Ann Thorac Surg 1997;64:628-633
© 1997 The Society of Thoracic Surgeons
Original Article: Cardiovascular
Creation of Chimeric Hearts: A Tool for Testing the "Passenger Leukocyte" Hypothesis
Simon D. Eiref, BA,
Wei Zhang, MD,
Sicco H. Popma, BS,
Lisa J. Shah,
Jonni S. Moore, PhD,
BruceR. Rosengard, MD
Division of Cardiothoracic Surgery, University of Pennsylvania Health System, Philadelphia, Pennsylvania
 |
Abstract
|
|---|
Background. Bone marrow-derived antigen-presenting cells (APCs) are thought to be a migratory component of organ allografts that activate the rejection response, and recently they have been postulated to play a critical role in tolerance induction. Our goal was to create chimeric hearts (organs with parenchyma and APCs of differing genotype) for use in models of transplantation to test the "passenger leukocyte" theory.
Methods. Murine bone marrow transplantations were performed in two fully major histocompatibility complex (MHC) mismatched strain combinations: C3H 
B10 and CBA BALB/c. Recipients were lethally irradiated (10 Gy) and then received 1 x 107 bone marrow cells intravenously. Bone marrow transplant survivors had their organ APCs isolated by digestion with collagenase D, followed by density gradient centrifugation. The APC-enriched fraction was stained with fluorescein-labeled monoclonal antibodies specific for either donor (I-Ak) or recipient (I-Ab/d) class II MHC antigens, which are expressed by all APCs but not by parenchymal cells. Donor and recipient class II expression was determined by flow cytometry.
Results. Sixty-nine of 100 (69.0%) of C3H B10 and 52/107 (48.6%) of CBA BALB/c bone marrow transplant recipients survived more than 100 days, whereas all B10 (n = 12) and BALB/c (n = 10) irradiation controls died within 14 days. Mortality appeared to be caused by engraftment failure as most recipients died before day 20. Flow cytometry demonstrated complete APC replacement in hearts (n = 17) and spleens (n = 40), as APC-enriched fractions stained only for donor class II MHC antigens.
Conclusion. Bone marrow transplantation leads to replacement of heart APCs in two murine models. Chimeric hearts are now being used to test the role of APCs in allograft rejection and in tolerance induction.
|
Introduction
|
|---|
Passenger leukocytes (PLs) are bone marrow-derived cells that are carried within the interstitium of organ allografts from donor to recipient. It is widely held that PLs migrate from organ transplants to the lymphoid tissue of recipients, where they stimulate the host immune response to the allograft. Recently, long-term acceptors of kidney [1] and liver [2] allografts have been found to have donor cellular elements in their peripheral lymphoid compartment. This observation has fueled speculation that PLs might also play an important role in the development of host unresponsiveness to donor antigens. This concept of posttransplantation microchimerism has led to clinical trials [3, 4] of solid organ transplantation with simultaneous infusion of donor bone marrow in hopes of increasing the number of PLs and the degree of microchimerism, and thereby decreasing the frequency and vigor of rejection episodes. More importantly, however, this hypothesis has refocused interest in posttransplantation cell-trafficking and the immunogenicity or tolerogenicity of PLs.
The notion that hematopoietic cells migrate from a transplant to regional lymph nodes and initiate an immune response was first suggested by Snell [5]. Subsequently, Steinmuller [6] showed that skin isografts taken from radiation chimeras were capable of sensitizing naive recipients against alloantigens expressed by the skin donor's hematopoietic system. It was then suggested that if PLs are capable of host sensitization, then perhaps they are required for rejection. Guttmann and colleagues [7] showed that kidney allografts from bone marrow chimeras had prolonged survival when transplanted into recipients presumably matched to the graft's PLs. Although conceptually sound, these experiments had several technical limitations: (1) hematopoietic reconstitution by bone marrow transplantation (BMT) was questionable as only 4 of 12 rats were clearly shown to be chimeric, (2) the degree of hematopoietic reconstitution could not be accurately determined at that time, (3) PL replacement within kidney by BMT was not examined, (4) organs from the 4 rats proven chimeric did not uniformly show preserved function after transplantation, and (5) some of the donor rats had graft-versus-host disease (GVHD) at the time of organ donation. Thus, solid conclusions could not be made from this study.
"Organ parking" is an alternative method for replacing PLs, in which organs are parked in an immunosuppressed, intermediate host before retransplantation into a secondary untreated recipient. The limitation of this approach was stated directly in the methods section of the first manuscript [8] describing organ parking: "after 60–300 days of residence in an intermediate host it was assumed that most if not all of the original passenger leukocytes would have migrated from the allograft." Subsequent articles in the literature using organ parking to assess the role of PLs in rejection have assumed that parking results in PL replacement. Studies using organ parking have shown that parked kidneys have prolonged survival when retransplanted into secondary recipients matched to the intermediate host [9, 10] and that a rapid rejection response to parked kidneys can be reconstituted by adoptive transfer of donor parenchymal-type dendritic cells at the time of transplantation [11]. These findings were taken as proof of the hypothesis that donor type antigen-presenting cells (APCs) are required to trigger allograft rejection, despite the fact that the studies neither confirmed APC replacement nor ruled out the transfer of graft resident suppressor cells [12] in parked hearts. Furthermore, all of these studies lacked retransplants of parked hearts into third-party hosts. Third-party controls are necessary to confirm that absence of donor PLs as opposed to the presence of recipient-type PLs in a chimeric organ is responsible for prolonged survival on retransplantation.
The goals of the present study were to develop models for creation of chimeric hearts using BMT and to quantitate the degree of APC replacement using monoclonal antibodies specific for donor or recipient class II major histocompatibility complex (MHC) antigens and flow cytometry. The creation of chimeric hearts with complete APC replacement would allow accurate testing of the hypothesis that PLs are critical for cardiac allograft rejection and tolerance induction. We report here on the successful development of two mouse BMT models (C3H B10 and CBA BALB/c), which produce chimeric hearts with complete APC replacement as assessed by flow cytometry.
|
Material and Methods
|
|---|
Experimental Animals
Male BALB/cByJ (H-2d), CBA/J (H-2k), C3H/HeJ (H-2k), and C57BL/10SnJ (H-2b) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Recipients were 6 to 8 weeks of age and donors were greater than 6 weeks of age at the time of BMT. Mice were housed in conventional cages with filter lids and maintained on a conventional diet. All animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication 85-23, revised 1985).
Bone Marrow Transplantation
ANTIBIOTICS.
Neomycin sulfate (40 mg/L) and polymyxin B sulfate (200,000 U/L) were added to the drinking water of BMT-recipient mice beginning 2 days before BMT and continuing until 14 days after BMT.
IRRADIATION.
Recipient mice received 10 Gy of total body irradiation from a cesium source (Gammacell 40; Atomic Energy of Canada Limited, Mississauga, Ont, Canada) 6 hours before BMT.
PREPARATION OF DONOR CELLS.
On the day of BMT, femora and humeri of donor mice were surgically removed and were cleaned of adherent tissue under sterile conditions. The ends of the bones were clipped, and bone marrow was flushed out with chilled Dulbecco's phosphate-buffered saline solution (Gibco, Grand Island, NY). Clumps were removed by passing the cell solution through a 70-µm filter. Red blood cells were lysed with ammonium-chloride-potassium buffer (Bio Whittaker, Walkersville, MD) and were washed three times with phosphate-buffered saline before final resuspension at a concentration of 1 x 107 cell/mL. Cell viability was determined by trypan blue dye exclusion and always exceeded 95%.
BONE MARROW INFUSION.
Six hours after irradiation, recipient mice received 1 x 107 bone marrow cells in 1 mL phosphate-buffered saline solution through tail vein injection.
Flow Cytometry
PREPARATION OF SINGLE-CELL SUSPENSIONS FROM HEART AND SPLEEN.
Bone marrow transplantation recipients surviving at least 100 days were killed by cervical dislocation. Excised hearts were placed in heparinized saline solution (10 U/mL) and spleens were placed in RPMI 1640 with 25 mmol/L HEPES and L-glutamine (GIBCO/BRL, Grand Island, NY). Fatty tissue was removed from the organs. Hearts and spleens were treated individually as follows. Each heart was quartered with a scalpel, washed vigorously three times in heparinized saline to remove any blood, and then chopped finely into less than 1-mm fragments. The heart was then digested for a period of 2 hours by sequential incubations at 37°C in collagenase D (lot #14361722, Boehringer Manheim, Indianapolis, IN) in Hank's balanced salt solution (GIBCO/BRL). The initial incubation contained 100 U/mL of collagenase D in 5 mL and then residual tissue fragments were transferred to 400 U/mL collagenase D in 3 mL. Each spleen was processed by injecting 3 mL of 100 U/mL collagenase D subcapsularly with a 27-gauge needle, then teasing the spleen into less than 1-mm fragments, and incubating the remaining solid tissue for 90 minutes at 37°C in 2 mL of 400 U/mL collagenase D. For both heart and spleen, the aliquots of 10 U/mL and 400 U/mL were then filtered through a 70-µm sieve and combined.
BOVINE SERUM ALBUMIN FRACTIONATION.
Bovine serum albumin (Fraction V, Intergen Co., Purchase, NY) was prepared as previously described [13] and the refractive index was adjusted to between 1.384 and 1.385 at 25°C. The digested organ fractions from the previous step were resuspended in 2.5 mL bovine serum albumin and overlaid with 1.5 mL RPMI in 10-mL centrifuge tubes (Oak Ridge 3119-0110, Nalgene, Rochester, NY). After centrifugation at 9500g for 15 minutes at 22°C, low-density cells were collected from the RPMI and RPMI/bovine serum albumin interface and washed in RPMI. The yield of low-density nucleated cells varied but typically ranged between 1 and 5 x 105 cells per heart and between 1 and 10 x 106 cells per spleen.
ANTIBODY STAINING.
The following fluoresceinated murine monoclonal antibodies were obtained commercially (Pharmingen, San Diego, CA): 25-9-17 (anti-I-Ab/d, specific for either bone marrow recipient), 11-5.2 (anti-I-Ak, specific for either bone marrow donor), and G155-178 (an immunoglobulin G2a isotype control). The cells were resuspended in wash buffer consisting of phosphate-buffered saline with 1% fetal calf serum (Hyclone Laboratories, Logan, UT) and incubated in the dark at 4°C for 35 minutes with fluorescein-labeled monoclonal antibodies specific for either donor (I-Ak) or recipient (I-Ab/d) class II MHC antigens or with an isotype-matched (IgG2a, k isotype standard) monoclonal antibody of irrelevant specificity as a negative control. After incubation, the cells were rinsed twice with 1 mL of wash buffer, and resuspended in either 0.5 mL of buffer for immediate flow cytometric analysis or in 0.5 mL of 1% paraformaldehyde (Sigma Chemical Co., St. Louis, MO) if flow cytometry was delayed. Flow cytometric analysis was performed on a FACSCAN (Becton Dickinson, San Jose, CA). Fluorescence intensity was determined at 530 ± 15 nm.
STATISTICS.
Actuarial survival curves were calculated by the Kaplan-Meier product-limited method [14]. The equality of survival curves was tested by the Breslow statistic [15], which is analogous to the Kruskal-Wallis (generalized Wilcoxon) test.
|
Results
|
|---|
Bone Marrow Transplantation
Autologous and allogeneic BMTs were performed in B10 (Fig 1
) and BALB/c recipients (Fig 2). C3H B10 BMT survival was 69% at 100 days, which was not significantly different (p = 0.406) from the survival of autologous BMTs (81%) using B10 marrow. Two thirds of the mortality occurred earlier than 20 days, suggesting engraftment failure, and the late deaths did not appear to be related to GVHD. Bone marrow transplantation in the CBA BALB/c model was not as successful and had a survival rate of 49% at 100 days. This was significantly (p = 0.008) different from the survival of autologous BMTs with BALB/c marrow (89%). In the CBA BALB/c model, the vast majority of mortality occurred within the first 2 weeks after BMT, consistent with engraftment failure. In both models, irradiation controls all died of aplasia by day 14 after treatment.
Flow Cytometry
Antigen-presenting cell–enriched fractions were isolated from hearts of at least 100-day-old chimeras (n = 17) and were stained with monoclonal antibodies specific for donor or recipient class II MHC antigens. Histograms for chimeric B10 hearts (Fig 3) and chimeric BALB/c hearts (Fig 4) demonstrate that there was complete replacement of class II-positive cells as the mean fluorescence intensity of the recipient-type (I-Ab/d) monoclonal antibody coincides with that of the isotype control, whereas staining with donor-type (I-Ak) antibody shows a population of cells with mean fluorescence intensity coinciding with the isotype control (nonspecific staining) as well as a second population with higher mean fluorescence (specific staining). Similar studies were performed on spleens (n = 40) harvested from BMT chimeras, and flow cytometry confirmed complete replacement of splenic APCs (Figs 5, 6). The shift in mean fluorescence was more dramatic in assays using splenocytes because the absolute number of class II-positive cells in the spleen is orders of magnitude greater than in the heart and the percentage of spleen cells that are class II-positive is also much higher than in the heart. Thus, the purity of the APC-enriched fraction was much higher in the splenocyte samples as compared with those from the heart. This explains a virtually complete rightward shift in the fluorescence intensity curve with splenocytes because there was increased specific binding of the anti-I-Ak monoclonal antibody as a result of increased class II expression. Furthermore, there was decreased nonspecific binding of the anti-I-Ak monoclonal antibody because of fewer dead and class II-negative cells, which bind nonspecifically to monoclonal antibody.
View larger version (20K):
[in this window]
[in a new window]
|
Fig 3. . Flow cytometric analysis of an antigen-presenting cell (APC)-enriched fraction isolated from a chimeric B10 heart (C3H B10 BMT): (a) Staining with fluoresceinated monoclonal antibodies G155-178 (dashed line), an immunoglobulin G2a isotype control of irrelevant specificity, and 25-9-17 (solid line), an anti-I-Ab/d(recipient B10 class II major histocompatibility complex [MHC] antigen) reagent. (b) Staining with fluoresceinated monoclonal antibodies G155-178 (dashed line), an immunoglobulin G2a isotype control of irrelevant specificity, and 11-5.2 (solid line), an anti-I-Ak (donor C3H class II MHC antigen) reagent.
|
|
View larger version (19K):
[in this window]
[in a new window]
|
Fig 4. . Flow cytometric analysis of an APC-enriched fraction isolated from a chimeric BALB/c heart (CBA BALB/c BMT): (a) Staining with fluoresceinated monoclonal antibodies G155-178 (dashed line), an immunoglobulin G2a isotype control of irrelevant specificity, and 25-9-17 (solid line), an anti-I-Ab/d (recipient BALB/c class II MHC antigen) reagent. (b) Staining with fluoresceinated monoclonal antibodies G155-178 (dashed line), an immunoglobulin G2a isotype control of irrelevant specificity, and 11-5.2 (solid line), an anti-I-Ak (donor CBA class II MHC antigen) reagent.
|
|
View larger version (21K):
[in this window]
[in a new window]
|
Fig 5. . Flow cytometric analysis of an APC-enriched fraction isolated from a chimeric B10 spleen (C3H B10 BMT): (a) Staining with fluoresceinated monoclonal antibodies G155-178 (dashed line), an immunoglobulin G2a isotype control of irrelevant specificity, and 25-9-17 (solid line), an anti-I-Ab/d (recipient B10 class II MHC antigen) reagent. (b) Staining with fluoresceinated monoclonal antibodies G155-178 (dashed line), an immunoglobulin G2a isotype control of irrelevant specificity, and 11-5.2 (solid line), an anti-I-Ak (donor C3H class II MHC antigen) reagent.
|
|
View larger version (21K):
[in this window]
[in a new window]
|
Fig 6. . Flow cytometric analysis of an APC-enriched fraction isolated from a chimeric BALB/c spleen (CBA BALB/c BMT): (a) Staining with fluoresceinated monoclonal antibodies G155-178 (dashed line), an immunoglobulin G2a isotype control of irrelevant specificity, and 25-9-17 (solid line), an anti-I-Ab/d (recipient BALB/c class II MHC antigen) reagent. (b) Staining with fluoresceinated monoclonal antibodies G155-178 (dashed line), an immunoglobulin G2a isotype control of irrelevant specificity, and 11-5.2 (solid line), an anti-I-Ak (donor CBA class II MHC antigen) reagent.
|
|
|
Comment
|
|---|
Interest in phenomena related to allograft passenger APCs has been rekindled by reports of long-term microchimerism in transplant recipients [1, 2]. This observation has led to a revised view of the PL hypothesis, which theorizes that graft resident APCs are a requirement for developing donor-specific unresponsiveness in recipients in contrast to the traditional view that migratory APCs are the primary immunogenic component of organ allografts and are required to trigger acute rejection. A possible dual nature of APCs [16] is an intriguing possibility and warrants further investigation. Unfortunately, the models that have been used in the past to study passenger cell phenomena have significant limitations. The primary approach has been to use "organ parking" protocols to replace APCs before retransplantation [9–11]. However, these studies have failed to examine organs before retransplantation to confirm APC replacement by either phenotypic or functional studies. Furthermore, organ parking studies in rats have failed to rule out intragraft transfer of suppressor T cells, which are well described in rat systems [17]. Efforts to produce chimeric organs by allogeneic BMT antedate organ parking experiments [7]. Unfortunately, these studies were done at a time when it was difficult to quantify APC replacement phenotypically because flow cytometry was in its infancy. Because our laboratory is interested in defining the role that APCs play both in organ rejection and in tolerance induction, we sought to create a reproducible method of generating chimeric hearts so that transplants could be performed in which either parenchyma or APCs are matched to the recipient.
This study details our method for producing chimeric B10 or BALB/c hearts using C3H B10 or CBA BALB/c BMTs. Both BMT models have acceptable survivals and the CBA BALB/c model shows no evidence of GVHD. Three animals in the C3H B10 model (n = 100) demonstrated hair loss and scaly skin, suggestive of GVHD. However, the late mortality observed in 10% of C3H B10 BMTs was not caused by GVHD and was likely related to infection as fully allogeneic radiation chimeras are relatively immunodeficient [18]. The virtual absence of GVHD in these models is important because end-organ injury as a result of alloreactive T cells could impair the function of chimeric grafts before transplantation, thus invalidating experiments in which chimeric hearts are used to assess the role of PLs in rejection or tolerance.
Monoclonal antibodies against class II MHC specificities were used to assess the degree of APC replacement in solid organs consequent to BMT, as class II MHC antigens are highly expressed on APCs but are not expressed constitutively on cardiac parenchymal cells [19]. APCs isolated from chimeric hearts stained positively for donor-type class II MHC antigens and negatively for recipient-type class II antigens, which suggests complete APC replacement. This represents phenotypic demonstration of cardiac APC replacement by BMT. The availability of chimeric hearts will permit studies examining the immunogenicity and tolerogenicity of APCs. Studies reexamining the role of APCs in cardiac allograft rejection are currently under way.
|
Footnotes
|
|---|
Presented at the Poster Session of the Thirty-third Annual Meeting of The Society of Thoracic Surgeons, San Diego, CA, Feb 3–5, 1997.
Address reprint requests to Dr Rosengard, Division of Cardiothoracic Surgery, University of Pennsylvania Health System, 6 Silverstein, 3400 Spruce Street, Philadelphia, PA 19104 (e-mail: brosenga{at}mail.med.upenn.edu).
|
References
|
|---|
- Starzl TE, Demetris AJ, Trucco M, et al. Chimerism and donor-specific nonreactivity 27 to 29 years after kidney allotransplantation. Transplantation 1993;55:1272–7.[Medline]
- Starzl TE, Demetris AJ, Murase N, Ildstad S, Ricordi C, Trucco M. Cell migration, chimerism, and graft acceptance. Lancet 1992;339:1579–82.[Medline]
- Pham SM, Keenan RJ, Rao AS, et al. Perioperative donor bone marrow infusion augments chimerism in heart and lung transplant recipients. Ann Thorac Surg 1995;60:1015–20.
- Shapiro R, Rao AS, Fontes P, et al. Combined simultaneous kidney/bone marrow transplantation. Transplantation 1995;60:1421–5.[Medline]
- Snell G. The homograft reaction. Ann Rev Microbiol 1957;11:439–58.[Medline]
- Steinmuller D. Immunization with skin isografts taken from tolerant mice. Science 1967;158:127–9.[Abstract/Free Full Text]
- Guttmann RD, Lindquist RR, Ockner SA. Renal transplantation in the inbred rat. IX. Hematopoietic origin of an immunogenic stimulus of rejection. Transplantation1969;8:472–84.[Medline]
- Stuart FP, Bastien E, Holter A, Fitch FW, Elkins WL. Role of passenger leukocytes in the rejection of renal allografts. Transplant Proc 1971;3:461–4.[Medline]
- Stuart FP, Garrick T, Holter A, Lynch A, Bastien E. Delayed rejection of renal allografts in the rat and dog by reduction of passenger leukocytes. Surgery 1971;70:128–34.[Medline]
- Hart DN, Winearls CG, Fabre JW. Graft adaptation: studies on possible mechanisms in long-term surviving rat renal allografts. Transplantation 1980;30:73–80.[Medline]
- Lechler RI, Batchelor JR. Restoration of immunogenicity to passenger cell-depleted kidney allografts by the addition of donor strain dendritic cells. J Exp Med 1982;155:31–41.[Abstract/Free Full Text]
- Rosengard BR, Ojikutu CA, Guzzetta PC, et al. Renal transplantation in miniature swine: preliminary evidence that graft infiltrating leukocytes suppress donor-specific cell-mediated lymphocytotoxicity in co-culture. Transplant Proc 1991;23:189–91.[Medline]
- Swiggard WJ, Witmer-Pack MD, Steinman RM, Inaba K. Enrichment of dendritic cells by plastic adherence and EA rosetting [CD-ROM]. In: Coligan JE, Kruisbeck AM, Margulies DH, Shevach EM, Strober W. Current Protocols in Immunology. New York: Wiley, 1996:Unit 3.7.
- Kaplan EL, Meier P. Nonparametric estimation from incomplete observations. J Am Statist Assoc 1958;53:457–81.
- Breslow N. A generalized Kruskal-Wallis test for comparing K samples subject to unequal patterns of censorship. Biometrika 1970;57:579–94.[Abstract/Free Full Text]
- Steinman RM, Inaba K, Austyn JM. Donor-derived chimerism in recipients of organ transplants. Hepatology 1993;17:1153–6.[Medline]
- Pawelec G. Suppressor cells in transplantation immunology: do recent advances in T cell immunobiology and cytokine networking contribute to the solution of an old conundrum? Transpl Immunol 1993;1:172–81.[Medline]
- Sachs DH. Transplantation tolerance. Ann Thorac Surg 1993;56:1221–7.[Abstract]
- Hansen TH, Beatriz MC, Sachs DH. The major histocompatibility complex. In: Paul WE, ed. Fundamental immunology. New York: Raven Press, 1993:577–628.
This article has been cited by other articles:
|
|
|
|
 
W. Y. Szeto, A. M. Krasinskas, D. Kreisel, S. H. Popma, and B. R. Rosengard
Donor antigen-presenting cells are important in the development of obliterative airway disease
J. Thorac. Cardiovasc. Surg.,
December 1, 2000;
120(6):
1070 - 1077.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
