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J Thorac Cardiovasc Surg 1998;115:998-1002
© 1998 Mosby, Inc.

CARDIAC AND PULMONARY REPLACEMENT

Triple immunosuppression reduces mononuclear cell infiltration andprolongs graft life in pig–to–newborn baboon cardiacxenotransplantation

Supported in part by a gift from Roche Pharmaceuticals.

Read in part at the Seventeenth Annual Meeting of the InternationalSociety of Heart and Lung Transplantation, London, United Kingdom, April 3-6,1997.

Received for publication April 22, 1997. Revisions requested August 11, 1997. Accepted for publication Dec. 5, 1997. Address for reprints: Robert E. Michler, MD, Karl P. Klassen Professorof Surgery, Chief, Division of Cardiothoracic Surgery, Doan Hall North, OhioState University Medical Center, 410 West 10th Ave., Columbus, OH 43210.

Abstract

Objective: Pig hearts transplanted intounmedicated newborn baboons do not undergo hyperacute rejection by preformedxenoantibody and complement. These grafts are rejected at days 3 to 4 inassociation with the infiltration of macrophages and natural killer cells. Weinvestigated whether an immunosuppressive regimen used widely in cardiacallotransplantation could reduce this cellular response and prolong xenograftlife.
Methods: Ten newborn baboonsunderwent heterotopic pig cardiac xenotransplantation. Five baboons wereimmunosuppressed with mycophenolate mofetil (100 mg/kg), methylprednisoloneacetate (0.8 mg/kg), and cyclosporine A (INN: ciclosporin; 10 mg/kg). Xenograftrejection was studied by light microscopy and immunofluorescence. The inducedhumoral response to porcine xenoantigens was documented by enzyme-linkedimmunosorbent assay using synthetic -1,3-galactosyl epitopes coupled tobovine serum albumin.
Results: Graft lifewas extended from a mean of 3.6 ± 0.5 days (n =5) to a mean of 6.2  ± 1.1 days (n =5, p = 0.01). In comparison with controls,explanted grafts from medicated baboons demonstrated reduced infiltration withnatural killer cells and macrophages, but increased evidence ofcomplement-mediated rejection substantiated by increased deposition ofimmunoglobulin M, complement, and fibrin. In all baboons receiving transplants,levels of both immunoglobulin M and immunoglobulin G anti-galactose weresignificantly increased after transplantation, with immunoglobulin G levelsremaining persistently elevated.
Conclusions:These results indicate that cyclosporine-based triple immunosuppressionmarginally prolonged xenograft survival and appears to have reduced the naturalkiller cell and macrophage infiltrates. The immunosuppressive protocol, however,was not adequate to prevent the induced immunoglobulin M humoral response andprevent complement-mediated graft injury. (J Thorac Cardiovasc Surg1998;115:998-1006)

Cardiac transplantation has progressed to the point at which it currentlyrepresents the best therapeutic option available to patients with end-stageheart disease. The supply of human donor hearts, however, remains inadequate tomeet the ever-increasing demand. The severe shortage of donor hearts isespecially acute in the pediatric population. This situation has been furtherexacerbated by the inability to use mechanical assist devices as "abridge," sustaining pediatric patients in need of a heart transplant untilan appropriate donor is located. This situation has prompted the investigationof xenografts as an alternative source of organs for human beings. The similarphysiology and size of pig and human organs, the ease with which pigs can bebred in large numbers, and their relative lack of pathogens capable of causinginfection in human beings make pigs an attractive source of organs.

Mature primates contain high levels of preformed immunoglobulin M (IgM)xenoantibody, and grafts transplanted into these animals undergo hyperacuterejection within minutes to hours. ¹These preformed xenoantibodies are primarily directed to the epitope, Gal--1-3-Galß-1-4-GlcNAc-R (-Gal). ²Expression of the -Gal residue on cells depends on the presence of afunctional gene encoding for the -1,3-galactosyltransferase glycosidationenzyme. The phylogenetically related group of primates, which includes humanbeings, apes, and Old World monkeys (the platyrrhines), contains an inactivated-1,3-galactosyltransferase gene and does not express -Gal on cellsurface structures. In contrast, all other mammalian species contain an intact-1,3-galactosyltransferase gene and express -Gal on endotheliumand other cell lines. ³

It is thought that anti-Gal antibodies initially develop in human beings,apes, and Old World monkeys after exposure to gut bacterial flora, which alsoexpress the -Gal epitope. ⁴Because the newborn gut is sterile, there is an immunologic window during whichnewborn primates do not have IgM anti-Gal antibodies and do have reduced levelsof IgG anti-Gal antibodies. Indeed, IgM xenoantibodies are nearly absent in theserum of newborn human beings and primates and progressively increase to reachadult levels by 2 months of age. As for xenoreactive IgG antibody levels, thosein newborn infants are approximately 25% of those in adults, reflectingpassive transfer of maternal IgG across the placenta. ^5,6Because newborn human beings and baboons have very low levels of anti-Gal IgMantibodies and reduced levels of anti-Gal IgG antibodies, newborns serve as anatural model of antibody depletion and pig hearts transplanted into baboons donot undergo hyperacute rejection. ⁷Rather, we have previously shown that these grafts are rejected at days 3 to 4.This rejection process is associated with an infiltration of mononuclear cells(predominately natural killer cells and macrophages) and an induced humoralresponse. ^8,9 The importance of natural killercells and macrophages was first observed in complement-depleted rat recipientsof heterotopic guinea pig cardiac xenografts. ¹⁰ This phenomena has been termed"delayed xenograft rejection" and is distinct in its time course andpathogenesis from T cell–mediated rejection seen in acute allograftrejection.

It has yet to be determined whether "delayed xenograft rejection"in primates can be abated with conventional immunosuppression. Therefore thisstudy sought to investigate the effects of a cyclosporine-basedimmunosuppressive protocol (INN: ciclosporin), analogous to those used inallotransplantation, on "delayed xenograft rejection" in newbornbaboon recipients of pig cardiac xenografts.

Materials and methods

Animals
Newborn baboons (Papio anubis) were bornat the Institute for Comparative Medicine (Columbia University, New York, N.Y.).Expectant mothers were purchased from Biologic Resources Laboratory, Chicago,Illinois. Day-old Yorkshire pigs weighing 1000 gm were purchased from AnimalBiotech Industries, Inc., Danboro, Pennsylvania, and used as donors. Approval bythe Animal Care and Use Committee of Columbia University was obtained beforethis study was begun. All animals received humane care in compliance with the"Guide for the Care and Use of Laboratory Animals" prepared by theInstitute of Laboratory Animal Resources and published by the NationalInstitutes of Health (NIH Publication No. 86-23, revised 1985). Primateswere housed in a facility approved by the American Association for LaboratoryAnimal Care.

Cardiac transplantation
Heterotopic cardiac xenotransplantations were performed as previouslydescribed. ¹¹ In brief,day-old piglets were sedated with intramuscular ketamine (Aveco, Fort Dodge,Iowa) and intubated. General endotracheal anesthesia was maintained withisoflurane (Anaquest, Madison, Wis.). After isolation of the great vessels, theinnominate artery was cannulated with a 20-gauge Angiocath catheter (DeseretCo., Sandy, Utah). After transection of the left inferior pulmonary vein andinferior vena cava, the heart was arrested with cold (4° C) University ofWisconsin solution (Du Pont Pharmaceuticals, Wilmington, Del.) and chilled withtopical cold saline solution. The heart was resected, and the left atrial freewall, as well as the venae cavae, was closed with 7-0 polypropylene suture. Thepulmonary artery was transected at its bifurcation and the aorta was transectedslightly shorter than the pulmonary artery. The heart was then stored in coldsaline solution as final preparation of the recipient was completed.

Newborn baboons weighing 950 to 1300 gm (average age 36 days control, 38days experimental) were sedated with ketamine, intubated, and maintained undergeneral isoflurane anesthesia. They underwent heterotopic cardiactransplantation, with anastomosis of the graft aorta and pulmonary artery to thecommon iliac artery and vein, respectively. Rejection of the graft wasdetermined by cessation of graft contraction. At the time of rejection the graftwas explanted and the baboon was returned to its mother. The nonparametricMann-Whitney test was used to determine a two-tailed pvalue.

Immunosuppression
All recipient baboons received cyclosporine (CsA, Sandimmune, SandozPharmaceuticals, East Hanover, N.J.) 10 mg/kg daily by intramuscular injectionor intravenously from the day of transplantation until rejection.Methylprednisolone sodium succinate (Solu-Medrol, The Upjohn Company, Kalamazoo,Mich.) 15 mg/kg was given intravenously on the day of transplantation. Dailysteroid therapy was then tapered to 1 mg/kg daily if intravenous access wasmaintained; otherwise it was switched to 0.8 mg/kg daily of methylprednisoloneacetate by intramuscular injection (Depo-Medrol, Upjohn). In addition to the CsAand steroids, baboons received mycophenolate mofetil (MMF, CellCept, RocheLaboratories, Nutley, N.J.). One week before transplantation, recipientsreceived MMF 100 mg/kg every other day by subcutaneous injection; once thetransplant operation was done, they received daily injections. Allimmunosuppression was stopped once the graft was rejected and explanted. CsA andsteroids were purchased through the pharmacy at Columbia-Presbyterian MedicalCenter. MMF was a gift of Roche Pharmaceuticals.

Serum samples
Recipient baboon serum was obtained from the baboons 1 week beforetransplantation (before immunosuppression), at the time of rejection, and aftergraft explantation and cessation of immunosuppression (postoperative days 14 and21). As a control for the induced anti-Gal response, additional serum sampleswere taken from a newborn baboon that underwent the same operation as thetransplant recipients, but did not receive a xenograft. Each sample was frozenand stored at –80° C. The samples were thawed for the first time atthe time of antibody measurement.

Enzyme-linked immunosorbent assay studies with -Gal–bovineserum albumin
Anti-Gal activity in the serum of the transplanted baboons was determinedin enzyme-linked immunosorbent assay (ELISA) using synthetic -galactosylepitopes coupled to bovine serum albumin (BSA) (Dextra, Reading, United Kingdom)as the solid-phase antigen. Fifty microliters of -gal–BSA at aconcentration of 10 mg/ml in carbonate buffer (pH 9.5) was placed in microtiterwells (3129 plates; Falcon, Oxnard, Calif.) and incubated for 2 hours at 37°C and overnight at 4° C. The plates were subsequently blocked with 1%BSA in carbonate buffer (pH 9.5) to decrease nonspecific binding ofimmunoglobulins. Fifty-microliter serum aliquots in twofold serial dilution inphosphate-buffered saline (PBS) solution containing 1% BSA and startingat a dilution of 1:100 were then placed in the wells. After 90 minutes'incubation at room temperature, the plates were washed five times with PBSsolution containing 0.05% Tween. Horseradish peroxidase-conjugated rabbitanti-human IgG or IgM (Dako, Carpinteria, Calif.) was added at a dilution of1:1000 to each well in 50 ml aliquots. The plates were further incubated for 1hour and washed with PBS-Tween. The color reaction was developed witho-phenylenediamine dihydrochloride (Sigma Chemical Co., St. Louis, Mo.), and theabsorbance was measured in an ELISA reader at 492 nm.

In our experience, the blocking of the ELISA plates cannot completelyprevent nonspecific human immunoglobulin binding. Therefore, to assess thespecific binding of anti-Gal, we performed a parallel ELISA assay using theneoglycoprotein N-acetyllactosamine (Gal-ß-1-4GlcNAc-R)–BSA as acontrol solid-phase antigen. Primates and other mammals usually lack antibodiesto this epitope, because it is normally expressed in various amounts onmammalian cells. The specific binding of anti-Gal was assessed at each serumdilution by subtracting the optical density measured with N-acetyllactosamine–BSAfrom those with -gal–BSA. ¹²

Light microscopy and immunofluorescence
Tissue specimens were placed in formalin, sectioned, and stained withhematoxylin and eosin, or they were placed in embedding medium (OCT compound;Miles, Naperville, Ill.), snap-frozen in liquid nitrogen, and stored at –80°C. Frozen tissue sections (4 m thick) were prepared in a Leica cryostat (Leica,Heidelberg, Germany). The sections were air-dried, fixed with acetone, andwashed with PBS solution. Deposition of IgG, IgM, C3, and fibrin was detected byincubating each section with fluorescein isothiocyanate–conjugated goatantihuman antibodies (Dako). After incubation, tissue sections were washed withPBS solution and mounted with p-phenyenediamine/glycerol solution. Sections wereevaluated with a Leitz DMRB epifluorescence microscope (Leica) and photographed.

Results

Survival
Triple immunosuppression increased average graft life from 3.6 ± 0.5 days (n = 5) to 6.2 ± 1.1 days (n = 5, p =0.01, Table I).All of the baboons tolerated the immunosuppression without complication. Aftergraft explantation the baboons were returned to their mothers. The baboons havenot had any long-term ill effects from the medication. They have developednormally, with the first baboon recipient now more than 1 year beyondtransplantation.

View larger version (136K): [in this window] [in a new window]   Fig. 1. Micrograph at low powerof a heterotopic pig cardiac xenograft placed into an unmedicated newbornbaboon. This specimen demonstrates a dense mononuclear cell infiltrate. Tissuewas obtained at the time of rejection (4 days). This specimen is representativeof all control grafts.

View larger version (134K): [in this window] [in a new window]   Fig. 2. Micrograph at highpower of a heterotopic pig cardiac xenograft placed into an unmedicated newbornbaboon. This specimen demonstrates a dense mononuclear cell infiltrate with onlya scattering of polymorphonuclear neutrophils. Tissue was obtained at the timeof rejection (4 days). This specimen is representative of all control grafts.

View larger version (129K): [in this window] [in a new window]   Fig. 3. Low-power micrograph ofa heterotopic pig cardiac xenograft placed into a medicated newborn baboon.Tissue was obtained at the time of rejection (6 days). This specimen isrepresentative of all medicated grafts. It demonstrates complement-mediatedrejection, with diffuse hemorrhage and edema. There is thrombosis of the arterysupplying the myocardium in the upper right corner.

View larger version (145K): [in this window] [in a new window]   Fig. 4. High-power micrographof a heterotopic pig cardiac xenograft placed into a medicated newborn baboon.Tissue was obtained at the time of rejection (6 days). This specimen isrepresentative of all medicated grafts. It demonstrates a small artery in crosssection, with destruction of the endothelium, thrombosis, and infiltration ofneutrophils.

View larger version (99K): [in this window] [in a new window]   Fig. 5. Immunopathology of aheterotopic pig cardiac xenograft placed into a immunosuppressed newborn baboon.Tissue samples were obtained at the time of rejection (6 days). These samplesare representative of all specimens obtained from the immunosuppressed baboons.There is significant deposition of IgM.

View larger version (104K): [in this window] [in a new window]   Fig. 6. Immunopathology of aheterotopic pig cardiac xenograft placed into a immunosuppressed newborn baboon.Tissue samples were obtained at the time of rejection (6 days). These samplesare representative of all specimens obtained from the immunosuppressed baboons.There is significant deposition of C3.

View larger version (139K): [in this window] [in a new window]   Fig. 7. Immunopathology of aheterotopic pig cardiac xenograft placed into a immunosuppressed newborn baboon.Tissue samples were obtained at the time of rejection (6 days). These samplesare representative of all specimens obtained from the immunosuppressed baboons.There is significant deposition of fibrin.

View larger version (65K): [in this window] [in a new window]   Fig. 8. Immunopathology of aheterotopic pig cardiac xenograft placed into a immunosuppressed newborn baboon.Tissue samples were obtained at the time of rejection (6 days). These samplesare representative of all specimens obtained from the immunosuppressed baboons.There is trace deposition of IgG.

IgG anti-Gal levels
Before transplantation all of the baboons had circulating anti-Gal IgGantibody titers ranging from 20 to 320. The titer of anti-Gal antibody wasdefined as the reciprocal of serum dilution which yields 1.5 O.D. (i.e., 50%of maximum binding). At the time of graft explantation, serum anti-Gal IgGantibodies were barely detectable. However, because IgG was detected byimmunofluorescence in the explanted graft, this finding suggested that themajority of circulating IgG antibody had bound to the xenograft. After theremoval of the graft, the anti-Gal IgG antibody titers rose in all of thebaboons that received transplants. In every instance the anti-Gal IgG titerremained elevated or continued to rise at 3 weeks. Table III describes thechanges in anti-Gal IgG antibody concentration in one of the immunosuppressedbaboons.These changes are representative of all baboon recipients of a pig xenograft. Inthe one newborn baboon that did not receive a xenograft, there was a gradualdecline in anti-Gal IgG antibody titer, consistent with the expected decrease inpassively acquired maternal antibody.

The results of this study indicate that CsA-based tripleimmunosuppression at a dose tolerable by a newborn baboon can increase graftsurvival. Despite barely detectable levels of IgM anti-Gal xenoreactive antibodyat the time of engraftment, this immunosuppressive regimen was not sufficient toprevent an induced IgM xenoreactive antibody response that led to graft loss ondays 6 to 7. Interestingly, it appears that the treated animals demonstrated aless significant macrophage and natural killer cell infiltrate than theuntreated animals.

Probably because of the greater duration of xenograft exposure, the serumantibody response was more pronounced in treated recipients than in unmedicatedanimals. This resulted in a delayed form of humoral rejection with destructionof the entire vascular bed resulting in ischemic infarction of the myocardium.The time course of the induced humoral response parallels that of a primary IgMresponse to a new antigen.

In previous studies aimed at elucidating the mechanisms of human naturalkiller cell lysis of porcine endothelium, we have observed two distinctactivation pathways—one involving binding of xenoreactive IgG to CD16 andanother resulting from activation with interleukin-2 (lymphokine-activatednatural killer cell activity). Interleukin-2 augmented lytic activity isapproximately twofold greater than antibody-dependent cellular cytotoxicity andis, therefore, likely to be of greater clinical significance. We thereforesuspect that the CsA-based immunosuppressive protocol used in this study mayhave reduced interleukin-2 production and consequently diminished natural killercell lysis.

An induced donor-specific antibody response can develop in somerecipients of allografts. Why these allografts usually are not rejected with thesame vigor as a xenograft may be attributed to several factors. Xenoreactiveanti-pig antibody is directed at the -Gal epitope, which is expressed asmillions of epitopes per cell in porcine cells of ectodermal and mesenchymalorigin, whereas allospecific antibody is primarily directed against majorhistocompatibility antigens, the distribution of which is less dense. On theother hand, ABO-incompatible allografts are at risk for complement-mediatedinjury. ¹³ Like pigxenografts, ABO-incompatible allografts express a carbohydrate epitope, not inas great a distribution as -Gal, but greater than the majorhistocompatibility antigens. The greater distribution of antigen, therefore,results in greater IgM binding and more efficient complement activation.Additionally, membrane-bound complement regulatory proteins (homologousrestriction factor, decay-accelerating factor, and membrane cofactor protein)are species-specific and therefore probably less effective in protecting axenograft from complement-mediated injury.

The immunosuppressive regimen used in this study is comparable with thatused in clinical allotransplantation. We have previously demonstrated that CsAgiven to baboons by intramuscular injection at a dose of 10 mg/kg daily willresult in serum trough concentration of 300 to 500 ng/ml. ¹⁴ The intraoperative steroid doseof 15 mg/kg is greater than that usually administered in clinicalallotransplantation, but it is equivalent to the dose used when ABO-incompatiblekidneys have been engrafted, the clinical situation most analogous to thisexperiment. ¹⁵

In an attempt to inhibit the induced anti-Gal xenoantibody response, weincluded an anti-B cell agent in the protocol. MMF is a potent, selective,noncompetitive, and reversible inhibitor of inosine monophosphate dehydrogenase.MMF can inhibit the de novo pathway of guanosine nucleotide synthesis. Since T-and B-lymphocyte proliferation is critically dependent on the de novo synthesisof purines, MMF has potent cytostatic effects on lymphocytes. MMF can alsoinhibit proliferative responses of T- and B-lymphocytes to both mitogenic andallogenic stimulation and potentially suppress antibody formation byB-lymphocytes. At doses of 100 mg/kg, MMF has been shown to inhibit lymphocyteproliferation while allowing for the replication of intestinal basal epithelialcells and germinal cells. ¹⁶

MMF was inadequate to prevent the marked induction of xenoreactiveantibody in this model. Additional strategies to prevent complement-mediatedxenograft injury will be required to achieve clinically relevant graftprolongation. Such strategies are presently being used in our laboratory andinclude porcine organs transgenic for human complement-regulatory proteins. Itis expected that these organs may one day be used as "a bridge" toallotransplantation, sustaining newborn human beings in need of a hearttransplant until an appropriate donor can be found.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map 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): Robert E. Michler Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Minanov, O. P. Articles by Michler, R. E. Search for Related Content PubMed PubMed Citation Articles by Minanov, O. P. Articles by Michler, R. E.