Chapman, F

Lee, PH

Makowka, L

Murase, N

Qian, S

Saunders, R

Starzl, TE

Todo, S

Zerbe, A

English

PubMed

Lee, P-H

Name not available

The effect of FK 506 on hyperacute rejection in presensitized rats

D-Scholarship@Pitt

The Effect of FK-506 on Hyperacute Rejection in Presensitized Rats L. Makowka, F. Chapman, S. Qian, A. Zerbe, P-H. Lee, N. Murase, R. Saunders, S. Todo, and T.E. Starzl T HE PATHOGENESIS of hyperacute organ rejection involves a specific ini-tiating immunologic reaction that consists of antibody-mediated injury directed against antigens on the vascular endothelium of trans-planted organs. I,2 This unleashes a very intense and complex nonspecific effector cas-cade that involves most of the classic mediator systems of the acute inflammatory process.3 The final response can be described as a failure or breakdown of the microvasculature that results in intravascular coagulation and ischemic necrosis of the organ, with a charac-teristic pathological appearance.4•6 Immunosuppressive agents, such as cyclo-sporine or FK-506, which inhibit classic cell-mediated rejection, should therefore not be expected to have a significant and consistent effect on hyperacute rejection when used alone. However, one new potential approach for controlling the effector cascade of hyper-acute rejection by pharmacological interven-tion exploits the role of acetyl glycerol ether phosphorylcholine or platelet-activating fac-tor (PAF) as a central candidate in the overall evolution of this inflammatory process.7,s There is now clear evidence that establishes the candidacy of PAF as a key biologic media-tor in the pathogenesis of antibody-mediated hyperacute allograft rejection.9- 11 Therefore, inhibition of PAF-induced reactions offers a potentially significant therapeutic approach to this entity. SRI 63-441 is currently the most potent and specific PAF receptor antagonist avail-able. 12 Our recent studies represent the first known report to describe the therapeutic effi-cacy of a PAF antagonist (SRI 63-441) in any model of hyperacute rejection. 13 The combi-nation of SRI 63-441 with either prostacyclin or prostaglandin EI resulted in a six- to nine-fold increase in kidney survival and a three- to 30-fold increase in urine output when com-pared with control animals in a pig-to-dog renal xenograft model. 13 Furthermore, subse-quent studies have demonstrated that a single bolus injection of SRI 63-441 alone, before revascularization, could significantly improve rat cardiac allograft survival in sensitized recipients (unpublished results). Histological evaluation of hearts that had functioned nor-mally and then ceased at three and four days demonstrated a very aggressive acute cellular rejection compared with a classic picture of hyperacute cardiac rejection consisting of myonecrosis and intravascular coagulation in control rats. These findings suggested that the addition of potent immunosuppression could further abrogate this entire process. The pres-ent studies evaluated combination therapy with FK-506 and SRI 63-441 on cardiac allograft survival in presensitized rats. MATERIALS AND METHODS Animals Commercially available inbred male Lewis (L) (RTll) and ACI (RTl') strain rats weighing 200 to 220 g were purchased from Harlan Sprague Dawley Co, Indianapo-From the Departments of Surgery and Pathology, University Health Center of Pittsburgh, University of Pittsburgh, and the Veterans Administration Medical Center, Pittsburgh. Supported by Research Grants from the Veterans Administration and Project Grant No. AM-29961 from the National Institutes of Health, Bethesda, MD. Dr Makowka is a recipient of a Centennial Fellowship from the Medical Research Council of Canada. Address reprint requests to L. Makowka, MD, PhD, Department of Surgery, 3601 Fifth Ave, Falk Clinic, 4 West, Pittsburgh, PA 15213. © 1987 by Grune & Stratton, Inc. 0041-1345/87/1905-6017$03.00/0 Transplantation Proceedings, Vol XIX, No.5, Suppl 6 (October). 1987: pp 79-83 79 80 lis. This strain combination was selected on the basis of a strong histoincompatibility to provide a reproducible and reliable model of hyperacute rejection. '4 Surgical Procedures Skin grafting. Presensitization of L recipients was carried out under ether anesthesia by removing full-thickness ventral abdominal skin from ACI donor rats. 14 A 2.5-cm circular skin graft was transplanted to the dorsum of the L recipient and fastened with Clay Adams wound clips. Casting material was then applied for four days after the grafting procedure to immobilize the tissue. Grafts were evaluated visually for rejection. A second and third graft were placed at intervals of 14 days in a similar manner. Heterotopic heart transplantation. Heterotopic heart transplantation was performed 17 to 20 days after the third skin graft. The animals were anesthetized with 40 mg/kg sodium pentobar-bital intra peritoneally and supplemented with methoxyflurane. Surgical procedures for primarily vascu-larized cardiac transplants in rats have been well described previously. IS Briefly, an end-to-side anastomo-sis is performed between the donor and recipient aorta as well as the donor pulmonary artery and recipient vena cava. Impulses of the transplanted hearts were monitored every 30 minutes for the initial 15 hours and then twice daily thereafter by palpation through the recipient abdominal wall. Rejection was considered complete when there was no palpable contractions and confirmed visually at laparotomy and by histological evaluation. All experi-mental animals were weighed daily. Drug Administration PAF antagonist. SRI 63-441 was supplied by Sandoz Research Institute (East Hanover, NJ). SRI 63-441 (molecular weight [mol wtl, 662) was reconstituted in 0.68% sodium acetate and 0.9% sodium chloride (pH 5.3) to a final concentration of 10 mg/mL and placed in a warm water bath (26 to 28OC) for five minutes. FK-506. Crystalline powder (mol wt, 882) was sup-plied by Fujisawa Pharmaceutical Co, Ltd, Osaka, Japan. A final concentration of 1.28 mg/mL was prepared in 0.9% sodium chloride. Experimental Design Contemporaneous controls were performed for each experimental group. All recipients (L) animals were sensitized to donor (ACI) antigen. Experimental groups evaluated were as follows: group I, control (vehicle only); group 2, FK-506 alone; group 3, SRI 63-441 alone (l0 mg/kg); group 4, SRI 63-441 (lO mg/kg) plus FK-506; and group 5, SRI 63-441 (I5 mg/kg) plus FK-506. MAKOWKA ET AL A single bolus injection of SRI 63-441 was adminis-tered via the inferior vena cava four minutes before revascularization. FK-506 was administered at a dosage of 1.28 mg/kg/d for a duration of 14 days (or until rejection) by intramus-cular injection commencing three days before transplan-tation. Histology Tissues were formalin-fixed, paraffin-embedded, cut at 3 /Lm, and stained with hematoxylin and eosin (H + E). Statistical Analysis Where available, mean survival times (MST) for experimental groups were compared by using the Wil-coxon rank sum test. A P value of < .05 was considered statistically significant. It should be noted that in group 4 and 5 many of the animals were alive with functioning grafts at the time of manuscript preparation. RESULTS Survival The survival rates for the five groups evalu-ated are listed in Table 1. Sensitized L control recipients consistently rejected ACI donor hearts in a hyperacute fashion (MST 0.27 ± 0.09 days). Treatment of sensitized animals with SRI 63-441 alone as a single intravenous bolus of 10 mgjkg before revascularization resulted in a significant prolongation of car-diac allograft survival (MST, 1.70 ± 0.66 days; P < .003) when compared with control rats. Of even greater significance was the response in cardiac allograft survival observed when SRI 63-441 treatment (at two dosage levels) was combined with immunosuppres-sion by FK-506 (groups 4 and 5). It is impor-tant to note that five of 11 animals were still alive with functioning grafts at the time of preparation of this manuscript, with three animals approaching I-month survival. In fact, out of the 11 animals evaluated, one was killed at nine days for histological evaluation (Fig 1), and four grafts ceased to function at 4, 8, 13, and 20 days after transplantation. These grafts demonstrated similar morpho-logical features to those shown in Fig 1. One . . FK-506 AND HYPERACUTE REJECTION Table 1 . The Effect of FK-506 and SRI 63-441 on Hyperacute Rejection of Rat Cardiac Allografts Group Treatment Control (vehicle on ly) 13 2 FK-506 alone 5 3 SRI 63-441 alone (10 mg/ kg) 5 4 SRI 63-441 (10 mg/ kg) + 5 FK-506 5 SRI 63-441 ( 15 mg/kg) + 6 FK-506 "P < .003 as compared with group 1 (Mann-Whitney). tDied with a functioning graft. tKilled with a functioning graft for histological evaluation . Graft Survival (d) 0002, 0005,0.007,0.01 , 0 .02,0.08 , 0.15,0 .24, 0,26,0.27 , 0.67,0.85, 0 .97 0 .06, 1.4, 2, > 3 , > 6 0.06,1 . 13, 1. 19,2.19, 4.0 4, 4 , t 13,21 , > 28 8,9t,>4.> 10. >22,> 24 MST '" SE 0.27 ± 0,09 1.70 ± 0 .66" 81 Fig 1. Myocardium (H + E; original magnification x 200) demonstrating slight mononuclear interstitial infiltrate and replacement by fibroblasts . This specimen is takan from a rat treated with SRI 63-441 and FK-506 and sacrificed at day 9 after transplantation with a functioning graft. It should be noted that a similar picture of minimal cellular rejection was evident in the grafts that ceased to function in similarly treated rats. 82 rat in group 4 died with a functioning graft four days after transplantation. This survival far exceeds allograft survival in nonsensitized recipients (6.3 ± 0.5 days). There was no significant change in weight in rats surviving long-term. Histologic Evaluation Tissue sections stained with H + E were reviewed without knowledge of treatment pro-tocols. Morphological features interpreted as cardiac hyperacute rejection included exten-sive interstitial hemorrhage, occlusion of ves-sels by platelets, endothelial cell damage, and myocyte necrosis. Polymorphonuclear cell infiltration was not a constant feature. Sec-tions exhibiting predominately a mononuclear cell infiltrate were interpreted as acute cellu-lar rejection. Typically, there was much less interstitial hemorrhage and little or no plate-let plugging of vessels noted. Myocyte de-generation and fibroblast proliferation were frequently extensive. No overlap with hyper-acute rejection was recognized. The native hearts of the animals in all groups were consistently without morphologi-cal change. The extent and quality of the mononuclear infiltrate observed in the grafts of those animals treated with SRI 63-441 alone and SRI 63-441 and FK-506 was quite different. The grafts of those animals treated with SRI 63-441 alone contained a more abundant mononuclear cell infiltrate, many of which exhibited features of blast formation. DISCUSSION The present study represents the first known report describing a therapeutic approach that completely overcomes the phe-nomenon of experimental hyperacute rejec-tion. We had already demonstrated that sig-nificant abrogation of a very rapid and violent form of hyperacute rejection in a xenograft model could be achieved solely by the phar-macological manipulation of the inflamma-tory mediator response. 13 This relied on the MAKOWKA ET AL antagonism of PAF, perhaps the most impor-tant inflammatory mediator to be nominated as a central candidate in the overall evolution of hyperacute rejection.9. 11 In a model of het-erotopic cardiac transplantation into sensi-tized rats, we were able to demonstrate that the phenomenon of hyperacute rejection, pre-viously thought to be refractory and uncon-trollable, could be converted into a potentially more manageable form of classic rejection by a single bolus administration of a PAF antag-onist, SRI 63-441, before revascularization. Indeed, hearts in treated animals that ceased to function at two to four days after transplan-tation demonstrated a very aggressive histo-logical picture of acute cellular rejection with marked edema and extensive mononuclear cell infiltration (unpublished results). This suggested the prospect of adding classic immunosuppression in an attempt to over-come the cellular rejection. It was clear that very potent immunosup-pression would be required because the cellu-lar rejection of hearts in the SRI 63-441-treated animals was much more aggressive and was occurring much earlier than is usually seen in the same donor-recipient com-bination without prior sensitization. This probably reflects the aggressive rejection of an organ made vulnerable by antibody attack. Other reports in this symposium have already demonstrated the potency and efficacy of FK-506 as an immunosuppressant in standard transplant models. The combination of FK-506 and SRI 63-441 treatment in this model of hyperacute cardiac rejection resulted in remarkable prolongation of graft survival in healthy animals. In fact, at the time of manu-script preparation, 45% of the grafts were still surviving at various times after transplanta-tion, with three grafts each approaching 30-day survival. These studies provide further insight into the complex immunologic and inflammatory reactions involved in the process of hyperacute rejection. Moreover, we introduce a novel and FK-506 AND HYPERACUTE REJECTION potentially effective therapeutic approach that relies on a combined attack on the very potent and unusually diverse spectrum of bio-logic activities of a key inflammatory media-tor as well as on a very aggressive form of classic cell-mediated rejection. REFERENCES 1. Giles GR, Boehmig JJ, Lilly J, et al: Transplant Proc 2:522, 1970 2. Boehmig HJ, Giles GR, Amerniya H, et al: Trans-plant Proc 3:1105,1971 3. Pinckard RN: Monogr Pat hoi 23:38, 1982 4. Starzl TE, Lerner RA, Dixon FJ, et al: N Engl J Med 278:642, 1968 5. Kissmeyer-Nielsen F, Olsen S, Petersen VP, et al: Lancet 2:662, 1966 83 6. Rosenberg JC, Broersrna RJ, Bullerner G, et al: Transplantation 8: 152, 1969 7. Venuti MC: Annu Rev Med Chern 20:193,1985 8. Braquet P, Varagaftig BB: Transplant Proe 18:\0, 1986 9. Ito S, Camussi G, Tetta C, et al: Lab Invest 51:148, 1984 10. Camussi G, Aglietta M, Malavasi F, et al: J Irnmunol131:2397,1983 II. Carnussi G: Transplant Proc 18:91, 1986 12. Handley DA, Tomeseh JC, Saunders RN: Thromb Haemost 56:40, 1986 13. Makowka L, Miller C, ChapChap P, et al: Ann Surg (in press) 14. Guttman RD: Transplantation 17:383, 1974 15. Ono K, Lindsey E: J Thorae Cardiovase Surg 57:225, 1969 

The effect of FK-506 on hyperacute rejection in presensitized rats.

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The effect of FK-506 on hyperacute rejection in presensitized rats.

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