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Ann Thorac Surg 1996;62:1360-1363
© 1996 The Society of Thoracic Surgeons

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Original Article: Cardiovascular

Fetal Inoculation With Donor Cells in Cardiac Xenotransplantation

Department of Cardiothoracic Surgery, The Medical College of Pennsylvania and Hahnemann University School of Medicine, Philadelphia, Pennsylvania

Accepted for publication May 25, 1996.

	Abstract

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Background. In utero fetal inoculation with allogeneic cells has produced subsequent tolerance to experimental cardiac allografts. We attempted to extend this observation to a model of xenogeneic cardiac transplantation.

Methods. Lewis rat fetuses were inoculated with Golden Syrian hamster thymocytes (n = 5) or whole spleen cells (n = 5) on the tenth day of intrauterine life. Six weeks after the birth of pretreated fetuses, heterotopic cardiac transplantation using a hamster donor was performed. Three to 4 weeks after parturition, we performed heterotopic cardiac transplantation using hamster donors in the female Lewis rats whose fetuses had been treated in utero.

Results. Animals treated in utero with either thymocytes or whole spleen cells had graft survival of 3 days, not different from that in untreated Lewis rats (n = 5) (p= not significant). Maternal Lewis rats whose fetuses were treated with thymocytes (n = 5) or whole spleen cells (n = 4) had markedly reduced survival of xenogeneic cardiac grafts (range, 3 to 20 hours; mean, 15 hours; p < 0.01; and range, 5 to 15 minutes; mean, 10 minutes; p < 0.01, respectively). Female Lewis rats without intrauterine inoculation (n = 5) had expected xenograft survival time (3 days) (p = not significant). Immunohistochemical staining of hyperacutely rejected grafts showed deposits of immunoglobulin M as well as immunoglobulin G and complement. In normally rejected xenografts, no immunoglobulin M was detected.

Conclusions. These studies reveal the surprising observation that fetal exposure to xenogeneic cells sensitizes the maternal rat without tolerizing the fetal rat as observed in an allograft model. In addition, whole spleen cells produce a more vigorous hyperacute rejection than thymocytes, suggesting that B cells or macrophages may be the sensitizing agents. The accelerated rejection observed has the characteristics of an immunoglobulin M antibody–mediated hyperacute rejection response with deposition of complement.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Cardiac transplantation is effective therapy for patients with end-stage heart disease, but one of the main limitations of its application is an inadequate donor supply. Among the solutions to this problem is xenotransplantation, or the use of organs from animal donors. Unfortunately, the immunologic barriers to xenotransplantation have yet to be overcome. A number of investigators have addressed the problem of hyperacute rejection in discordant species combinations [1]. This process, which is believed to be due to preformed antibody, leads to rapid destruction of xenografts within minutes to hours. Even in concordant species combinations in which hyperacute rejection does not occur, long-term xenograft survival has been achieved infrequently, and genuine tolerance to a vascularized xenograft has not been produced [2–7].

We have demonstrated that exposure of prospective recipients to donor-specific splenocytes can produce subsequent tolerance to an experimental cardiac allograft when rat fetuses are treated at day 14 of gestation [8]. Analogous treatment of rat fetuses with hamster splenocytes, a concordant species combination, does not produce any effect when pretreated rats undergo cardiac xenografting with a hamster donor [9]. We hypothesized that intrauterine treatment at an earlier phase of gestation might produce subsequent xenograft tolerance in pretreated rats. The present experiments were designed to investigate this hypothesis and to investigate the biology of intrauterine pretreatment by also transplanting hearts into the mothers of rats treated in utero. These studies were designed to address our growing conviction that the mechanisms of xenograft rejection, and therefore the procedures that will be necessary to produce xenograft tolerance, are fundamentally different from these processes in allografts.

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Animals
Pregnant female Lewis rats were obtained from Harlan Sprague-Dawley Company (Indianapolis, IN). Cardiac donors were male and female Golden Syrian hamsters from Charles River Laboratories (Wilmington, MA) weighing 110 to 120 g. Animals were acquired without viral infestation and were kept in an environment free of virus in the animal facility at The Medical College of Pennsylvania. All animals were handled in a humane fashion in accordance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Institutes of Health (NIH publication 86-23, revised 1985).

Preparation of Donor Cells
Spleens were removed from hamster donors, pressed through a screen, and suspended in RPMI 1640 medium by repetitive pipetting. Whole spleen cells were washed with RPMI 1640 medium three times and resuspended before inoculation. Thymocytes were prepared in the same manner.

Intrauterine Inoculation
Laparotomy was performed on day 10 of gestation in gravid female rats under general anesthesia. The normal rat gestation period is 21 days. Fetal injections were performed by identifying each fetus and directing a 30-gauge needle cephalad between the tail and hind limb. The intrafetal location was confirmed by ensuring movement of the fetus upon movement of the needle. Xenogeneic cells (25 x 10⁶) were injected in a volume of approximately 0.1 mL. Pressure was applied to the puncture site to prevent leakage of amniotic fluid from the fetal membranes.

Heart Transplantation
Hearts were transplanted heterotopically in the manner described by Ono and Lindsey [10]. Transplant function was determined by daily abdominal palpation and, if findings were indeterminate, by direct visualization. Rejection was marked by the complete absence of ventricular contractions and was confirmed histologically.

Experimental Design and Procedure
Lewis rats were recipients in all experiments. Pretreated animals were divided into two groups: group A was injected with hamster whole spleen cells, and group B was injected with hamster thymocytes. This treatment causes a 50% attrition rate of rat fetuses. Live-born pretreated rats were allowed to mature and then received a cardiac transplant from a hamster donor at 6 weeks of age. Three to 4 weeks after parturition, heart transplantation using a female hamster donor was performed in the maternal Lewis rats whose fetuses had been treated in utero. For control, heart transplantation using a male or female hamster donor was carried out in untreated male or female Lewis rats.

Immunohistochemical Staining
Heart xenografts in untreated and pretreated recipients were removed as soon as rejection caused cessation of graft function. Transverse ventricular sections were embedded in Tissue-Tek II (Miles Scientific, Naperville, IL) and snap-frozen in liquid nitrogen. Samples were stored at -70°C until study. Frozen heart tissue sections (4 µm thickness) were prepared in a Lipshaw cryostat under conditions of constant humidity (30%) and temperature (25°C). Tissue sections were air-dried, fixed in acetone, and washed with phosphate-buffered saline solution, pH 7.4. Sections were stained with fluorescein isothiocyanate–conjugated goat monoclonal antibodies specific for rat immunoglobulin (Ig)G, IgM, and complement (C3) (Organon Teknika, Durham, NC), and the reagent was absorbed with goat serum. Tissue sections were washed with phosphate-buffered saline solution after incubation with reagent and were mounted in PermaFluor Aqueous Mounting Medium (Immunon/Lipshaw Corp, Detroit, MI). Control sections of normal, untransplanted hearts from hamsters were stained as described previously. Slides were examined with an Olympus (BH.2, Hg light source) microscope (Olympus Optical, Japan) equipped for epifluorescence. Exposures were taken for 30 to 40 seconds with ASA 400 Ektachrome film (Eastman Kodak, Rochester, NY).

Histopathologic Studies
The rejected hearts also were examined using routine histopathologic staining. Sections of rejected hearts were fixed in 10% buffered formalin and embedded in paraffin. Staining was carried out with hematoxylin and eosin, and slides were examined by light microscopy.

Statistical Analysis
Groups were compared using the nonparametric Mann-Whitney U test.

	Results

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Lewis rat fetuses pretreated in utero with either hamster thymocytes or whole spleen cells rejected hamster cardiac xenografts in 3 days. This is not different from rejection in untreated Lewis rats (p = not significant). Hyperacute rejection of cardiac xenografts was seen in the maternal Lewis rats whose fetuses had been treated. The survival of xenogeneic cardiac grafts was 3 to 20 hours (mean, 15 hours; p < 0.01) in maternal Lewis rats whose fetuses were treated with thymocytes and 5 to 15 minutes (mean, 10 minutes; p < 0.01) in those whose fetuses were treated with whole spleen cells. There were significant differences in survival time between these groups and compared with controls (p < 0.01). Female Lewis rats without treatment had the expected cardiac xenograft survival time (3 days, p = not significant). Survival of the hearts is summarized in Table 1.

There were also distinct differences in the immunohistochemical staining of xenogeneic hearts rejected hyperacutely by maternal Lewis rats whose fetuses were inoculated and the immunohistochemical appearance of hearts normally rejected by treated fetuses. Immunofluorescence staining of hyperacutely rejected xenografts revealed deposits of IgM as well as substantial amounts of IgG and complement. In normally rejected xenografts, no IgM was detected, and IgG and complement C3 staining was significantly weaker. The deposits of IgM were localized to the endothelium of coronary vessels including capillaries, and IgG and complement C3 were deposited in both the walls of small vessels and the interstitial spaces of the myocardium.

	Comment

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Xenogeneic heart transplantation is not effective therapy in humans because insurmountable barriers have thus far prevented the use of animal donors [11]. Most currently used immunosuppressive agents have not proved effective in experimental xenotransplantation, although some success has been achieved in certain models with combined therapy [2–7]. We previously extended our immunologic manipulation methods, which are effective in models of cardiac allografting, to a rodent model of concordant xenografting, but the immunologic barriers cannot be overcome by these methods.

Specifically, pretreatment of fetuses in utero at day 14 of gestation [9], intrathymic inoculation of neonatal rats [9], and a brief course of systemic immunosuppression therapy combined with intrathymic pretreatment in adult rats [12], all of which produce allograft tolerance, have had no effect or have produced hyperacute rejection in the hamster to rat xenograft model. The rat gestation period is 21 days. The present experiments were designed to test the hypothesis that inoculation with xenoantigen at day 10, at a considerably earlier phase of immune ontogeny, would produce subsequent tolerance to a cardiac xenograft. Furthermore, to begin to develop an understanding of the biology of xenoreactivity and intrauterine pretreatment, we investigated the immunologic effects of this procedure on the gravid mother herself.

Our data demonstrate that exposure of graft recipients to xenogeneic cells in utero at an early stage of gestation does not produce tolerance to subsequent cardiac xenografts. However, in the maternal rat whose fetuses are pretreated, a hyperacute rejection response develops that appears to be mediated by IgM and complement. This result raises the question of why immunomodulation that is effective in an allograft model fails in concordant xenograft recipients. The finding suggests that the basic processes of xenoantigen rejection, and therefore the path to tolerance induction, must be fundamentally different from these phenomena in allotransplantation. Alternatively, it must be acknowledged that another explanation of our findings is that successful xenogeneic inoculation produced fetal death and that the inoculum missed live-born fetuses and led to maternal sensitization. The only counter to this hypothesis is our experience with allogeneic inoculation, although we do not have data on injection at 10 days' gestation.

We used splenocytes for xenogeneic inoculation because of our previous experience with allotransplantation. Hematopoietic cells such as bone marrow or fetal liver cells theoretically might be superior, and such experiments are planned.

The finding that the maternal rat of pretreated fetuses becomes sensitized to xenogeneic cells is somewhat surprising. We presumed that intraamniotic inoculation of xenogeneic cells would not expose the mother to the xenoantigens. This was clearly not the case. The accelerated rejection response had the characteristics of IgM-mediated hyperacute rejection [13–15]. This is similar to what we have observed in adult rats pretreated with antilymphocyte serum and intrathymic inoculation of xenogeneic cells [12]. It is possible that there was spillage of xenoantigen outside the amniotic sac, thus exposing xenoantigen to the maternal immune system. At present, we have no other hypotheses to explain how xenoantigen was able to escape from what we believed to be an immunologically privileged site and cause sensitization of the maternal rat.

We were also interested to note that whole spleen cells produced a more vigorous hyperacute rejection response than thymocytes. Thymocytes consist predominantly of T cells, but almost surely include some thymic stromal cells as well. Splenocytes include T and B lymphocytes and macrophages. Splenocytes appear to produce a much more vigorous hyperacute rejection response, with grafts destroyed uniformly in less than 1 hour, which suggests a sensitizing role for B cells and macrophages. In contrast, the thymocyte-induced hyperacute response produces graft destruction over several hours. The importance of this observation is unclear, but it suggests avenues for further research. Both IgM and complement appear to be the mediators of both rejection responses.

Deposition of IgM was not detected in xenografts rejected by untreated rats or rats treated as fetuses. However, these hearts did show deposition of IgG and complement, suggesting that antibody plays a role even in nonhyperacute concordant xenograft rejection. This may be part of the critical difference between allograft and concordant xenograft rejection, and may therefore be another clue as to what must be addressed to produce xenograft tolerance. That is, even in a nonhyperacute rejection response, macrophages, B cells, antibody, and complement play important roles. This may explain the failure of intrauterine, neonatal, and intrathymic inoculation to produce xenograft tolerance. These treatment strategies may be effective only in T-cell–mediated delayed hypersensitivity–type responses, and not in presumably B-cell–mediated antibody or other responses.

The results of our study suggest, first, that exposure of xenograft recipients to xenogeneic antigens, even early in gestation, does not lead to subsequent tolerance to a cardiac xenotransplant. Second, and somewhat surprisingly, intrauterine inoculation of the fetus appears to produce a hypersensitivity response in the mother. The hyperacute rejection response observed in the mothers of pretreated fetuses has the characteristics of an IgM- and complement-mediated acute rejection response. For reasons that remain to be investigated, splenic cells appear to induce a more vigorous hyperacute rejection reaction than thymocytes alone. These experiments confirm our growing conviction that the basic mechanisms of concordant xenograft rejection and, therefore, strategies for producing concordant xenograft tolerance, must be fundamentally different from these processes in allotransplantation.

	Footnotes

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 
Address reprint requests to Dr DiSesa, Cardiothoracic Surgery, Medical College of Pennsylvania Hospital, 3300 Henry Ave, Philadelphia, PA 19129.

	References

Top Footnotes Abstract Introduction Material and Methods Results Comment References

 

1. Bach FH. Revisiting a challenge in transplantation: discordant xenografting. Hum Immunol 1991;30:262–9.[Medline]
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3. Steinbruchel DA, Madsen HH, Nielsen B, et al. Treatment with total lymphoid irradiation, cyclosporin A and a monoclonal anti-T cell antibody in a hamster to rat heart transplantation model: graft survival and morphological analysis. Transplant Int 1990;3:36–40.[Medline]
4. Kemp E, Dieperink H, Jensenius JC, et al. Hope for successful xenografting by immuno-suppression with monoclonal antibody against CD4, total lymphoid irradiation and cyclo-sporin. Six months' survival of hamster heart transplanted into rat. Scand J Urol Nephrol 1990;24:79–80.[Medline]
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8. Kline GM, Shen Z, Mohiuddin M, Ruggiero V, Rostami S, DiSesa VJ. Development of tolerance to experimental cardiac allografts in utero. Ann Thorac Surg 1994;57:72–5.[Abstract]
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11. Auchincloss H Jr. Xenogeneic transplantation: a review. Transplantation 1988;46:1–20.[Medline]
12. Mohiuddin M, Kline GM, Shen Z, Ruggiero V, Rostami S, DiSesa VJ. Experiments in cardiac transplantation. J Thorac Cardiovasc Surg 1993;106:623–5.
13. Wu GD, Gramer DV, Chapman FA, et al. Comparative immunopathologic study of hyperacute rejection in naive and sensitized rodent cardiac xenograft models. Transplant Proc 1993;25:464–5.[Medline]
14. Nielsen B, Steinbruchel DA, Lillevang ST, Salomon S, Kemp E. Natural history of hamster heart rejection when transplanted to rat: a pathoanatomical study including immunohistochemistry. Transplant Proc 1994;26:1024–5.[Medline]
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This Article Abstract Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Zhenya Shen Verdi J. DiSesa Permission Requests Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Shen, Z. Articles by DiSesa, V. J. Search for Related Content PubMed PubMed Citation Articles by Shen, Z. Articles by DiSesa, V. J.