Path. Res. Pract. 187,957-962 (1991)

Apolipoproteins and Immunohistological Differentiation of Cells in the Arterial Wall of Kidneys in Transplant Arteriopathy 1 Morphological Parallels with Atherosclerosis E. Vollmer, A. Bosse, J. Bogeholz, A. Roessner, S. Blasius, A. Fahrenkamp and W. Boeker Gerhard-Domagk-Institut fOr Pathologie, Westfalische Wilhelms-Universitat MOnster, FRG

c. Sorg Institut fOr experimentelle Dermatologie, Westfalische Wilhelms-Universitat MOnster, FRG

SUMMARY 22 nephrectomy specimens of renal allografts in chronic rejection after periods between 3 and 96 months, were studied immunohistologically. Various cell types in the arterial wall were characterized with antibodies specific against different cells of the mononuclear phagocyte system, against smooth muscle cells, and against differentiating lymphoid cells. In addition, the metabolism of lipoproteins was investigated using appropriate antibodies against several apolipoproteins. Subendothelial plaques of foam cells were found to consist of macrophages in foamy transformation. At the stage of intimal fibrosis the smooth muscle cells are more prominent. Lymphatic infiltration consists almost exclusively of T-Iymphocytes. Apolipoprotein analysis reveals deposits of Apo A j , A2 and BJ, most of them extracellular. According to these results, it is not only immunologic factors that are involved in arterial wall reactions during chronic transplant arteriopathy, but disorders of the lipoprotein metabolism - probably due to endothelial dysfunction - are also playing an important role like in atherosclerosis.

Introduction So far, the pathogenesis of transplant arteriopathy has not been explained and understood in all its aspects. It was repeatedly suggested that cellular reactions in the arterial wall might be related to the morphogenesis of arteriosclerosis 2,3,5,15,27. Today, the principal concept is that some

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Supported by DFG grant VO 386/1.

© 1991 by Gustav Fischer Verlag, Stuttgart

immunologic damage to the vascular endothelium is responsible for accelerated changes in the arterial walP. Recent studies of the atherosclerotic plaque have shown that, correlated to the consecutive stages, deposits of Apolipoproteins Al and B are found in both intra- and extracellular location4, 7,10,11,27. The present study comprises investigations of 22 nephrectomy specimens in chronic rejection. Our main interest focused on the analysis of different cell populations in the arterial wall, and on a comparison with corresponding conditions in the atherosclerotic plaque, with additional analysis of the lipoprotein metabolism. 0344-0338/9110187-0957$3.5010

958 . E. Vollmer et al.

Material and Methods 22 nephrectomy specimens in chronic transplant rejection were available for our investigation. The transplants had remained in situ for periods from 3 months up to 96 months. The age of donors was 5 to 50 years, that of recipients 12 to 64 years. Renal tissue was prepared for immunohistochemistry immediately after explant. Samples were fixed in buffered 4% paraformaldehyde and carefully embedded in paraffin. Since primary antibodies could be applied to paraffin sections, the reactions were carried out on thin paraffin sections, a method offering the advantage of distinctly better preserved cytologic structures. Antigens were demonstrated with the modified AP AAP method 6• The scale of primary antibodies used is listed on Table 1. Monoclonal antibodies were used in dilutions up to 1: 10, polyclonal antibodies 1: 20000. The latter required an extra incubation step, i.e. an additional mouse-antisheep antibody 1: 125 (DIANOV NHamburg) was used between the primary and the link antibody (rabbit-antimouse 1: 30, DAKOlHamburg). The APAAP complex (1: 100, DAKO/Hamburg) as well as the previously applied antibodies were maintained on the slides for 30 min each. All the incubation steps alternated with 5 min thorough washing of the samples in tris-buffer. Color development required 30 min incubation in

Table 1. Primary antibodies used in the investigation Directed against

Name

References

a) smooth muscle cells

anti-muscle-actin 1 Dako-desmin D 33 2

24,25, 21

b) macrophagesl monocytes

25-F-9.3 27-E-I03

29 30

c) lymphocytes

Dako-UCHL 12 (T) Dako-L 26 2 (B)

22 18

d) apolipoproteins

anti-h-Apo A14 anti-h-Apo A24 anti-h-Apo B4

27,10 27,10 27,10

Suppliers: IOrtho (Enzo)/Heidelberg; 2DakolHamburg; 3Institute for Experimental Dermatology, University of Munster; 4Boehringer/Mannheim. a, b, c = monoclonal mouse antibodies, d = polyclonal sheep antibodies. naphthol AS-BI phosphoric acid/dimethyl formamide together with a neofuchsin solution. At the same time, endogenous alkaline phosphatase was suppressed by levamisole.

Fig. 1. Strong staining of foam cells with antibody 25-F-9 against mature tissue macrophages (a) in contrast to the monocyte marker 27-E-1O (b), which confirms the macrophage nature of foam cells (x 60).

Apolipoproteins in Transplant Arteriopathy . 959

Results Even in transplants that had been in situ for only 3 months interlobular arteries showed subendothelial areas of foam cells obliterating the vascular lumen almost completely. Staining with antibody 2S-F-9 (against mature tissue macrophages) revealed strong positivity in all foam cells (Fig. 1a). In comparison, staining with antibody 27-E-10 (against blood monocytes) failed to give significant positive results (Fig. 1b). Only a few isolated infiltrating cells are seen among the foam cells, and so these may be supposed to be immigrating monocytes of the recipient, which will later be maturing to macrophages, and then be transformed into foam cells. Lymphoid cells lie between these foamy macrophages. Stained with antibodies against T-cells they appear as a distinct infiltrate (Fig. 2a), whereas immunohistochemistry fails to yield positive results for B-cells in the arterial intima (Fig. 2 b). Apolipoproteins (APO) Al and A2 are known as the main protein components of high density lipoproteins (HDL). Staining with antibodies against these reveals extracellular deposits in the arterial intima, and an intra-

cytoplasmic positivity in foam cells is also observed (Fig. 3a). Staining with antibodies against Apolipoprotein B (chief protein component of low density lipoproteins = LDL) demonstrates predominantly extracellular deposits of that substance (Fig. 3 b). Advanced arterial alterations always manifest distinct intimal fibrosis. The amount of foam cells is receding, and numerous spindle cell formations are seen instead. Staining with the antibody against the epitope of a muscle-specific actin isotope elicits distinct positivity in those spindle cells (Fig.4a), thereby confirming their smooth muscle cell nature. These transformed smooth muscle cells are capable of producing various substances of the extracellular matrix, predominantly collagens, proteoglycans, and elastic lamellae. Quite another picture results from staining with the antibody against desmin: there will be no significant positivity for this muscle-specific intermediate filament in the transformed smooth muscle cells of the arterial intima (Fig. 4 b). Particular interest is due to the behavior of apolipoproteins in stages with advanced intimal fibrosis. Stained with antibodies against Apo Al and Az, these areas will show

Fig. 2. Distinct labelling of T cells (a) in contrast to B cells (b) of the lymphocytic infiltration between foam cells of the arterial intima (x 120).

960 . E. Vollmer et al.

Fig. 3. Deposits of Apolipoprotein AI within foam cells and in extracellular spaces (a) in contrast extracellular deposits (b) (x 120). extracellular deposits which, however, are also found in transformed smooth muscle cells obviously also involved in the HDL metabolism. Apo B also shows predominant extracellular positivity. Discussion Transplant arteriopathy is the most severe complication affecting immunologic reactions in the transplanted organ. Quite often, the disease responds poorly or not at all to therapy and will eventually lead to complete loss of transplant functions 2, 14, 15 • Transplants in chronic rejection are particularly liable to changes associated with complete obliteration of vascular lumina. Seen under formal pathogenetic aspects, these stages of vascular obliteration display very clear relationships with the stepwise development of arteriosclerosis as described by several authors 15, 18, 19. There is indeed a principal similarity, but according to the results presented here, the picture of foam cell areas is much more clearly manifested in transplant arteriopathy than in the development of arterio-

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Apolipoprotein B with only

sclerosis. In the latter, the nature of the appearing foam cells is fully determined: most of them are in fact macrophages in foamy transformation entering the arterial intima 1,22. With the aid of distinctive monoclonal antibodies against cells of the mononuclear macrophage system, analogous results could be verified in the subendothelial foam cell areas observed in transplant arteriopathy. Stained with antibodies against mature tissue macrophages 29 , the foam cells revealed strong positivity, but staining with an antibody against blood monocytes 30 could reveal only a few isolated infiltrating cells between foam cells. This suggests that blood monocytes first immigrate into the arterial intima, where they mature into histiocytes, and are finally transformed into foam cells. The question why the cells of the mononuclear macrophage system should invade the arterial intima at all, leads us to the problems of lipoprotein metabolism in the arterial wall. It is known from heart transplantation that such foam cells will contain numerous cholesterol esters3 . It may be inferred that, in analogy to the process in arteriosclerosis, there must be an insudation of cholesterolbearing LDL into the arterial intima. Accordingly, Apo B,

Apolipoproteins in Transplant Arteriopathy . 961

Fig. 4. Advanced vascular changes with marked intimal fibrosis. Spindle-shaped cells distinctl y expressing actin (a) in contrast desmin (b) (X 330).

chief component of LDL, could be demonstrated immunohistologically in the arterial intima even in transplant vasculopathy, as it had been confirmed previously in arteriosclerosis4, 7,10,17,27. According to current knowledge, activation and alteration of endothelial functions may be of special importance for the cellular adhesion of Iymhocytes and monocytes 15 • This may also lead to an enhanced influx of lipoproteins which in turn evokes further cell reactions, and the eventual transformation of those macrophages into foam cells 16 • Moreover, the subendothelial areas of foam cells would also show a strong positive reaction of Apo Aland A2, the chief structural proteins of HDL. Similar conditions are known from the atherosclerotic plaque 4, 27, but the true role of Apo Al and A2 is as yet unclear. Since higher blood levels of Apo Al are known to exert a protective effect, it might be able to delay atherogenesis in the plaque 11 , too. Despite the unexplained role, the rather similar distribution pattern of apolipoproteins in transplant arteriopathy and in the atherosclerotic plaque is apt to enhance the pathogenetic correspondences between the two lesions and their development.

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Macrophages are known to secrete certain factors that induce the proliferation of smooth muscle cells 9 , 12, 13. The same mechanism may be supposed to favor the proliferation of smooth muscle cells in transplant arteriopathy, which eventually leads to advanced stages with obliterating intimal fibrosis. The smooth muscle cell nature of the spindle cells obviously forming the extracellular matrix can be readily verified by immunohistological methods. The muscle-specific anti-actin antibody24,25 shows strong positivity in the transplant arteries with intimal fibrosis, thereby confirming the smooth muscle cell nature of those spindle-shaped cells. Smooth muscle cells in the atherosclerotic plaque will show alterations in the expression of intermediate filaments, favoring vimentin of desmin which is manifested poorly or not at a1l 8 • In the same way no significant staining of spindle cells with antibodies against desmin was found in many areas of transplant arteriopathy specimens. The distinctly different distribution of T- and Blymphocytes in the arteriosclerotic plaque, and their functional impact on the pathomechanism of atheroscle-

962 . E. Vollmer et a1.

rosis itself, are still under controversial discussion 26 • The results of our study revealed that besides monocytes and macrophages, T-lymphocytes seem to play an important role in obliterating transplant vasculopathy with considerable bearing on the entire immune mechanism. In agreement with parallel findings in atherosclerosis, these observations suggest that, judging by many functional and morphological congruences, transplant vasculopathy may be partly interpreted as an accelerated variant of arteriosclerosis. References 1 Aquel NM, Ball RY, Waldmann H, Mitchinson MJ (1985) Identification of macrophages and smooth muscle cells in human atherosclerosis using monoclonal antibodies. J Pathol 146: 197-204 2 Bohle A, Gartner HV, Laberke HG, Kruck F (1984) Die Niere. Struktur und Funktion. F. K. Schattauer, Stuttgart-New York 3 Billingham ME (1987) Cardiac transplant atherosclerosis. Transpl Proc XIXl4, Supp1. 5: 19-25 4 Carter RS, Siegel RJ, Chai AU, Fishbein MC (1987) Immunohistochemical localization of apolipoproteins Al and B in human carotid arteries. J PathollS3: 31-36 5 Cerilli J (1987) The role of autoantibody to vascular endothelial cell antigens in atherosclerosis and vascular disease. Transpl Proc XIX/4 Suppl 5: 47-49 6 Cordell JL, Falini B, Erber WN, Ghosh AK, Abdulaziz Z, MacDonald St, Pulford KAF, Stein H, Mason DY (1984) Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). J Histochem Cytochem 32: 219-229 7 Feldmann DL, Hoff HF, Gerrity RG (1984) Immunohistochemical localization of apoprotein B in aortas from hyperlipemic swine. Arch Pathol Lab Med 108: 817-822 8 Gabbiani G, Schmid E, Winter S, Chaponnier C, Chastonay DEC, VanderkerckhoveJ, Weber K, Franke WW (1981) Vascular smooth muscle cells differ from other smooth muscle cells: Predominance of vimentin filaments and a specific alpha-type actin. Proc Natl Acad Sci USA 78: 298-302 9 Glenn KC, Ross R (1981) Human monocyte-derived growth factor(s) for mesenchymal cells: Activation of secretion of endotoxin and concavalin A. Cell 23: 603-615 10 Harrach B, Robenek H, Vollmer E, Roessner A, Bosse A, Drescher H, Boeker W (1989) Immunelektronenmikroskopische Lokalisation von Apolipoproteinen in Zellkulturen und dem arteriosklerotischen Plaque. Verh Dtsch Ges Path 73: 675 + 11 Hoff HF, Lie JT, Titus JL, Bajardo RJ, Jackson RL, Debakey ME, Gotto AM (1975) Lipoproteins in atherosclerotic lesions. Arch Pathol 99: 253-258 12 Leibovich SJ, Ross R (1976) A macrophage-dependent factor that stimulates the proliferation of fibroblasts in vitro. Am] Pathol 84: 501-514 13 Martin BM, Gimbrone MA Jr, Unanue ER, Cotran RS (1981) Stimulation of nonlymphoid mesenchymal cell proliferation by a macrophage-derived 'growth factor. ] Immunol 126: 1510-1515

14 Mihatsch MJ, Zollinger HU, Gudat F, Schuppler ], Riede UN, Thiel G, Brunner F, Enderlin F (1975) Transplantation arteriopathy. Path Microbiol 43: 219-223 15 Muller-Hermelink HK, Dammrich JR (1989) Die obliterative Transplantatvasculopathie: Pathogenese und Pathomechanismen. Verh Dtsch Ges Path 73: 193-206 16 Munro JM, Cotran RS (1988) Biology of disease. The pathogenesis of atherosclerosis: Atherogenesis and inflammation. Lab Invest 58: 249-261 17 Niendorf A, Rath M, Wolf K, Peters S, Arps H, Beisiegel U, Dietel M (1990) Morphological detection and quantification of lipoprotein (a) deposition in atheromatous lesions of human aorta and coronary arteries. Virchows Archiv A 417: 105 -111 18 Norton A], Isaacson PG (1987) Monoclonal antibody L 26: an antibody that is reactive with normal and neoplastic B lymphocytes in routinely fixed and paraffin-wax-embedded tissue.] Clin Pathol40: 1405-1412 19 Roessner A, Herrera A, Honing HJ, Vollmer E, Zwadlo G, Schurmann R, Sorg C (1987) Identification of macrophages and smooth muscle cells with monoclonal antibodies in the human atherosclerotic plaque. Virchows Archiv A 412: 169-174 20 Ross R (1986) The pathogenesis of atherosclerosis - an update. N Engl J Med 314: 488-500 21 Schmid E, Osborn M, Rungger-Brandle E, Gabbiani G, Weber K, Franke WW (1982) Distribution of vimentin and des min filaments in smooth muscle tissue of mammalian and avian aorta. Exp Cell Res 137: 329-340 22 Smith S, Brown H, Marrion H, Rowe D, Callards RE, Beverley PCL (1986) Functional subsets of human helper-inducer cells defined by a new monoclonal antibody, UCHL 1. Immunology 58: 63-70 23 Stary HC (1987) Macrophages, macrophage foam cells, and eccentric intimal thickening in the coronary arteries of young children. Atherosclerosis 64: 91-108 24 Tsukada T, Tippens D, Gordon D, Ross R, Gown AM (1987) HHF 35, a muscle actin-specific monoclonal antibody. I. Immunocytochemical and biochemical characterization. Am J Pathol126: 51-60 25 Tsukada T, McNutt MA, Ross R, Gown AM (1987) HHF 35, a muscle actin-specific monoclonal antibody. II. Reactivity in normal, reactive, and neoplastic human tissues. Am J Pathol127: 389-402 26 Vollmer E, Maurer Th, Roessner A, Bosse A, Winde G, Boeker W (1988) Immunohistologische Untersuchungen zum Lymphozyteninfiltrat in unterschiedlichen Stadien der humanen Arteriosklerose. Verh Dtsch Ges Path 72: 600+ 27 Vollmer E, Brust J, Roessner A, Bosse A, Harrach B, Robenek H, Herrera A, Boeker W (1989) Immunohistochemische Untersuchungen zur Verteilung von Apolipoproteinen in der arteriosklerotischen GefaRwand menschlicher Arterien. Verh Dtsch Ges Path 73: 445 + 2S Zollinger HU, Mihatsch MJ (1978) Renal Pathology in Biopsy. Springer, Berlin-Heidelberg-New York 29 Zwadlo G, Brocker EB, v. Bassewitz DB, Feige U, Sorg C (1985) A monoclonal antibody to a differentiation antigen present on mature human macrophages and absent from monocytes.] Immunol134: 1487-1492 30 Zwadlo G, Schlegel R, Sorg C (1986) A monoclonal antibody to a subset of human monocytes found only in the peripheral blood and inflammatory tissues. J Immunol 137: 512-518

Received August 29, 1990 . Accepted December 17, 1990

Key words: Transplant artheriopathy - Apolipoprotein distribution - Cell differentiation - Atherosclerosis Dr. Dr. Ekkehard Vollmer, Gerhard-Domagk-Institut fur Pathologie, Domagkstr. 17, W-D 4400 Munster, FRG

Apolipoproteins and immunohistological differentiation of cells in the arterial wall of kidneys in transplant arteriopathy. Morphological parallels with atherosclerosis.

22 nephrectomy specimens of renal allografts in chronic rejection after periods between 3 and 96 months, were studied immunohistologically. Various ce...
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