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The Journal of Immunology, 1999, 163: 2850-2857.
Copyright © 1999 by The American Association of Immunologists

Accommodated Xenografts Survive in the Presence of Anti-Donor Antibodies and Complement That Precipitate Rejection of Naive Xenografts1

Yuan Lin2,*, Miguel P. Soares*, Koichiro Sato*, Ko Takigami*, Eva Csizmadia*, Neal Smith{dagger} and Fritz H. Bach*

* Immunobiology Research Center, Department of Surgery, Beth Israel Deaconess Medical Center, Boston, MA 02215; and {dagger} Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02215


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hamster hearts transplanted into transiently complement-depleted and continuously cyclosporin A (CyA)-immunosuppressed rats survive long-term despite deposition of anti-donor IgM Abs and complement on the graft vascular endothelium. This phenomenon is referred to as "accommodation." The hypothesis tested here is that accommodated xenografts are resistant to IgM Abs and complement that could result in rejection of naive xenografts. After first hamster hearts had been surviving in cobra venom factor (CVF) + CyA-treated rats for 10 days, a time when the anti-donor IgM Ab level was maximal and complement activity had returned to approximately 50% of pretreatment levels, naive hamster hearts or hamster hearts that had been accommodating in another rat for 14 days were transplanted into those rats carrying the surviving first graft. The naive hearts were all hyperacutely rejected. In contrast, a majority of regrafted accommodating hearts survived long-term. There was widespread Ab and activated complement deposition on the vascular endothelium of accommodating first hearts, second accommodating hearts, and rejected second naive hearts. However, only the rejected naive hearts showed extensive endothelial cell damage, myocardial necrosis, fibrin deposition, and other signs of inflammation. Accommodating first and second hearts but not rejected second naive hearts expressed high levels of the protective genes A20, heme oxygenase-1 (HO-1), bcl-2, and bcl-xL. These data demonstrate that accommodated xenografts become resistant to effects of anti-donor IgM Abs and complement that normally mediate rejection of xenografts. We hypothesize that this resistance involves expression by accommodated xenografts of protective genes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Hyperacute rejection of discordant xenografts, such as in the pig-to-primate combination, is caused by activation of complement following deposition on the graft endothelium of xenoreactive natural Abs. If xenoreactive natural Abs are depleted before transplantation, hyperacute rejection no longer occurs (1, 2). Upon exposure to xenoantigens expressed on the graft, host B cells synthesize elicited anti-donor Abs that precipitate delayed xenograft rejection (DXR),3 typically occurring within 3–5 days (1, 2, 3). DXR occurs in T cell-deficient recipients suggesting that this type of rejection can proceed independently of T cells (4), which is also reflected by a lack of ability of T cell-directed immunosuppressants to suppress DXR (5, 6).

Under certain circumstances, when anti-donor Abs and complement-mediated immune responses are inhibited for a few days, grafts survive indefinitely despite the return of anti-donor Abs and complement, a phenomenon we refer to as "accommodation" (7, 8). Accommodation may provide a potential approach to clinical application of xenotransplantation, since it may eliminate the need for continuing depletion or inhibition of anti-donor Abs and complement. In addition, further understanding of the mechanism(s) of accommodation will provide clues to potential therapeutic manipulations of the donor or recipient that could improve the survival of xenografts.

We and others have studied accommodation in concordant models, such as hamster hearts transplanted to rats (9, 10). Rejection in this model occurs around 3–4 days, initiated primarily by elicited anti-donor IgM Abs generated in a T cell-independent manner and associated complement activation (11, 12, 13, 14, 15), closely mimicking DXR as seen in discordant models. Long-term graft survival can be achieved with brief inhibition of complement by cobra venom factor (CVF) plus daily and continued treatment with cyclosporin A (CyA) (9, 10). Surviving hamster hearts function in the presence of anti-hamster IgM Abs, even following the return of complement activity (9, 10), consistent with the definition of accommodation (7, 8).

We suggested that achieving accommodation may in part involve changes in the endothelium such that the graft becomes resistant to Ab and complement-mediated rejection (7, 8). Those changes may include expression in the graft endothelial cells (EC) and smooth muscle cells of a number of "protective genes" such as A20, bcl-2, bcl-xL, and heme oxygenase-1 (HO-1) (10). Expression of protective genes suppresses apoptosis and the proinflammatory response associated with EC activation that may otherwise lead to graft rejection (16, 17). We have recently shown that the expression of HO-1 by the mouse heart xenograft endothelium is essential to ensure xenograft accommodation (18).

Previous data from our laboratory and those of others showed that accommodated grafts are resistant to adoptively transferred anti-donor sera (10, 19, 20), and survive in ~1/3 of cases when transplanted to second recipients receiving only CyA, which by itself does not protect naive xenografts from rejection (21). These experiments do not, however, establish the extent to which accommodated xenografts survive under the pathophysiological conditions that would lead to rejection of a naive xenograft. In the present study, we tested these questions by transplantation of a second hamster heart to rats already carrying an accommodating hamster heart when high levels of elicited IgM Abs and significantly restored complement activity were present. The naive hearts were all rejected whereas the accommodating hearts continued to beat. We also tested whether reperfusion injury contributed to the rejection of the naive second hearts by comparing the survival of the second naive hearts with the survival of second hearts that had already survived for 14 days in a different rat. The majority of the latter grafts survived long-term. We conclude that accommodated xenografts are resistant to Ab and complement-mediated injury that can readily induce rejection of a naive xenograft.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Golden Syrian hamsters, weighing 60–80 g, were used as organ donors. Inbred Lewis rats (RT1l) (Harlan Sprague Dawley, Indianapolis, IN), weighing 100–200 g, were used as recipients. All animals were housed in accordance with guidelines from the American Association for Laboratory Animal Care. The research protocols were approved by the International Animal Care and Use Committees of the Beth Israel Deaconess Medical Center.

Heterotopic heart transplantation

Cervical heterotopic hamster-to-rat heart transplantation was performed using a technique described previously (11). A second hamster heart was placed to the contralateral side of the recipient neck or to the abdominal cavity at day 10 following first xenotransplantation. Second grafts included hamster hearts that had been surviving for 14 days in a first recipient rat. The function of the grafts was monitored by daily inspection and palpation. Rejection was diagnosed by cessation of visible and palpable ventricular contraction, and confirmed by histology.

Immunosuppressive agents

CVF (purity >=85% by SDS Page; Quidel, San Diego, CA) was diluted in distilled water and administered by i.p. injection at day -1 (60 U/kg) and day 0 (20 U/kg) of transplantation. CyA (Novartis Pharma, Basel, Switzerland) was diluted in normal saline and administered by daily i.m. injection (15 mg/kg/day, starting from day -1).

Assay of elicited XAb

The IgM and IgG isotype anti-hamster XAb were measured by flow cytometry using hamster PBMC as target cells as described previously (11). Briefly, hamster PBMC were depleted of B lymphocytes by panning cells to an anti-hamster IgG (Cappel, Aurora, Ohio)-coated petri dish (22) to reduce the cross-binding by secondary Ab-FITC directed against rat Igs. Aliquots of 5 x 105 cells were incubated for 30 min at 4°C with 100 µl of 1:10 diluted serum taken from recipient rats at various time intervals after transplantation. To determine the titers of anti-hamster IgG, the cells were further incubated with mAb directed against rat IgG1 (MARG1-2), IgG2a (MRG2a-1), IgG2b (MARG2b-8), and IgG2c (MARG2c-5) (mouse IgG1, a gift of Prof. Bazin, University of Louvain, Belgium) or a mixture of those Abs. After addition of FITC goat-anti-rat IgM antiserum (Cappel) or FITC rat-anti-mouse IgG1 antiserum (Zymed, South San Francisco, CA), the cells were examined by flow cytometry using CellQuest software (FACScan; Becton Dickinson, Mountain View, CA). Results were expressed as the mean channel fluorescence of stained cells divided by the mean channel fluorescence of cells incubated with control serum and FITC Abs.

CH50 assay

Hemolytic complement (CH50) of the classical pathway was determined using a modification of the technique of Kabat and Mayer (23). Briefly, a cell dose of 1 µl of 50% (v/v) Ab-sensitized SRBC (~1 x 107 cells) (Cappel) was added to serially diluted serum (1/50, 1/100, . . . 1/1600) in 200 µl of complement fixation diluent (Sigma, St. Louis, MO). Experiments were performed in duplicate wells in 96-well round-bottom microtiter plates. After incubation for 30 min at 37°C, the plates were centrifuged for 5 min at 200 x g. A volume of 100 µl of the supernatant was transferred to another 96-well plate and estimated for hemoglobin in a spectrophotometer ({lambda} = 415 nm). One hundred percent hemolysis and 0% hemolysis were included by incubation of the cells with 200 µl of ACK lysing buffer (0.15 M NH4Cl, 1.0 mM KHCO3, 0.1 mM Na2EDTA) and 200 µl of complement fixation diluent, respectively. The 50% hemolytic end point is usually in the region of 1/200 to 1/400 (i.e., about 1000 to 2000 CH50/ml).

Serum transfer

Serum was prepared from pooled blood from untreated rats 10 days following transplantation or from rats carrying an accommodating hamster heart transplanted 10 days earlier. A various doses of the serum were injected into naive rats through the tail vein 30 min following hamster heart transplantation.

Histopathology and immunohistochemistry

Graft samples for histology were fixed in 10% formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E) for light microscopy. Graft samples for immunohistochemistry were snap frozen in prechilled isopentane and stored at -70°C. The frozen specimens were cut into 4-µm sections in a cryostat at -25°C and air dried. Rat anti-hamster Igs in the xenografts were detected by mAb directed against rat IgM (MARM-4) (a kind gift from Prof. H. Bazin). Intragraft complement deposition was detected by staining with an anti-rat C3 mAb (ED11) (Serotec, Oxford, U.K.). Expression of cytoprotective genes was analyzed using rabbit polyclonal Abs to rat Bcl-2, Bcl-xL (Santa Cruz Biotechnology, Santa Cruz, CA), A20 (a kind gift from S. Grey from our center), and HO-1 (StressGene Biotechnologies, Victoria, B. C., Canada). Cryostat sections were fixed in paraformaldehyde-lysine-periodate for demonstration of activation Ags and humoral reactants, or fixed in acetone for localization of cytoprotective genes as described (10). Isotype-matched mAbs or purified Ig and a control for residual endogenous peroxidase activity were included in each experiment.

Statistics

The results were statistically analyzed by the Student t test, or by the Fisher exact test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Graft survival

Survival of hamster hearts in rats is shown in Table IGo. Consistent with previous data (9, 10, 21), CyA monotherapy did not significantly influence (p > 0.05) graft survival (3.3 ± 0.5 days) when compared with untreated control rats (3.4 ± 0.5 days). CVF alone resulted in some prolongation of graft survival (6.3 ± 1.3 days; p < 0.05 vs untreated controls). CVF + CyA treatment resulted in 100% of grafts surviving long-term.


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  		Table I. Survival of hamster hearts transplanted in rats

 
Survival of fresh hamster hearts in naive rats receiving adoptive serum transfer is shown in Table IIGo. Sera were taken 10 days following transplantation from untreated rats that had rejected a hamster heart in 3–4 days or rats carrying an accommodating heart. Consistent with previous studies (10), transfer of 0.5 ml of the serum from untreated rats provoked hyperacute rejection of fresh hearts in naive rats (group 1). In contrast, sera from rats carrying an accommodated heart could precipitate hyperacute rejection only when high doses of the serum, i. e. up to 4 ml, were given (group 2).


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  		Table II. Survival of fresh hamster hearts in naive rats receiving immune serum taken from rats carrying an accommodating heart versus untreated rats

 
Table IIIGo depicts survival of second hamster hearts when transplanted into rats carrying a surviving first graft for 10 days. Those rats hyperacutely rejected naive second hearts (20 min to 6 h), while the first hearts continue to survive, i.e., accommodate (Group 1). When the second graft was a heart that had been already surviving for 14 days, i.e., was accommodating, in a different rat, the majority of the second hearts (8/11) survived long-term, with the remaining 3 hearts being rejected within a few days (4–9 days) (group 2, p < 0.001 vs group 1).


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  		Table III. Survival of second accommodating hearts versus second naive hearts in rats carrying a surviving first hamster heart transplanted 10 days earlier

 
Anti-donor Ab formation and complement activity

Anti-donor Ab formation in rats following hamster heart transplantation is shown in Fig. 1Go. In agreement with our previous data (11, 12), rats normally have very low titers of anti-hamster Abs, but rapidly produced anti–hamster IgM Abs that reached highly significant levels within 4 days following transplantation and were associated with graft rejection (Fig. 1GoA). CVF or CyA alone (data not shown) or in combination did not significantly (p > 0.05) influence the IgM Ab formation (Fig. 1GoB) as compared with untreated rats, consistent with the concept that those IgM Abs are primarily T cell independent (11). The elicited IgM Abs reached peak levels by day 8 to day 10, followed by a gradual decline to pretransplantation levels by day 30. Elicited anti-donor IgG Abs were completely suppressed by CyA in CyA alone (data not shown) or CVF + CyA treated rats (Fig. 1GoB), suggesting that those IgG Abs are T cell dependent (11).



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  		FIGURE 1. Anti-donor Ab formation and complement activity in rats after hamster heart transplantation. A, Anti–donor Abs in untreated rats; (B) anti-donor Abs in CVF + CyA treated rats. The data are shown as the mean value ± SD of six individual rats in each group. C, Complement activity (CH50) in rats treated with CVF on days -1 and 0. The data are shown as the mean value ± SD of four individual rats in each group.

 
Lytic complement activity (CH50) is shown in Fig. 1GoC. Administration of CVF on days -1 and 0 inhibited complement activity to undetectable levels until day 4–6 posttransplantation, followed by a progressive recovery to normal levels around day 14. This change of CH50 levels is correlated with the change of C3 levels (12).

Isotype of anti-donor Abs in transferred serum

Fig. 2Go shows the isotype of anti-donor Abs in sera taken 10 days following transplantation from rats carrying an accommodating hamster heart or from untreated rats that had rejected their grafts. Both sets of sera contained high titers of anti-donor IgM Abs (Fig. 2Go, A and G). In contrast to the sera taken from the rats carrying the accommodating hamster hearts, the sera from untreated rats displayed high titers of anti-donor IgG (Fig. 2Go, B and H). These IgG Abs contained IgG1, IgG2a, IgG2b, and no IgG2c subclass Abs (Fig. 2Go, C-F and I-L).



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  		FIGURE 2. Isotype of anti-donor Abs in transferred sera taken 10 days following transplantation from rats carrying an accommodated heart vs untreated rats. IgM and IgG, as well as IgG subclass in sera from accommodating rats (A–F) and untreated rats (G–L). The data show representative one of three separate tests in each group. Sera from naive rats (open histogram) were used as controls.

 
Anti-donor Abs in rats with simultaneous first and second xenografts

We monitored Ab titers in rats simultaneously carrying an accommodating first heart and a freshly transplanted second graft (Fig. 3Go). Sera were taken from animals immediately after rejection of the naive second heart or at the same time point carrying an accommodating first heart and a second accommodating heart. The two sets of sera exhibited a comparable level of anti-donor IgM Abs (Fig. 3Go, A and B) without IgG (Fig. 3Go, C and D).



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  		FIGURE 3. Anti-donor Abs in rats carrying simultaneously an accommodating first and freshly transplanted second heart. Anti-donor IgM and IgG in sera taken from rats immediately after rejection of a naive second heart (A and C) or at the same time carrying an surviving second accommodating heart (B and D). The data show representative one of four separate tests in each group. Sera from naive rats (open histogram) were used as controls.

 
Histopathology and immunohistochemistry

Histology of accommodating hearts harvested 10 days posttransplantation exhibited healthy appearing cardiac fibers and vascular endothelium (Fig. 4Goa). In contrast, rejected second naive hearts showed widespread endothelial destruction, hemorrhage, edema, and myocardial necrosis (Fig. 4Gob). In both accommodating first hearts and rejected second naive hearts, vascular deposition of rat IgM (Fig. 4Go, c and d) and C3 (Fig. 4Go, e and f) were observed with essentially no IgG (data not shown). In contrast to accommodating first hearts, rejected second naive hearts showed extensive fibrin deposition along vessel walls (Fig. 4Go, g and h). Accommodating first hearts, but not rejected second naive hearts, expressed in the vasculature high levels of the protective genes: A20 (Fig. 5Go, A and E), bcl-2 (Fig. 5Go, B and F), bcl-xL (Fig. 5Go, C and G), and HO-1 (Fig. 5Go, D and H). Accommodating hearts that continued to survive in the second recipients showed a similar histology picture as seen in accommodating first grafts. The loss of three of such second grafts (3 of 11) was associated with histopathological changes as seen in fresh grafts rejected in untreated naive rats (10).



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  		FIGURE 4. Histology and immunoperoxidase staining for Igs, complement, and fibrin in accommodating first hearts vs rejected second naive hearts. Grafts were harvested after rejection or 10 days posttransplantation. H&E-stained paraffin sections of accommodating first hearts (a) and second naive hearts that underwent hyperacute rejection (b). Vascular deposition of rat IgM, C3, and fibrin in accommodating first hearts (c, e, and g) and rejected second naive hearts (d, f, and h).

 


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  		FIGURE 5. Immunoperoxydase staining for protective gene expression in accommodating first hearts vs rejected second naive hearts. Grafts were harvested after rejection or 10 days posttransplantation. A20, bcl-2 bcl-xL, and HO-1 expressed in accommodating first hearts (A–D) and rejected second naive grafts (E–H).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We and others have argued that the continued survival of an immediately vascularized xenograft in the presence of anti-donor Abs and complement, i.e., accommodation, likely reflects the fact that the graft has become resistant to Ab and complement-mediated rejection (8, 24, 25). Two lines of evidence have been used to support this hypothesis. First, accommodated hamster hearts are not rejected when a given dose of rat anti-hamster serum is administered to the recipient, whereas a naive graft is rejected rapidly (10, 19). Second, a hamster heart that has been accommodating in a first recipient survives in one-third of cases when re-transplanted to a second naive recipient being treated only with CyA, a therapy that cannot prevent rejection of a fresh hamster heart in naive rats (21). However, the ability of accommodated grafts to resist rejection has not, until now, been tested directly under pathophysiological conditions where naive grafts undergo rejection.

In the case of adoptively transferring anti-hamster serum to the rat carrying the accommodated hamster heart (10, 19), one has to be unsure whether the level (dose) of anti-donor Abs administrated was appropriate. Although a serum dose chosen by others and ourselves cause rejection of only naive hearts, higher doses of the same serum could also lead to the rejection of accommodated hearts (10). Further, since the transferred sera contain both IgM and IgG Abs (taken from untreated rats 7 days after transplantation) (11, 12), we did not know whether the accommodated heart succumbed to the action of IgM plus complement and/or to IgG-mediated mechanisms (26, 27, 28). To some extent, previous studies have suggested that the accommodated graft becomes resistant to both the effects of IgM and IgG, a conclusion that has been only indirectly tested (10, 29). Moreover, it is not clear whether the balance of IgM and IgG in the sera that have been injected (10, 19) reflects the physiological situation that allows survival of the accommodated heart but causes rejection of a naive graft.

In the second protocol previously used, we showed that an accommodated heart could survive when transplanted to a second recipient that was being treated only with CyA; one-third of accommodated hearts survived whereas all naive hearts were rejected by day 3–4 (21). However, the retransplanted, accommodating hearts encountered a situation in which the anti-donor Abs were gradually elicited as they are by a naive heart. We have speculated in the past that one reason why accommodation occurs under certain circumstances is the gradual exposure of the heart to increasing amounts of anti-donor Abs, providing a "window of opportunity" for grafts to adapt to host immune responses, i. e., during which they express inducible protective genes (21, 30). Thus, the experimental protocol in which the accommodated hearts were transplanted to second fresh recipients receiving only CyA did not test whether those hearts could resist the sudden onslaught of preexisting anti-donor Abs plus complement.

In an attempt to test the rejection potential of the serum anti-donor IgM Abs to which the accommodated heart becomes resistant, we investigated whether adoptive transfer of these sera could precipitate rejection of freshly transplanted hamster hearts in naive rats. Rejection occurred only when high doses (up to 4 ml) of the sera were injected, in contrast to the sera from untreated rats (0.5 ml) (Table IIGo). This difference is likely due to a high level of both IgM and IgG in the sera of untreated animals, whereas the sera of rats carrying the accommodating hearts contain only IgM (see Fig. 2Go), making the latter sera relatively poorly resistant to transfer-induced dilution of Ab concentration. We previously showed that these xenoreactive IgG Abs exhibit potent activity in mediation of complement-dependent rejection (12, 28).

Given these caveats that are needed to interpret past experiments, we performed second hamster heart transplantation in rats carrying an accommodating graft at the time when a high level of anti-donor Abs and returned complement activity were present, i.e., 10 days following the first xenografting.

Short-term administration of CVF plus daily CyA resulted in temporary complement inactivation and continuous T cell inhibition, with essentially an intact T cell-independent IgM response (Fig. 1Go). We previously showed that CVF + CyA partially decreased anti-donor IgM Ab production (12), whereas this effect was not profound in the present study. This discrepancy may be explained by the different methods (FACS vs cellular ELISA) and different target cells (fresh PBMCs vs renal epithelial cells) used to detect the Ab in the two experimental systems.

Complement activity begins to recover after 4–6 days. Although pretreatment levels of CH50 are not routinely achieved until day 14 (Fig. 1GoC), previous studies demonstrated that reconstitution of complement activity to 10% of the normal level in complement-deficient rats enables hyperacute rejection of xenografts to occur in these animals (28, 31). The second naive hamster hearts were hyperacutely rejected in all cases, whereas the first graft continued to survive. Immunohistopathology demonstrated vascular deposition of IgM and complement in the accommodating first hearts and the second naive hearts to approximately the same extent, whereas tissue injury occurred only in the rejected second naive hearts (Fig. 4Go). Thus, the anti-donor Abs and complement that normally destroy a naive graft do not cause rejection of the accommodated graft.

A factor that could contribute to rejection of the naive second heart is ischemia-reperfusion injury that may render grafts susceptible to rejection (32, 33, 34), a factor not present for the initial transplants on day 10 of their survival. We tested whether a hamster heart that has been accommodating in another rats would survive in the second recipients under conditions that lead to rejection of the naive heart. The second accommodating hearts all survived more than 4 days with 8/11 of grafts surviving long-term (Table IIIGo). The loss of the remaining 3 grafts was presumably due to injury associated with the complicated procedure of retransplantation, which requires significantly longer than a normal transplant and thus has a correspondingly longer ischemia time.

Survival of the accommodating hearts in their second recipients may be due in part to their resistance to ischemia-reperfusion injury by expression of a protective gene, i.e., HO-1 (35, 36). Our data, however, suggest that the immunological factors, i.e., Abs and complement, of the host determine rejection of the second transplants. We previously showed that, when the second fresh hearts were transplanted 30 days following the first graft that had been accommodating, the second hearts also accommodated in a majority of cases (10). Moreover, whereas HO-1-deficient (knockout; HO-1-/-) mouse hearts fail to survive and accommodate in CVF + CyA-treated rats as the wild-type HO-1+/+ mouse hearts do, the HO-1-/- hearts do survive in T and B cell-deficient Rag 2 mice (18). In the present study, we found that complement inactivation during the second xenografting prevented hyperacute rejection and resulted in long-term survival of second naive hearts (Lin et al., manuscript in preparation). These findings would suggest that the accommodated hearts withstand the immunological factors that normally cause rejection of xenografts.

The time points of second transplantation may determine the rejection or acceptance of the second grafts. These results may be explained by a change in the host immune response during the progress of graft accommodation (20, 37, 38, 39). By day 30 following transplantation, elicited anti-donor IgM Abs that normally have a short half-life usually within a few days (40) had declined to very low levels (Fig. 1Go). B cells fail to produce IgM Abs in response to the second, xenoantigen-specific challenge by a second xenograft, indicating a state of xenospecific B cell hyporesponsiveness (20, 38, 39). In addition, the acceptance of the second hearts may be enhanced by a host Th2 immune deviation (10) that is strongly present by day 30. The Th2 cytokines may have beneficial effects on graft survival by suppressing Th1 cell immune response (41) and enhancing the expression of protective genes (42).

Accommodating first hearts and second accommodating grafts expressed the protective genes A20, bcl-2, bcl-xL, and HO-1, whereas these genes were expressed at an undetectable or very low level in the rejected, naive second hearts (Fig. 5Go). Others’ and our previous work showed that these protective genes are induced under inflammatory conditions associated with EC activation (16, 43, 44), which underlies the mechanism of xenograft rejection (16). Using a HO-1-/- mouse-to-rat heart transplantation model, we recently obtained direct evidence that expression of the HO-1 gene products is essential for graft survival and accommodation (18). In a time-course analysis, we previously detected that up-regulation of some of these genes occurred as early as 12 h posttransplantation (18). The hyperacute rejection of the second naive hearts, which did not allow the induction of the protective genes and perhaps other protective factors, may at least in part explain the loss of those grafts. This balance between "rejection" and "protection" responses may also account for the rejection within a few days of initially transplanted hamster hearts in untreated rats or rats treated with CyA alone.

In the present study, we have established the resistance of accommodated hamster hearts to rat anti-hamster IgM Abs and complement under pathophysiological conditions that normally lead to rejection of xenografts. This resistance may depend in part on the expression in graft EC and smooth muscle cells of a number of protective genes that we have been studying.


   Acknowledgments
 
We thank Drs. Simon Robson, Josef Anrather, Robert Lindemann, and others in our research center for discussion of these results and reading of the manuscript.


   Footnotes
 
1 This work is supported by National Institutes of Health Grant 1R01 HL58688. F.H.B. is the Lewis Thomas Professor at Harvard Medical School, and a paid consultant to Novartis Pharma, Basel, Switzerland. This is a paper 762 from our laboratory. Back

2 Address correspondence and reprint requests to Dr. Yuan Lin, Immunobiology Research Center, Beth Israel Deaconess Medical Center, Harvard Medical School, 99 Brookline Avenue, Boston, MA 02215. E-mail address: Back

3 Abbreviations used in this paper: DXR, delayed xenograft rejection; CVF, cobra venom factor; CyA, cyclosporin A; EC, endothelial cell; HO-1, heme oxygenase-1; H&E, hematoxylin and eosin. Back

Received for publication February 19, 1999. Accepted for publication May 27, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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