IMMUNOSUPPRESSIVE AGENTS IN TRANSPLANTATION:MECHANISMS OF ACTION AND CURRENTANTI-REJECTION STRATEGIES

VIJAY S. GORANTLA, M.D., 1 JOHN H. BARKER, M.D., Ph.D., 1

JON W. JONES, JR., M.D., 3 KAUSTUBHA PRABHUNE, M.D., 1

CLAUDIO MALDONADO, 1 M.D., and

DARLA K. GRANGER, M.D. 2*

Over the past century, the concept of interfering with theimmune response at various sites by blocking the formation,stimulation, proliferation, and differentiation of lymphocyteshas led to relentless development of new immunosuppres-sive drugs. These agents are associated with reduced risk ofshort- and long-term toxicity and have dramatically improvedallograft and patient survival, especially in recipients of solidorgan transplants. Current protocols in such patients arenearly all calcineurin-inhibitor based, using cyclosporine ortacrolimus, as part of dual, triple, or sequential therapy. Thisreview focuses on agents currently in clinical use at trans-

plant centers in United States. The drugs are described interms of their basic mechanisms of action, therapeutic uses,clinical studies, and adverse effects. In addition, the efficacyand toxicity of a few promising new therapeutic approachesare examined. Finally, important challenges regarding phar-macological immunosuppression as it relates to solid organand composite tissue allotransplantation are discussed.

© 2000 Wiley-Liss, Inc.

MICROSURGERY 20:420–429 2000

At one time, organ failure meant certain death. The adventof transplantation brought the possibility of a second chanceat life for those who faced this prospect. Currently, tens ofthousands of people are alive because of the miracle oftransplantation. The United Network for Organ Sharing(UNOS) reports that 1-year graft and patient survival ratescontinue to increase each year for all organ types. Novelimmunosuppressants, careful monitoring, and ever-improved technology can be credited for this continuingsuccess. However, despite tremendous advances in surgicaltechniques, management of infectious disease, and overallmedical care over the past few decades, manipulation of theimmune response remains the major barrier to successfulorgan transplantation.

A major focus of current transplantation research is thedevelopment of strategies to induce donor-specific toler-ance. However, the quest for this “Holy Grail” has beenlong and tedious. Unprecedented progress in understanding

the cellular and immune mechanisms of the alloimmuneresponse has successfully resulted in tolerance induction insmall animal models, while obviating the need for immu-nosuppressive drugs. Nevertheless, it has been difficult toreproduce such success in humans or even in primate mod-els. Further, achieving long-term graft survival and over-coming chronic rejection remain the chief obstacles in trans-plantation today. While we strive toward this ultimate goalof tolerance, we still need to invest our knowledge of theimmune system in gaining insight into novel mechanisms ofimmune manipulation using drugs.

Immunosuppressive drugs have always been the mainline of defense against the alloimmune reactions triggeredby transplantation and will probably always remain as abackup support system to suppress immune responses in theevent that alternate methods of tolerance induction fail.

HISTORY

The clinical practice of transplantation spans relativelyfew decades, despite the fact that mythology has describedtransplantation as a cure for disease for centuries. Pharma-cological treatment to facilitate graft survival was describedas early as the 5th centuryBC.1 The advent of modern trans-plantation brought with it the inevitable need for immuno-suppression, and later advances in transplantation wereintegrally related to developments in the field of immuno-suppression. In 1944, it was Medawar who described the

1Division of Plastic and Reconstructive Surgery, University of Louisville, Lou-isville, Kentucky

2Department of Surgery, University of Louisville, Louisville, Kentucky

3Department of Transplant Surgery, Carolinas Medical Center, Charlotte,North Carolina

Grant sponsor: Jewish Hospital in Louisville, Kentucky.

*Correspondence to: Darla K. Granger, M.D., Department of Surgery, Univer-sity of Louisville, Louisville, KY 40292.

Received 10 September 2000; Accepted 10 September 2000

© 2000 Wiley-Liss, Inc.

immunological basis for rejection of foreign tissues. Thediscovery of the human leukocyte antigen (HLA) system byDausset in 1958 contributed greatly to our understanding ofhow rejection occurs. From 1910 to 1950, radiation (totalbody or total lymphoid) was used to destroy cells in theimmune system responsible for rejection. While radiationwas effective in doing so, unfortunately it was nonselectiveand also destroyed many other cells in the organism thatreproduce rapidly.

The concept of suppressing the immune response atvarious sites by blocking precursor cell formation, immu-nocompetent cell stimulation, and proliferation or differen-tiation of lymphocytes (through inhibition of nucleic acidsynthesis) led to the development of new immunosuppres-sive drugs. During the late 1950s, immunosuppressive drugtherapy consisted of using cytotoxic drugs and antimetabo-lites, which were originally developed and were being usedat the time to control the proliferation of neoplastic cells. Atthis time, the discovery of the anti-proliferative drug 6-mer-captopurine (6-MP) was the harbinger of the modern era ofimmunosuppression. Other cytotoxic agents that proved tobe of value in suppressing the immune response were thealkylating agents (cyclophosphamide) and purine analogues(azathioprine or AZA). A derivative of 6-MP, AZA poten-tiated the activity of cyclophosphamide by improving itsbioavailability. However, like radiation, in addition to at-tacking immune cells and holding rejection at bay, thesedrugs acted indiscriminately, damaging normal cells essen-tial for survival, particularly hematopoietic cells. The era ofimmunosuppression that followed employed procedures orlymphocytotoxic drugs to eliminate immunocompetentcells, mainly T cells. These included total lymphoid irradia-tion (TLI), thymectomy, splenectomy, antilymphocyte se-rum, and corticosteroids.

Corticosteroids are naturally occurring hormones se-creted by the adrenal cortex, which intervene at many levelsin the immune response. They affect T cells and inhibitcytokine production. Corticosteroids were added to AZA toimprove efficacy and reduce toxicity. During the 1960s and1970s, immunosuppressive therapy consisted of the com-bining corticosteroids and AZA, with or without anti-lymphocyte globulin. Although this combination was mod-erately successful in prolonging allograft survival, it alsocaused a variety of toxic side effects. These included over-whelming and sometimes-fatal infections, direct organ tox-icities, impaired wound healing, anemia, diabetes, and ma-lignancies. Using this regimen, the average 1-year kidneysurvival rate from all transplantation centers reached only50%. At this time, liver transplantation was still consideredto be an experimental procedure and heart transplantation,while experiencing a brief and transient period of successduring the late 1960s, was abandoned at all but three centersworldwide.

The combination of corticosteroids and azathioprine

continued in use as standard therapy until the 1980s her-alded a new era of immunopharmacology. This new ap-proach was characterized by using drugs to more selectivelyregulate defined subpopulations of immunocompetent cells.From this new approach, in 1972, came Cyclosporine A(CsA), which was the first transplant-specific drug devel-oped to specifically target the effector T cells to preventrejection. The Food and Drug Administration (FDA) in1983 approved CsA for patient use in the United States. Stillused widely today, CsA changed the entire field of trans-plantation by significantly increasing survival times of alltransplanted organs.

Over the past decade, the development of several newdrugs continues to extend transplanted organ survival timesand reduce systemic drug toxicity. These new drugs accom-plish this by specifically targeting T-cell subsets and modu-lating interleukins and cytokines. These drugs include:monoclonal antibodies, calcineurin inhibitors such as tacro-limus (FK506), inhibitors of nucleic acid synthesis such asmycophenolate mofetil (MMF), and inhibitors of signals forT-cell differentiation, such as sirolimus. These new drugshave led to a significant reduction in acute rejection ratesand have improved short-term allograft survival while at thesame time causing minimal systemic toxicity. Conse-quently, these advances have led to an explosion in the fieldof transplantation. In the past decade, the field of transplan-tation has gone from only transplanting kidneys and heartsto now transplanting livers, heart and lung, kidney and pan-creas, small bowel, and most recently composite tissue.

The timeline of development of immunosuppressiveagents reflects a progression from the use of agents that actnonselectively on a variety of tissues (radiation, alkylatingagents, purine analogues, and steroids), to those that selec-tively inhibit certain pathways or intracellular sites (Fig. 1).This article reviews the mechanisms of action and the cur-rent and future role of some of these novel immunosuppres-sive agents that have made and will continue to make thissuccess possible.

CURRENTLY USEDIMMUNOSUPPRESSIVE DRUGS

Currently no ideal immunosuppressive drug has beendiscovered or developed. Current research focusing on im-munosuppressive drugs seeks to identify/develop drugs thateffectively suppress rejection while at the same time causeminimal toxic side effects. The primary goals sought inthese agents are to:

Suppress the onset of immune responses as well as re-verse alloreactive responses

Significantly reduce therapeutic drug dosages and toxic-ity through the use of combination drug therapy

Specifically suppress the immune system’s response to

Transplantation Immunosuppression 421

the transplanted organ/tissue and not suppress the en-tire immune system

Eliminate the need for corticosteroids (there is universalagreement in the transplant community that corticoste-roids, despite being the mainstay of immunosuppres-sion for decades, cause the greatest long-term morbid-ity in transplant patients)

So, the ideal immunosuppressive drug should help the hostbecome tolerant or adapt to the foreign antigens in the trans-planted donor organ/tissue, either through clonal deletion orother mechanisms. Clonal deletion is the inactivation ofdonor cells reactive against the recipient by the recipientthymus after transplantation. In current clinical practice, im-munosuppressive drugs are used after transplantation forthree purposes: induction, maintenance, and treatment.

Induction

The goal of induction therapy, although not always suc-cessful, is to switch off the immune system for approxi-mately 2 weeks posttransplant to reduce the likelihood of

immediate rejection (within 10 days after the transplant) andacute rejection (within 3 months after the transplant). Drugsused for induction are very potent and are administeredimmediately after transplant and continued for up to 14days. Currently there are primarily four agents used for thispurpose: polyclonal anti-thymocyte globulins (ATG); anti-interleukin-2 (IL-2) receptor monoclonal antibodies, dacli-zumab and basiliximab; and anti-CD3 monoclonal antibod-ies such as OKT3.

Maintenance

Maintenance agents are used to reduce the immune sys-tem’s ability to recognize and reject the foreign organ ortissue. To accomplish this, combinations of immunosup-pressants are often used, with each drug inhibiting the im-mune system at different site(s). The overall effect of com-bining different drugs that act by different mechanisms isthat a very powerful immunosuppressive effect is achieved.This makes it possible to administer low doses of eachindividual drug and thus reduce the drug-related toxicity.

Figure 1. Schematic overview of mechanism and sites of action of immunosuppressive agents. Phase-specific actions of immunosuppres-sive drugs on T-cell activation, proliferation and differentiation are shown. Note that ATG is non-phase-specific and depletes all T cells bycoating them.

422 Gorantla et al.

Another benefit of using this approach is that since theimmune system is not non-specifically suppressed, it is ableto maintain sufficient host defenses to protect itself againstinfections and malignancies. During the past 10 years, sev-eral new maintenance drugs have been developed and cometo market. Notably, the FDA approved the use of tacrolimus(FK506) in 1994, mycophenolate mofetil (MMF) and a newmicroemulsion form of cyclosporine in 1995, and sirolimus(rapamycin) in 1999.

Treatment

Specific agents are used to treat episodes of acute re-jection. These include the same powerful agents used forinduction (ATG and OKT3) and corticosteroids primarily.Corticosteroids are the first line of treatment in acute rejec-tion episodes. When they are unsuccessful in suppressingrejection, antibody therapy with OKT3 or ATG is usuallystarted. Rejection episodes have also been successfullytreated with high-dose tacrolimus and sirolimus (rapamy-cin). The use of anti-IL-2R antibodies to treat rejection epi-sodes has not been established. Topical corticosteroids andtopical tacrolimus have been used successfully to treat re-jection in composite tissue recipients.

Combination Immunosuppressive Regimens

Current immunosuppression protocols used in solid or-gan transplantation are nearly all calcineurin-inhibitorbased, using CsA or tacrolimus, as part of dual, triple, orsequential combination therapy.2 For reasons mentionedearlier, these regimens are designed to ultimately increasegraft and host survival, while simultaneously decreasing theoccurrence of toxic side effects. There are countless varia-tions in the combination of drugs used, dosing, administra-tion, and monitoring guidelines.

Immunosuppressive drugs currently used clinically inthe United States (in combination with CsA or tacrolimus)are the following:

Interleukin-2 receptor (IL-2R) inhibitors: basiliximab,daclizumab

Anti T-cell or lymphocyte depletion agents: anti-CD3monoclonal antibody (OKT3), antithymocyte globulin(ATG)

Purine biosynthesis inhibitors: azathioprine (AZA), my-cophenolate mofetil (MMF)

Cytokine gene expression blockers: prednisoneTarget of rapamycin (TOR) inhibitors: sirolimus (rapa-

mycin)

Calcineurin Inhibitors (CsA and Tacrolimus)

Calcineurin inhibitors continue to be the cornerstone ofsuccessful long-term immunosuppressive regimens. Theyexert their effects through regulation of cytokine produc-tion. CsA became the prototype agent in this class. The

introduction of CsA was the first major advance in trans-plantation since the synthesis of azathioprine and predni-sone during the early 1950s. Tacrolimus (FK506), also inthis category, was released in Japan in 1984 and underwentclinical trials in 1989.

Cytokine production in T cells is regulated by a series ofsteps consisting of phosphorylation and dephosphorylationof specific proteins. Both CsA and tacrolimus are prodrugs,because they must first form a complex with cellular pro-teins called “immunophilins” before exerting their effects.The immunophilins occur in two classes, the tacrolimus(FK506) binding proteins (FKBP) that bind tacrolimus andsirolimus, and the cyclophilins that bind CsA. CsA binds tocyclophilin (CyP) and tacrolimus binds to tacrolimus bind-ing protein (FKBP). Calcineurin is a phosphatase that iscrucial for intracellular events leading to IL-2 gene tran-scription and release by T cells. Once the drugs bind to therespective immunophilin, the drug-immunophilin complexinhibits the activity of calcineurin, thereby reducing the pro-duction of IL-2 and other cytokines.Cyclosporine A. CsA is a fungal metabolite extractedfrom Tolypocladium inflatum gams(Trichoderma polyspo-rum).3 It is highly specific for T cells and inhibits the acti-vation of both mature helper T cells (CD4+) and killer Tcells (CD8+) impairing their development in the thymus.The immunosuppressive action of CsA is due to suppressionof IL-2 production by T cells. These effects are reversibleupon discontinuation of the drug. CsA blocks cellular acti-vation only after mature T cells have received their activa-tion signal through an antigen or antigen-presenting cell(APC). This may involve steps such as lymphokine secre-tion, cell proliferation, and differentiation. Thus, no neweffector T cells are formed, and those already in circulationare inhibited. On the other hand, hematopoiesis and matu-ration of lymphoid stem cells to antigen-sensitive precursorcells are not affected. CsA cannot block lymphocyte acti-vation due to exogenous lymphokines and has no effect onIL-2 receptor expression or signaling but spares suppressorcells and phagocytic function.

Numerous kidney and other solid organ transplantationtrials have shown that CsA demonstrates potent immuno-suppressive action without myelosuppression. It has beenshown that CsA has a synergistic effect when used withcorticosteroids in blocking IL-2 release through differentsites of action. Multi-center trials demonstrated that CsAtherapy produced a 20% increase in graft survival whencompared to AZA and prednisone.4–6

Sandimmune, the first form of CsA developed, can beadministered either as an intravenous injection or oral medi-cation. It is a potent and selective immunosuppressive drug;however, intestinal absorption is erratic. This form of CsAis absorbed slowly and incompletely, with bioavailabilityranging from 20% to 50%. To overcome this problem, anew microemulsion formulation was developed and ap-

Transplantation Immunosuppression 423

proved for clinical use in the United States in 1995. CsAmicroemulsion has been accepted as a superior vehicle fororal administration and produces more linear, rapid, con-stant and complete absorption of CsA than previous formu-lations.7,8 Microemulsion decreases both interpatient andintrapatient variability of CsA levels,9,10 resulting in morepredictable pharmacokinetics.11

Tacrolimus (FK506). Tacrolimus is structurally distinctfrom CsA and is derived from the soil fungusStreptomycestsukubaensis.12 It is also a calcineurin inhibitor, but in vitroit has been shown to be 100 times more potent than CsA.13

In its actions on the immune system tacrolimus resemblesCsA. The four known intracellular binding proteins (immu-nophilins) for tacrolimus are FKBP-12, 13, 25, and 52. Ofthese the most important protein crucial for T-cell activationis FKBP-12. Binding of tacrolimus to this receptor proteinprevents activation of T cells. It is also a potent suppressorof B-cell activation.14

In vivo, tacrolimus has been shown to prolong survivalof transplanted hearts, kidneys, livers, and even skin. Theintroduction of tacrolimus during the 1980s improved out-comes after small intestine transplantation, allowing theseprocedures to become a more viable clinical alternative.Tacrolimus has been shown to be useful as rescue therapyfor recurrent acute rejection in kidney transplants.15 Currentdata suggest that both tacrolimus and CsA microemulsionproduce comparable graft outcomes, a finding consistentwith their common molecular sites of action.16–18

Comparison of adverse effects of tacrolimus andCsA microemulsion. Both tacrolimus and CsA havesimilar renal and hepatic toxicities but differ in other toxicside effects, presumably because of differences in the bio-logical actions of their binding proteins (Table 1). Neuro-toxicity and diabetes can complicate the use of tacrolimus.19

A recent multicenter trial comparing tacrolimus with CsAmicroemulsion in kidney transplant patients reveals no sig-nificant differences in the incidence of nephrotoxicity be-tween these two drugs.20 Tacrolimus is associated with anonstatistically significant trend toward a lesser risk of hy-pertension.21 CsA microemulsion can cause hypercholester-olemia, thus significantly increasing the risk of cardiovas-cular complications in recipients on maintenance

immunosuppression.22 Both tacrolimus and CsA are me-tabolized by cytochrome P-450 enzymes. Drugs that affectthese enzymes can dramatically affect their therapeutic win-dow.Effects of tacrolimus unique to composite tissueallotransplantation (CTA). Tacrolimus has been shownto promote nerve regeneration in small animal models afternerve injury.23 This neurotrophic influence of the drug ap-pears not to be mediated by FKBP-12. Instead, these effectsseem to be related to actions of multiple neuroimmunophilinligands. Even though the exact components that mediateneurite growth are unclear, the most likely candidates seemto be FKBP-52, hsp-90, and p23.24

The development of non-FKBP-12 binding compoundslacking immunosuppressant activity and that are selectivefor FKBP-52 in an attempt to augment nerve regeneration inisolation is possible. Such agents may be of potential use inCTA such as hand transplants, where sensory recovery iscrucial for overall function of the transplanted limb in theface of prolonged survival. In the event that researchers aresuccessful in tolerance induction using mixed allogeneicchimerism or other methods of immunomodulation, a non-immunosuppressant neurotrophic drug may improve nerveregeneration and function, if it does not interfere with theunderlying tolerance mechanisms.

Interleukin−2 Receptor Antagonists

The IL-2 receptor is a complex of several transmem-brane polypeptide chains, of which three chains (a, b, andg) have been characterized. These chains are noncovalentlylinked to form the high-affinity binding site for IL-2. Theachain is also called cluster determinant region 25 (CD25)and is present on activated T cells, some activated B cells,and APCs. IL-2 receptor (CD25) antagonists inhibit T-cellactivity by blocking the binding of thea chain or the IL-2receptor without activating it. Two agents in this class, basi-liximab and daclizumab, became available as inductionagents. These agents are well tolerated and can be admin-istered via peripheral vein. Neither results in cytokine re-lease syndrome as seen with OKT3 or serum sickness asseen after ATG use. In clinical trials, neither basiliximabnor daclizumab resulted in increased incidence of malig-nancy or opportunistic infections when compared to pla-cebo.

Lymphocyte-Depleting Agents

Induction immunosuppression using biological immu-nosuppressants makes use of polyclonal antilymphocyte an-tibodies (antithymocyte globulin) or monoclonal antibodies(anti-CD3). The potency of these agents is comparable inboth the induction phase as well as in the treatment ofsteroid-resistant or refractory rejection.25,26 Antibody re-agents are an attractive immunosuppressive strategy be-cause they enable rapid reduction of lymphoid cells (periph-

Table 1. Comparison of Adverse Effects of Cyclosporine(Microemulsion) and Tacrolimus

Adverse effect

Risk

CsA Tacrolimus

Risk of hypertension Higher Less highLDL cholesterol levels Higher Less highPosttransplant diabetes Less high HigherNeurologic toxicity Less high HigherGingival hyperplasia Common Not seenAbnormal hair growth Hirsutism Alopecia

CSA, Cyclosporine A; LDL, low-density lipoprotein.

424 Gorantla et al.

eral and thymic) as well as suppress the function of specificT-cell populations. The powerful nature of these agents,with their concomitant increased risk of malignancy andopportunistic infection, limits their use to less than 21 days.Anti-CD3 antibody (OKT3). OKT3 is a mouse mono-clonal antibody against human CD3. It binds to the CD3glycoprotein on the T-cell surface, in close association withthe T-cell receptor (antigen recognition complex). By bind-ing to CD3, OKT3 prevents antigen binding to the antigenrecognition complex, and cell-mediated cytotoxicity isblocked. The T-cell receptor complex is internalized and nolonger expressed on the cell surface. The actions of OKT3are rapid, with peripheral circulating T cells depleted withinminutes of administration of the agent. However, once theuse of OKT3 is stopped, new T cells appearing in the bloodre-express T-cell receptor complexes, and CD3+ cells rap-idly return to normal levels. Thus, T-cell involvement in theimmune response is curtailed only during the active use ofOKT3.

OKT3 has been used to prevent acute rejection in heart,kidney, and liver transplants. OKT3 has been shown to re-verse acute rejection in 94% of recipients of primary cadav-eric kidney transplants when compared with 75% reversalwith high-dose steroids.27 It has also been used to deplete Tcells from donor bone marrow prior to bone marrow trans-plantation.

Administration of the first dose of OKT3 that results inbinding to CD3 can cause the sudden release of cytokinesfrom activated T cells (cytokine release syndrome). Cyto-kines such as tumor necrosis factor-a (TNF-a) have beenimplicated as the major cause of this syndrome. Manifesta-tions range from a mild flu-like illness to a life-threateningshock-like reaction. This adverse reaction can be minimizedby prophylaxis with high doses of steroids a few hoursbefore administration of the drug.28 Other toxic side effectsinclude encephalopathy, nephropathy, and hypotension.Some cases of irreversible graft failure have been reportedbecause of thrombosis. Human antibodies against the mu-rine-derived monoclonal antibody can develop, resulting indecreased efficacy with subsequent administration. Variantsof anti-CD3 antibodies with different isotype or epitopespecificities have been developed to attempt to limit toxici-ties.29

Antithymocyte globulin (ATG). ATG and antilympho-cyte globulin (ALG) are the active globulin fractions ofheterologous antilymphocyte serum (ALS). Commerciallyavailable purified immunoglobulin is prepared from hyper-immune serum of horses (ATGAM) and rabbits (thymo-globulin) that were immunized with human thymic lympho-cytes.

ATG binds to the surface of T cells and exerts potentimmunosuppressive activity by severely depleting both cir-culating T cells and those within lymphoid organs. ATGinterferes most with cell-mediated reactions, including al-

lograft rejection and graft-versus-host disease (GVHD). Thelymphocyte-depleting effects of ATG are caused by coatedT cells that are cleared by the reticuloendothelial macro-phages of the spleen and liver.

Since its clinical debut in 1965,30 ATG has been used totreat acute rejection in solid organ transplantation, includingkidney and heart. ATG has also shown promise in bonemarrow transplantation where pretreatment of the recipientnot only suppresses anti-donor responses and GvHD, butalso enlarges the marrow space. In addition, ATG has beenused in the prophylaxis of allograft rejection.31,32 Studiesshow that patients with fewer rejection episodes in the firstmonth have a better prognosis. Both ATG and ALG wereused prophylactically to prevent rejection in cardiac trans-plantation until the introduction of CsA. Results in variousstudies reveal that even though prophylactic use of ATGreduces the number of rejection episodes in the first monthposttransplant, it does not have any effect on the frequencyof subsequent rejection episodes.33 Some studies haveshown superior renal allograft survival rates with ATG,34,35

whereas others have opined that the adverse effects of ATGoutweigh its beneficial aspects. A 10–50% improvement in1-year renal graft survival rates has been shown with theprophylactic use of ATG.36, 37Thymoglobulin has a longerimmunosuppressive effect in vivo when compared to horseATG.38 ATG has also been found to be effective in steroid-resistant rejection, allowing a dose reduction of steroidswith resulting lower morbidity.39

Since ATG is a heterologous serum prepared againsthuman tissue, it can cross-react with other tissue antigensand cause anaphylactic reactions in the recipient. The majortoxicities result from ATG being recognized as a foreignprotein, leading to flushing, fever with chills, anaphylaxis,and serum sickness. Other toxic effects include phlebitis,leukopenia, thrombocytopenia, and nephritis. The incidenceand severity of these adverse reactions is reduced in thepresence of other immunosuppressive drugs that are used asa part of combination regimens in transplantation.

Purine Biosynthesis Inhibitors(Antiproliferative Agents)

Antiproliferative agents inhibit the division and differ-entiation of immunocompetent lymphocytes once antigen isencountered. Their structure resembles that of essential cel-lular metabolites; they combine with critical components ofthe cell, such as DNA. Incorporation of these substances inthe DNA results in production of faulty molecules, thusinterfering with molecular function. For optimal efficacy,these agents must be administered at the time of transplan-tation and continued for the life of the allograft to inhibitongoing cellular and humoral immune responses.Azathioprine (AZA). This purine analogue is the imid-azole derivative of the cytotoxic drug 6-mercaptopurine (6-MP). It was synthesized by Hitchings40 and first used clini-

Transplantation Immunosuppression 425

cally in 1963.41 AZA is converted in the liver to 6-MP,which then enters the cell to form 6-MP ribonucleotide. Thismolecule resembles inosine monophosphate, which is animportant precursor in nucleic acid synthesis. Because ofthis structural similarity, it causes fraudulent feedback in-hibition of the early enzymes, catalyzing the cellular syn-thesis of DNA, RNA, and other cofactors. It acts early dur-ing the proliferative cycle of effector T- and B-cell clonesand is a powerful inhibitor of primary (but not secondary)immune responses. AZA also suppresses neutrophil produc-tion and macrophage activation.

For more than two decades, AZA had been the mainstayof anti-rejection regimens. Recently, it has been eliminatedfrom many protocols or has been replaced by mycopheno-late mofetil. Although application of AZA diminishes theincidence and intensity of rejection episodes, it is of novalue in the therapy of ongoing rejection.

In addition to its effects on immune cells, AZA non-specifically affects rapidly growing cells, including bonemarrow and the lining cells of the intestinal tract. This re-sults in leukopenia, thrombocytopenia, and gastrointestinaltoxicity. In addition, AZA can cause hepatotoxicity becauseof the high rate of RNA synthesis by hepatocytes.Mycophenolate mofetil (MMF). MMF is an ester ofmycophenolic acid (MPA). There are two pathways in cellsto synthesize purines that are essential components of DNA.One is the de novo pathway, dependent on the enzymeinosine monophosphate dehydrogenase (IMPDH), and theother is the salvage pathway, dependent on the enzymehypoxanthine-guanine-phosphoribosyl-transferase (HG-PRtase). MMF is a potent, reversible, noncompetitive in-hibitor of IMPDH. Since T and B cells depend solely on thisenzyme for DNA synthesis, MMF selectively blocks pro-liferation of T cells and suppresses antibody formation by Bcells.42 Bone marrow cells and parenchymal cells, whichrely more on the salvage pathway for purine synthesis, aretherefore spared by MMF.

The use of MMF, in a multicenter, randomized clinicaltrial resulted in reduction of biopsy-confirmed acute rejec-tion to below 20% and the frequency of resistant rejection to5%.43 Therefore, MMF is quickly replacing AZA as part ofthe traditional triple therapy in many centers. Even thoughMMF is maximally effective when started at the time oftransplantation, it also reduces the risk of acute rejectionwhen instituted in the posttransplant phase.

It is well known that chronic rejection is the most im-portant cause of late graft loss.44 Small animal and limitedstudies in humans have shown that MMF can slow the pro-gression of chronic rejection or induce tolerance to donortissue. These effects are the most promising aspects of thedrug, but controlled clinical trials have shown that risk ofchronic rejection still remains at 20% at 3 years.45

Adverse effects include diarrhea, esophagitis, and gas-tritis. The risk of leukopenia and opportunistic infections

with cytomegalovirus is similar in recipients who are onMMF or AZA.

Cytokine Gene Expression Blockers

Adrenal glucocorticoids (corticosteroids). Cortico-steroids are the most common agents used clinically intransplantation. Prednisone, the prototypic agent used, isanalogous to the major endogenous corticosteroid, cortisol(hydrocortisone). However, it is four times more potent inefficacy than cortisol.

The actions of corticosteroids are mediated by subcel-lular hormone receptors that form steroid receptor com-plexes. These complexes bind to DNA and affect expressionof specific genes that drive protein synthesis and cellularprocesses. In particular, they affect cytokine gene transcrip-tion and inhibit the secretion of IL-1, IL-6, interferon-g(IFN-g) and TNF-a by lymphocytes and macrophages. Inaddition, IL-2 production and binding to its receptor is alsoinhibited. Corticosteroids block the effects of migration in-hibition factor and macrophage activation factor on macro-phages and suppress prostaglandin synthesis. They inducelymphocytopenia, which is due to redistribution of lympho-cytes from the intravascular space to the lymphoid space,presumably because of cell surface alterations. This effect isgreater on T cells than on B cells.46 Corticosteroids wereinitially used to reverse acute rejection in recipients on AZAmaintenance. Currently, it is common practice to use much-reduced doses of corticosteroids in combination with otherdrugs for maintenance therapy or short courses of highdoses for treatment of acute rejection. GVHD after bonemarrow transplantation is usually treated with low-dose cor-ticosteroids. They are also used to minimize hypersensitiv-ity reactions due to induction immunosuppressants such asmonoclonal antibodies and antithymocyte globulin. Studieshave confirmed that triple drug therapy with corticosteroids,AZA, and CsA minimizes the adverse effects of each ofthese drugs, while achieving good rejection control.47

Multiple adverse reactions are common with the use ofthese drugs, especially with high doses aimed at treatmentof ongoing rejection episodes. Among the possible toxicside effects, the most serious include hypertension, diabetes,weight gain, osteoporosis, peptic ulcers, gastrointestinalbleeding, opportunistic infections, cataracts, and poorwound healing.Adverse effects of corticosteroids unique to CTA.CTA involves transplantation of multiple tissues that, unlikesolid organ transplants, are made up predominantly of skinand connective tissue. A major adverse effect of corticoste-roid use in transplantation is deranged wound healing.48,49

The anti-rejection actions of corticosteroids are intrinsicallylinked to the factors that influence wound healing.

The two-pronged effects of corticosteroids on transplantrejection and wound healing are caused by nonspecific ac-tions on cytokines, adhesion molecules, growth factors, and

426 Gorantla et al.

cellular receptors. IL-1 is a potent stimulator of epithelial-ization. IL-6 and IL-8 increase the proliferation of keratino-cytes. CD8+ T-lymphocytes are a subset of cytotoxic cells,which not only play a key role in allograft rejection but alsoinhibit wound healing.50,51 The overall scenario of cortico-steroid use in composite tissue allotransplantation is thus acomplex one. Due to their detrimental effects (Table 2),corticosteroids should only be used with caution.

Rapamycin (TOR) Inhibitors

Sirolimus (rapamycin): Sirolimus, a macrolide antibiot-ic that is structurally similar to tacrolimus, is extracted fromthe soil fungusStreptomyces hygroscopicus.52 It binds tothe immunophilin FKBP-12, but unlike tacrolimus, it doesnot bind calcineurin. It affects the G1 phase of the cell cycleby acting on a unique cellular target called mammalian tar-get of rapamycin (mTOR). Sirolimus blocks calcium-independent events during the G1 phase (for comparison,CsA inhibits calcium-dependent lymphokine synthesis by Tcells during the G0 → G1 phase transition (Fig. 1). In ad-dition, sirolimus also blocks the second signals delivered byIL-2, IL-4, and IL-6 to T cells. Thus, sirolimus blocks thelate events in the immune cascade caused by the down-stream effects of IL-2 signaling, while sparing cytokinegene expression.53

The addition of sirolimus to combination regimens hasallowed for the reduction of CsA dosages and steroid with-drawal. Because they compete for the same binding protein(FKBP-12), sirolimus and tacrolimus should be expected toantagonize each other. This is true in vitro, but not so invivo, where a synergistic or additive effect is observed.54

On the other hand, synergistic interaction between sirolimusand CsA may be related to their sequential molecularmechanisms of action. Recent phase II trials have shownthat the incidence of acute rejection in kidney transplants

was reduced by 20% when both agents were used incombination.55, 56 Since sirolimus has been proven to pre-vent chronic vascular injury in rat models, this drug mayhave potential use in prevention of chronic allograft rejec-tion and accelerated atheromatous disease in transplant re-cipients.57 The most fascinating role for sirolimus is in thetransplantation of human islets. The so-called Edmontonprotocol of daclizumab, tacrolimus, and sirolimus withoutsteroids has shown great promise in making human islettransplantation a clinical reality.58

Multicenter trials have shown that when different com-bination regimens using AZA, CsA, prednisone, and siroli-mus were compared, the incidence of malignancy and in-fection were similar in all groups, but hypercholesterolemiawas more common with sirolimus. This particular toxic sideeffect increases the cardiovascular risk in combination regi-mens with CsA. Leukopenia and thrombocytopenia can oc-cur with high doses of sirolimus, but hypertension, nephro-toxicity, and hepatotoxicity are not common.

DISCUSSION

As has been the case throughout the history of organtransplantation, the main obstacle standing in the way ofwidespread clinical use of composite tissue allotransplanta-tion is immunological rejection. The question then arises:Why has solid organ transplantation advanced quickly tobecome standard care while composite tissue allotransplan-tation is still only performed experimentally? The answer tothis question is that until now reconstructive surgeons havefelt that the risks posed by the immunosuppressive drugsnecessary to prevent rejection outweighed the benefits ofthese non-lifesaving reconstructive procedures.

Skin is often an important component of a compositetissue allograft and is considered among the most immuno-genic of tissues. Because of the skin’s high degree of im-munogenicity, until recently it was widely assumed that thedosage of immunosuppressive drugs required to prevent re-jection were too high to be used safely in the clinical setting.Our own clinical experience59 and that of others60 usingtacrolimus-based combination immunotherapy in clinicalhand transplantation tends to refute this assumption (see thearticle by Francois et al.,this issue.)

The rapid advances in immunobiology and immuno-pharmacology witnessed over the past decade have resultedin the release of new, powerful immunosuppressive agents.The choice of agents currently available increases the avail-able options for posttransplant immunosuppression. Al-though CTA is still in its infancy, the preliminary outcomesin various tissue transplants, including cartilage (larynx),bone (knee)61 and hand59 are encouraging. These positiveoutcomes have been made possible through the use of mod-

Table 2. Adverse Effects of Glucocorticoids (Prednisone) onWound Healing

Adverse effectsPrednisone

(mg/day) Major mechanism of action

Suppression ofinflammation

<10 ↓ of chemotaxis, ↓ fibroblastand monocyte/macrophageinflux

Decreased woundstrength

10–30 ↓ collagen and groundsubstance(glycosaminoglycansynthesis) and ↓ collagenaseactivity

Inhibition of woundcontracture

>40 ↓ endothelin 1, ↓ transforminggrowth factor (TGF-b), and ↓platelet-derived growth factor(PDGF)

Delayedepithelialization

>40 ↓ IL-1, ↓ keratinocyte growthfactor (KGF) expression

↓, decreased.Compiled from Anstead.49

Transplantation Immunosuppression 427

ern immunosuppressive drugs administered using the latestinduction, maintenance, and treatment regimens.

The specific drugs highlighted in this review, while suc-cessful in preventing allograft rejection, can cause wide-ranging toxic side effects to the other organs in the body.Because of the limitations of individual drugs, present strat-egies seek to use synergistic drug combinations to potentiaterejection prophylaxis. More importantly, such combinationsalso permit reduction of doses of individual drugs whilebroadening their therapeutic window. The introduction ofcalcineurin inhibitors has markedly reduced the incidence ofacute rejection and has dramatically prolonged organ sur-vival rates. CsA microemulsion, tacrolimus, and MMF haveincreased the available options for posttransplant mainte-nance immunosuppression.

Continued research by the pharmaceutical industry hasensured an increase in the number of immunosuppressantoptions over the years. Despite the addition of many noveldrugs, no drug has yet been proved to prevent the onset ofchronic rejection or reverse its progression. Chronic rejec-tion continues to be the curse of transplantation today re-sulting in late graft loss in transplant recipients. Researchmust be refocused to determine the relevant targets for long-term immunosuppression. Presently, optimal long-termtherapy is still controversial and the risks of chronic rejec-tion in composite tissue allotransplantation are unknown.

In conclusion, the ideal immunosuppressive strategywould be a combination of agents that are selective andspecific in function, synergistically active for maximal ef-fectiveness, free of toxic reactions, easy to administer, andinexpensive. As it stands today, optimal drug therapy re-quires careful clinical management of the patient includingproper dosage strategies, drug level monitoring in the blood,and graft monitoring by biopsy.

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