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Right arrow Transplantation - heart

Ann Thorac Surg 2004;77:354-362
© 2004 The Society of Thoracic Surgeons

Review

Drug immunosuppression therapy for adult heart transplantation. Part 1: immune response to allograft and mechanism of action of immunosuppressants

Xavier M. Mueller, MDa*

a Department of Cardiovascular Surgery, Centre Hospitalier Universitaire de Sherbrooke, Sherbrooke, Quebec, Canada

* Address reprint requests to Dr Mueller, Department of Cardiovascular Surgery, Centre Hospitalier Universitaire de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, Quebec J1H 5N4, Canada
e-mail: xavier.mueller{at}usherbrooke.ca


   Abstract
Top
 Abstract
Introduction
 Definition of rejection
 Immunologic response to...
 Mechanism of action of...
 Corticosteroids
 Calcineurine inhibitors
 Antiproliferative agents
 Antibodies
 References
 
In the early days of transplantation, immunosuppression therapy was rather broad and nonspecific, mainly using high-dose corticosteroids and azathioprine. Thereafter we progressively narrowed the target of immunosuppressive strategy starting with polyclonal antibodies. The introduction of cyclosporine, OKT3, and tacrolimus further narrowed the target on the T-cell pathways. More recently mycophenolate mofetil progressively took the place of azathioprine with its higher lymphocyte specificity and sirolimus and interleukin-2 receptor antibodies were introduced. In this field in constant movement the aim is to find a drug or a regimen that provides optimal immunosuppression therapy with minimal side effects, in other words to find the right balance between overimmunosuppression and underimmunosuppression therapy. This review is divided into two parts. The first part will provide a basic understanding of the immunologic response to allograft and explain how conventional and recently introduced immunosuppressive agents work. The second part will describe the clinical application of immunosuppressive drugs to provide practical information for those in charge of heart transplant recipients.


   Introduction
Top
 Abstract
 Introduction
Definition of rejection
 Immunologic response to...
 Mechanism of action of...
 Corticosteroids
 Calcineurine inhibitors
 Antiproliferative agents
 Antibodies
 References
 
Over the past 40 years immunosuppressive drug regimens have evolved greatly and transformed heart transplantation into a routine clinical procedure. The revolution came with the introduction of cyclosporine in the early 1980s, which significantly improved the clinical outcome in heart transplantation. According to the International Society of Heart and Lung Transplantation registry, 3-year survival from 1975 to 1981 (precyclosporine era) was 40% compared with 70% in the cyclosporine era from 1982 to 1994 [1]. However current immunosuppressive protocols are far from ideal. Despite the clear progression of the survival rate only 50% of transplant patients in large series are alive at 10 years [2], most of them showing substantial chronic side effects of long-term immunosuppression therapy [3], and almost all of these allografts will have failed by 20 years.

Graft loss is mainly related to chronic allograft vasculopathy, which has been linked to insufficient immunosuppression therapy. No specific therapy against chronic rejection has been established yet. Because recurrent or severe episodes of allograft rejection have been repeatedly correlated with the occurrence of allograft vasculopathy [4–6], the attempt to limit this ominous problem is currently limited to improving the prevention and to reducing the rate of acute rejection episodes. Conversely a significant proportion of the late deaths are the consequence of infection and malignancy that are secondary to excess of immunosuppression. Therefore nowadays the aim of immunosuppressive regimen is to find the optimal trade-off between overimmunosuppression and underimmunosuppression therapy. This unsatisfactory compromise stimulates the search for better immunosuppressive agents and strategies.

In the early days of transplantation, immunosuppression therapy was rather broad and nonspecific mainly using high-dose corticosteroids and 6-mercaptopurine or azathioprine. Thereafter we evolved to a progressive narrowing of the target of immunosuppressive strategy starting in the 1970s with antithymocyte globuline (ATG). In the 1980s the introduction of cyclosporine, OKT3, and tacrolimus further narrowed the target on the T-cell pathways. In the 1990s mycophenolate mofetil progressively took the place of azathioprine with its higher lymphocyte specificity, and sirolimus and interleukin-2 (IL-2) receptor antibodies, targeting downstream effects of the IL-2 receptor, were introduced. Despite these advances in immunosuppressive therapy rejection rates and short-term survival rates have not changed over the past decade. However the effects of most recently introduced drugs such as sirolimus and IL-2 receptor antibodies remain to be determined. The tremendous redundancy in the human immune system explains in great part the failure to develop a drug truly specific for the alloreactive reactive cells and this complexity renders unlikely the development of such a drug in the short term.

Nevertheless with the explosion in knowledge of molecular immunology, major advances have been made in the understanding of the cellular and molecular mechanisms that underlie the immunologic response to allograft. The purpose of the first part of this review is to provide a basic understanding of the immunologic response to allograft and to describe how conventional and recently introduced immunosuppressive agents work. The second part will report the clinical application of immunosuppressive drugs to provide practical information for the medical community in charge of heart transplant recipients.


   Definition of rejection
Top
 Abstract
 Introduction
 Definition of rejection
Immunologic response to...
 Mechanism of action of...
 Corticosteroids
 Calcineurine inhibitors
 Antiproliferative agents
 Antibodies
 References
 
Before addressing the various aspects of immunosuppression therapy a clear definition of its target—rejection—is mandatory. Rejection involves cell- and antibody-mediated organ injury occurring as the result of recognition of allograft as nonself. This process is categorized histologically and immunologically into three major types: hyperacute, acute, and chronic.

Hyperacute rejection results when an abrupt loss of allograft function occurs within minutes to hours after circulation is established in the allograft. This process is mediated by preexisting antibodies to allogeneic antigens on the vascular endothelial cells within the donor organ. These antibodies fix complement thereby promoting intravascular thrombosis and leading to rapid occlusion of graft vasculature and rapid rejection of the graft. Donor recipient human leukocyte antigen (HLA) and ABO blood-group cross-matching are used to prevent hyperacute rejection [7].

Acute rejection is primarily a cell-mediated process that most commonly occurs from the first week to several years after transplantation. It is characterized by necrosis of parenchymal cells within the donor organ. After heart transplantation [8], 61% of patients remain rejection free after the first posttransplantation month, 38% of patients are without rejection by 6 months, and 34% of patients are without rejection by 12 months. The hazard function for initial rejection peaks at approximately 1 month.

Chronic rejection, or late graft failure, is an irreversible gradual deterioration of graft function that occurs in many allografts months to years after transplantation. It is characterized by intimal thickening and fibrosis leading to luminal occlusion of the graft vasculature [7]. Cardiac allograft vasculopathy has been detected angiographically in 44% of heart transplant recipients at 3 years [4]. This form of rejection involves a variety of immune-system components: T cells, cytokines, macrophages, and adhesion molecules [9]. Both immunologic and nonimmunologic factors appear to be involved in the ultimate impairment of organ functions. Acute rejection episodes, inadequately treated acute rejection, insufficient long-term immunosuppression therapy, preservation injury, lipid abnormalities, and infection have all been associated with chronic rejection [10].


   Immunologic response to allograft
Top
 Abstract
 Introduction
 Definition of rejection
 Immunologic response to...
Mechanism of action of...
 Corticosteroids
 Calcineurine inhibitors
 Antiproliferative agents
 Antibodies
 References
 
This field is in constant evolution. For the description of such a complex phenomenon only the most commonly accepted mechanisms are reported, keeping in mind that some are subject to controversy. Figure 1 illustrates the successive steps of the response to the allograft.



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  		Fig 1. Stages of CD4 T-cell activation and cytokine production with identification of the sites of action of different immunosuppressive agents. Antigen fragment-major histocompatibility complex (MHC) II molecule complexes are responsible for initiating the activation of CD4 T cells. These MHC-peptide complexes are recognized by the T-cell recognition complex (TCR), which consists of transmembrane proteins associated with the CD3 molecule. Engagement of the TCR/CD3 complex and CD4 coreceptors in conjunction with a costimulatory signal initiates signal transduction with activation of second messengers. Downstream the cytoplasmic Ca2+ concentration increases through an influx of extracellular Ca2+. The Ca2+ dependent enzymes, one of which is the calcineurine, are activated. Calcineurine removes phosphates from the nuclear factors (NFAT-P) allowing them to enter the nucleus. These nuclear factors specifically bind to an interleukin-2 (IL-2) promoter gene facilitating IL-2 gene transcription. Interaction of IL-2 with its receptor (IL-2R) on the cell membrane surface induces cell proliferation and production of cytokines specific to the T cell, which in turn act by binding to their respective receptors. The thick arrows indicate the target of different immunosuppressive drugs. (APC = antigen-producing cells; MMF = mycophenolate mofetil.)

 
Acute rejection can be initiated when graft injury produces an up-regulation of adhesion molecules on endothelial cell lining blood vessels in the graft [11]. Host T-cell receptors bind to these adhesion molecules increasing their transit time through graft vessels and promoting their migration into allograft tissue. Subsequently two varieties of cells—T lymphocytes and antigen-presenting cells (APCs)—are recruited [12]. The professional APCs are critical to the immune response. They include dendritic cells and macrophages that bind antigen and present it to T cells [13].

The key event in both the initiation and the coordination of the rejection response is T-cell activation. Antigen recognition by T cells is the initial stimulus for their activation, proliferation, cytokine production, and performance of regulatory or cytolytic effector function. Antigen-specific T lymphocytes do not recognize antigens in the free form or in soluble form but only as short peptides, products of protein digestion, that are bound to a special molecule—the major histocompatibility complex (MHC)—of recipient or donor APCs. Moreover direct recognition of allo-MHC molecules by host cells plays a major role in the initiation of acute rejection as well. Major histocompatibility complex is one set of the many histocompatiblity molecules that has a predominant influence on tissue compatibility and is by far the most polymorphic [14]. Actually the MHC molecules and the peptides they hold are the strongest antigens present in transplantation. The MHC in humans occupies a large region on the short arm of chromosome six [15]. The gene products of the MHC molecules in humans are called human leukocyte antigens (HLA). There are two main types of MHC molecules that are important for allograft transplantation: class I and II [16]. The HLA class I molecules are present on all nucleated cell surface to display antigenic peptides to the cytotoxic cells; however their expression can be increased by cytokines. This is important as an amplification mechanism. In contrast class II molecules are found almost exclusively on cells associated with the immune system: the professional APC found in lymphoid tissues and activated T cells. Resting T cells do not express class II molecules. For the most part class I MHC molecules present peptides from proteins that originate inside the cell such as those from virally infected, transformed, or otherwise abnormal cells [16]. Conversely class II MHC molecules hold peptides that were outside the cell, that were internalized, and were degraded in lysozymes [17]. All lymphocytes are restricted to one of these two classes.

All T cells express on their surface a specific T-cell receptor (TCR), which is the site for antigen binding. There are also transmembrane proteins (CD3) associated with the TCR. Collectively these complexes compose the TCR complex. The intracellular segment of the CD3 molecule initiates the intracellular signal transduction events that follow antigen recognition [16, 17]. In addition to acquiring TCR complex, T cells also acquire differentiation receptors called cluster of differentiation (CD) antigens. The best known CD markers are CD4 and CD8 and they are exclusive markers of circulating T cells. These molecules act as accessory coreceptors and play an important role in initiating the early events of receptor activation by binding in an antigen-independent fashion to the MHC class II and I respectively [18]. These two coreceptors define the two major types of T cells. Cells bearing the CD8 molecule can directly lyse a foreign cell and become antigen-specific cytotoxic cells. In contrast there are CD4 positive cells that activate the response to a given antigen, helper cells. Moreover when a T cell encounters alloantigen for the first time, a second signal is required in addition to the TCR-CD3 interaction before T-cell activation can proceed. Important pathways identified involve T-cell surface molecule such as CD28 and CD40 [19].

Engagement of the TCR/CD3 complex and CD4 or CD8 coreceptors in conjunction with the costimulatory signal initiates signal transduction with activation of second messengers such as protein tyrosine kinases [16]. Downstream the cytoplasmic Ca2+ concentration increases through an influx of extracellular Ca2+. The Ca2+ dependent enzymes, one of which is the calcineurine, are activated [17]. Calcineurine removes phosphates from nuclear factors of activated T cells (NF-AT), allowing these factors to enter the nucleus, where they specifically bind to an IL-2 promoter gene, facilitating IL-2 gene transcription [16]. Interaction of IL-2 with its receptor (IL-2R) on the cell membrane surface induces cell proliferation and production of cytokines specific to the T cell [16].

Cytokines are intracellular chemical communicators that include molecules such as interleukins and interferons. These are soluble proteins or glycoproteins that are effective across short distances and that in turn amplifiy the response and activate other cells [20]. Unlike hormones most cytokines act locally and are not usually found in the circulation in large quantities because of their short half-life [16]. Cytokine/receptor binding triggers intracellular signal transduction with activation of second messengers such as protein tyrosine kinases. Signal transduction leads to transcription of selected genes with production of transcriptional regulatory proteins and subsequently a second wave of transcription leading to production of cytokines among which is IL-2 as well as its receptor IL-2R. Interleukin-2 is one of the best characterized cytokines. It is a key growth factor that is required for the expansion of T cells during a T-cell-mediated response. Interleukin-2 is produced by CD4 cells and to a lesser extent by CD8 cells and it exerts both an autocrine and a paracrine response. Only those T cells that have been activated by their specific antigens and express IL-2R can respond to IL-2. During an acute rejection episode IL-2 stimulates the clonal expansion of alloantigen-activated T cells thereby increasing the number of T cells that are capable of destroying the graft. In addition IL-2 triggers the production of other cytokines and stimulates the cytotoxic activity of CD8 cells and natural killer (NK) cells and the growth of B cells. Thus the amount of IL-2 produced by activated CD4 cells appears to be one of the major determinants of the magnitude of the immune response to a donor allograft [21].


   Mechanism of action of immunosuppressants
Top
 Abstract
 Introduction
 Definition of rejection
 Immunologic response to...
 Mechanism of action of...
Corticosteroids
 Calcineurine inhibitors
 Antiproliferative agents
 Antibodies
 References
 
The following section will describe the present understanding of the immunosuppressive mechanisms and toxic effects of both the conventional agents and the newer agents being used in allograft transplantation today. The immunosuppressant drugs will be divided into four categories: (1) corticosteroids, (2) calcineurine inhibitors, (3) antiproliferative agents, and (4) polyclonal and monoclonal antibodies. The targets of the different immunosuppressive drugs are found on Figure 1.


   Corticosteroids
Top
 Abstract
 Introduction
 Definition of rejection
 Immunologic response to...
 Mechanism of action of...
 Corticosteroids
Calcineurine inhibitors
 Antiproliferative agents
 Antibodies
 References
 
Corticosteroids are nonspecific anti-inflammatory agents. Lymphocyte depletion is the main antilymphocyte action of steroids. They reversibly block T cell and APC derived cytokine and cytokine-receptor expression that otherwise would occur as a consequence of T-cell activation [22, 23]. By the nature of their hydrophobic structures corticosteroids easily diffuse into cells and bind to specific cytoplasmic receptors [24]. The corticosteroid-receptor complexes translocate to the nucleus, where they attach to DNA sites thereby inhibiting the transcription of certain cytokine genes [24, 25]. Moreover they bind to glucocorticoid response elements in the promoter regions of cytokine genes. Cytokine production of macrophages is also inhibited with subsequent inhibition of their chemotaxis and phagocytosis properties. As a result of these actions steroids affect elements involved in all stages of the T-cell activation process beginning with the inhibition of endothelial-cell adhesion molecule up-regulation and infiltration of allograft tissue. Owing to their profound immunosuppressive, anti-inflammatory, and hormonal actions on numerous target tissues steroids have innumerable side effects: cushingoid appearance, hypertension, dyslipidemia, weight gain with central obesity, peptic ulcer formation and gastrointestinal bleeding, pancreatitis, personality changes, cataract formation, hyperglycemia progressing to steroid diabetes, and osteoporosis with avascular necrosis of bone.


   Calcineurine inhibitors
Top
 Abstract
 Introduction
 Definition of rejection
 Immunologic response to...
 Mechanism of action of...
 Corticosteroids
 Calcineurine inhibitors
Antiproliferative agents
 Antibodies
 References
 
Cyclosporine (Sandimmun, Neoral; Novartis, St Louis, MO), a small fungal cyclic peptide, and tacrolimus-FK506 (Prograf; Fujisawa, Deerfield, IL), a macrolid antibiotic, have become the cornerstone of immunosuppressive therapy in solid organ transplantation. Each agent forms complexes with cytosolic protein called immunophilins [26]. Cyclosporine binds to a group of immunophillins called cyclophilins, which are important biologic molecules present in all tissues, and tacrolimus complex with FK506 binding proteins (FKBP). These complexes bind to calcineurine, a pivotal enzyme in T-cell IL-2 production [26] thereby inhibiting cytokine transcription by the CD4 cell. Blockade of cytokine production and cytokine-receptor expression inhibits T-cell proliferation and differentiation so that the various effector arms of the immune response are not activated. Compared with nonimmune cells lymphocytes show greater sensitivity to cyclosporine and tacrolimus, which may be due to the limiting amounts of calcineurine in immune cells compared with noninmmune cells [27].

The adverse effects of cyclosporine are many owing in part to the ubiquitous tissue distribution of cyclophylins. Nephrotoxicity has been the greatest concern. Its incidence has been reported between 40% and 70% [28]. Acute nephrotoxicity occurs secondary to intrarenal vasoconstriction and is reversible. Chronic nephrotoxicity is likely a long-term secondary sequela of persistent renal vasoconstriction and ischemia and is irreversible with obliterative vasculopathy and interstitial fibrosis on histology. There has also been a question as to whether cyclosporine increases the risk for the development of transplant coronary artery disease. That has been suggested, as the incidence of transplant coronary artery disease has not decreased with the use of cyclosporine despite having lower rejection incidence. However as pointed out by Haverich and colleagues [29] this relation is questionable because hypertension and nephrotoxicity observed with cyclosporine therapy are both probably related to a disturbed vasomotor tone and a pathologic reaction to physiologic vasoconstrictive and vasolilative agents. These effects are dose related and reversible. Therefore it is unlikely that this pathologic response is operative in precipitating or promoting coronary artery disease. That is further supported by results of in vivo and in vitro studies as well as by comparisons of immunosuppressive protocols including cyclosporine or not. Moreover if cyclosporine-related damage were to occur in coronary arteries independent from immunologic factors the incidence of coronary disease should be similar among heart, lung, liver, and kidney transplant recipients.

Cardiovascular adverse effects include hypertension and complex effects on intravascular coagulation with an increase in the incidence of deep venous thrombosis [30]. A variety of neurologic complications have been reported with cyclosporine use including tremor, headache, convulsions, and various paresthesias of the limbs. Hyperkaliemia is common in patients on cyclosporine [31] and is reversible by lowering the dosage. Hyperglycemia may occur and is reversible too. Elevated serum urate levels may occur as a result of tubular defect associated with cyclosporine nephrotoxicity. This hyperuricemia may lead to gout. Urate levels may require to be controlled with allopurinol and the leukocyte count needs to be monitored carefully if the patients is also on azathioprine. Together with steroid therapy cyclosporine has been implicated in contributing to dyslipidemia [32] and conversion to tacrolimus as been reported to lower cholesterol, low-density lipoprotein, and apolipoprotein B in hyperlipidemic patients [33]. Hepatotoxicity had been observed in patients on cyclosporine and manifests as an elevation of liver function tests that regresses on dosage reduction. Importantly no associated histologic change has been described. The most common mucocutaneous side effects of cyclosporine are gingival hyperplasia and hypertrichosis, which are usually reversible with conversion to tacrolimus.

A significant problem with the original standard oil-based formulation of cyclosporine (Sandimmune) is poor and variable oral absorption resulting in large variability between patients in total cyclosporine exposure. That led to the development of a microemulsion formulation of cyclosporine, called Neoral, which has better and more consistent bioavailability with more reproducible daily exposure to the drug. This improved pharmacokinetic profile has been reported to result in lower acute rejection rates [34].

Tacrolimus is more potent than cyclosporine with an in vitro 50 to 100 times greater inhibition of lymphocyte proliferation [27] presumably owing to a greater affinity of its complex with FKBP to calcineurine [35]. Its side-effect profile is at variance with that of cyclosporine: although their nephrotoxicity is equivalent tacrolimus induces a higher incidence of diabetes mellitus and neurotoxic reaction but a lower incidence of hypertension, hyperlipidemia, hirsutism, and gingival hyperplasia [36] and as described above conversion from cyclosporine to tacrolimus should be considered to decrease these side effects.


   Antiproliferative agents
Top
 Abstract
 Introduction
 Definition of rejection
 Immunologic response to...
 Mechanism of action of...
 Corticosteroids
 Calcineurine inhibitors
 Antiproliferative agents
Antibodies
 References
 
This category includes agents that directly or indirectly inhibit the expansion of alloactivated T-cell and B-cell clones. Whereas 6-mercaptopurine, cyclophosphamide, and metotrexate all effectively inhibit lymphocyte proliferation their role in solid organ transplantation is limited primarily because of their toxicity. Azathioprine (Imurek; Glaxo Wellcome, Pittsburg, PA), mycophenolate mofetil (CellCept; Roche, Nutley, NJ), and sirolimus-rapamycin (Rapamune; Wyeth-Ayerst, St Davids, PA) are the currently used antiproliferative agents. The first two agents have similar mechanism of action in that each acts by interfering with purine synthesis [26].

Azathioprine
Before cyclosporine use conventional immunosuppression therapy was provided by azathioprine and corticosteroids. Azathioprine is an antimetabolite, 6-mercaptopurine (6-MP) with an additional side chain. Usually administered orally it is well absorbed by the gastrointestinal tract. It is primarily metabolized in the liver where its side chain is removed. The side chain allows azathioprine to be less toxic than 6-MP. Azathioprine can also be administered intravenously; generally the intravenous dose is half the oral dose.

As a purine analogue azathioprine can be incorporated into nucleic acids where it causes feedback inhibition in the early stages of purine metabolism and slows the whole process. In addition it inhibits several enzymes leading to blockage or reduction in the synthesis of cellular DNA, RNA, certain profactors, other nucleotides, and protein. Through these actions azathioprine blocks most T-cell functions, inhibits primary antibody synthesis, and decreases the numbers of circulating monocytes and granulocytes [26, 37]. Because it has little effect on established immune responses azathioprine is effective in the prevention but not the treatment of acute rejection [38].

The major dose-related adverse effect of azathioprine is bone marrow depression, which primarily manifests as leukopenia, but anemia and thrombocytopenia may also occur [26, 37]. Gastrointestinal sequelae include pancreatitis and less commonly nausea, vomiting, and diarrhea [39]. Hepatotoxicity is occasionally seen but is not dose related. The increased incidence of neoplasia is due to immunosuppression and associated reduction in immunosurveillance [26].

Mycophenolate mofetil
Mycophenolate mofetil is a selective inhibitor of the de novo pathway of purine biosynthesis thereby providing more specific and potent inhibition of T-cell and B-cell proliferation. When administered orally mycophenolate mofetil is converted into mycophenolic acid then to its glucuronide and is finally secreted into the bile [26]. The relative selectivity of this agent for lymphocytes is possibly related to the higher dependency of activated lymphocytes on both salvage and de novo synthesis of guanosine nucleotides than other cell types [26, 40].

Side effects include gastrointestinal upsets and diarrhea and an increased risk of tissue invasive cytomegalovirus infection. The most common hematologic disorders are leukopenia and thrombocytopenia with occasional pancytopenia. Interestingly clinical studies could not demonstrate a reduced incidence of bone-marrow suppression compared with azathioprine, with a similar frequency of leukopenia and opportunistic infections [40, 41]. Based on the mechanism of action of mycophenolate mofetil leukopenia was unexpected but may have been the result of coadministration of ganciclovir for concurrent cytomegalovirus infection [40]. Its long-term safety in humans has already been demonstrated when used for psoriasis as leukopenia was a very rare event with doses similar to those used in transplantation [41, 42].

Sirolimus (rapamycin)
Sirolimus, a macrocyclic antibiotic, is a highly potent immunosuppressive agent that has recently undergone phase III trials in kidney transplantation [43]. It is structurally related to tacrolimus and forms a complex with FKBP but its mechanism of immunosuppression differs. Whereas tacrolimus like cyclosporine interferes with early events of T-cell activation by blocking calcineurine activity, sirolimus prevents progression of T cell from the G1 to the S phase by blocking signaling downstream IL-2R [44]. On the one hand, calcineurine inhibitors block T-cell production of cytokine and sirolimus inhibits T-cell response to these cytokines. On the other hand, conversely sirolimus blocks lymphocytes proliferation at a point upstream from the antiproliferative agents discussed above. Hence sirolimus should be synergistic with those antiproliferatives as well as the calcineurine inhibitors. Secondary effects include hyperlipidemia, thrombocytopenia, and leukopenia [45].


   Antibodies
Top
 Abstract
 Introduction
 Definition of rejection
 Immunologic response to...
 Mechanism of action of...
 Corticosteroids
 Calcineurine inhibitors
 Antiproliferative agents
 Antibodies
References
 
Polyclonal antibodies
Polyclonal antibody preparations or polyclonal antilymphocyte globulin are gamma-globulin fractions of serum derived from animals inoculated with human lymphocytes, thymocytes or cultured lymphoblasts: namely rabbit antibodies to human lymphocytes (antilymphocyte globulin) and horse or rabbit antibodies to human thymocytes (horse antithymocytes globulin [ATGAM], rabbit antithymocyte globulin [RATG], and rabbit antithymocytes sera [ATS]) [46–48]. Polyclonal antibodies may exert their effects by several mechanisms. The IgG fraction contains variable amounts of specific antibodies directed against T-cell molecules. After polyclonal antibodies bind to these antigens, circulating T-cell levels are depleted. Proposed mechanisms for lymphocyte depletions include complement-dependent cytolysis and cell-mediated opsonization.

However polyclonal reagents have several drawbacks. Because of the production technique each polyclonal antibody preparation varies in constituent antibodies. This unpredictability is associated with variable efficacy and adverse reaction. There is an increased risk of infection and lymphoproliferative disease due to overimmunosuppression [49]. The toxicity of any heterologous serum prepared against human tissue depends on two factors: (1) its cross reactivity with other tissue antigens and (2) the ability of the patient to make antibodies against the foreign protein. Although serum sickness is a potential complication of these preparations it fortunately is rarely observed [50]. The presence of antibodies specific for nonlymphocyte antigen may cause thrombocytopenia, leukopenia, or anemia [49, 51].

Monoclonal antibodies
In 1975 Kohler and Milstein [52] developed the technology for stomatic cell hybridization, which could establish immortalized B-cell lines that each secrete a single, or monoclonal antibody in limitless supply [52]. This revolutionary technology provided reagents that are reactive only with T cells or even more importantly with specific T-cell subsets or other molecular targets such as TCR, CD3, CD4, and IL-2R.

The first monoclonal antibody (MAb) available for therapeutic use in the treatment and prevention of allograft rejection was OKT3 (Orthoclone OKT3; Janssen, Titusville, NJ), a murine MAb directed against the {epsilon} chain of CD3 molecule, which is part of the TCR complex and functions to modulate the receptor and inactivate T-cell function. By engaging the TCR complex OKT3 blocks not only the function of naive T cells but also the function of established cytotoxic T cells [53]. Currently it is the only MAb that has widespread use in clinical transplantation. Despite its extensive use a number of adverse reactions can occur after its administration. Cytokine release syndrome occurs in almost all patients and may be life-threatening in a few. This phenomenon, termed the first-dose effect, is the result of a massive albeit transient release of T-cell cytokines. It is characterized by fever, chills, general weakness, and mild hypotension. Less commonly patients experience vomiting, diarrhea, and rarely bronchospasm or severe hypotension [47, 54]. As with any agent overimmunosuppression can result in opportunistic infections particularly viral. An increase incidence of Herpes group virus infections including cytomegalovirus and the Epstein-Barr virus-associated posttransplant lymphoproliferative disorder has been reported after treatment with OKT3 [55]. Lymphomas have included B cell, T cell, large cell, Burkitt, and other cell types. However the progression to lymphoma is related to the intensity of the immunosuppression rather than specifically to any particular agent. For instance an increased posttransplant lymphoproliferative disorder incidence was noted in a group of 154 heart transplant patients after 2 weeks of OKT3 prophylaxis was introduced without the reduction of the concomitantly administered triple drug therapy [55] whereas 1 month of prophylaxis in combination with azathioprine and steroids but without cyclosporine resulted in no lymphoma in 150 renal transplant patients [56].

Because OKT3 is a murine antibody that is highly immunogeneic it commonly induces a human antimurine antibody (HAMA) response. Human antimurine antibody can rapidly inactivate and eliminate mouse MAb from the circulation. As a result most mouse MAbs have a half-life of only 1 to 2 days [57]. Human antimurine antibody inevitably develops after 10 to 14 days of treatment.

Given the central role of the CD4 cell in allograft rejection most new immunosuppressive strategies have sought to inhibit CD4 cell activation. Several ligand/receptor interactions occur between the T cell and the APC during antigen presentation. While some simply mediate cell-cell adhesion others transduce activation signals to either the T cell or the APC. A range of MAb is being developed to block these interactions. Because a discussion of all of the MAb currently undergoing investigation in the management of allograft transplantation is beyond the scope of this review the discussion will be limited to the only commercially available agents, the IL-2R blockers. These drugs have the potential to provide more specific immunosuppression therapy as the IL-2R is expressed only by activated lymphocytes.

In initial clinical studies rodent antibodies to the IL-2R administered as induction therapy were as effective as and better tolerated than ATG [58] and allowed safe use of a low initial dose of cyclosporine [59]. The short plasma half-life of these antibodies and the generation of HAMA by the recipient limited the efficacy of these preparations to several days. Circumvention of the HAMA response to the xenogeneic murine MAb has been accomplished using hybridoma technology and genetic-engineering techniques for the generation of unique chimeric and humanized antibody structures that contain both human and murine components.

The structure of MAb is Y-shaped with two distinct regions: (1) the ends of the Y, or variable regions, that bind antigen, and (2) the stem of the Y, the constant region, or Fc region, that serves as a structural support, activates complement, and permits the antibody molecule to attach to Fc receptors on neutrophils, lymphocytes, and monocytes. The HAMA is directed primarily to the nonspecific constant region of the mouse MAb. Chimeric and humanized antibodies are constructed so that the constant and nonhypervariable regions are of human origins thus greatly reducing immunogenicity. Chimeric antibodies combine the entire variable region of the murine MAb with human heavy and light chain constant regions. Humanized antibodies contain only the murine complementarity determining regions (hypervariable region) while the rest of the antibody molecule is human [60, 61].

The IL-2R is a multimeric complex of three transmembrane proteins ({alpha}, ß, and {gamma}). Noncovalent association of these chains forms the high-affinity binding site for IL-2. In contrast to the ß and {gamma} chains the {alpha} chain is not expressed on resting T cells but is induced after activation and is necessary for the formation of the signal-transducing, high-activity IL-2R [62]. Because only activated T cells express the Tac antigen (IL-2R{alpha} chain or CD25 antigen), anti-Tac IL-2R MAb are expected to target graft-reactive lymphocytes immediately after transplantation in an induction or sequential immunosuppression therapy protocol. The only activated T cells present at that point would be destroyed. However an important characteristic of this MAb is that it does not inhibit IL-2 binding to the IL-2R ß chain. Although the cytokine cannot bind to the isolated {gamma} chain it is possible for the ß and {gamma} chains to be linked by the IL-2 without the participation of the {alpha} chain if sufficiently high concentrations of IL-2 are present for long enough. The consequence is that this MAb is designed to be used with a calcineurin-blocking agent such as cyclosporine or tacrolimus to decrease the amount of IL-2 that would be available for any unblocked IL-2 receptors.

Whereas targeting IL-2/IL-2R pathway with anti-CD25 MAb induction therapy has been reported to clearly reduce the number of acute rejections after clinical transplantation it does not prevent T-cell reactivity completely because some of the treated allograft recipients still reject their graft [63, 64]. Recently IL-2 negative rejections or anti-CD25 blocked rejections have been shown to express other members of the IL-2 family [65]. These cytokines, IL-7 and IL-15, may be alternatives for IL-2 in allogeneic responses [66].

Two new anti-Tac MAb became available in 1998. Basiliximab (Simulect; Novartis, St Louis, MO) is a chimeric antibody retaining the murine elements of the variable portion of the immunoglobulin chain. Daclizumab (Zenapax; Roche, Nutley, NJ) is more completely humanized retaining a smaller region of the murine antibody. The immunogenicity of these molecules as measured by in vivo circulating half-life and by the appearance of antibodies against the agents is significantly reduced when compared with strictly murine anti-CD25 MAb. Phase III clinical trials were performed with kidney transplantation [67, 68]. Both drugs were compared with placebo in combination with standard cyclosporine and corticosteroids and with azathioprine in the daclizumab trial. The results were strikingly similar: both antibodies were well tolerated and reduced the incidence of infection or other adverse events within the 1-year follow-up period. Although these agents are given only for a limited period after transplantation effective IL-2R blockade is achieved for several weeks after the last dose thereby covering the critical period when acute rejection is most common.


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 Abstract
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 Definition of rejection
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