Endothelial activation and cytokine expression in human acute cardiac allograft rejection

Pathology

ISSN: 0031-3025 (Print) 1465-3931 (Online) Journal homepage: https://www.tandfonline.com/loi/ipat20

Endothelial activation and cytokine expression in human acute cardiac allograft rejection Ruth N. Salom, Julie A. Maguire & Wayne W. Hancock To cite this article: Ruth N. Salom, Julie A. Maguire & Wayne W. Hancock (1998) Endothelial activation and cytokine expression in human acute cardiac allograft rejection, Pathology, 30:1, 24-29 To link to this article: https://doi.org/10.1080/00313029800169625

Published online: 06 Jul 2009.

Submit your article to this journal

Article views: 15

View related articles

Citing articles: 1 View citing articles

Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=ipat20

Pathology (1998), 30, pp. 24-29

Pathology (1998), 30, February

ENDOTHELIAL ACTIVATION AND CYTOKINE EXPRESSION IN HUMAN ACUTE CARDIAC ALLOGRAFT REJECTION RUTH N. SALOM*t, JULIE A. MAGUIRE* AND WAYNE W . HANCOCK~

Department of Pathology and hnmunology, Monash Medical School, Prahran*, Dorevitch Pathology, Fairfieldt, Victoria Australia, and Sandoz Center of Immunobiology, Harvard Medical School, Boston, Massachusetts, USA$

Summary By extrapolation from the responses of cultured human umbilical vein endothelial cells (EC) and bovine aortic EC to short-term cytokine stimulation, EC activation is postulated as a likely component of the host response in acute allograft rejection and cardiac transplant-associated accelerated arteriosclerosis. To investigate the extent to which EC activation occurs in vivo in humans and to identify potential targets for therapeutic interventions, we compared the phenotypic characteristics of vascular EC as seen during clinicopathologically significant vs non-significant acute cardiac altograft rejection. We used monoclonal and monospecific polyclonat antibodies to coagulation molecules [tissue factor, thrombomodulin (TM), antithrombin 111 (AT-Ill), fibrinogen/fibrin, cross-linked fibrin and von Willebrand factor (vWF)], adhesion molecules (P-selectin, ICAM-1) and major histocompatibility complex (MHC) class I and II molecules. In addition we sought evidence of local cytokine production (IL-1, tL-2R, IL-4, IL-6, IL-7, IL-8, TNF-c~, PDGFAA, PDGF-BB), which might mediate alterations in expression of these proteins. We found that in clinically significant grades of cardiac allograft rejection requiring increased immunosuppression, in contrast to lesser grades of rejection not requiring clinical intervention, there was increased microvascular EC activation and differential expression of cytokines. EC changes associated with more extensive cardiac allograft rejection requiring treatment included: (i) disruption of the normal anticoagulant state with downregulation of TM and AT-Ill, upregulation of tissue factor and vWF expression, and associated extensive fibrin deposition; (ii) upregulation of MHC class 1 antigens, which are potential targets for host cytotoxic T lymphocytes; (iii) increased expression of the leucocyte adhesion molecules P-selectin and ICAM-1; (iv) expression of the proinflammatory cytokines IL-lfl and TNF-cq and (v) increased expression of PDGF-AA and BB, which are known to promote migration and proliferation of intimal cells, and hence may contribute to development of transplant-associated atherosclerosis. Collectively these findings suggest that immune events resulting in EC surface changes and/or production of key cytokines play a role in the pathogenesis of acute transplant rejection and may contribute to the longterm complication of accelerated arteriosclerosis in allograft coronary arteries.

Key words: Endothelium, cytokines, allograft, cardiac. Abbreviations: AT-Ill, antithrombin III; ATGAM, anti T celt irnmunoglobulin; EC, endothelial cells; EMB, endomyocardialbiopsies; IL,

interleukin; mAbs, monoclonal antibodies; MHC, major histocompatibility complex; PC, protein C; PDGF, platelet-derived growth factor; PECAM, platelet endothelial cellular adhesion molecule;PS, protein S; TM, thrombomodulin; TN~e, tumor necrosis factor-alpha; vWF, yon Willebrand factor; XL-, cross linked. Accepted 21 July 1997

INTRODUCTION Endothelial cells (EC) provide the initial array of allogeneic molecules that stimulate a host alloresponse and are the first targets of allo-injury. In addition, EC are thought to play an active role in the development of allograft rejection, 1'2 arteriosclerosis 3'4 and other diseases with an important inflammatory component, since stimulation of EC, at least in vitro, results in multiple responses which collectively are termed endothelial activation. 5 These in vitro responses include induction of leucocyte adhesion molecules, production of multiple pro-inflammatory molecules including certain cytokines, and conversion of endothelial surfaces from an anticoagulant to a procoagulant state. 5'6 While no unequivocal systemic marker of EC activation in vivo has been established, these events may be visualised directly in tissue sections using appropriate monoclonal antibodies (mAbs) to antigens whose expression is either up- or downregulated following EC activation. 7m Studies of cell adhesion molecules have shown differences in the level of basal expression of different molecules and their inducibility by various stimuli. Vascular adhesion molecules, such as ICAM-1, platelet endothelial cellular adhesion molecule (PECAM), and VCAM-1 are constitutively expressed at low levels in normal human heart, but rapidly increase in cardiac allografts undergoing rejection. 2 In vitro these molecules mediate the binding of leucocytes to E C 5'6 and facilitate their extravasation at the inflammatory site. The effects of endothelial activation on the normally anticoagulant surface of vascular EC were only recently recognised. 5 The anticoagulant state of resting EC is maintained by antithrombin III (AT-III), heparin co-factor II and the thrombomodulin (TM)/protein C (PC)/ protein S (PS) system, which together inhibit the activity of procoagulant factors such as factors Va, VIIIa, XIIa, XIa and IXa. EC also produce inhibitors of platelet activation (eg prostacyclin) and fibrinolytic proteins. 5 Thus, EC contribute to the local regulation of clotting, platelet activation and clot dissolution. Knowledge of the mediators of such EC activation is still largely derived from in vitro studies;

0031-3025/98/010024-6 © 1998 Royal College of Pathologists of Australasia

HUMAN ACUTECARDIACALLOGRAFTREJECTION 25 TABLE 1 lmmunosuppression and timing and histologic grade of rejection Patient

Sex

Immunosuppression

GR

M

CsA/Aza/Prednisone

FPa

M

CsA/Aza/Prednisone

EW

F

CsA/Aza/Pred/ATGAM

AK

M

CsA/Aza/Prednisone

SB

M

CsA/Aza/Pred/ATGAM

PM

M

CsA/Aza/Prednisone

BU

M

CsA/Aza/Prednisone

DM b

M

CsA/Aza/Prednisone

Weeks Post-Tx

Grade

2 3 6 2 4 5 6 9 2 4 6 7 6 7 18 26 28 18 22 25 77 81 126

1B 3A 3A 3A 1B 1B 1B 1B 3A 1B IB 1B 1B 3A 3A 1B 1B 1B 1B 1B 1B 1B 3B

a Initial convalescence was complicated by colonisation of central venous catheter by Staphylococcus epidemidis, S. aureus and Actinobacter; systemic antibiotics were given from days 10 to 23. b Post-mortem cardiac tissue from patient who died from acute rejection after voluntarily stopping his immunosuppression.

additional 4-5 biopsies were sent for routine histological assessment) and was transferred to the laboratory in PBS, snap-frozen in liquid nitrogenchilled isopentane and stored at - 7 0 ° C until use. Cryostat sections (4 #m) were fixed in paraformaldehyde-lysine-periodate for demonstration of leucocytes and activation-associated antigens, or in acetone for studies of cytokines and coagulation molecules, and stained by a 4-layer (for mAbs), or a 3-layer (for polyclonal antibodies) peroxidase-antiperoxidase method) 2 Briefly, this involved overnight incubation at 4°C with primary antibodies, treatment with methanol/H202 to block endogenous peroxidase, 30 min incubations at room temperature with subsequent antibody layers, prolonged incubation with the substrate diaminobenzidine, hematoxylin connterstain and mounting. All EMB sections were stained at the same time under the same conditions for each antibody examined.

Antibodies Sections were stained using mAbs to leucocytes (CD45; Dako, Carpinteria, CA), IL-lfl (Olympus, Piscataway, NJ), TNF-c~ (courtesy of Professor I. MacKenzie, Melbourne, Australia), IL-7 (Genzyme, Boston, MA), IL-2R (CD25; Dako), and polyclonal Abs to IL-4, IL-6, IL-7, IL-8, PDGF-AA and PDGF-BB (Genzyme). Endothelial cell activation was assessed using mAbs to class II antigens (HLA-DR), ICAM-1 (CD54; Dako), TM (courtesy of Dr H. Salem, Melbourne), yon Willebrand factor (vWF; Dako), AT-III (AT-III; Dako) and tissue factor (American Diagnostica, Sydney, NSW), and a polyclonal Ab to P-selectin (Dr M. Berndt, Melbourne). A mAb to a shared fibrinogen/fibrin determinant and a mAb specific to cross-linked (XL-) fibrin were obtained from Dr B. Kudryk (Center for Blood Research, New York, NY).

Evaluation of immunoperoxidase labelling c y t o k i n e s s u c h as i n t e r l e u k i n - 1 (IL-1) a n d t u m o r n e c r o s i s f a c t o r - a l p h a ( T N F - c 0 b i n d to, a n d activate, EC, 5 w h i l e others, s u c h as I L - 4 a n d IL-13, c a n b l o c k i n d u c t i o n o f t h e p r o c o a g u l a n t state. 1° T o d e t e r m i n e t h e e x t e n t a n d clinical utility o f E C activ a t i o n in vivo, w e a n a l y s e d a series o f e n d o m y o c a r d i a l b i o p s i e s ( E M B ) f r o m cardiac allograft r e c i p i e n t s in w h i c h h i s t o l o g i c e v i d e n c e o f acute r e j e c t i o n w a s sufficient to w a r r a n t i n c r e a s e in t h e i r i m m u n o s u p p r e s s i o n c o m p a r e d to t h o s e i n w h i c h n o rejection, or o n l y m i n o r r e j e c t i o n n o t r e q u i r i n g a l t e r a t i o n in i m m u n o s u p p r e s s i o n , w a s o b s e r v e d . Specifically, w e e x a m i n e d (a) E C e x p r e s s i o n o f a d h e s i o n , c o a g u l a t i o n a n d M H C m o l e c u l e s ; a n d (b) the e x t e n t to w h i c h a l t e r a t i o n in p r o t e i n e x p r e s s i o n w a s a s s o c i a t e d w i t h t h o s e c y t o k i n e s t h a t p r o m o t e E C a c t i v a t i o n in vitro.

MATERIALS AND METHODS Tissues EMB (n 22) and one failed cardiac allograft from 8 cardiac transplant recipients were studied (Table 1). Each patient received triple therapy with cyclosporine A, azathioprine and prednisolone. One patient also received anti T cell immunoglobulin (ATGAM) for the first 14 d posttransplantation. No additional immunosuppressive therapy was received within 2 wks prior to the EMB studied. EMB were obtained at the time of clinically indicated sampling for histopathologic assessment of rejection. Diagnosis and grading of rejection were based on examination of corresponding paraffin-embedded tissues according to the standardised cardiac biopsy grading of the International Society for Heart Transplantation (Table 2). 11 Control non-transplant cardiac tissues ( n = 4 ) were obtained at postmortem, within 24 h of death.

Immunohistology One EMB was obtained at each clinically indicated biopsy procedure (an

Tissue labelling was evaluated independently of the clinical or histologic diagnosis and the entire EMB sample was examined microscopically using a × 40 objective. Labelling was graded semiquantitatively due to the presence of reaction products beyond individual cells (cytokines), or the extensive, often continuous labelling of EC. Expression of each antigen on the EC surface was graded for the intensity and extent of EC labelling. Intensity was assessed from 0 to 5 + , whereby 0 indicates no staining, 1 fine granular staining just visable, 2 fine granular staining easily visable, 3 coarse granular staining, 4 intense coarse granular staining, 5 intense staining with coalescence of granules. The extent of EC labelling was graded as negative, < 10%, 10-75%, > 75% in each EMB section.

RESULTS Histology S i x t e e n o f the 22 E M B studied w e r e classified as s h o w i n g n o n - s i g n i f i c a n t r e j e c t i o n ( g r a d e 1B) n o t w a r r a n t i n g a n y i n t e r v e n t i o n or c h a n g e i n f o l l o w up, a n d six b i o p s i e s w e r e clasgified as significant r e j e c t i o n a s s o c i a t e d w i t h m y o c y t e n e c r o s i s ( g r a d e 3A) a n d n e c e s s i t a t i n g clinical i n t e r v e n t i o n a n d i n c r e a s e d i m m u n o s u p p r e s s i o n . T h e failed allograft s h o w e d g r a d e 3B rejection. T h e r e w a s n o e v i d e n c e o f intravascular coagulation in any EMB.

Immunohistologic analysis o f untransplanted cardiac tissue S e c t i o n s o f c o n t r o l n o r m a l h u m a n c a r d i a c tissue c o n t a i n e d few l e u c o c y t e s ( < 1/field) a n d l a c k e d a n y c y t o k i n e expression. N o l a b e l l i n g for P - s e l e c t i n w a s o b s e r v e d , a n d d e t e c t i o n o t~ I C A M - 1 a n d v W F w a s restricted to large v e s s e l EC. B y contrast, l a r g e v e s s e l s a n d all o f the m i c r o v a s c u l a t u r e s h o w e d s t r o n g s t a i n i n g for A T - I I I a n d T M . A b o u t 3 0 % o f E C s h o w e d l a b e l l i n g for M H C class I a n d class II antigens. M H C class II w a s also d e t e c t e d o n o c c a s i o n a l interstitial d e n d r i t i c cells w i t h i n the m y o c a r d i u m , b u t n o l a b e l l i n g o f m y o c y t e m e m b r a n e s for

26

SALOMet at.

Pathology (1998), 30, February

TABLE 2 Standardisedsystem of the InternationalSociety for Heart Transplantationfor grading of EMB11 Grade

Description

0 IA IB II III

No rejection Focal perivascularor interstitialinfiltratewithout myocyte damage Diffuse, sparse perivascularand/or interstitialinfiltratewithout myocyte damage Single focus of aggressive infiltrationand/or focal myocytedamage A = Multifocal aggressive infiltratesand/or myocyte damage B = Diffuse inflammatoryprocess with myocyte damage Diffuse polymorphicinfiltrateand edema, hemorrhage, vasculitisand necrosis

IV

TABLE 3 Semiquantitativeassessmentof immunoperoxidaselabellingof cytokines, adhesion, coagulationmad MHC molecules on vascular EC in varying grades of cardiac allograft rejection Protein Intensity IL-lfl IL-4 IL-6 IL-7 IL-8 TNF-c~ PDGF-AA PDGF-BB MHC class I MHC class II ICAM-1 P-selectin Tissue factor TM Fibrinogerdfibrin XL-fibrin AT-III vWF

0 3 4 0 3 0 1 1 4 4 2 3 3 5 4 4 5 5

Grade 1 Involvement(%)

Grade 3A Intensity Involvement(%)

0 > 75 > 75 0 > 75 0 < 10 < 10 10-75 10-75 < 10 > 75 < 10-75 > 75 > 75 > 75 > 75 > 75

2 1 2 2 2 2 2 2 4 5 3 4 4 2 4 5 3 5

either class of MHC molecule was observed. Other proteins were not detected in normal tissue apart from focal, occasional EC labelling for tissue factor.

Immunohistologic analysis of cardiac allografts In our previous analysis of these and additional EMB, 12 the absolute number of leucocytes, consisting primarily of T cells and macrophages, increased with increasing grade of rejection, though the proportions of each cell type remained similar; comparable results were found with respect to the numbers of activated (IL-2R + ) intragraft mononuctear cells. The current study examined the effects of intragraft mononuclear and/or endothelial cell immune activation by analysing immunostaining for cytokines, adhesion molecules, MHC and coagulation proteins. The results are summarised in Table 3, which lists the mean value for each grade of rejection examined, and illustrated in Fig. 1.

Cytokines: Grade 1B rejection was associated with minimal or no staining for IL-1/~ or TNF-c~, whereas in grade 3A and 3B rejection, these cytokines were detected on and adjacent to the surfaces of intragraft leucocytes, as well as the membranes of most EC and vascular smooth muscle within the media of small arteries and arterioles. Focal myocardial cell staining for TNF-c~ was also detected in grade 3A and 3B but not grade 1B cardiac rejection. Like TNF-c~, IL-7 labelling was present on mononuclear cells, vascular EC and on focal myocytes during grade 3A and 3B rejection but not during grade 1B rejection. In contrast,

< 10 10-75 10-75 < 10 10-75 < 10 > 75 10-75 > 75 > 75 10-75 > 75 > 75 10-75 > 75 > 75 10-75 > 75

Grade 3B Intensity Involvement(%) 3 1 1 2 1 5 4 2 5 5 3 5 5 2 4 5 3 5

10-75 10-75 < 10 < 10 < 10 > 75 > 75 t0-75 > 75 > 75 10-75 > 75 > 75 10.75 > 75 > 75 10-75 > 75

staining for IL-4, IL-6 and IL-8 was maximal in grade 1B rejection and showed little or no staining in higher grades. Labelling for both isoforms of PDGF (platelet-derived growth factor) were increased in the higher grades of rejection, though the expression of P D G F - A A was always more intense and extensive than that of PDGF-BB (Fig. 1 m and n).

MHC molecules: There was an increase in EC expression of class I and class II antigens with increasing grades of allograft rejection. In addition, during grade 3B rejection, there was dense labelling for MHC class I antigens on myocardial cell membranes (Fig 1 k and 1). Mononuclear cells were consistently positive for both MHC class I and class II antigens.

Adhesion molecules: With increasing rejection there was increased expression of ICAM-1 on EC and extensive labelling of essentially all of the microvasculature (Fig. 1 a and b). P-selectin expression, which reflected both intragraft platelets and EC, also increased with advancing grade of rejection.

Coagulation proteins: Increasing rejection was associated with a marked downregulation of capillary and microvascular TM and AT-III expression, and concomitant upregulation of tissue factor (Fig 1 g and h). There was strong staining of all EC for TM expression during grade 1B rejection, whereas TM was markedly decreased during grade 3A and 3B rejection (Fig. 1 c and d); this decrease

HUMAN ACUTE CARDIAC ALLOGRAFT REJECTION

27

Fig. 1 Paired photomicrographs of immunoperoxidase labelling for cytokines, adhesion and coagulation molecules and MHC antigens in serial cryostat sections of human cardiac allografts diagnosed as grade I rejection (left side panels: a, c, e, g, i, k, m) and grade 3 rejection (right side panels: b, d, f, h, j, 1, n). Increased rejection resulted in greater expression of the adhesion molecule ICAM-1 (a/b); disruption of the normal anticoagulant state with downregulation of the constitutive endothelial anticoagulant molecules, TM (c/d) and AT-III (e/f) and upregulation of the procoagulant molecule, tissue factor (g/h); upregulation of vWF (i/j); upregulation of MHC class I antigens on myocardial cell membranes, mononuclear cells and vascular endothelium (k/l); and increased expression of PDGF-AA by mononuclear cells and vascular endothetinm (m/n). (Cryostat sections, hematoxylin counter stain, original magnifications × 630).

28

Pathology (1998), 30, February

SALOM et aL

was particularly observed on microvascular but not venular EC. Comparable changes were seen in the intensity and distribution of labelling for AT-III (Fig. 1 e and f). Increasing rejection was also associated with increased EC labelling for vWF (Fig. 1 i and j) and XL-fibrin. XL-Fibrin was densely deposited on EC and adjacent to mononuclear cells in most EMB examined.

DISCUSSION This study of EMB from cardiac allograft recipients showed that clinicopathologically significant cardiac allograft rejection was associated with EC activation as evidenced by: (a) upregulation of the leucocyte adhesion molecules ICAM-1 and P-selectin; (b) development of a net procoagulant state at the EC surface with downregulation of TM and AT-III,-upregutation of tissue factor and vWF, and considerable local fibrin deposition; and (c) upregulation of MHC class I and class II molecules. In addition, compared to grade 1B rejection, intragraft EC activation occurring during higher grade rejection was accompanied by increased expression of the cytokines IL-I[~, TNF-a and PDGF-AA and BB on mononuclear cells and vascular EC, and a decrease in IL-4, IL-6, IL-7 and IL-8. These results suggest that immune events resulting in EC surface changes and/or production of key cytokines play an active role in the pathogenesis of clinically significant acute all0graft rejection. Furthermore, the release of mediators, growth factors and cytokines during repeated bouts of rejection, if accompanied by associated EC activation, could contribute to the proliferative response of -various cell types within the vascular wall and the development of accelerated atherosclerosis, which remains a common problem in cardiac allograft recipients. in a previous study of these and further EMB, 12 we found that increasing grades of acute myocardial rejection were associated with an increase in mononuclear cell infiltration, including activated (IL-2R + ) and proliferating mononuclear cells. IL-2R + mononuclear cells generate a plethora of cytokines, which may well induce many of the features of EC activation documented in the current study, although, as reviewed,5 local hypoxia, arachidonic acid metabotites and other factors could also contribute. Changes in the EC surface phenotype such as downregulation of TM and AT-III can, in vitro, be induced by exposure to IL-1/~ and TNF-ct. 5 We know that these cytokines are produced locally by infiltrating mononuclear cells during experimental cardiac allograft rejection8 and atherosclerosis] 3 and neutralization of TNF-e in vivo using an anti-TNF-e Ab preserves TM expressionJ 4 Moreover, TNF-c~ is expressed during human cardiac ~5 and renal allograft rejection] Indeed, during analysis of sequential renal transplant biopsies, TNF-c~ expression was inversely associated with alterations in the EC expression of TM during rejection episodes and following response to increased immunosuppression. 7 Hence, TNF produced by activated host mononuclear cells is a prime candidate for modulation of TM expression as witnessed in the current study, with consequent capacity for local fibrin generation and tissue ischemia. By contrast, whether TNF-e or other inflammatory mediators regulate AT-III expression by EC is unknown. Other previously documented effects of TNFc~ that are relevant to the current results include: direct

injury to adjacent myocardial cells; ~6 T cell activation, proliferation and production of cytokines such as IL-1; 17 augmentation of procoagulant properties by inducing tissue factor and downregulation of plasma or tissue levels of each component of the TM/PC/PS pathway; 7 increase in MHC class I and II expression; ~8 induction of E-selectin and increased expression of ICAM moleculesP In light of the present data, the observed increase in TNF-c~ production with increased grades of cardiac allograft rejection may well contribute to the increased IL-1 expression, the conversion of vascular endothelial surfaces from anticoagulant to procoagulant state and the induction/upregutation of MHC class I and II molecules. The observed induction of MHC I molecules on the myocardial membrane, and its upregulation on the vascular endothelium, would in turn act to increase the susceptibility of these targets to cytotoxic damage by LFA-1 + , CD8 + T cells. Lastly, the induction of MHC II molecules, which are recognised by T cells, could further increase the release of cytokines which enhance leucocyte adhesion and migration across EC into the graft. Increased EC and leucocyte labelling for the cytokine IL-4 in the absence of rL-1 and TNF-e, during nonsignificant rejection (grade 1B), may reflect the adequacy of the immunosuppressive treatment. In vivo studies show IL-4 to inhibit expression of IL-1 and TNF-c~ by mononuclear phagocytes and protect the endothelium and the monocyte surface against inflammatory mediator-induced procoagulant changes. 1° Similarly, IL-6, which is known to inhibit the production of TNF in vitro and in vivo, 19 was increased in non-significant rejection (grade 1B). PDGF has recently been implicated as a mediator of atherosclerosis and tissue fibrosis. 9'2°'21 It remains to be investigated as to whether the observed increase in PDGFAA and BB expression with increasing grades of rejection results in significant smooth muscle recruitment and intireal proliferation, as seen during the early phase of vascular atherosclerosis. The current findings provide new insights into the complexities of EC responses during clinical allograft rejection. These studies show that EC activation and cytokine production differ in significant compared to non-significant cardiac rejection, and suggest that EC activation plays a significant role in the development of acute and, potentially also, chronic cardiac altograft rejection. We suggest that vascular leaking resulting from cytokine-mediated reorganisation, and/or from EC injury, as well as the induction of a net procoagulant state at the EC surface, produces blood stasis, allowing optimal leucocyte-endothelial adhesion to occur, and facilitates subsequent cell transmigration. Furthermore, when the subendothelium is exposed to the circulating blood, platelet adherence and release of growth factors, particularly PDGF, may lead to migration of cells into the intima and intimal proliferation. These insights into the extent of clinical EC activation in vivo suggest multiple opportunities for diagnostic applications and therapeutic intervention during the management of cardiac allograft rejection. ACKNOWLEDGEMENTS This work was supported by the Alfred Hospital (Melbourne, Australia) Research Grant Z 9211. Address for corre.~pondence: W.W.H., Harvard Medical School,Boston,

Massachusetts, USA.

HUMANACUTECARDIACALLOGRAFTREJECTION 29 References 1. Hengstenberg C, Rose ML, Page C, Taylor PM, Yacoub MH. Immunocytochemical changes suggestive of damage to endothelial cells during rejection of human cardiac allografts. Transplantation 1990; 49: 895--9. 2. Taylor PM, Rose ML, Yacoub MH, Pigott R. Induction of vascular adhesion molecules during rejection of human cardiac allogr~ts. Transplantation 1992; 54: 451-7. 3. Wood KM, Cadogan MD, Ramshaw AL, Parums DV. The distribution of adhesion molecules in human atherosclerosis. Histopathology 1993; 22: 437-44. 4. Li H, Cybulsky MI, Gimbrone MA, Libby P. An atherogenic diet rapidly induces VCAM-1, a cytokine-regulatable mononuclear leukocyte adhesion molecule, in rabbit aortic endothelium. Arterioscler Thromb 1993; 13: 197-204. 5. Pober JS, Cotran RS. The role of endothelial cells in inflammation. Transplantation 1990; 50: 53744. 6. Springer TA. Adhesion receptors of the immune system. Nature 1990; 346: 425. 7, Tsuchida A, Salem H, Thomson N, Hancock WW. Tumor necrosis factor production during human renal allograft rejection is associated with depression of plasma protein C and free protein S levels and decreased intragraft thrombomodutin expression. J Exp Med 1992; 175: 81. 8. Hancock WW, Sayegh MH, Sablinski T, Knt JP, Kupiec-Weglinski JW, Milford EL. Blocking of mononuclear cell accumulation, cytokine production and endothelial activation within rat cardiac allografts by CD4 monoclonal antibody therapy. Transplantation 1992; 53: 1276-80. 9. Hancock WW, Adams DH, Wyner LR, Sayegh MH, Karnovsky MJ. CD4 + mononuclear cells induce cytokine expression, vascular smooth muscle proliferation and carotid occlusion following endothelial injury. Am J Pathol 1994; 145(5): i008-14. 10. Herbert JM, Savi P, Laplace MC. IL-4 and IL-13 exhibit comparable abilities to reduce pyrogen-indnced expression of procoagulant ac-

11.

12.

13. 14.

15.

16. 17. 18.

t9. 20. 21.

tivity in endothelial cells and monocytes. FEBS Lett 1993; 328: 268-70. Billingham ME, Cary NRB, Hammond EH. A working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: heart rejection study group of the International Society for Heart Transplantation. Heart Transplantation 1990; 9: 587-93~ Salom RN, Maguire JA, Esmore D, Hancock WW. Analysis of proliferating cell nuclear antigen expression aids histological diagnosis and is an indicator predictive of progression of cardiac allograft rejection. Am J Pathol 1994; 145, 876-82. Tipping PG, Hancock WW. Production of tumor necrosis factor and interleukin-1 by macrophages from human atheromatons plaques. Am J Pathol 1993; 142: 1721-8. Hancock WW, Tanaka K, Salem H, Tilney NL, Atkins RC, KupiecWeglinski JW. TNF as a mediator of cardiac rejection and effects on the protein C/protein S/thrombomodulin pathway. Transplant Proc 1991; 23: 235-7. Arbustini E, Grasso M, Diegoli M, Bramerio M, Folieni AS, Albertario M, et al. Expression of tumor necrosis factor in human acute cardiac rejection. Am J Pathol 1991; 139: 709-15. Odeh M. Tumor necrosis factor-alpha as a myocardial depressant substance. Int J Cardiol 1993; 42: 3. Tracey KJ, Cerami A. Tumor necrosis factol~a pteiotmpic cytokine and therapeutic target. Annu Rev Med 1994; 45: 491. Collins TL, Lapierre LA, Fiers W, Strominger JL, Pober JS. Recombinant tumor necrosis factor increases mRNA levels and surface expression of HLA-A, B antigens in vascular endothelial cells and dermal tibroblasts in vitro. Proc Natl Acad Sci USA 1986; 83: 446. Aderka D, Le J, Vitcek J. IL-6 inhibits lipopolysaccharide induced TNF production in cultured human monocytes, U937 cells and in mice. J Immunol 1989; 143: 3517. Casscells W. Smooth muscle cell growth factors. Prog Growth Fac Res 1991, 3: 177. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature t993, 362: 801.