Mechanisms of action of cyclosporine and effects on connective tissues

Mechanisms of Action of Cyclosporine and Effects on Connective Tissues By Graham Russell, Robert Graveley, Janet Seid, Abdul-Karim

Al-Humidan,

Cyclosporine is a potent immunomodulatory agent with an increasing number of clinical applications. Its major mode of action is inhibition of the production of cytokines involved in the regulation of T-cell activation. In particular, cyclosporine inhibits the transcription of interleukin 2. Although cyclosporine’s major actions are on T cells, there is some evidence that it produces direct effects on other cell types. Its immunosuppressive action is closely linked to its binding of cyclophilin, a member of a family of high-affinity cyclosporine-binding proteins widely distributed in different cell types and in different species. The cyclophilins have been shown to have peptidyl-prolyl cis-rrans isomerase enzyme activity that is blocked by cyclosporine. Although this may be a factor in cyclosporine’s selective inhibition of cytokine gene

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YCLOSPORINE IS a cyclic uncadecapeptide, originally isolated from Tolypocladium injlatum Gums (Fig 1). ’ The compound has very potent immunomodulatory properties, and its highly specific immunosuppressive activity has been responsible for significant advances in the clinical treatment of organ transplant patients.’ The therapeutic value of cyclosporine now ex-

From the Department of Human Metabolism and Clinical Biochemistry, University of Shefield Medical School, Shefield, England; and Rigshospitalet, University Hospital, Copenhagen, Denmark. Dr Skjodt is supported by the Danish Rheumatism Association (Gigtforeningen). The authors are also gratefulfor support from the Arthritis and Rheumatism Council, The Nu@ield Foundation, the Science and Engineering Research Council, and Sandoz Pharmaceuticals. Graham Russell, MD, PhD, FRCP, FRCPath: Professor, University ofShe@eld Medical School; Robert Graveley, BSc: Research Student, University of Shefield Medical School; Janet Seid, BSc, PhD: Research Scientist, University of Shefield Medical School; Abdul-Karim Al-Humidan, BSc: Research Student, University of Shefield Medical School; Hemik Skjodt, MD, PhD: Lecturer in Medicine, Rigshospitalet. University Hospital, Copenhagen. Address reprint requests to R.G.G. Russell, MD, PhD, FRCP, FRCPath, Department of Human Metabolism and Clinical Biochemistry, University ofShe@eld Medical School, Beech Hill Road, Sheffield SlO ZRX, England. Copyright 0 1992 by W.B. Saunders Company 0049-0172/92/2106-3003$S.O0/0 16

and Henrik Skjodt transcription, it is still unclear whether inhibition of this activity is the mechanism through which cyclosporine exerts its effects on target cells. The ubiquitous presence of cyclophilins raises the question of why cyclosporine has major effects on T cells. Perhaps the critical proteins affected are transcriptional regulators restricted in their tissue distribution. The effects of cyclosporine on T cells and, directly or indirectly, on connective tissue cells, all of which can produce a range of cytokines, are of interest in relation to the tissue changes that occur in such inflammatory conditions as rheumatoid arthritis. Copyright 0 1992 by W. 6. Saunders Company INDEX WORDS: Cyclosporine; cytokines; T lymphocytes; bone; arthritis.

tends to many other disorders in which altered immunoregulation may play a part, including psoriasis, 3-5rheumatoid arthritis, 6,7the nephrotic syndrome,8 and inflammatory bowel disease. There have been many excellent reviews of the pharmacology and clinical uses of cyclosporine and related agents.7-22 Despite fascinating advances in knowledge of the actions of cyclosporine, the precise biochemical processes by which it works remain only partially understood. Its major pharmacological effect appears to be inhibition of early events in the activation of T cells by antigens.23 These early effects give rise to various secondary events on B cells, macrophages, and other cells that are dependent on the cytokines and lymphokines produced by activated T cells.24,25It is likely that the agent has effects on other cells, but understanding the extent to which T-cell and non-T-cell actions contribute to its therapeutic value remains a difficult challenge. CYCLOSPORINE, CYTOKINE PRODUCTION, AND IMMUNOREGULATION

Cyclosporine selectively inhibits the production of several cytokines involved in regulating the cellular activity of the immune system. Within the immune system, cyclosporine’s spectrum of activity appears to be more or less restricted to lymphocytes. All sustained immune

Seminars in Arthritis and

Rheumatism, Vol2 1, No 6, Suppl3 (June), 1992: pp 16-22

MECHANISMS

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17

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responses involve activation of T lymphocytes. Cyclosporine’s inhibition of T-cell priming is a key event, and in particular, cyclosporine inhibits the production of messenger RNA (mRNA) for some early-activation genes (eg, C-myc). It also inhibits the de novo synthesis of interleukin 2 (IL-2) interleukin 3 (IL-3) interleukin 4 (IL-4) and interferon gamma as shown at the mRNA level in activated T cells. 26-30In contrast, synthesis of granulocyte-macrophage colony-stimulating factors (GM-CSF) in T cells appears to be resistant to cyclosporine. Cyclosporine also may partially inhibit the expression of cell surface receptors for IL-2 on T cells. The combined effect of these changes retards the proliferation of T cells as promoted by the autocrine and paracrine effects of IL-2. The initial stimulus for the production of IL-2 from T cells is exposure to interleukin 1 (IL-l) derived from macrophages during the process of antigen processing and presentation. The action of cyclosporine probably is not restricted to T cells. For example, certain B-cell responses also are sensitive to the compound (reviewed by Klaus in 19883’). Although IL-1 production by macrophages apparently is unaffected by cyclosporine, the production of tumor necrosis factor alpha (TNF-a) may be sensitive to the agent independent of any effect on the mRNA levels for TNF-LU.32 Antigen processing by antigen-presenting cells (including monocytes, dendritic cells, and B cells) also appears to be unaffected by cyclosporine, but this subject is controversial.33.34 Even so, the induction of the

major histocompatibility complex (MHC) class II determinants necessary for antigen presentation by macrophages and other cells is inhibited by cyclosporine.35 A possible explanation for this effect is that it is mediated through the inhibition of production of interferon gamma and IL-4 by T cells. Interferon gamma is particularly important in the induction ofthese class II determinants involved in antigen presentation. The complexity of the cytokine network means that multiple cytokines may act concurrently on many cell types. producing distinct responses that include further stimulation of production of cytokines and their receptors. It is possible that cyclosporine modulates many pathological responses by inhibiting transcription of T-cell cytokines, thus inhibiting many of the secondary responses to cytokines in different cell types. Also, the fact that many cytokines are not produced under basal conditions may restrict the activity of cyclosporine to certain phases of cell responsiveness. BIOCHEMICAL MECHANISM OF ACTION

Although surface receptors for cyclosporine have been described,36 the lipophilic cyclosporine molecule is generally thought to gain access to the cytoplasm of any cell type by simple diffusion across the membrane. Toxic effects described in hepatocytes and renal tubular and endothelial cells may be distinguishable from the immunosuppressive properties of cyclosporine. Within cells, cyclosporine binds to specific proteins known as cyclophilins.37 The binding of cyclosporines to such proteins, with dissociation constants (kDs) in the nanomolar range, seems closely linked to the immunosuppressive action of cyclosporines. Recently, cyclophilins have been shown to possess enzyme activity and, thus, to act as peptidyl-prolyl ci.struns isomerases: 78-40This activity changes the conformation of proline residues within proteins, thereby regulating protein folding during cell protein synthesis. The peptidyl-prolyl cis-truns isomerases, also known as rotamases, may play an important role in defining the functional state of many cell proteins. Cyclosporine blocks peptidyl-prolyl cis-truns isomerase activity, and this action may be important in its selective inhibition of cytokine-gene transcription. However, not all cyclosporine analogues suppress enzyme action. even though they are immunosuppressive. Fur-

18

RUSSELL

thermore, it appears that the amounts of cyclosporine found within cells are probably insufficient to provide full occupancy of cyclophilin and therefore to inhibit completely the associated enzyme activity. However, the ubiquitous presence of cyclophilins and peptidyl-prolyl cis-truns isomerases raises the question of whether cyclosporine can inhibit cytokine production or otherwise alter protein synthesis in nonimmune cells. In terms of identifying proteins whose function may be altered as cyclosporine binds to cyclophilin with its possible resultant effects on rotamases, nuclear proteins that regulate gene transcription are obvious potential candidates (Fig 2). Indeed, there is evidence that cyclosporine may alter the function of several such proteins (eg, NF-AT, AP-3, and NF-LB), all of which are involved in the regulation of the transcription of the IL-2 gene.4’ So

RNA pol II lead to formation of active transcription complex

ET AL

far, NF-AT appears very restricted in its cellular distribution. If it is present only in T cells, as seems possible, this would help explain why cyclosporine’s actions may be restricted to T cells. Consequently, cyclosporine’s inhibition of individual gene expression in different cell types may be highly dependent on cell type. The action of other immunomodulatory agents may involve similar mechanisms. For example, fujimycin (IX 506), a macrolide structurally unrelated to cyclosporine,‘9,42-44 also binds to a specific binding protein called FK-BP (FK-binding protein).2’,45 It is an I 1.8-kilodalton protein, and although it is quite distinct from the protein cyclophilin ( 17 kilodaltons) it also possesses rotamase activMoreover, rapamycin, which has ity. 20,40,46-49 structural homology with FK 506, also binds to FK-BP but not to cyclophilin.50,5’ Therefore, both FK-BP and cyclophilin belong to a family of high-

II (_)

Fig 2: Possible biochemical modes of action of cyclosporine and its effects on T-cell activation. Cyclosporine reduces T-cell proliferation by inhibiting the production of IL-2. It may also prevent transduction of the mitogenic activation signal from the cell surface to the nucleus. This may be influenced by cyclosporine’s binding to the intracellular receptor protein, cyclophilin. CS, cyclosporine; IL-2, interleukin 2; mRNA,

messenger

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cell; MHC,

major histocompatibility

complex; AG,

antigen; IL-2R, IL-2 receptor; RNA pol II, RNA polymerase; CD, cluster designation; TCR, T-cell receptor. (Reprinted with permission.*)

MECHANISMS

OF ACTION

19

OF CYCLOSPORINE

affinity binding proteins, also termed immunophilins.50%“’Nevertheless, rapamycin appears to act by different intracellular mechanisms than cyclosporine and FK 506 and to interfere with intracellular signalling pathways. Reports that FK-BP may have inhibitory activity toward protein kinase C remain to be confirmed. Other drugs, such as glucocorticoids (GCs), may also exert some of their immunomodulatory and anti-inflammatory effects by altering the transcriptional regulation of specific cytokine genes. Indeed, the GC receptor belongs to a superfamily of DNA-binding proteins that includes receptors for estrogens, androgens, thyroid hormones, retinoids, and vitamin D metabolites, all of which have major effects on the regulation of specific genes. SIGNIFICANCE OF CYTOKINE ACTIVITY

IN BONE

AND OTHER CONNECTIVE TISSUES

Although production of some cytokines, including IL-2, appears restricted to cells ofthe immune system (eg, CD4-positive T-helper cells). many cytokines originally defined as immune cell mediators are produced in a wide variety of nonimmune cells. Cytokines probably play an important role in regulating connective tissue turnover, particularly under pathological conditions.“i-55 A number of cytokines modulate bone cell activity in vitro. In particular, IL-l, TNF-(U (cachectin), and tumor necrosis factor beta (TNF-fi; lymphotoxin) are potent stimulators of bone resorption and affect osteoblast metabolism.‘“62 Interferon gamma preferentially inhibits this cytokine-stimulated resorption of bone. In addition, colony-stimulating factors, including GM-CSF, are involved in the induction of osteoclasts in hematopoietic marrow.63 Bone cells may be a local source of many cytokines, including TNF-cu and colony-stimulating factors released during cell culture. The study of a putative local bone cytokine network awaits determination of cytokine mRNA expression in bone surface cells. In addition to bone stromal cells and osteoblasts, hematopoietic cells, osteoclasts, and immune system cells are present in the microenvironment of the bone remodeling surface. We recently described expression of MHC class II determinants (HLA-DR and DQ) by a subset of human osteoblastlike bone surface cells in long-term culture. Moreover, human bone cells

stimulate proliferation of allogeneic and autologous peripheral blood mononuclear cells in coculture and function as antigen-presenting cells. The MHC class II expression by human bone cells is enhanced by interferon gamma and 1,25dihydroxyvitamin D3. Activation of T cells, including release of osteotropic cytokines, could be involved in some regulatory pathways within bone and be important in causing the local and systemic bone changes associated with such diseases as rheumatoid arthritis. ACTIVITY

OF CYCLOSPORINE ON BONE

The interactions between bone stromal cells and immune cells, including cytokine production, have prompted interest in the possible modulation of bone cell metabolism by cyclosporine. In two different bioassays of bone resorption, cyclosporine inhibited the bone-resorbing activity of IL- 1. 1.25dihydroxyvitamin D3. parathyroid hormone, and prostaglandin E2.h4-67This effect is reversible and appears to be independent of any significant cytotoxicity. Moreover. the agent has no apparent effect on basal calcium release from the organ cultures or on the endogenous induction of IL-l by other cytokines. However, cyclosporine antagonizes normal cellular HLA-DR expression as well as interferon gamma-stimulated expression by human bone cells, suggesting a possible inhibition of MHC class II-stimulating factors in the cultures68.6y (Skjodt, unpublished). In addition, the stimulation of prostaglandin El production by human bone cells as well as human articular chondrocytes in culture exposed to IL- I is inhibited by cyclosporine.h5.66 However, cyclosporine’s inhibitory effect on the action of bone-resorbing factors is not mediated by inhibition of prostaglandin E, because it is still present after the addition of indomethacin. Interestingly, the nonimmunosuppressive analogue cyclosporine H has no effect in the assays. In contrast. in vivo studies show apparently conflicting results in terms of cyclosporine inhibition of bone resorption and stimulation of bone formation.‘” Induction of marked bone resorption in rats given high doses (> 15 mg/kg) of cyclosporine has been reported.5’,7’.7’ The mechanism responsible for these effects and their potential significance are unclear. At high doses, cyclosporine appears to enhance renal production

20

RUSSELL

of 1,2%dihydroxyvitamin D3 and might be a factor.73,74In other studies the compound has had little effect on bone mass in experimental animals.75 This finding may reflect different dose regimens and stresses the point that in vivo modulation by cyclosporine of a complex cytokine network is not tested in in vitro models. The agent has marked protective effects against the bone loss that occurs in association with adjuvant arthritis in rats, probably because of inhibition of T-cell-driven activation of inflammatory tissue destruction. The effects on human bone have not been evaluated fully. 76-78Assessment is difficult because the underlying disease and other drugs (especially GCs) may influence bone metabolism.79 The demonstration that cyclosporine can prevent the progression of erosive changes in rheumatoid arthritis6 indicates that the agent’s effects on bone may have considerable clinical significance. Cells that are targets of cyclosporine action in bone have not been identified. Although T cells may be present in the bone organ cultures, no markers of immune cells (monocytes, T and B lymphocytes) have been found in human bone cell cultures. Our studies have not shown reproducible inhibition of expression of mRNA for cytokines such as IL-l, IL-6, and GM-CSF by human bone cells or MG-63 osteosarcoma cells in culture, even under conditions in which T-cell production of cytokines is inhibited. Whether cyclosporine modulates stromal bone cells directly and/or acts through immunosuppressive

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Possible sites of action of cyclosporine in

intercellular interactions in bone. Within the local bone environment gen-presenting

cyclosporine may inhibit anti-

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rogation of MHC class II expression. Cyclosporine may also affect the function of osteoclasts by the inhibition of cytokines,

such as tumor necrosis

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sponsible for the activation of resorption, and/or by inhibiting cytokines that are inhibitors of resorption, such as interferon gamma (IFN-7). granulocyte-macrophage (GM-CSF),

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factor

and interleukin 4 (IL-4).

effects in bone remains to be determined. Some of the possible effects of cyclosporine on T cells and bone are summarized in Fig 3. Further studies on the modulation of cytokine production by cyclosporine in bone and in other connective tissue cells are needed.

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MECHANISMS

OF ACTION

21

OF CYCLOSPORINE

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22

binant human interferon gamma: In vitro effects on basal and IL 1 stimulated proteinase production, cartilage degradation and DNA synthesis. Biochim Biophys Acta 1012: 128-l 34, 1989 54. Bunning RAD, Russell RGG: The effect of tumour necrosis factor alpha and interferon gamma on prostaglandin E production and caseinase activity in human articular chondrocytes and the resorption of human articular cartilage. Arthritis Rheum 32:780-784, 1989 55. Van Damme J, Bunning RAD, Conings R, et al: Characterization of granulocyte chemotactic activity from human cytokine-stimulated chondrocytes as interleukin-8. Cytokine 2:106-l II, 1990 56. Bertolini DR, Nedwin GE, Bringman TS, et al: Stimulation of bone resorption and inhibition of bone formation in vitro by human tumour necrosis factor. Nature 319:516518, 1986 57. Evans DB, Bunning RAD, Russell RGG: The effect of recombinant human interleukin 1betaon cellular proliferation and the production of prostaglandin E2 plasminogen activator, osteocalcin and alkaline phosphatase by osteoblast-like cells derived from human bone. Biochem Biophys Res Comm 166: 208-216, 1990 58. Evans DB, Bunning RAD, Russell RGG: The effects of recombinant human granulocyte-macrophage colonystimulating factor (rhGM-CSF) on human osteoblast-like cells. Biochem Biophys Res Comm 160:588-595, 1989 59. Evans DB, Bunning RAD, Van Damme J, et al: Natural human IL I beta exhibits regulatory actions on human bonederived cells in vitro. Biochem Biophys Res Comm 159:12421248, 1989 60. Heath JK, Saklatvala J, Meikle MC, et al: Pig interleukin 1(catabolin) is a potent stimulator of bone resorption in vitro. Calcif Tissue Int 37:95-97, 1985 6 I. Gowen M, Chapman K, Littlewood A, et al: Production of tumor necrosis factor by human osteoblasts is modulated by other cytokines, but not by osteotropic hormones. Endocrinology 126:1250-1255, 1990 62. Ralston SH, Russell RGG, Gowen M: Estrogen inhibits release of tumor necrosis factor from peripheral blood mononuclear cells in postmenopausal women. J Bone Miner Res 5:983-988, 1990 63. Elford PR, Felix R, Cecchini M, et al: Murine osteoblast-like cells and the osteogenic cell MC3T3-El release a macrophage colony-stimulating activity in culture. Calcif Tissue Int 41:151-157, 1986 64. Klaushofer K, Hoffmann 0, Stewart PJ, et al: Cyclosporin A inhibits bone resorption in cultured neonatal mouse calvaria. J Pharmacol Exp Ther 243:584-590, 1987 65. Skjodt H, Beresford JN, Wood DD, et al: Interleukin 1 and cyclosporin A modulate actions of l,25-dihydroxyvi-

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ET AL

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