| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J Thorac Cardiovasc Surg 1994;107:1001-1005
© 1994 Mosby, Inc.
CARDIAC AND PULMONARY TRANSPLANTATION |
Buffalo, N.Y.
Supported in part by the Veterans Administration.
Address for reprints: Steven M. Peterson, MD, Department of Surgery, State University of New York at Buffalo, D. K. Miller Building, 462 Grider St., Buffalo, NY 14215.
Abstract
Although untreated Lewis rat recipients will reject a transplanted hamster heart in 3 days, accommodation of heart xenografts can be induced by treatment with cyclosporine and splenectomy, improving graft survival to greater than 50 days. Both humoral and cellular arms of the immune system may be involved in the mechanisms responsible for the prolongation of graft survival. Our objective was to study the impact of cyclosporine and splenectomy on the deposition of antibodies, complement, or both within the graft. We also compared the cellular component of inflammation in treated recipients with that in untreated controls. Inbred male Lewis rats given cyclosporine 15 mg/kg per day were splenectomized 2 days after they had received heterotopic heart transplants from Golden Syrian hamsters. Recipients of syngeneic grafts or untreated xenografts served as controls. Plasma interleukin-6 activity was measured in a standard proliferation assay with 7TD1 hybridoma cells. Deposition of immunoglobulin M, immunoglobulin G, and complement in heart tissue was evaluated by immunofluorescence. Cells infiltrating the graft that expressed major histocompatibility complex class II antigens were identified by immunohistochemical staining with OX6 antibodies. In xenograft recipients receiving immunosuppression, interleukin-6 activity, immunoglobulin M and complement deposition were significantly reduced, graft infiltration was mild, and cardiac function was good compared with the results in those without treatment 3 and 10 days after implantation. Inflammatory cells expressing major histocompatibility complex class II antigens were significantly reduced in immunosuppressed xenograft recipients (2.8 ± 0.4 cells/high power field) compared with those in xenogeneic controls (9.5 ± 0.6 cells/high power field; p < 0.0005). The significant decrease in deposition of humoral components (immunoglobulin M and complement), interleukin-6 plasma levels, and expression of major histocompatibility complex class II antigens by inflammatory cells within the nonrejecting grafts suggests that the synergistic benefit of cyclosporine and splenectomy depends on the attenuation of both cellular and humoral mechanisms of xenograft rejection. (J THORACCARDIOVASCSURG1994;107:1001-5)
The rejection of xenogeneic transplanted organs is characterized by endothelial hyperplasia, interstitial mononuclear cell infiltration, thrombosis, and necrosis of vessel walls and myocytes. 
1 These characteristic features of rejection have been attributed to the preformed heterophile antibodies directed against xenogeneic tissue antigens, 2 unlike allograft rejection which is primarily cell mediated. The ability of T-cell depleted recipients to reject xenografts provides strong evidence for the overwhelming importance of humoral factors in the rejection process. 3
Interleukin-6 (IL-6) is a pleiotropic cytokine that was initially described as a B cell differentiation factor. 4 It has been suggested that IL-6 may contribute to the activation of cytotoxic T cell production, IL-2 production, or both during cardiac allograft rejection. 5-7 IL-6 may also be important in the rejection of renal 8 and hepatic allografts. 9 Although the action of IL-6 has been implicated in the mechanisms leading to acute allograft rejection, the role of IL-6 in xenograft rejection has not yet been well studied.
New immunosuppressive regimens have targeted both the humoral and cellular arms of the xenograft response to control the concordant xenogeneic rejection reaction. This article describes the effect of cyclosporine and delayed splenectomy on both the cellular and humoral response to xenogeneic cardiac grafts.
MATERIALS AND METHODS
Animals
Rats and hamsters were purchased commercially (Harlan Sprague Dawley, Indianapolis, Ind.). Animals were housed and fed in compliance with National Institutes of Health standards. Inbred male Lewis rats (175 to 250 gm) were recipients of hearts obtained from male Syrian Golden hamsters. In control experiments, male Lewis rats were the syngeneic heart donors.
Heart transplants
Heterotopic heart transplantation was performed as described by Ono and Lindsey, 10 with minor modifications. Splenectomy was performed through a left flank incision 2 days after heart transplantation. Cyclosporine diluted from a standard stock solution in olive oil, was given to recipients beginning at day 0 (day of implantation) at 15 mg/kg per day intramuscularly. Graft contractility was evaluated daily by abdominal palpation. Cessation of beating was confirmed at laparotomy. The different experimental groups are listed in Table I.
|
Immunohistopathologic studies
Cardiac tissue was snap-frozen in liquid nitrogen and 4 µm sections were cut on a cryostat (Leica Incorporated, Deerfield, Ill.). Fluorescein isothiocyanate–conjugated antibodies to rat immunoglobulin G (IgG) (Nordic Immunological Laboratories, Capistrano Beach, Calif.) and complement (C3) (Cappel Laboratories, Cochranesville, Pa.) were used for direct immunofluorescence, whereas goat anti-rat IgM (Cappel) was coupled with a secondary fluorescein isothiocyanate–conjugated antibody, rabbit anti-goat IgG (Sigma Chemical Co., St. Louis, Mo.). Specific binding within the graft was graded as negative (-), weak (+), moderate (++), and strong (+++) for the extent of binding within the tissue. An indirect immunoperoxidase method was used for the identification of class II major histocompatibility antigen with the use of the antibody OX6 (Bioproducts for Science, Indianapolis, Ind.). Positive cells were counted in a minimum of 10 high power fields (1000x oil-immersion) randomly distributed within the specimen. In addition, a biventricular section of each sample fixed in buffered formalin was embedded in paraffin and stained with hematoxylin and eosin.
RESULTS
Immunosuppression
The impact of different therapeutic regimens graft survival is graphically represented in Fig. 1. A synergistic effect of cyclosporine and splenectomy on graft survival was observed.
|
IL-6
Plasma IL-6 activity was significantly higher in untreated recipients of xenografts than in recipients of syngeneic grafts (p < 0.005) at day 1 (Fig. 2). The activity of IL-6 in plasma remained elevated in untreated xenograft recipients at end-stage rejection at day 3 (p < 0.01). In contrast, in the syngeneic recipients, plasma levels of IL-6 did not change from day 1 to day 3. Plasma levels of IL-6 in xenograft recipients treated with cyclosporine and splenectomy were not different from the syngeneic controls at day 3.
|
Immunohistopathologic studies
Normal hamster hearts did not stain for major histocompatibility complex MHC class II antigen with the OX6 antibody. In untreated xenograft recipients, infiltration of the hamster heart with OX6 positive mononuclear cells from the recipient increased as rejection progressed, similar to those recipients treated only with splenectomy. The number of OX6 positive cells in the grafts from the syngeneic controls was less than in untreated xenograft recipients at day 3. In recipients treated with cyclosporine and splenectomy or cyclosporine alone, infiltration of OX6 positive cells was significantly reduced compared with the cells in untreated xenograft recipient controls at day 3 (p < 0.0005; Fig. 3). Interestingly, an increase in OX6 infiltrating cells seen at day 10 in syngeneic grafts was not seen in the hamster hearts from the recipients treated with cyclosporine and splenectomy.
|
We have confirmed that combined treatment with cyclosporine and delayed splenectomy synergisticly improves graft survival by suppressing both cellular activation and decreasing antibody synthesizing capabilities, as shown by others. 11-13 The prolonged survival of the hamster heart in the treated rat recipients provided an opportunity to investigate the response of the host's immune system during acute xenograft rejection. This is the first report describing elevated levels of IL-6 during acute xenograft rejection. In addition, increased numbers of inflammatory cells expressing the MHC class II antigen, and increased deposition of IgM and C3 were also observed in the untreated xenograft recipients.
The vascular endothelium has been shown to be a primary stimulus and the main target of heterophile antibodies. 14-16 The activation of the endothelium by antibody induces production of bioactive factors, including IL-6. Immunofluorescence analysis of IgM and C3 showed an early granular pattern within the xenograft vasculature correlating closely with the significant elevation of IL-6 plasma activity. The continued elevation of IL-6 at end rejection implies a continued production of the cytokine. In both nonrejecting models, isografts and treated xenograft recipients, the plasma activity of IL-6 was significantly lower than in the rejecting cardiac xenografts. Our data suggest that IL-6 may be an important marker and mediator for xenograft rejection. Its biologic activity leading to the induction of B-cell terminal differentiation and activation of macrophages possibly plays a role in graft destruction.
In conclusion, the significant decreased deposition of humoral components (IgM and C3), decreased IL-6 plasma levels, and decreased infiltrating cells expressing MHC class II antigen in the nonrejecting grafts suggests that the synergy of cyclosporine and splenectomy depends on the attenuation of both cellular and humoral mechanisms involved in xenograft rejection. This synergistic effect may be partially due to a change in the presentation of antigen, cytokine production, or both. The success of xenografts for clinical use will depend on the understanding of the interaction between the humoral and cellular arms of the rejection process and the combination of therapies to suppress both T- and B-cell reactions.
Appendix: DISCUSSION
Dr. Bruce A. Reitz (Stanford, Calif.).
Have you done studies in which you have looked at the group with splenectomy alone and the group with cyclosporine alone with IL-6 levels to see which is important?
Dr. Peterson.
Study of the groups receiving only splenectomy or cyclosporine is currently in progress, although, because of the inherent problems with bioassays, we have continued these studies looking at IL-6 messenger RNA expression within the grafts.
Dr. Reitz.
Is there a monoclonal antibody against IL-6?
Dr. Peterson.
To my knowledge, there is no antibody specific for rat IL-6 commercially available. Dr. Gauldie (McMaster University, Hamilton, Ontario Canada) was kind enough to supply us with a sample of his polyclonal antibody for IL-6. A protocol was initiated with the use of his antibody that met with limited success in our in vivo system.
Footnotes
From the State University of New York at Buffalo, Department of Surgery, a Department of Microbiology, b Veterans Administration Medical Center, Medical Research Building, Buffalo, N.Y. 
Read at the Seventy-third Annual Meeting of The American Association for Thoracic Surgery, Chicago, Ill., April 25-28, 1993.
References
This article has been cited by other articles:
|
|
 |
|
  D. R. Meldrum Tumor necrosis factor in the heart Am J Physiol Regulatory Integrative Comp Physiol, March 1, 1998; 274(3): R577 - R595. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ANN THORAC SURG | ASIAN CARDIOVASC THORAC ANN | EUR J CARDIOTHORAC SURG |
| J THORAC CARDIOVASC SURG | ICVTS | ALL CTSNet JOURNALS |
