Prevention and Treatment of Renal Allograft Rejection: New Therapeutic Approaches and New Insights Into Established Therapies1
Christopher V. Lu,2 Stanley Carlos Sicher, and Miguel A. Vazquez
“ hemotherapeutic agents are not only CV. Lu, S.C. Sicher, M.A. Vazquez, The University Trans- . e #{149} ends In themselves but also serve as plant Program at Parkland Memorial Hospital, Division tools for unlocking doors and probing Nature’s mys- of Nephrology, Department of Internal Medicine, Uni- terles. . versity of Texas Southwestern Medical School, Dallas, Gertrude B. Elion, lecture upon receiving the Nobel TX Prize for Physiology or Medicine (1) (J. Am, Soc. Nephrol. 1993; 4:1239-1256) There is a dynamic interaction between clinical transplantation and basic immunology. On the one hand, new insights by basic scientists have resulted ABSTRACT in new therapeutic agents that may totally revolu- Renal transplantation is the preferred treatment mo- tionize therapy in the next decade. On the other hand, dality for patients with ESRD who are good surgical in solving clinical problems, a new understanding of risks and able to comply with chronic immunosup- basic immunologic processes has emerged. The pur- pressive medications. Clinical transplantation has pose of this review is not only to discuss new agents that may be part of the coming revolution in clinical advanced significantly, with most transplant centers transplantation, but also to discuss what these reporting 1-yr renal allograft survival rates of better agents have taught us about basic immunologic proc- than 80%. Nevertheless, rejection and a progressive esses. We will also examine new insights into several loss of allogratts over time continue to occur. The established agents. In the near future, the challenge immunosuppressive agents currently used may lead to transplant nephrologists and surgeons will be to to the development of life-threatening infections, use new, powerful agents intelligently. This will re- malignancies, and advanced atherosclerosis as a quire understanding their mechanisms of action. consequence of some of their side effects. This re- view examines the mechanisms involved in allograff MECHANISMS OF RENAL ALLOGRAFT rejection as currently understood. The recent knowl- REJECTION edge into the mechanism of action of cyclosporine, Before discussing the new immunosuppressive FK506, and rapamycin on T cell activation is pre- drugs and monoclonal antibodies, we briefly outline sented. Information recently available on some of the mechanisms of allograft rejection. Processes in- the established therapies such as steroids, antime- hibited by specific drugs or monoclonal antibodies tabolites and monoclonal antibodies as well as the will be discussed in greater detail later in this review. newer agents is also discussed. The interaction be- Two different classes of T lymphocytes are neces- tween clinical transplantation and basic research in sary for the most vigorous allograft rejection (2). One is the CTL or cytotoxic T lymphocyte. These T cells immunology continues to result in exciting advances have a cell-surface marker called CD8. An Interac- in both fields. tion of a naive CTL with its target cell is by Itself not Key Words: Transplantation, immunosuppression, rejection, sufficient to result In lysis of the target cell. The aio graft. kidney naive CTL must receive accessory signals from a helper T cell. The latter cells have a cell-surface Received February 4, 1993. Accepted July 2. 1993. 2Coffespondence to Dr. C. Lu, University Transplant Program at Parkiand Me- marker called CD4. These accessory signals include mortal Hospital, Division of Nephrology, University of Texas Southwestern Medi- interleukin (IL)-2, interferon gamma, and IL-6. After cal School, 5323 Harry Hines Boulevard, Dallas, TX 75235-8856. receiving these accessory signals, the naive CTL be- 1046-6673/0406- 1239$03.00/0 comes fully competent to kill the allograft kidney cell. Journal of the American Society of Nephrology Copyright © 1993 by the American Society of Nephrology The accessory signals are secreted by helper T cells.
Journal of the American Society of Nephrology 1239 Prevention and Treatment of Renal Allograft Rejection
Note that two signals are necessary to stimulate helper T cells (for a review, see references 9 and 10). craft cell There is an antigen-specific interaction between the T cell receptor and Its specific antigen. There must also be stimulation of the T cell by accessory signals
such as B7 or IL- 1 . The activated T cell then secretes additional lymphokines, including IL-2, IL-4, granu- locyte-macrophage colony stimulating factor, and in- terferon gamma. These lymphokines activate mac- rophages and B cells and change the biology of the allograft so that it is more susceptible to rejection. As an example of the latter, interferon gamma increases the amount of MHC antigens on the allograft and thus makes it better able to stimulate host T cells. In addition, the lymphokines stimulate bone marrow to stimulate bone marrow produce more inflammatory cells. Thus, the activated to produce inflammatory cells activate rnacrophages T cell performs many activities that coordinate the activate B cells complex process of allograft rejection. In addition, increase MHC ags on allograft the entire process is amplified when the activated T cell proliferates. DRUGS INTERFERING WITH BIOSYNTHESIS & THUS DNA SYNTHESIS AND PROLIFERATION AZATHIOPRINE. RS61443. IMMUNOSUPPRESSIVE DRUGS MIZORIBINE Drugs Binding lmmunophilins: Cyclosporines, Figure 1 . Outline of allograft rejection-activation of CD4 FK5O6, Rapamycin, and Their Analogs helper T cells. Reprinted with permission from reference 151. Ia, class II MHC antigen (ag). See Text. IFN, interferon The story of this family of drugs and how they gamma; IL2R, 11-2 receptor; GM-CSF, granulocyte-macro- work is an elegant example of the dialogue between phage colony-stimulating factor; IcR, I cell receptor. clinical transplantation and basic immunology. This dialogue is resulting not only In new, possibly safer
In Figure 1 , we review the activation of the helper and more effective, immunosuppressive drugs, but T cell in greater detail. As shown at the top of Figure also in a more profound understanding of T cell
1 , host helper T cells may be activated by interacting activation. directly with Class II major histocompatibility anti- These drugs may be grouped together because they gens (Class II MHC or Ia) on allograft cells. This Is the all bind cytosolic proteins called Immunophilins so-called “direct” pathway of T cell activation and (1 1 , 1 2). This binding Is critical for the immunosup- may occur either when host T cells encounter allo- pressive effect. The active intracellular inhibitor is graft cells in the allograft kidney (“peripheral sensi- not the drug alone, but a drug-immunophilin corn- tization”) or when donor dendritic cells (“passenger plex. It is therefore appropriate to briefly review the leukocytes”) emigrate to the host spleen and encoun- immunophilins in the next paragraph. ter host T cells at that site (“central sensitization”) Cyclosporines bind a group of immunophilins (3,4). Alternatively, antigens from the allograft may called cyclophilins. FK506 and rapamycin bind a be internalized by host macrophages or dendritic different group called FK506-binding proteins (ab- cells and digested into short peptides. These peptides breviated FKBP). In this review, we will focus on associate with host Ia molecules and are displayed cyclophilin A (12) and the 1 2-kd FKBP because they on the cell surfaces of these host cells. These then are the best understood of each immunophilin fam- stimulate the helper T cell. This is the so-called “in- ily. Proteins with amino acid sequences similar to direct” pathway of T cell activation (for example, see that of cyclophilin are present throughout nature references 5 and 6). The relative importance of these from Eschertchta colt to humans. This suggests that two alternative pathways in the activation of helper cyclophilin has such an important function that it T cells is not well established at this time. Note that, has been conserved through eons of evolution. Cyclo- in addition to presenting antigens to helper T cells, philins are present in all tissues, further suggesting macrophages also secrete important accessory mole- an important biologic function. The immunophilins cules, including IL-2, IL-6, and tumor necrosis factor- are peptidyl, prolyl, cis-trans isomerases. This alpha (TNF-ce). Among the large number of effects of means that they catalyze cis-trans conformations of these cytokines is their ability to activate the allograft peptides, perhaps allowing them to fold Into their endothelium (7,8). This results in the recruitment of active conformations and be transported to the ap- host lymphocytes, macrophages, and polymorpho- propriate places in the cell. For example, this occurs nuclear leukocytes into the allograft. In the development of the Drosophila eye. In this
1240 Volume 4 Number 6. 1993 Lu et al
model system, mutations in the cyclophilin called calcineurin (17,18). Calcineurin is a calcium-acti- “ninaA” prevent the transport of rhodopsin Rh 1 from vated phosphatase. Its inhibition may prevent T cell the endoplasmic reticulum to the appropriate place activation in the following way. Normally, after the In the photoreceptor cell, and visual impairment re- T cell receptor is stimulated, intracellular calcium suits (13). rises. This activates calcineurln, which removes The discovery that cyclosporine, FK506, and ra- phosphate(s) from the nuclear factor of activated T pamycin inhibit the isomerase activity of immuno- cells (NFAT). This dephosphorylatlon allows NFAT to philins led to the idea that these drugs inhibited T enter the nucleus, where it combines with another cell activation by inhibiting isomerase activity. How- protein to form an active nuclear factor. The active ever, this idea is not true for the following reasons NFAT binds to and activates the gene for IL-2. ThIs (14,15). One, the immunophilins are broadly distrib- ultimately results In IL-2 secretion ( 1 4, 1 9). By Inhib- uted, and if these drugs acted by inhibiting isomerase iting the phosphatase activity of calcineurin, cyclo- activity, they should inhibit the activation of all cells. sporine-cyclophilin and FK506-FKBP complexes pre- Yet, the immunophilin-binding drugs usually only vent NFAT from moving from the cytoplasm to the inhibit T cells. Two, the concentrations of cyclospor- nucleus. Activation of the IL-2 gene does not occur me or FK506 required to inhibit T cells are far below (1 7, 1 8). Supporting this hypothesis is the correlation the concentrations needed to inhibit isomerase actlv- between the inhibition of T cell activation by analogs ity. Three, there is no correlation between the ability of FK506 or cyclosporine and the ability of the ana- of analogs of cyclosporine and FK506 to inhibit iso- log-immunophilin complex to inhibit calcineurin merase activity and the analog’s ability to inhibit T phosphatase activity (20). This hypothesis also ex- cell activation. Four, this idea fails to explain the plains the specificity of cyclosporine and FK506 for absence of correlation between the biochemIcal T cell activation because the nuclear binding factor structure and the immunosuppressive ability of these NFAT is found only in T cells. drugs. In other words, cyclosporine, a cyclic polypep- Also supporting this hypothesis is an observation tide, and FK506, a macrolide antibiotic, have com- of potential clinical importance. FK506 and rapa- pletely different biochemical structures, yet they in- mycin inhibit each other’s immunosuppressive ac- hibit the same step in T cell activation. The inhibited tivities (11,12,14). This is illustrated in Figure 3. step Is activation through the T cell receptor. In con- When FK506 binds to FKBP, the isomerase Is inhib- trast, rapamycin and FK506 are structurally related macrolide antibiotics and inhibit the same isomerase. However, despite their biochemical similarities, ra- pamycin and FK506 inhibit completely different steps of T cell activation (Figure 1). FK506 inhibits T cell activation via the T cell receptor, but not via the IL-2 receptor. On the other hand, rapamycin inhibits 7 Component X inhibited. activation via the IL-2 receptor but not via the T cell FKSO6 1-cell response to stimulation of IcR receptor (16). inhibited. No binning A hypothesis that explains all of the currently isomerase inrsbited to component Y available data states that the active intracellular im- 506BD munosuppressant is not the immunophilin-binding > cornponentsX or drug itself, but a drug-immunophilin complex i-n r’---’ -i isomerase inhibited (11,12,14) (Figure 2). Thus, complexes of FK506- FKSO6 binding ______protein IFKBP) FKBP or cyclosporine-cyclophilin bind to and Inhibit Component Y inhibited. rapamycin T-cell response to lL-2 ANTIGEN inhibited. Binding to component X prevented. TcR
isomerase inhibited CALONEURIN rt L NT’ i T i NFAT.A8-.-I.ACflVATE 1-2 Figure 3. The complex of FK5O#{212}and FKBP inhibits I cell activation via the I cell receptor (IcR), whereas the com- Figure 2. The complex of cyclosporine (cya) and cyclo- plex of rapamycin and FKBP inhibits I cell activation via the philin inhibits I cell activation by inhibiting calcineurin. IcR, 11-2 receptor. This model illustrates how FK5O6 and rapa- T cell receptor; CA, calcium; Pi, phosphorus; NFAT-AB, ac- mycin might antagonize each other’s immunosuppressive tive heterodimer of nuclear factor of activated I cells. See activity. Component V. calcineurin. Reprinted with permis- Text. sion from reference 151. See Text.
Journal of the American Society of Nephrology 1241 Prevention and Treatment of Renal Allograft Rejection
ited. However, the important fact, as discussed Steroids above, is that the FK506-FKBP complex binds to and inhibits calcineurin (“component X” in Figure 3). On Steroids remain important immunosuppressive the other hand, when rapamycin binds to FKBP, the drugs. Although they are old agents, there is a new rapamycln-FKBP complex binds to a different pro- understanding of how steroids work. Steroids have tein, “component Y.” This inhibits the T cell response significant inhibitory actions at a number of differ- to stimulation via the IL-2 receptor, but not via the T ent stages of allograft rejection. cell receptor. By binding up all of the FKBP, rapa- As discussed in the previous section, cyclosporine mycin may prevent the formation of FK506-FKBP and FK506 prevent the activation of the IL-2 gene by complexes and thus prevent the inhibition of calci- preventing the formation of NFAT, the nuclear factor neurin (“component X” in Figure 3). This explains that activates this gene. Steroids also prevent the the ability of rapamycin to antagonize the immuno- activation of the IL-2 gene, but by an entirely differ-
suppressive effect of FK506 and vice versa (2 1 ). The ent mechanism (32,33). Steroids act, not by prevent- effects of 506BD, an analog of FK506, are particu- ing the formation of NFAT and other nuclear factors, larly instructive In this regard. 506BD binds to the but by preventing appropriate amounts of these flu- isomerase and InhIbits It. However, the 506BD-FKBP clear factors from activating the IL-2 gene. complex does not bind to either calcineurin (“compo- Steroids also inhibit T cell activation indirectly by nent X”) “component Y.” Thus, 506BD inhibits the inhibiting macrophage functions. Steroids inhibit the isomerase but has no immunosuppressive activity. expression of Ia molecules on macrophage cell sur- Indeed, by binding up to FKBP, 506BD prevents the faces (34-36). Thus, macrophages ingest antigens formation of FK506-FKBP complexes and thus an- shed by allograft cells, process these antigens, and tagonizes the immunosuppressive effects of FK506 then present the antigenic peptides to T cells in the (16,2 1). context of Ia molecules. This is the “indirect” pathway
Because cyclosporine binds to different immuno- of T cell activation (Figure 1 ) (5,6). T cell activation philins than FK506 and rapamycin, cyclosporine requires that the T cell receptor interact with the should not be antagonized by FK506 or rapamycin. antigen in the context of Ia. If steroids inhibit the Furthermore, because cyclosporine and rapamycin expression of Ia molecules, they will also inhibit the inhibit different stages in T cell activation (Figure 1), ability of macrophages to present antigen to and these two drugs are predicted to synergize with one activate T cells.
another. This prediction is valid in animal models Steroids inhibit the production of TNF-a IL- 1 , and (22). eicosanoids by macrophages (37,38). This has impor- “Component Y” in Figure 3, the target of the rapa- tant implications (8,39). A major event during allo- mycin-FKBP complex, has not been Identified. How- graft rejectIon is the Infiltration of allograft tissue by ever, rapamycin does selectively inhibit the activa- host inflammatory cells. In other words, the inflam- tion of p70 S6 kinase by IL-2 (23). This kinase may matory cells that are in the host’s bloodstream must be important in the response of T cells to IL-2. interact with the endothelium, such that the T cells, We have focused on the effects of cyclosporine, neutrophils, and monocytes translocate across the FK506 and rapamycin on T cell activation because endothelium into the allograft tissue, where rejection
these are the best-understood immunosuppressive then occurs. TNF-a, IL-i , and eicosanoids (prosta- activIties of these drugs. These drugs also have mul- glandins, leukotrienes, thromboxanes, and lipoxins) tiple other effects on the immune system, but a dis- activate aliograft endothelial cells such that they ex- cussion of them is beyond the scope of this review. press adhesion molecules, such as intercellular adhe- The reader is referred to several excellent recent sion molecule 1 (ICAM- 1), and selectins (for a review, reviews (15,24,25). see references 8, 40, and 41). These molecules cause This review is focused on the immunology of im- inflammatory cells to adhere to the endothelium and munosuppressive agents, and the clinical use of cy- then to move across it. In addition, eicosanoids are closporine, FK506, and rapamycin is not discussed. chemotactic agents that attract leukocytes to sites of There are several excellent recent reviews on the inflammation. Indeed, the antiinflammatory effects clinical use of cyclosporine (26-28). Cyclosporin G of nonsteroidal antiinflammatory agents, such as (29), an analog of cyclosporin A, may have similar aspirin and indomethacin, are the result of their immunosuppressive activities but less nephrotoxic- ability to inhibit the production of the eicosanoid ity. FK506 has been used successfully in transplan- prostaglandin E2. tation (30) but has not yet been compared with cyclo- The mechanisms by which steroids inhibit the pro- sporine in randomized, blinded clinical trials. Rapa- duction of the above proinflammatory molecules is mycin has not yet been tested in humans, but there now understood at a molecular level. Steroids inhibit is extensive preclinical experience (for a review, see both the transcription and the translation of the gene reference 31). encoding phospholipase A2 (42). Phospholipase A2 is
1242 Volume 4 Number 6 1993 Lu et al
important because it is the first enzyme in metabolic immunodeficiency occurring In patients whose pur- pathways leading to the production of all eicosanoids. inc metabolism is disrupted by an inherited defi- The idea that steroids act on lipocortin to inhibit ciency of adenosine deaminase or purine nucleoside phospholipase A2 (43) is not consistent with some phosphorylase (5 1 ,52). recent evidence (44,45). In addition, steroids also Azathioprine. The Nobel Prize-winning research Inhibit the gene for prostaglandin synthetase at the of Elion, Hitchings, and Black demonstrated that 6- translational level (46,47). The major inhibitory ef- mercaptopurlne was an effective antileukemic agent fects of steroids on the production of TNF-a and IL-i by virtue of its ability to interfere with the purine occur after these genes have been transcribed into metabolism of rapidly dividing leukemic cells. Robert mRNA. Steroids inhibit the translation of the mRNA Schwartz realized that lymphoblasts formed during into TNF-a (37) and IL-i fi (48,49) protein and de- an immune response resembled leukemic lympho- crease the stability of mRNA for IL-i f3 (38). cytes and demonstrated that 6-mercaptopurine was also an effective immunosuppressive drug. Roy Calne successfully applied this insight to kidney transpian- Drugs Interfering With Purine and Pyrimidine tatlon (for reviews, see references 1 and 57). Metabolism Subsequent studies showed that azathioprine, the This class of drugs includes azathioprine, which is i -methyl-4-nitro-5-imidazolyl derivative of 6-mer- a well-established immunosuppressive agent, and captopurine, was even more effective than the parent the newer experimental drugs RS6 1443, mizoribine, compound (1). Azathioprine is slowly converted into and brequinar. These drugs all interfere with the 6-mercaptopurine and methylnitrolmidazole by glu- metabolism of either purine or pyrimidine nucleo- tathione and other sulfhydryl-containing molecules sides. Their effects may not be limited simply to the In red blood cells. This results in more sustained inhibition of DNA synthesis and the proliferation of levels of 6-mercaptopurine and methylnitroimidazole immune cells. As shown in Figure 4, nucleotides are in serum, both of which are important in the immu- important in a number of additional cellular proc- nosuppressive effect of azathioprlne. The effect of esses (50). UDP and GDP are Important in the glyco- the 6-mercaptopurine moiety of azathioprine will be sylation of proteins. Such glycoproteins Include the discussed first. integrins and other adhesion molecules Important in Although the metabolic pathways are intricate, allograft rejection (see “Antibodies to Adhesion Mol- they must be reviewed because they have Important ecules” section). ATP is the universal currency of clinical implications. In an initial critical step. 6- energy for cellular processes. ATP and GTP, via its mercaptopurine is converted to 6-thioinosinic acid effects on G proteins, are important intermediates in (TIMP) by the enzyme hypoxanthine-guanine phos- intracellular signalling. The importance of purines phoribosyltransferase (HGPRT) (Figure 5). TIMP is in lymphocyte function is illustrated by the severe then converted to thionucleotides, which inhibit the
FORMATiON OF DNA A.SD I THUS LYMPHOCYTE PROLIFERATION
/ I FORMATIONOFGTP AD E NUCLEOTIDLS I’ #{188} ARE COMPONENES OF TIlE COV ZYMES NAD+, FAD, & REGULATES LYMPHOCYTE & POWERS MOVLML OF CoA) ThIPORTANT i MACROPHAGE ACTIVATION MACROMOLECULES METABOLISM OF PROTE 4S. VIA “G-PROTE S T4 CELLS CARBOHYDRATES, & FATS
Figure 4. Importance of purine and pyrimidine metabolism on various cellular functions. CoA, coenzyme A; FAD, flavin adenine nucleotide. See Text.
Journal of the American Society of Nephrology 1243 Prevention and Treatment of Renal Allograft Rejection
RThOWS’PHOSPHATE of 6-mercaptopurine ( 1 ). The mechanisms underlying the greater sensitivity of lymphocytes and neutro- R1$OSE PHOSPHATE PYROPBOSPHOIC 4ASE phils to azathioprine, as opposed to other rapidly (PR?? SYN’I’IlETASE) replicating cells such as gut epithella, are not known. It is important to emphasize the potential clinical importance of the salvage pathway (see HGPRT in Figures 5 and 6) in the immunosuppression by 6- mercaptopurine. After prolonged therapy with aza- thioprine, some T lymphocytes mutate and become deficient in HGPRT. These T lymphocytes do not convert 6-mercaptopurine to TIMP and thus are not inhibited. The appearance of such mutated HGPRT- deficient lymphocytes may result in acute rejection (53). In a similar fashion, patients with Lesch-Nyhan syndrome, who lack HGPRT on a genetic basis, also SALVAGE PATHWAYr resist the immunosuppressive effects of 6-mercap- HYPOXANTRP4E-GUANP4E PHOSPHORIBOSYL
TRANSFERASE (HGPRT) topurine (53,54). I 6-Mercaptopurine may be inactivated in two ways ADENP4E GUANPIE before it is converted by HGPRT to TIMP (Figure 6). ( 6.MERCAFTOI’UR1NE (6MP) j 6-Mercaptopurine may be inactivated by the enzyme thiopurine methyltransferase (TPMT). This is clini- C or&