostaining in the glomeruli ranged from very weak to moderately strong, but was still elevated when compared with the non-transplanted kidneys (F = 5.02; P = 0.036). The blood vessels stained moderately positive and differed from non-transplanted kidney (F = 12.10; P = 0.0041). A distinct immunostaining pattern was seen with the TGFp2 antibody, which stained the vascular smooth muscle cells strongly (Figure 1F). However, the staining intensity was only slightly increased when compared with the normal vascular cells in the non-transplanted kidneys (Figure 1E) which also expressed the protein (F = 6.16; P = 0.020). In the allograft the TGF-P2 immunoreactivity was more intense in all compartments except in the tubuli which were essentially negative. The largest increase in the allografts occurred in the interstitium (F = 17.57; P = 0.0003). A significant increase was also observed in the glomeruli (F = 13.58; P = 0.0018). TGF-p3 showed the most intense immunostaining and a slightly larger difference between the transplanted and nontransplanted kidneys (Figure lG, H) when compared to the other members of the TGF-P family. The most prominent increase in the TGF+3 immunoreactivity (Figure 1H) occurred in the interstitium of the transplanted tissue (F= 48.16; P = O.OOOl), closely followed by the mesangial cells of the glomeruli (F = 10.72; P = 0.0035) and the blood vessels (F= 5.98; P = 0.028). Small blood vessels were in some cases hard to distinguish from the interstitium in the transplants, since the TGF+3 antibody stained the interstitium equally strongly.
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FXgure 1 Immunohistochemical staining of TGF-ps in a non-transplanted kidney (A, C,E, G) and a transplanted kidney (B, D, F, H). Note the increased immunoreactivity of LTBP (B) and TGF-@l (D) in the inters&km and the glomeruli of the transplanted kidney compared with the nontransplanted kidney (A, C), and the less marked increase of TGF-p2 immunoreactivity in the glomeruli and interstitium of the transplanted kidney (F). Blood vessels stained positive in both cases (E, F). Strongly enhanced TGF-/33 immunoreactivity is detected in the interstitium and the glomeruli as well as in the blood vessels of a transplanted kidney (H). In the non-transplanted kidney only the internal lamina elastica of the vessels stained positive. Arrowheads point to the glomeruli, and arrows point to the lymphocyte aggregates which are essentially negative. Original magnifications: x 66.
Transplant
Immunology
1996; 4: 209-214
Transforming growth factor+
Discussion In our search for the role of TGF-Ps in transplanted kidneys we have studied the in viva expression of TGF-Ps and LTBPl in biopsies from transplanted kidneys with or without rejection episodes, as well as from non-transplanted, morphologically intact kidneys. With regard to the expression of any of the three TGF-P isoforms and LTBPl, we were unable to find any significant differences between rejected kidneys of different types, as classified by the clinical course (acute or chronic) oy by the histological features of the biopsy specimens (vascular, interstitial reactions). None of these classifications revealed any significant difference in the expression of TGF-P. One clear difference was found, however, between transplanted and non-transplanted kidneys. Non-transplanted kidneys were mostly from non-tumorous tissue obtained in conjunction with removal of tumours, or in a few cases, donor kidneys without any kidney disease. Thus, all transplanted kidneys exhibited significantly stronger TGF+ expression than non-transplanted kidneys. Different types of rejection did not
demonstrate any difference in expression of TGF-p, either in clinical or in histological classifications. We also found a weak significant difference in the TGF-P expression between kidneys derived from diabetic and non-diabetic recipients. A diabetic environment can induce TGF-P expression according to several in vitro and in vivo studies.20*21This is not likely to be the cause of the TGF-P induction noted in this study, however, since the difference disappeared when the non-transplanted kidneys were excluded. This distinct difference between transplanted kidneys and non-transplanted kidneys supports the idea that transplantation itself rapidly induces the expression of TGF-P. In our previous study of heterotopic cardiac transplantations in rats, we could unambiguously show a strong and clear induction of TGF-PI and LTBPI, and to a lesser extent TGF-P2, in monocytes as well as in thickened blood vessels in allogeneic transplants.z2 CyA has been reported to induce TGF-@:3 and might be a factor affecting the increased expression in the transplanted kidneys. However, in our recent study on transplanted aortas in rat we noted significant differences in the expression of TGF-Ps between the allogeneic and syngeneic combinations, although none of those rats received CYA.~~Furthermore, the induction seemed to be very rapid in the allogeneic transplants since no significant increase in TGF-P expression was seen with observation time after transplantation. In the present clinical materials all specimens were allogeneic transplants. It is thus possible that a similar mechanism would be responsible for the induction of the TGF-P in the transplanted kidneys. The expression patterns of the various TGF-P isoforms were clearly different in our material. Thus, the strongest immunoreactivities were found for TGF-P3 in both golmeruli and interstitium. Similar distributions but weaker reactivities were detected for TGF-PI. A distinct and strong expression for TGF-/32 was found in blood vessels as well as in the glomeruli, although a similar pattern of weaker expression was seen in the non-transplanted kidneys. This is also concordant with our previous findings in various tissues, suggesting that the TGF-P2 might play an important physiological role in normal blood vessels. The LTBPl immunoreactivity was diffusely located in both glomeruli and interstitium, indicating that this protein might be present in association with extracellular matrix which is known to be produced in an increasing amount in nephritis and other inflammatory diseases of the kidney. The Transplant Immunology 1996; 4: 209-214
in human kidney transplants
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extracellular LTBPl might be able to regulate the activation of TGF-p.” Several experimental studies attempting to block the TGFp activities in the kidneys by means of in vivo administration of TGF-p antagonists demonstrated dramatic improvements in kidney function, sometimes being able to halt the course of disease.26*27Thus, the negative effects of TGF-f3 in kidney disease seem to be established.28 Despite the favourable effects of TGF-p in transplantation through suppression of immune reactions, the overall results of XF-B in the kidney seem to be negative. Whether this phenomenon is restricted to kidney transplantation remains to be further examined.
This work was supported in part by grants from the Ingrid and Fredrik Thuring Foundation, the Selander Foundation and the Swedish Medical Research Council.
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