Use of immunosuppressive agents in ocular diseases

0278-4327(93)E0004-N CHAPTER 3

Use of Immunosuppressive Agents in Ocular Diseases M A N A B U M O C H I Z U K I *~

and M A R C DE S M E T t

*Department o f Ophthalmology, Kurume University School o f Medicine, Kurume Japan +National Eye Institute, National Institutes of Health, MD, USA CONTENTS 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

479

2. Mode of Action of Conventional Immunosuppressive Agents and Immunophilin Ligands . . . . 2.1. Corticosteroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Cytotoxic Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Alkylating agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Antimetabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Immunophilin Ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

480 480 483 484 484 484

3. Immunophilin Ligands in Uveitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Use of Immunophilin Ligands in Experimental Uveitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Clinical Use o f Immunophilin Ligands in Refractory Uveitis . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. Cyclosporine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2. FK506 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

486 486 487 490 490 491

4. Immunophilin Ligands in Corneal Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Use of Immunophilin Ligands in Experimental Corneal Transplantation . . . . . . . . . . . . . . 4.3. Clinical Use of Immunophilin Ligands in Corneal Transplantation . . . . . . . . . . . . . . . . . . .

492 492 493 497

5. Immunophilin Ligands in Allergic Conjunctivitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Use of Immunophilin Ligands in Experimental Allergic Conjunctivitis . . . . . . . . . . . . . . . . 5.3. Clinical Use of Immunophilin Ligands in Allergic Conjunctivitis in H u m a n . . . . . . . . . . . .

498 498 499 500

6. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

500

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

501

1. I N T R O D U C T I O N

homeostasis and health, although it may also cause pathologic reactions to the host in response to exogenous or endogenous pathogenic agents. This pathologic reaction is classically known as the immune hypersensitivity reaction. It consists of four types of hypersensitivity reaction: (1) type

Recent advances in immunology and molecular biology have provided us with new insights into the pathogenesis o f a variety of diseases. The immune system plays a central role in maintaining

*Corresponding author: Department of Ophthalmology, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830, Japan.

Progress in Retinal and Eye Research Vol. 13 No. 2 Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved.

479

480

M. MOCHIZUKIand M. DE SMET TABLE 1. Anti-inflammatory and Immunosuppressive Effects ol" Corticosteroids Anti-inflammatory effects: Stabilization of the vascular bed Decreased expression of adhesion molecules Decreased granulocyte and monocyte accumulation in inflammatory loci Impairment of granulocytes and monocyte activation Immunosuppressive effects: Decrease in circulating lymphocytes and monocytes Decrease in lymphocyte and monocyte activation Decrease in immunoglobulin and complement levels

I reaction mediated by antibodies, especially IgE, and mast cells; (2) type II reaction mediated by cytotoxic antibodies; (3) type III reaction mediated by the immune complex consisting of antigen, antibody and complement; and (4) type IV reaction mediated by T-cells and lymphokines. The type IV reaction is termed a cell-mediated immune reaction, whereas the other three hypersensitivity reactions are humoral. In the eye, immune-mediated hypersensitivity reactions play essential roles in the pathogenesis of a number of ocular disorders such as uveitis, allograft rejection in corneal transplantation, allergic conjunctivitis, and the like. Immunomodulation to affect or manipulate the immune system at its critical points causes a change in the expression of disease and can provide effective strategies for the therapy of immune-mediated ocular diseases. Strategies for immunomodulation studied in experimental models for uveitis include the use of immunosuppressive agents (Nussenblatt et al., 1981; Mochizuki et al., 1985; Kawashima et al., 1988; Roberge et al., 1993). Therapy with anti-Ia antibody which interferes with the level of the antigen-presenting cells and thereby the initial activation of T-cells and the recruitment of autoaggressive cells into the eye (Wetzig et al., 1988) has also been used. A fusion molecule of interleukin-2 (IL-2) and toxin (PE40) which affects activated lymphocytes expressing IL-2 receptors (Roberge et al., 1989) has proved to be effective as well as T-cell vaccination using a low dose of T-cell line specific to retinal soluble antigen (S-antigen) (Beraud et al., 1992); and induction of tolerance to S-antigen induced-EAU by oral administration of S-antigen (Nussenblatt

et al., 1990) has been shown. Among these strategies, approaches to immunomodulation that already have moved on to the clinical arena are the use of immunosuppressive agents (Nussenblatt et al., 1983; Mochizuki et al., 1991, 1993) and oral induction of tolerance with S-antigen (Robert Nussenblatt, written communication, 1992). This paper reviews the basic and clinical studies of the immunological activity of immunosuppressive agents with special attention to a new generation of immunosuppressants, the immunophilin ligands, which inhibit specific pathways, of signal transduction that lead to T-cell activation and thereby selectively suppress T-cell-mediated immune responses.

2. MODE OF ACTION OF CONVENTIONAL IMMUNOSUPPRESSIVE AGENTS AND IMMUNOPHILIN LIGANDS 2.1. Corticosteroids

Corticosteroids are clearly the most widely used agents in the treatment of inflammatory eye disease. Despite their widespread use and a substantial amount of research regarding their mode of action, their precise mechanisms of action still remains a mystery. Corticosteroids have effects on many stages of the immune process, and at times, these effects appear antagonistic. In its simplest form, the effect of corticosteroids can be divided into an antiinflammatory effect and an immunosuppressive effect (see Table 1). At a cellular level, these

IMMUNOSUPPRESSANTS IN OCULAR DISEASES

481

TABLE2. Glucocorticoid Effects on Leukocyte Movement in Humans Lymphocytes: Circulating lymphopenia 4 - 6 hours after administration of glucocorticoid Depletion of recirculating lymphocytes Selective depletion of T-lymphocytes more than B-lymphocytes

Monocytes-Macrophages: Monocytopenia for 4 - 6 hours as a result of redistribution Inhibition of the accumulation of cells at the site of inflammation

Neutrophils: Circulating neutrophilia Accelerated release of PMN from the bone marrow Decrease accumulation of PMN at the site of inflammation

Eosinophiis: Circulating eosinopenia secondary to redistribution Decreased migration of eosinophils into skin test sites

effects can be divided into effects on cell movement and on cell functional capacity (Parrillo et al., 1979). In addition, endogenous corticosteroids and, in particular, the response of the pituitary-adrenal axis to 'immunologic stress' appears to play a critical role in the induction of disease. Most of the information presented here is derived from animal experiments. Care must be taken in extrapolating experimental data to humans. For example, steroids are cytolytic in many animal species including the rat, mouse and rabbit (Claman, 1972), but this is generally not the case in humans, except when the cells are malignant. Also, many effects have been described as a result of in vitro experiments using supraphysiologic steroid doses, or doses that can only be maintained in humans for a short time (Fauci et al., 1976). Despite these limitations, a generalized picture of the action of steroids has evolved. Its understanding can help to place the role of corticotherapy in perspective both as a sole therapeutic agent and as an adjunct with other more selective immunosuppressants such as cyclosporine and FKS06. Corticosteroid effects on cell movement: Perhaps the major mechanism whereby corticosteroids exert their anti-inflammatory effect in man is by preventing the accumulation of lymphocytes at the site of inflammation (Selye, 1953). This phenomenon is in part dependent on an altered expression of adhesion molecules by both the leukocytes themselves and the underlying

endothelial cells. On their cell surfaces, leukocytes express adhesion molecules which mediate their specific localization to sites of inflammation (CD1 la-c/CD18, L-selectin, LECAM-I) (Smith et al., 1991; Larson et al., 1990). In addition, endothelial cells express complementary adhesion molecules which help to regulate the traffic of leukocytes into inflamed and infected areas [GMP140, endothelial-leukocyte adhesion molecule 1 (ELAM-1)], intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule (VCAM)) (Springer, 1990; Geng etal., 1990; Walz et ai., 1990). Glucocorticoids are able to inhibit expression of ICAM-1 and ELAM-1 by endothelial cells, and can achieve this effect at nanomolar concentrations (Cronstein et al., 1992). In addition to preventing the influx of leukocytes at the inflammatory site, corticosteroids cause a redistribution of all leukocyte classes by affecting both their intravascular and their extravascular distribution. At reasonable pharmacological doses, corticosteroids cause the release into the circulation of young neutrophils from the bone marrow (Dale et al., 1975), and increase the half-life of PMNs already in circulation (Parrillo and Fauci, 1979; Athens et ai., 1961) (Table 2). In contrast, corticosteroids lead to a transient eosinopenia and lymphopenia. Lyrnphopenia is maximal at four to six hours after oral or parenteral administration of the drug, with a return to normal counts at 24 hours. This effect is not cumulative and is present even in patients

482

M. MOCHIZUKI and M. DE SMEF TABLE 3. Glucosteroid Effects on Leukocyte Function in Humans

Lymphocytes: Inhibits production of IL-2, IFN-y Shift of response from Thl to Th2 Decreased activity of NK cells Induction of apoptosis in immature T- and B-cells, and in activated cells

Macrophages: Inhibits antigen processing and presentation to T-cells (down regulation of Class 11 expression) Inhibits cytotoxic functions Inhibits the synthesis of cytokines such as IL-I, TNF-a, IL-6

receiving steroids for years (Fauci et al., 1976). Maximal lymphopenia appears to be reached at doses in the range of 6 0 - 80 mg of prednisone per day, with little further decrease in the lymphocyte count at higher doses. It is noteworthy that at these doses, there is little suppression in the functional capabilities of the lymphocytes left in circulation. Thus, one of the major effects of corticosteroids on lymphoid cells in doses commonly used in uveitis is not functional suppression, but a quantitative depletion of lymphocytes from the circulation, making them less readily available at the inflammatory site. This may explain why the combination of steroids and agents such as cyclosporine are so effective when given simultaneously, since the former prevents leukocytes from reaching the site of inflammation, and the latter prevents further activation and recruitment at that site. Suppression of function by steroids only occurs at higher doses and will be discussed in the next section. Lymphocyte depletion occurs as a result of the redistribution of cells out of the intravascular space to the extravascular lymphocyte pool, composed mainly of lymphatics, the spleen and the bone marrow (the reticuloendothelial system). The flow of cells, in particular T-cells, between the vascular space and the reticuloendothelial system is a normal physiological mechanism that allows for immune surveillance (Stoolman, 1989). In fact, T-cells and in particular helper/inducer T-cells are depleted to a greater extent from the circulation than are other types of lymphocytes (Parrillo and Fauci, 1979). The.exact mechanisms underlying this redistribution have not yet been well studied. What is known is that redistribution even among helper T-cells is not uniform.

Different states of activation lead to a greater or lesser degree of margination (Schlesinger and Israel, 1974). Binding interactions between lymphocytes and endothelial cells are dependent on the state of activation of T-cells. Non-activated T-cells use LFA-1/ICAM-1 independent pathways, while activated T-cells are more dependent on LFA-1 and ICAM-1 (Tamatani et al., 1991). Steroids can inhibit the expression of ICAM-1 by endothelial cells (Cronstein et al., 1992); thus, preventing the redistribution of certain types of T-cells. Similarly, steroids may promote the margination of lymphocytes through the activation of yet undefined adhesion pathways. Corticosteroid effects on cell function: It is possible to interfere with virtually every functional capability of the different classes of leukocytes by using sufficiently high in vitro concentrations of corticosteroids (Table 3). However, the relevance of these effects at physiological doses in humans remains questionable. In monocytes and macrophages, however, the effect is substantial. Migration of monocytes to the inflammatory site, cytotoxic (antimicrobial) functions, antigen presentation and synthesis of pro-inflammatory cytokines are all inhibited by physiological doses of steroids (Waage et al., 1990; Zembowicz and Vane, 1992). The high sensitivity of monocytes to the effect of corticosteroids is well demonstrated in vivo by the dramatic response sometimes observed in diseases such as sarcoidosis, or in the reactivation of granulomatous diseases such as tuberculosis. Macrophages are closely linked to the activation of T-lymphocytes (Unanue and Cerottini, 1989; Harding et al., 1988), and much of the inhibition of lymphocyte function attributed to steroids may be mediated by the action of

IMMUNOSUPPRESSANTS IN OCULAR DISEASES

macrophages on the lymphocyte. However, at high enough doses, corticosteroids have the potential to directly suppress the functional capability of lymphocytes. They can inhibit the synthesis of several lymphokines including IL-2 and IFN-y (Vacca et al., 1992), decrease the activity of NK cells (Parrillo and Fauci, 1978), and induce apoptosis in immature T- and B-cell precursors (Cohen et al., 1992) as well as in some mature T-cells populations (Zubiaga et al., 1992; Gonzalo et al., 1993). This latter capability, the induction of cell death or apoptosis, may be one of the major mechanisms through which steroids exercise their effect on lymphocytes. Resting mature T-lymphocytes appear to be resistant to lysis, but activated lymphocytes such as those that are present in immunologically-mediated disease are more susceptible. The elimination of specific subsets of lymphocytes has been demonstrated in some animal models and may also be operative in humans (Gonzalo et al., 1993). Role o f endogenous glucocorticoM production: Basal and stress-induced levels of corticosteroid appear to play an important role in the susceptibility to autoimmune disease. This association has been proposed in several experimental models to explain the difference in susceptibility to autoimmune diseases between different rat strains (Villas et al., 1991; Sternberg et al., 1989; Mason et al., 1990). This difference is particularly striking in the Lewis and Fisher rats. These two strains share major histocompatibility antigens including class II, and thus should have a similar sensibility to the induction of disease. However, both in experimental autoimmune encephalitis (EAE) and in EAU, the Fisher rat is fairly resistant while the Lewis rat is highly susceptible to disease induction (de Smet et al., 1993). This difference in susceptibility has been linked in the Lewis rat to a defect at the level of the hypothalamic - pituitary- adrenal (HPA) axis (Sternberg et al., 1989). Corticosterone is normally released early in the course of an inflammatory episode, in part through the stimulation of the HPA by IL-I (Del Rey et al., 1987). Response to IL-1 and other HPA stimuli are depressed in the Lewis rat, thus preventing the normal physiological release of glucocorticoids and the induction of the anti-inflammatory

483

mechanisms described above. Levels of endogenous corticosteroids are also important for spontaneous recovery from EAE, and is dependent on the importance of the HPA axis in patients with uveitis. However, it is not uncommon to find patients who have been on steroids for months that develop a flare-up upon tapering the steroid dose below a critical level, often around 10- 15 mg of prednisone per day.

2.2. Cytotoxic Agents

Cytotoxic agents differ from the other immunosuppressive agents discussed in this review in that their immunosuppressive ability is directly linked to their ability to destroy or damage cells. In neoplasms, there is often an increased susceptibility to these agents as compared to normal cells based on a higher mitotic rate or aberrant metabolism. In the case of normal immunocompetent cells, the therapeutic window between immunosuppression and toxicity is often much more limited. The effect of cytotoxic agents is greatest in rapidly dividing cells; this is the basis of their action in inflammatory disease, but this action is not limited only to activated immune cells but will also affect precursor cells and other cells with a rapid turnover. Among leukocytes, neutrophils have a high turnover rate, and they are often the first to drop in number, making the patients more susceptible to infection. Another important consideration with these agents is the fact that they are often administered chronically over an extended period of time. Since their effect on the immune system is non-specific, they will always have some effect on the normal host defense and immune surveillance mechanisms, placing patients at risk of infection and also of malignancy. The latter can occur even years after termination of therapy. Nevertheless, these agents have an important role to play in the therapy of uveitis. Cytotoxic drugs have the potential of eliminating sensitized and activated lymphoid cells. They can eliminate lymphoid cells that have been secondarily recruited into an aberrant inflammatory site, and they can suppress the functional capabilities of surviving cells. Because of their potency

484

M. MOCHIZUKI and M. DE SMET

though, it is very important to set reasonable goals for the use of these cytotoxic drugs in the treatment of uveitis (Nussenblatt and Palestine, 1989).

2.2.1. ALKYLATINGAGENTS Alkylating agents have profound effects on the immune system. They work by inserting alkyl radicals into other molecules. Biologically effective alkylating agents are usually polyfunctional, allowing for the covalent linking of different sites. In DNA, this link is formed at the level of the 7-nitrogen position in guanine. By cross linking cellular DNA, replication is prevented; the cell cannot properly divide, ultimately leading to cell death (Roberts et al., 1971). Of the existing alkylating agents, cyclophosphamide is the best studied, and the most potent agent. Another agent commonly used is chlorambucil, but it is generally felt to be less potent than cyclophosphamide with regard to its immunosuppressive effects. Cyclophosphamide acts primarily during S phase of the cell cycle and has a profound effect on dividing cells, but it has an effect on cells in all phases of the cell cycle. With chronic administration in humans, the most consistent finding is lymphocytepenia of both T- and B-lymphocytes but there is a more profound effect on B-cells (Hurd and Giuliano, 1975). There is suppression of antibody responses and cutaneous hypersensitivity responses to new antigens with a relative sparing of the response to established cutaneous-delayed hypersensitivity. Higher doses are thought to acutely affect B-cell function, but when given at a moderate dosage and through chronic administration, they also affect T-cell function. Paradoxically, in animals, low dose administration results in a loss of suppressor cells; this has not been noted in humans.

2.2.2. ANTIMETABOLITES Grouped under this heading, one usually finds the purine analogues and the folic acid

antagonists. Commonly used purine analogues include 6-mercaptopurine (6MP) and azathioprine (a pro-drug of 6MP). These agents work by the incorporation of the analogue into cellular DNA with subsequent inhibition of nucleic acid synthesis. They can also alter the synthesis and function of RNA. The most commonly used folate antagonist is methotrexate. Methotrexate is able to bind with very high affinity to dihydrofolate reductase, preventing the formation of tetraphydrofolate. This causes an acute deficiency in folate coenzyme needed in the de n o v o synthesis of purine nucleotides (Segal et al., 1990). Azathioprine and 6MP inhibit both cellmediated and humoral immunity. They cause a total lymphocytopenia of both T- and B-cells but appear to have a greater effect on inhibiting B-cell proliferation (Yu et al., 1974). Delayed-type hypersensitivity to established antigens is not inhibited, but the induction of a response to new antigens is suppressed. Azathioprine also inhibits monocyte production; thus the number of monocytes that can be recruited into an inflammatory site (Gassman and van Furth, 1975; Phillips and Zweiman, 1973). Overall though, the activity is less than that of the alkylating agents and is quite variable in patients with uveitis, but these agents have fewer side effects. Methotrexate appears to only have a marginal effect on humoral and cellular immune responses. Its activity, particularly at the lower doses that are currently being proposed, appears to be directed mainly at neutrophils and macrophages (Segal et al., 1990). It appears to be able to reduce chemotaxis and accumulation of neutrophils at the site of inflammation and decrease IL-1 production and HLA class II expression by macrophages (Hu et al., 1988). This effect on cytokine production and chemotaxis points to a greater effect as an anti-inflammatory agent rather than as an immunosuppressant. This is probably its major mode of action in inflammatory disease.

2.3. lmmunophilin Ligands Cyclosporine, FK506, and rapamycin are inhibitors of specific signal transduction pathways

IMMUNOSUPPRESSANTSIN OCULAR DISEASES Me Me ~ N

Immunophlllnligands

".

,

485 ~

--

Me Me Me ~ N N

~ .x..,

o

Me N 0

~=o

Me Me Me~lde

Cyclosporine MeO

O

....

IL-2

~

MeO Me T OH1Me ~t~ H/"~. IMe ~ ~M e O

T lymphocyte (C04 +)

F1G. 1. Mode of action of immunophilin ligands (cyclosporine and FKS06) in the T-cell receptor-mediated signal transduction pathway (Mochizuki, M., Acta Soc. Ophthalmol. Jap. 96: 1608- 1634 (1992) published with permission).

that lead to T-cell activation. This group of immunosuppressive agents bind with high affinity to cytoplasmic receptors termed 'immunophilin', immunosuppressant binding proteins, and therefore are now termed immunophilin ligands (Bierer et al., 1990a; Schreiber, 1991). Figure 1 illustrates the mode of action of cyclosporine and FK506 on the T-cell receptor-mediated signal transduction pathway. The importance and significance of immunophilin ligands in medical science is as follows: (1) since immunophilin ligands act on very specific pathways of the immune system, the agents can be used as a tool to dissect the immune system and biological processes of a disease; (2) their profound suppressive effects on T-cellmediated immune responses are beneficial in the therapy for a large spectrum of human disorders such as allograft rejection in organ transplantations and autoimmune diseases. Cyclosporine (sandimmune) is a natural product of several fungi such as Tricoderma polysporum rifai and Cylindrocarpon lucidom booth. The compound is a cyclic endecapeptide with a molecular weight of 1202 (Fig. 2). It was reported that cyclosporine had a powerful ability to suppress lymphocytes, as demonstrated by the suppression of the appearance of plaque-forming cells, delayed skin graft rejection and graft-versushost disease in mice, and prevention of

~

FK506

M~. O~H." 1

H'" O

OMe

OMe Me~. Me OMeMe Me Rapamycin

FIG. 2. Structure of immunophilin ligands.

experimental allergic encephalitis (Borel et al., 1976). The immunosuppressive activity of cyclosporine is far more restricted to the immune system than any of the conventional immunosuppressive agents, corticosteroids and cytotoxic agents. Namely, cyclosporine affects the factors that promote T-cell activation and recruitment. Recent studies on the mode of action of cyclosporine have clarified the cellular and molecular level of its effects on the immune system. After cyclosporine enters the cells, it binds to a specific cytosolic binding protein, cyclophilin (Handschumacher et al., 1984; Harding and Handschumacher, 1988). Later studies have shown that cyclophilin is identical to peptidylprolyl isomerase, an enzyme that catalyses the cistrans isomerization of proline residues in proteins and peptides (Takahashi et aL, 1989). The cyclophilin-cyclosporine complex inhibits a T-cell receptor-mediated signal transduction pathway which results in the transcription of early T-cell activation genes such as interleukin-2 (Reem et al., 1983; Elliott et al., 1984; Kronke et al., 1984; Gunter et al., 1989). Accordingly, cyclosporine has intense effects on T-cell-mediated immune responses. FK506 (Fig. 2) is an antibiotic of the macrolide family with a molecular weight of 822 isolated from the fermentation broth of Streptomyces tsukubaensis (Kino et al., 1987a). FKS06 inhibits T-cell activation by mechanisms that are similar to those of cyclosporine, but FK506 is 1 0 - 100 times as potent (Kino et ai., 1987b). Like cyclosporine,

486

M. MOCH1ZUKI and M. DE SME'I

FK506 has a specific cytosolic binding protein, FK506 binding protein (FKBP) (Siekierka et al., 1989a, 1989b; Bierer et al., 1990b; Schreiber, 1991). FKBP is distinct from cyclophilin, though both have peptidyl-prolyl isomerase activity. Like the cyclophilin-cyclosporine complex, the complex of FKBP and FK506 inhibits the expression of the early phase of T-cell activation genes. The mRNAs affected by FK506 included IL-2, 3 and 4, gamma interferon, alpha tumor necrotizing factor and granulocyte-macrophage colony stimulating factor. This results in the inhibition of the production of IL-2 and other lymphokines; thus, the suppression of T-cell-mediated immune responses. Rapamycin is an anti-fungal antibiotic with a molecular weight of 914 which was isolated from the fermentation broth of Streptomyces hydroscopicus more than a decade ago (Vezina et al., 1975; Sehgal et al., 1975). After the recognition of the unique activity of FK506 on the immune system, it was suggested that rapamycin might have immunosuppressive activities similar to FK506 for the following two reasons: (1) rapamycin has a structural similarity to FK506 as shown in Fig. 2; and (2) more interestingly, the cytosolic binding protein of FKS06, FKBP, was shown to be the predominant rapamycin binding protein with an even higher affinity of rapamycin to FKBP than that of FKS06 (Bierer et al., 1990a). Studies on the immunosuppressive properties of rapamycin showed that it does, in fact, suppress T-cell activation, but by a pathway distinct from that of FK506 (see a review by Schreiber, 1991). Whereas rapamycin has no effect on the production of IL-2, the agent strongly inhibits the response of T-cells to IL-2. Accordingly, rapamycin inhibits the late lymphokine receptor-associated signal pathway. In contrast, FKS06 and cyclosporine are potent inhibitors of the T-cell receptor-mediated signal transduction pathway, as evidenced by their ability to inhibit the transcription of early T-cell activation genes for IL-2 and the like. 3. IMMUNOPHILIN LIGANDS IN UVEITIS 3.1. Background Uveitis is a sight-threatening inflammatory disorder affecting the iris or ciliary body (anterior

uveitis), the posterior segment of the eye (posterior uveitis), the vitreous and pars plana (intermediate uveitis) or the whole globe (panuveits). It is classified as exogenous when it is caused directly by infectious agents (toxoplasmosis, cytomegalovirus, syphilis, tuberculosis, etc.) or endogenous when it occurs without apparent inciting pathogens. The pathogenesis of endogenous uveitis is still controversial, but accumulating data indicate that endogenous uveitis is immune mediated. Examples of classical immune-mediated uveitis are sympathetic ophthalmia and Vogt - Koyanagi - Harada's disease, which are considered to be autoimmune diseases against melanocyte antigen. Pathologically, the eyes with endogenous uveitis are infiltrated by a number of inflammatory cells. Immunopathological examinations disclosed that the intraocular tissues with endogenous uveitis were infiltrated by CD4 T-cells, CD8 T-cells and macrophages. In an early stage of sympathetic ophthalmia, CD4 positive cells expressing IL2 receptors or MHC class II antigens were predominant, suggesting that activated T-cells and immune-mediated mechanisms play a significant role in the disease processes (Chart et al., 1986). A similar disorder to endogenous uveitis in humans can be induced in experimental animals by immunization with autoantigens isolated from the retina, i.e. retinal soluble antigen (S-antigen) (Wacker et al., 1977) and interphotoreceptor retinoid-binding protein (IRBP) (Gery el al., 1986b). The disease, experimental autoimmune uveoretinitis (EAU), is considered to be an animal model for endogenous uveitis in humans and is essentially a T-cellmediated autoimmune disease of the eye (see a review by Gery et al., 1986a). Interestingly, a significant number of patients with endogenous uveitis (Behqet's disease, V o g t - K o y a n a g i Harada's disease, ocular sarcoidosis, and birdshot retinochoroiditis) exhibit cellular responses to these retinal autoantigens (Nussenblatt et al., 1980 and 1982; Hirose et al., 1988; de Smet et al., 1990). This suggests that lymphocytes sensitized to the retinal autoantigens play a role in the pathogenesis of endogenous uveitis. These clinical and experimental data thus indicate that the immune-mediated mechanisms play an essential role in the pathogenesis of endogenous uveitis.

487

IMMUNOSUPPRESSANTSIN OCULARDISEASES Accordingly, immunomanipulation, particularly by immunosuppressive agents, is considered to be the major strategy for the therapy of endogenous uveitis. Much effort have been made to establish an efficient immunotherapy with agents having profound immunosuppressive activities and minimal toxicity. The first success of such effort was achieved with the use of cyclosporine. In 1981, Nussenblatt and his colleagues (Nussenblatt et al., 1981) first demonstrated the profound activity of cyclosporine to inhibit S-antigeninduced uveitis in Lewis rats. Based on these animal studies, they treated patients with refractory uveitis with cyclosporine and obtained satisfactory therapeutic results (Nussenblatt et al., 1983). More recently, other immunophilin ligands, FK506 (Kawashima et al., 1988; Kawashima and Mochizuki, 1990; Fujino et aL, 1991) and rapamycin (Rogerge et aL, 1993), were studied for their efficacy in uveitis using the same experimental model. In the next section, the immunopharmacological properties of these immunophilin ligands in uveitis are discussed based on our recent studies.

~

78

I

We have studied the efficacy of a variety of immunosuppressive agents having different modes of action using S-antigen-induced experimental autoimmune uveoretinitis (EAU) in the rat (Mochizuki et al., 1983, 1984; Fujino eta/., 1988; Kawashima et al., 1988, 1990; Mochizuki and Kawashima, 1990; Mochizuki, 1992). Immunosuppressive agents used in our study included (1) dexamethasone, a corticosteroid, (2) cyclophosphamide, an alkylating agent of cytotoxic agents, (3) mizoribine, an imidazole nucleotide isolated from Eupenicilium brefeldianum, which is an antimetabolite of cytotoxic agents and is effective in suppressing allograft rejection in renal transplantations (Uchida et al., 1979; Inou et al., 1980), (4) 15-deoxyspergualin, a chemical derivative of spergualin isolated from Bacillus laterosporus that has an intense anti-tumor activity (Ochiai et al., 1987; Hori et al., 1987), (5) cyclosporine, FK506 and rapamycin (immunophilin ligands). The Lewis rats were immunized

~='~IZOR

\

\

"6 50 ==3o-

\

\\

0.1

\

\

\ \\ \

\\\

0.01 0.03

\1\

0.3

1

/

3

10

30

100

D o s e ( mg/Kg/day )

FIG. 3. Effects of systemic immunosuppressive agents on EAU in the rat (days 0 - 1 4 postimmunization) DEX: dexamethasone, RAPA: rapamycin, CY: cyclophosphamide, CYA: cyclosporine, 15DSG: 15-deoxyspergualin, MIZOR: mizoribine (Mochizuki, M., Acta. Soc. Ophthalmol. Jap. 96: 1608- 1634 (1992) published with permission).

Therapy 100[- Drug ......................

80!

~" 3.2. Use of lmmunophilin Ligands in Experimental Uveitis

CY 15DSG

108,

l mizoribine4

"o¢O m~°40 60/

~.,~ /

ir

! ~i5DSG _ ooooIIICY ........... oo

20

,-,........ l b t ~

........t~-'.....

0

3

1

2

4

5

WeekafterImmunization

6

.

FK506

FIG. 4. Development of EAU after the cessation of systemic immunosuppressive agents. The systemic therapy with immunosuppressants was discontinued 2 weeks after S-antigen immunization and followed up without treatment to study if the animals developed EAU. DEX: dexamethasone, 15DSG: 15-deoxyspergualin, CY: cyclophosphamide, CYA: cyclosporine (Mochizuki, M., Acta Soc. Ophthalmol. Jap. 9 6 : 1 6 0 8 - 1 6 3 4 (1992) published with permission).

with S-antigen in complete Freund's adjuvant and were treated with systemic administration of the immunosuppressive agents from the day of immunization, from day 7 postimmunization, or after the onset of EAU. When given from the day of immunization, the tested immunosuppressive agents inhibited EAU

488

M. MOCHIZUKI and M. DE SMFr

TABLE 4 . EDso o f Various Immunosuppressants on EAU

lmmunosuppressant

ED~0~mol/Kg/day)

Dexamethasone (corticosteroid) FK506 (immunophilin ligand) Rapamycin (immunophilin ligand) Cyclosporine (immunophilin ligand) Cyclophosphamide(cytotoxic agent) 15-deoxyspergualin(cytotoxic agent) Mizoribine (cytotoxicagent)

0.2 0.2 0.2 2.7 3.8 11.3 57.9

development in a dose-dependent manner (Fig. 3). The immunosuppressive activities of these agents were checked for the following: the EDs0, the day of EAU onset after the discontinuation of drug administration, the immune response to S-antigen in drug-treated animals, and the immune cells infiltrating in the ocular tissues. Based on the EDs0 (~mol/Kg/day) of each agent, dexamethasone, FK506 and rapamycin are most effective in suppressing E A U induction; 1 0 - 2 0 times more intense than cyclosporine and cyclophosphamide, and 7 0 - 3 0 0 times more intense than 15-deoxyspergualin and mizoribine (Table 4). Cyclosporine inhibited the lymphocyte proliferative response to S-antigen, but not the antibody production specific to the antigen. On the other hand, dexamethasone and cytotoxic agents suppressed both the lymphocyte response and the antibody to S-antigen. These data thus indicate that immunophilin ligands inhibit EAU development by selective suppression to T-cell-mediated cellular immune response to S-antigen, while corticoster-

oids and cytotoxic agents inhibit EAU by nonspecific immunosuppressive effects. Animals treated with dexamethasone or cytotoxic agents developed EAU in 1 - 2 weeks after the cessation of the therapy. In contrast, animals treated with immunophilin ligands did not develop EAU long after the drug was discontinued. This is even more impressive since the drug concentration in the blood became undetectable by one week after drug cessation (Fig. 4). It, therefore, seemed probable that immunophilin ligands might induce immunological tolerance. This hypothesis was further examined as summarized in Table 5. Rats immunized with S-antigen and treated with cyclosporine ( 1 0 m g / k g / d a y ) or FK506 (1 m g / k g / d a y ) on days 0 - 1 4 postimmunization did not develop EAU even after a secondary immunization with S-antigen on day 30 (Groups D and G in Table 5). Similarly treated rats were highly susceptible to another autoimmune disease, experimental allergic encephalitis (EAE), after the secondary immunization with myelin basic protein (MBP) (Groups E and H in Table 5). These data indicate that treatment with immunophilin ligands initiated at the time of primary immunization induces immunological unresponsiveness specific to the primary antigen. On the other hand, cyclophosphamide did not cause immunological unresponsiveness (Group I). The possible involvement of suppressor cells in the immunological unresponsiveness was indicated by experiments both in vivo and in vitro. The enriched fraction of CD8 positive cells (suppressor cells), but not of CD4 positive cells, from rats unresponsive to

TABLE 5. Induction o f Immunological Tolerance by Immunophilin Ligands* Group

Primary immunization (day 0)

Drug treatment (mg/Kg/day) (days 0 - 14)

A

--

--

B

--

--

C D E F G H I

-S-antigen S-antigen -S-antigen S-antigen S-antigen

cyclosporine (10) cyclosporine (10) cyclosporine (10) FK506 (1) FK506 (1) FK506 (1) cyclophosphamide (10)

Secondary immunization (day 30) S-antigen MBP S-antigen S-antigen MBP S-antigen S-antigen MBP S-antigen

*Fujino et al. Clin. Immunol. lmmunopathol. ,~i: 234-248 (1988). Kawashima et al. Invest. Ophthalmol. Vis. Sei. 29: 1265-1271 (1988).

Disease following the secondary immunization EAU rats/total 15/15 6/6 9/29 10/10 0/10 7/7

EAE rats/total 5/:5 6/6 5/5

IMMUNOSUPPRESSANTS IN OCULAR DISEASES

Percent Suppression (%) Respondercells Additionalcells censltlzedIo (CDS) treatedwith 0 20 40 60 80 100 I

S-antigen

PBS

~-

CYA (10)

I

FKS06 (1)

I

I

I

I

I

Drug (mg/kg/dey) CYA Bucillamine o I

**

** P < 0.01

IRBP

PBS

CYA (10) FK506 {1)

Incidence of EAU ( % ) 2O

40

60

80

lOO

|

I

I

I

I

I

2 I

489

[3[3" [3-

FIG. 5. Antigen specific suppressor activity by CD8 positive T-lymphocytes from rats treated with immunophilin ligands. The CD8 positive T-lymphocytes were fractionated from rats which were immunized with S-antigen and treated with immunophilin ligands (cyclosporine or FK506) on days 0 - 1 4 postimmunization. The CD8 positive T-lymphocytes suppressed the proliferative response of S-antigensensitized lymphocytes to the antigen, but had no effect on the lymphocyte response to IRBP. (Mochizuki, M., Acta Soc. Ophthalmol. Jap. 9 6 : 1 6 0 8 - 1 6 3 4 (1992) published with permission).

induction of S-antigen-induced EAU after cyclosporine treatment or FK506 treatment, were found to inhibit the specific mitotic response of lymphocytes to S-antigen; they had no effect, however, on the lymphocyte response to another retinal antigen, IRBP (Fig. 5). In vivo, injection of such an enriched suppressor cell fraction into naive syngenic rats inhibited or delayed the development of EAU following immunization with S-antigen (Kawashima et al., 1990). Therefore, it is suggested that specific unresponsiveness plays a role in the suppression of EAU by cyclosporine or FK506 and that the unresponsiveness is mediated in part by specific suppressor cells. Immunophilin ligands are effective not only on the afferent limb of the immune response, but also on the efferent limb. Treating animals with a high dosage of cyclosporine (Mochizuki et al., 1983) or FK506 (Kawashima and Mochizuki, 1990) from day 7 after S-antigen immunization was found to be effective in inhibiting EAU induction. Furthermore, FK506 was effective in reducing the intensity of EAU even when it was given only after the onset of EAU. Adoptive transfer of EAU by S-antigen-sensitized lymphocytes was also suppressed by treating only recipient animals with

•"--

--

I

20

[

200

I

2

20

2

200

I 7-q

I I I

FIG. 6. Effects of combined therapy (cyclosporine and bucillamine) on EAU in the rat (Mochizuki, M., Acta Soc. Ophthalmol. Jap. 96:1608 - 1634 (1992) published with permission).

cyclosporine (Mochizuki et ai., 1983), FK506 (Kawashima and Mochizuki, 1990), or rapamycin (Roberge et al., 1993). These data thus indicate that immunophilin ligands are effective in suppressing ongoing processes of the immune response and inflammation. Histopathological examination of eyes with EAU without treatment disclosed a marked lymphocytic infiltration in the uveal tract, retina, vitreous, subretinal space and anterior chamber. The identification of inflammatory cells in EAU eyes and drug-treated rats were intensively investigated by Chan and her colleagues (Chan et al., 1985a, 1985b, 1987; Ni et ai., 1990). The majority of inflammatory cells bore T-cell markers, particularly CD4 (helper/inducer T-cell marker) in the early stage and CD8 (suppressor/ cytotoxic T-cell marker) in the late stage of the disease. These cells stained positively for MHC class II antigens and a proportion (10-20070) of the cells expressed IL-2 receptors. Expression of MHC class II antigens was also identified on some ocular resident cells (retinal vascular endothelium, retinal pigment epithelium, corneal keratocytes, and ciliary epithelium). Suboptimal dosages of dexamethasone, 15-deoxyspergualin, cyclosporine and FK506 delayed the cellular kinetics during the course of EAU. Among these immunosuppressants, FK506 had the greatest effects on the kinetics of T-cell subsets by causing the greatest increase in the recruitment time of the CD8 positive T-cells. Expression of IL-2 receptors on T-cells and expression of MHC class II antigens

490

M. MOCH1ZUKI and M. DE SMr~

on ocular resident cells were most greatly suppressed by treating animals with FK506. A therapy combining with low dosages of two immunosuppressive agents having different modes of action can be beneficial for additive immunosuppressive effects while minimizing adverse side effects of each drug. We examined two combination therapies in the EAU model in the rat: (1) cyclosporine and bucillamine, and (2) FK506 and dexamethasone (Mochizuki, 1993). Bucillamine is an antirheumatic drug developed in Japan (Fujimura et al., 1980), which we have now demonstrated to suppress the antigen presentation activity of macrophages (Mochizuki, 1992). A combination therapy combining suboptimal dosages of cyclosporine (2 mg/kg/day) and bucillamine (20 mg/kg/day) initiated on the day of S-antigen immunization suppressed EAU induction, whereas therapy with cyclosporine alone or bucillamine alone was much less effective (Fig. 6). The additive effects on EAU suppression might be attributed to the different modes of action of these two drugs: one (cyclosporine) on an early pathway of T-cell activation and the other (bucillamine) on antigen presentation by macrophages. Similarly, a combination therapy combining suboptimal doses of FK506 (0.1 mg/kg/day) and dexamethasone (0.01 mg/kg/day) had a marked effect on EAU induction. Again, the combination was much more effective than single therapy with each drug.

3.3. Clinical Use of lmmunophilin Ligands in Refractory Uveitis

3.3.1. CYCLOSPORINE Nussenblatt and his colleagues (Nussenblatt et al., 1983) first reported successful therapeutic effects of cyclosporine in eight patients with endogenous uveitis with retinal involvement that had required therapy with corticosteroids or cytotoxic agents. The previous therapy had been unsuccessful, either because of ineffectiveness of therapy or side effects. All patients started with 10 mg/kg/day of cyclosporine; this was later adjusted depending upon the efficacy and side

effects of the drug. Seven of the eight patients had both an improvement in visual acuily and a decrease in ocular inflammation on cyclosporine. As for side effects of cyclosporine: (1) renal impairment seen in one patient recovered to normal by reducing the dose of the drug; (2) the severe gum swelling and pain cleared with good dental hygiene; (3) tingling sensation in the extremities and around the lips, fatigue and muscle weakness were transient. Following the first report by Nussenblatt, a number of clinical studies done in many countries have confirmed that cyclosporine therapy is beneficial in endogenous uveitis with involvement of the posterior segment of the eye such as Beh~et's disease. Behc;et's disease is characterized by vasculitis in many systemic organs with major symptoms of uveitis, oral aphthous ulcer, dermal manifestations, and genital ulceration. The disease is frequent in Mediterranean and Asian countries. In Japan, Beh(;et's disease is the leading cause of endogenous uveitis and 12% of acquired blindness in Japanese adults may be due to the disease. Treatment of Behqet's disease is particularly important in this country. A double-masked clinical trial was performed in Japan to evaluate the efficacy of cyclosporine in Behqet's disease in comparison to conventional therapy with colchicine (Masuda et al., 1989). Colchicine is an antiinflammatory drug which decreases migration of polymorphonuclear cells and has been widely used in Japan to prevent the inflammatory episodes of Beh~;et's disease (Matsushima and Mizushima, 1975). Cyclosporine (10 mg/kg/day) was significantly more effective than colchicine (1 mg/day) in treating the ocular manifestations of Beh~et's disease as demonstrated by a significant decrease of the frequency of ocular inflammatory episodes and by better visual prognosis. Cyclosporine was also better than colchicine in controlling systemic problems such as oral aphthous ulcers and dermal symptoms. However, the frequency of side effects, particularly renal dysfunction, was much higher for cyclosporine than for colchicine. They concluded that cyclosporine is beneficial for the therapy of refractory uveitis in Behg:et's disease. Indication, dosage and side effects of cyclosporine in uveitis: Cyclosporine therapy is indicated for patients with endogenous uveitis

IMMUNOSUPPRESSANTS IN OCULAR DISEASES

with active inflammation in the posterior segment of the eye. A contraindication for the initiation of therapy is those individuals with renal impairment, liver dysfunction, poorly controlled hypertension, systemic infection and malignancy. The starting dosage of cyclosporine used in the earlier studies was 10 mg/kg/day; this was effective in reducing the inflammatory activity of uveitis although it caused a variety of side effects in many cases. Later studies have demonstrated that lower starting dosage, such as 3 - 5 mg/kg/day, is as effective as the higher dosage, but with fewer side effects (BenEzra et al., 1988; Nagata et al., 1992). Therefore, the high starting dosage (10 mg/kg/day) is no longer acceptable. BenEzra and his co-workers have proposed a therapeutic scheme for the treatment of sight-threatening bilateral endogenous uveitis with cyclosporine. Their recommendation is that cyclosporine dosage should be initially about 5 mg/kg/day with a maximum dosage of 7 mg/kg/day, given either once or twice a day. If the initial dosage is not effective, then the therapy should be changed to either (1) a higher dosage (7 mg/kg/day) of cyclosporine or (2) combination therapy with low doses of predonisone (0.2-0.4 mg/kg/day) and cyclosporine (5 mg/kg/day). Our most current dosage for Behqet's disease is 3 - 5 mg/kg/day given twice a day; the dosage is then adjusted based on the effects and side effects, with a maximum dosage of 7 mg/kg/day. When the therapy causes intolerable side effects, the dosage should be reduced in a stepwise manner. A quick reduction of the dosage often causes a severe inflammatory attack in Beh~et's disease. The side effects seen in 136 Japanese patients treated with various dosages of cyclosporine were: renal impairment in 22°70, hirsutism in 19%, neurolotoxicity in 17°70, gastrointestinal symptoms in 13070, gum swelling in 9°70, general fatigue in 9o70, hypertension in 5o70, and hepatic dysfunction in 2°70.

3.3.2. FK506 An open clinical trial to evaluate the efficacy of FK506 in refractory uveitis was currently carried out by the Japanese FK506 Study Group

491

(Mochizuki et al., 1991, 1993). Male and female patients were included in the study if they were between 16 and 76 years of age and had refractory uveitis of endogenous etiology with active inflammation in the posterior segment of the eye. Patients with hypertension, renal impairment, liver dysfunction, diabetes mellitus, systemic infection, and malignancy were excluded from the study. During the study period (December 1990April 1992), 53 patients (37 men and 16 women) were studied (41 patients with Behqet's disease; 5 with Vogt - Koyanagi - Harada's disease; 4 with idiopathic retinal vasculitis; and 3 with other forms of uveitis). The age of the patients varied from 21 to 67 years and the mean (_ standard deviation) was 41.1 +_ 12.0 years. Fifty-one patients had been treated with various immunosuppressive agents (colchicine in 33 patients, cyclosporine in 22 patients and cyclophosphamide in 6 patients). The therapy was changed to FK506 because of either ineffectiveness or intolerable side effects. After washing out the previous medication, FK506 was given orally twice a day at an initial dose of 0.05, 0.10, 0.15, or 0.20 mg/kg/ day. FK506 was found to improve uveitis in a dose-dependent manner. An initial dosage of 0.05 mg/kg/day was effective only in three of 8 patients (38o70), a higher dosage (0.10 mg/kg/ day) was effective in 9 of 15 patients (60o70), and the dosages of 0.15 and 0.20 mg/kg/day were effective in 10 of 12 patients (83%) and 11 of 14 patients (79%), respectively. Criteria for the improvement of uveitis by this therapy were defined as follows: (1) increase in the bestcorrected visual acuity of more than two lines: (2) decrease in uveitis activity, that is, disappearance of retinal exudates and haemorrhages, decrease of macular edema and vitreous opacities; and (3) decrease in the number of ocular inflammatory episodes in patients with Behqet's disease. After the dosage adjustment, uveitis was improved in 76.5°7o of treated patients, and deteriorated in only two patients (3.9O7o). In the other ten patients (19.6o70), uveitis was unchanged by the therapy. It is of note that FK506 was effective even in 7 of 11 patients (63.6o70) who were resistant to the previous therapy with cyclosporine. The visual acuity of 96 eyes in the patients was compared before FK506 treatment

492

M. MOCHIZUKIand M. DV~SMFr

and at the 12th week of FK506 therapy. The visual acuity was improved in 53 of the 96 (55.2%), remained unchanged in 17 eyes (17.7070), and deteriorated in 25 eyes (27.1%). Furthermore, FK506 therapy significantly decreased the frequency of ocular inflammatory episodes in Behqet's disease: the mean (__ standard deviation) number of ocular inflammatory episodes for 12 weeks before and after FK506 administration was 3.6 (4-2.5) and 2.7 (4- 2.0), respectively (/9<0.05). Adverse side effects were observed in 26 of 53 patients (49.1°70) treated with FK506 during the 12-week study period. The most common adverse effect was renal impairment (15 of 53 patients, 28.3°70). The second most common side effect was neurological symptoms (11 of 53 patients, 20.8°7o) which consisted of tremor, abnormal sensation, headache, insomnia, or meningoencephalitis. The third most common side effect was gastrointestinal symptoms (10 of 53 patients, 18.9%), including nausea, diarrhoea, appetite loss, and abdominal pain. Hyperglycemia was seen in seven patients (13.2°70). The incidence of these adverse effects depended on the dosage of FK506. The results suggest that efficacy of FK506 in uveitis is essentially very similar to those of cyclosporine. FK506 and cyclosporine did not cause myelotoxicity and suppression of reproductive organs, not like cytotoxic agents. The immunophilin ligands at high dosage, however, induced nephrotoxicity which was the most common and serious side effect of these agents.

4. IMMUNOPHILIN LIGANDS IN CORNEAL TRANSPLANTATION 4.1. Background

Corneal graft rejection is a major problem in corneal transplantation. Patients with corneal neovascularization and/or a history of previous graft rejection have a rate of graft failure due to rejection of more than 50°70, in spite of conventional treatment with corticosteroids or cytotoxic agents. Therefore, more effective immunosuppressive drugs are needed for the management of such high risk corneal grafts.

Recently, a number of new immunosuppressants including cytotoxical agents and immunophilin ligands have been studied in various organ transplantations models. Among them, immunophilin ligands have been extensively investigated. Comparison of the immunosuppressive activities of cyclosporine and FK506 in experimental rejection models is shown in Table 6. Cyclosporine has contributed to a dramatic improvement in organ transplantations. After the development of cyclosporine and its clinical application, a number of resistant rejection episodes in renal, cardiac, liver, pancreas, and bone marrow transplantations have been successfully treated. As summarized in Table 6, FKS06 is effective in suppressing experimental rejections at doses 10-20 times lower than cyclosporine, indicating that the effect of FKS06 on allograft rejection is 10-20 times more effective than cyclosporine on a mg per mg basis. Ochiai et al. (1987a) were the first to describe the immunosuppressive effects of FK506 using a cardiac transplantation model in rat. The 2-week administration of 0.32 mg/kg/day of FK506 produced permanent survival (more than 100 days) in most animals. Transfer of lymphocytes or serum from FK506induced longterm heterograft-bearing rats prolonged survival in non-treated rats (Ochiai et al., 1987b). Suppression was not produced in a third-party donor heart, suggesting that donor strain-specific suppressor cells are induced by treatment with FK506 in the presence o1' the allograft. But other investigators (Murase et al., 1987, 1990a; Lim et al., 1987) have used a similar heterotopical cardiac transplantation model in rats, and, even though prolongation of graft survival was achieved, permanent survival was not observed. Different models of transplantation rejection that included skin (Lagodzinski et al., 1990), limb (Arai et al., 1989), kidney (Ochiai et al., 1987c; Todo et al., 1987a; Calne et al., 1987), liver (Todo et al., 1987b), Langerhans islet (Yasunami et al., 1990), lung (Saitoh et al., 1990) and small intestine (Hoffman et al., 1990), have also shown that the immunosuppressive effect of FK506 does not produce permanent graft survival. Therefore, constant administration of FK506 was required to prevent rejection. Murase et al. (1990b) have shown that treatment begun one day

IMMUNOSUPPRESSANTS IN OCULAR DISEASES

before the rejection was able to prolong cardiac and liver survival in rats. A dramatic ability of FK506 to stop advanced and refractory rejection was observed in human transplantation of liver (Starzl et al., 1989; Fung et al., 1991), kidney (Shapiro et al., 1991), pancreas (Tze et al., 1991), heart (Armitage et al., 1991), and small bowel (Todo et al., 1991).

4.2. Use of Immunophilin Ligands in Experimental Corneal Transplantation

Cyclosporine administered systemically (Coster et al., 1979; Shepherd et al., 1980; Bell et al., 1982; Hunter et al., 1982; Liu et al., 1989) or topically (Salisbury et al., 1981; Foets et al., 1985; Williams et al., 1987; Hill et al., 1988) has been reported to be beneficial in the prolonged survival of corneal allografts in experimental animals. Because the ocular surface is the site of immunological activity in corneal transplants, topical instillation of cyclosporine is considered to be more beneficial compared with systemic cyclosporine from the point of view of eliminating the risk of systemic toxicity while maintaining a therapeutic level of local immunosuppressive activity (Belin et al., 1990). Pharmacokinetics o f topical cyclosporine: The pharmacokinetics of topically-applied cyclosporine has been studied in experimental animals to determine whether topical application gives high concentrations of the drug in the avascular cornea. Using a polyclonal radioimmunoassay (RIA), Mostteller et al. (1985) measured the penetration of 10°70cyclosporine ointment into the rabbit eye. In this study, the average cyclosporine concentrations one hour after application in the cornea, aqueous humor, and serum were 432, 168, and 55 ng/ml, respectively. The drug concentration in the cornea reached a peak level (926 ng/ml) in three hours and was maintained at a high level (672 ng/ml) even 24 hours after application. On the other hand, the drug concentration in the serum was undetectable after 24 hours. The pharmacokinetics of topical cyclosporine varied depending on the solvent used. Following a single dosage (10/al) of 2°7o

493

cyclosporine in caster oil, a maximum concentration of 900-1400 mg/ml was detected in the cornea 6 hours later. The concentration was still high (600-900 ng/ml) at 48 hours, although the concentrations in intraocular tissues (iris, vitreous, uvea/retina) were very low (<45 ng/ml) (Wiederholt et al., 1986). Following one drop (0.7 tal) every 15 minutes for 6 applications of 1°70 cyclosporine in olive oil (a total dose of 0.84 mg or 0.25 mg/kg), tissue levels of more than 50 ng/g were achieved within one hour. These high levels were maintained for 24 hours in the cornea and sclera, whereas much lower levels were observed in the aqueous humor, iris, cilicary body, vitreous, and retina (Kaswan, 1988). More currently, Akiyama et al. (1993) studied the penetration of cyclosporine eye drops in alpha-cyclodextrin. A single application of 0.025°70 cyclosporine in alpha-cyclodextrin gave high concentrations in the cornea (4133 ng/g wet tissue), but no detectable drug in aqueous humor and serum. These studies on the pharmacology of topical cyclosporine indicate that ointment and eye drops (caster oil, olive oil or alpha-cyclodextrin) are effective in promoting high concentration of the drug in the cornea, sclera, and conjunctive while only low levels are seen in the intraocular tissues and serum. Effects of topical cyclosporine on allograft rejection in rabbit corneal transplants: Hunter et al. (1982) used eye drops of cyclosporine (l°70 in arachis oil) in rabbit allograft transplants for 4 weeks or 13 weeks post-operation. The corneal grafts treated with 1°70 cyclosporine eye drops survived significantly longer than untreated control grafts. Furthermore, cyclosporine instillation for 13 weeks was more effective in prolonging graft survival than the 4 weeks-therapy. Four of nine allografts treated with cyclosporine for 13 weeks remained clear for more than 90 days after the cessation of the therapy, suggesting that specific immunological tolerance might be induced. Hill and Maske (1988) demonstrated that graft survival in high-risk keratoplasty in rabbits was improved by topical cyclosporine; systemic cyclosporine, however, produced a better graft survival rate than topical cyclosporine. More currently, effects of cyclosporine eye drops (0.025°70 in alpha-cyclodextrin) were studied in

494

M. MOCHIZUKI and M. Dt~ SMF~J

FIG. 7. Allograft rejection in the penetrating keratoplasty in the rat two weeks after the surgery without treatment (nontreated control rat) (Mochizuki, M., Acta Soc. Ophthalmol..lap. 96: 1608- 1634 (1992) published with permission).

rabbit keratoplasty (Akiyama et al., 1993). The cyclosporine eye drops (0.025°70, 0.05%) given four times a day for 50 days post-operative significantly suppressed allograft rejection of corneal transplantation in a dose-dependent manner. An interesting finding in their study was that one half of the grafts treated with topical cyclosporine (0.05%) remained transparent as long as 100 days post-operation, i.e. 50 days after discontinuation of drug application. These data thus indicate that cyclosporine is capable of suppressing immunological events in allograft corneal transplantation when given not only

systemically but also topically. Effects o f immunophilin ligands (systemic" or topical) on rat corneal transplants: The experimental studies discussed above were performed in rabbits. We studied the effect of cyclosporine and FK506 on a penetrating keratoplasty model in the rat (Herbort et al., 1989; Nishi et al., 1993; Mochizuki, 1992). The rat has several advantages for immunological studies compared with the rabbit: (1) availability of monoclonal antibodies specific to surface markers of immune cells and (2) availability of inbred strains. The orthotopic penetrating keratoplasty technique was used in

IMMUNOSUPPRESSANTS IN OCULAR DISEASES

495

FIG. 8. Immunopathology in the allograft corneal rejection in the rat two weeks after the corneal transplantation (Fisher to Lewis) without treatment (nontreated control rat) CTL: negative control, Mo: macrophages, Th/i: helper/inducer Tlymphocytes, Ts/c: suppressor/cytotoxic T-lymphocytes, Ia Ag: MHC class II antigen, IL-2R: interleukin 2 receptor (Mochizuki, M., Acta Soc. Ophthalmol. Jap. 96: 1608- 1634 (1992) published with permission).

our studies to induce 100°70 acute graft rejection. An inbred strain of Lewis rats (Rtl Ie) was used as the recipient and an inbred strain of Fisher rats (Rtl Iv') as the donor. Fisher and Lewis rats differ

only in their medial and minor histocompatibility antigen (Gill et al., 1987). A primary mixed lymphocyte reaction between naive rats o f the two strains did not proliferate. However, Lewis

496

M. MOCHIZUK1and M. DE SME'r Cell Marker

100 -

Grading Score in AIIograft 0

0.5 i

A

o~ 80 --

6oi

-

4o

-

~20

J.v-" '-~ ........ • ........ Q.FK506 (3)

cY,

j !

•

FKS06'(ii!

0 I

CD8

~z~-

IL-2R

~////////~....

I-7, P';'**

I~ .............. "

B cell M+

~///~,¢,_...

MHC-class I

~,a--

i

:

I

I

I

I

3 4 5 6 7 Weeks after Keratoplasty

I

8

FIG. 9. Effects of systemic immunophilin ligands on the corneal graft survival in the rat (Mochizuki, M., Acta Soc. Ophthalmol. Jap. 96: 1 6 0 8 - 1 6 3 4 (1992) published with permission).

lymphocytes primed to Fisher antigen after penetrating keratoplasty demonstrated significant proliferation when mixed with Fisher splenocytes. This stimulation was due to allograft sensitization because proliferation did not occur in Lewis to Lewis syngenic (Nishi et al., 1993). This combination of rats caused 100°70 acute graft rejection in two weeks after keratoplasty as demonstrated by (1) clinical observations with opacity and neovascularization in the grafts (Fig. 7) and (2) immunohistochemical observations. Figure 8 shows examples of immunohistochemical staining of grafts at the time of acute rejection (day 14 post-operation). The grafts were heavily infiltrated by cells stained for OX42, a surface marker for macrophages. Even in this early stage of rejection, macrophages were present in high number, suggesting that, in this model, a delayed type of hypersensitivity is one of the mechanisms involved in the rejection. As for T-iymphocytes, W3/25-bearing cells (helper/ inducer lymphocytes) were predominant compared with OX8-bearing cells (suppressor/ cytotoxic lymphocytes). Furthermore, the figure clearly shows strong staining for Ia antigen (MHC class II antigen) and for IL2 receptors. Systemic administration of cyclosporine (10 mg/kg/day) or FK506 (0.3, 1.0 and 3.0 mg/kg/day) was given to recipient rats from

i

~//~////d]

!I T--'--f :

om o, l

1.5

i

CD4

•

,i ........ , ---" ,_ k

1.0

MHC-class II ~///.~..._

I

2.0 I

**

I-**

I ** I **

FIG. 10. Effectsof systemicimmunophilinligandson the immunopathologyin the allograftcornealtransplantation (Mochizuki, M., Acta Soc. Ophthalmol. Jap. 96: 1068- 1634 (1992) published with permission).

the day of operation through day 15 post-operation. All untreated control recipients developed rejection at the third post-operative week, whereas administration of the immunophilin ligands prolonged the graft survival in a dose-dependent manner (Fig. 9). All allografts treated with immunophilin ligands were, however, eventually rejected after the discontinuation of systemic administration of these agents, suggesting that immunophilin ligands do not cause permanent graft survival in corneal transplantation. This is similar to other transplantation models, but not similar to S-antigen-induced experimental autoimmune uveitis. Immunological activation of cells infiltrating the corneal graft was markedly suppressed by treatment with immunophilin ligands as demonstrated by immunohistochemical examination (Fig. 10). Also, effects of topical immunophilin ligands on corneal rejection was studied using FK506 eye drops. The FK506 eye drops used in our study was a special suspending and tonicity agent at pH 5.25 provided by Fujisawa Pharmaceutical Co. (Osaka, Japan). To minimize nonspecific post-operative inflammatory reaction, systemic FK506 (0.3 mg/kg/day) was given to all recipients in both control and experimental groups on day 0 - 6 post-operation. Then, from day 7 through to day 20 post-operation, subjects in the experimental group and the control group were treated with topical FK506 (0.3°/o) and vehicle,

497

IMMUNOSUPPRESSANTS IN OCULAR DISEASES TABLE 6. C o m p a r i s o n o f t h e I m m u n o s u p p r e s s i v e

Response

Organ

A c t i v i t i e s o f F K 5 0 6 a n d C s A in E x p e r i m e n t a l R e j e c t i o n Models

Route

suppressed Allograft in: Rat:

Mouse:

FK506

CsA

heart

IM*

0 . 3 2 - 1.28

l0

skin liver

IM IM

0.3 0 . 3 2 - 1.28

32 --

L. islet limb small intestine skin heart

IM IM IM IP IP PO SC IM PO PO IM IM

0.32 2 - 10 2.0 0.1-1 0.7 4 0.1 0.16 1.5 1.0 0.2 0.1

-->20 3 . 5 - 35 10 21 ---20 20 --

IM IM

0.1 1- 2

---

liver pancreasduodenum mouse skin

PO PO

l0 10

---

IM

3.2

--

mouse heart

IM

3.2

--

Rabbit: Dog:

cornea kidney

Primate:

liver pancreas tracheal reconstr. lung kidney

Xenograft in: Rat:

Dose (mg/Kg/day)

References

Starzl et al. (1989) Ochiai et al. (1987) Murase et al. (1990) Inamura et al. (1988) Murase et al. (1990) Tsuchimoto et al. (1989) Yasunami et al. (1990) Arai et al. (1989) H o f f m a n et al. (1990) Lagodzinski et al. (1990) Morris et aL (1990) Morris et al. (1990) Herbort et al. (1989) Ochiai et al. (1987) Todo et al. (1987) Todo et al. (1987) Sato et al. (1989) Moriyama (1989) Saitoh et al. (1990) Calne et al. (1987) Imventarza et al. (1990) Monden et al. (1990) Ericzon et aL (1990) Inamura et al. (1988) Ochiai et al. (1987) Ochiai et al. (1987)

*IM = intra-muscular; OP = oral; IP = intra-peritoneal; SC = sub-conjunctival; L. islet = Langerhans' islet; tracheal reconstr. = tracheal reconstruction.

respectively. 10/al of instillation was given every 4 hours daily in the inferior fornix using a micropipette. All recipients treated with vehicle alone demonstrated graft rejection in 14 days post-operation, whereas all grafts in the experimental group survived to the time of discontinuation of topical FKS06 and developed graft rejection only thereafter (Fig. 11). Immunohistochemical observations confirmed that topical FKS06 markedly suppressed the delayed-type hypersensitivity to allograft mediated by Tlymphocytes. A significant decrease was observed in helper/inducer T-lymphocytes, suppressor/ cytotoxic T-lymphocytes, macrophages, IL-2 receptor-expressing cells and cells bearing MHC class I or class II antigen (Fig. 12). Because the blood concentration of FK506 in rats treated with topical FK506 was at or below the level

of detection, the effects were considered to be local effects. Presumably, application of topical immunophilin ligands is able to stop the ongoing interaction between T-lymphocytes and other immune cells at the local site.

4.3. Clinical Use of Immunophilin Ligands in Corneal Transplantation

Miller et al. (1988) reported successful engraftment of high-risk corneal allografts with shortterm immunosuppression with cyclosporine. In their study, a total of 15 high-risk recipients received corneal transplants accompanied by systemic cyclosporine (5 mg/kg/day) supplemented by topical application of corticosteroids.

498

M. MOCHIZUKI and

Systemic FK506 Eye Drop .rST....l. ..... i 100 i 0 . 3 0 FK506 eye drop 80n-

60

~40-

I

1

Placebo eye drop

~ 200-

.°

I

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I

I 20 Days after Keratoplasty

I 30

oL--//7 10

FIG. 11. Effects of topical FK506 on the corneal graft survival in the rat (Mochizuki, M., Acta Soc. Ophthalmol. Jap. 96: 1608- 1634 (1992) published with permission).

Cell Marker

Number of positive cells / 0.015 mm2 9 2s 5o 75 100

CD4

~ ~ . _ _

CD8

~

*

IL°2R

~

,

Me

~/2~---

MHC-class II ~

I

~

'

,

* t-~**

FIG. 12. Effects of topical FK506 on the immunopathology in the allograft corneal transplantation (Mochizuki, M., Acta Soc. Ophthalmol. Jap. 96: 1608- 1634 (1992) published with permission).

Systemic cyclosporine was continued for a total of 12 weeks. Twelve of 15 patients had long-term graft survival during a mean observation period of 25 months. A comparative study of systemic cyclosporine, topical corticosteroids, and topical plus systemic corticosteroids in high-risk keratoplasty demonstrated that systemic cyclosporine

M. DF SMET

was significantly more effective in prolonging the high-risk corneal grafts than the other' two therapies (Hill, 1989). Later studies (Irschick et al., 1989; Maeda et al., 1992) together with these earlier reports clearly indicate that systemic cyclosporine is beneficial in high-risk corneal transplantation. On the other hand, systemic cyclosporine causes a variety of adverse side effects. Therefore, topical use of cyclosporine would be more beneficial granted that it has the same immunosuppressive effects, observed in animals studies. Belin et al. (1989) used topical cyclosporine (2% in olive oil) in 11 high-risk corneal transplant patients. The topical cyclosporine was given 2 4 - 4 8 hours before surgery through 4 days after surgery with 1 drop every 2 hours while awake. Subsequently, patients received 1 drop of topical cyclosporine and 1 drop of topical corticosteroids 4 times a day for the first 3 months; the dosage of topical cyclosporine and corticosteroids was reduced thereafter. With this treatment schedule, 10 of the l l grafts (91%) remained clear without evidence of graft rejection or failure during the observation period (average: 16 months, range: 6 - 2 4 months). No patients had abnormal blood chemistry including blood urea nitrogen or serum creatinine. The whole blood level of cyclosporine measured by H P L C in these patients ranged from 14 to 64 ng/ml (average: 35 ng/ml), which was consistently below the blood trough level of 8 0 - 250 ng/ml recommended for renal transplant patients receiving oral cyciosporine. These data thus indicate that topical cyclosporine is beneficial even in high-risk corneal transplantation with minimal or no systemic adverse side effects. At present, no clinical studies have been performed to test the effects of systemic or topical FK506 in allograft rejection in human corneal transplants.

5. I M M U N O P H I L I N LIGANDS IN A L L E R G I C CONJUNCTIVITIS 5.1. B ac k gr ou n d

Studies of the actions and interactions of the immunophilin ligands (cyclosporine, FK506, and

IMMUNOSUPPRESSANTSIN OCULAR DISEASES

~ ~ + E

~

~P "~ Immunophilin ~

I

i,, ~ iVl i"*

|

l

No.of eyes

Absorbance at 620 nm ( mean 4- S.E. ) 0

0.2

0.4

0.6

0.8

1.0

I

I

I

I

I

I

Ligands

Ca2~-"7/~°esterase'~.). ' "N~ I ? "~ esterase .,/ f .... n

Eye drop at - 6, -4 & -2 hr.

499

/

NN~

Vehicle (control)

12

I

FK506

(0.03%)

10

[

+**

(0.1%)

10

~

**

(0.3%)

10

I

Betamethasone

10

I

exocytosis /

O

(0.1,/,)

+

-'~ -]-

**

oscG (2%) 10 I .... Mast cell, basophil FIG. 13. Mode of action of immunophilin ligands in the IgE-mediated exocytosis (Mochizuki, M., Acta Soc. Ophthalmol. Jap. 96:1608 - 1634 (1992) published with permission).

rapamycin) suggest that complexes of cyclosporine with cyclophilin or of FK506 with FK506binding protein (FKBP) inhibit the T-cell receptormediated signal transduction that results in the transcription of interleukin 2 and other lymphokines. Recent studies on the immunophilin ligands have provided a new insight into the actions and interaction of cyclosporine and FK506. Hultsch and co-workers (Hultsch et al., 1990, 1991) have demonstrated that cyclosporine and FK506 inhibits IgE receptor-mediated exocytosis (serotonin release) from a rat basophilic leukemia cell line with IC50 of 200 nM for cyclosporine and 2 nM for FK506. These results are similar to those reported for T-cell receptor-mediated activation of T-cells. Paulis et al. (1991) investigated the effects of FK506, rapamycin and cyclosporine on the release of preformed (histamine) and de n o v o synthesized (leukotriene C4) inflammatory mediators from human peripheral basophils. FK506 inhibited histamine release from basophils activated by either D e r p I (the relevant allergen of D. p t e r o n y s s i n u s ) , anti-IgE, or the Ca 2÷ ionophore A23187 in a dose-dependent manner (1 -300 riM). The inhibitory effects of FK506 on histamine release were more potent than those of cyclosporine. Rapamycin (30- 1000 nM) selectively inhibited only IgE-mediated histamine release from basophils. FK506 also inhibited de n o v o

--'l--

FIG. 14. Effects of topical FK506 on the experimental allergic conjunctivitis in the rat (lwaki, Y., Ocular Immunol. Inflamrn. 1:79-85 (1993) published with

permission).

Rats

FK506 No.of (0.1%) eyes

Percent of degranulated mast calls In the conJunctlva (%) 0 I

.aiv

No

20 I

40 I

60 I

t

80 I

100 I

---I tt

.t

,,,erg,°

I

÷

___1

Fie. 15. Effects of topical FK506 on the mast cell degranulation in the rat conjunctiva (Iwaki, Y., Ocular Imrnunol. Inflamm. 1 : 7 9 - 8 5 (1993) published with permission).

synthesis of leukotriene C4 from human basophils challenged with anti IgE. These in vitro studies thus suggest that cyclosporine and FK506 are capable of inhibiting IgE receptor-mediated signal transduction that results in the exocytosis of secretory granules and the release of inflammatory mediators from basophils and mast cells. Figure 13 illustrates the hypothetical mode of action of cyclosporine and FK506 on IgE receptormediated immune reactions.

5.2. Use of lmmunophilin Ligands in Experimental Allergic Conjunctivitis

No in vivo studies have been carried out to investigate the effects of immunophilin ligands on

500

M. MOCHIZUKIand M. DE SMET

IgE-mediated hypersensitivity reactions. Therefore, we performed a series of in vivo experiments to examine whether topical FK506 was effective in suppressing allergic conjunctivitis in the rat (Iwaki et al., 1993). Passive anaphylaxis was induced in Wister rats by injecting anti-ovalbumin IgE into the conjunctiva followed by intravenous injection of antigen (ovalbumin) and Evans blue. Topical FK506 (0.03-0.10/0) significantly suppressed passive anaphylaxis in the rat conjunctiva when given at 15 and 5 minutes, or 6, 4 and 2 hours before the antigen challenge (Fig. 14). The efficacy was significantly better than that of 2°70 disodium cromoglycate. Mast cell degranation in the conjunctiva was also significantly suppressed by topical FK506 compared to non-treated rats with allergic conjunctivitis (Fig. 15). Our preliminary experiments have shown an identical spectrum of activities of topical cyclosporine in allergic conjunctivitis in the guinea pig (Hikita et al., 1992). These data in experimental models for allergic conjunctivitis thus suggest that topical immunophilin ligands should be potentially useful in the therapy of allergic disorders in human eyes, particularly those of a refractory type.

cessation of the cyclosporine; the other children demonstrated a rapid recurrence of the vernal symptoms. None of the 12 patients treated with topical cyclosporine had systemic and local side effects of the drug. More recently, Secchi et aL (Secchi et al., 1990) reported an open trial of longterm therapy of topical cyclosporine ( 4 - 9 months) and a double-masked study in vernal keratoconjunctivitis. In their open study, 11 patients with severe vernal keratoconjunctivitis were treated with 2°7o cyclosporine eye drops in castor oil 4 times a day. No significant side effects occurred throughout the study. Within the first 2 weeks, symptoms and signs of the disease improved significantly; these results were maintained to the end of the therapy, although relapse of the disease occurred 2 - 4 months thereafter. A double-masked study of 9 patients (2°7o cyclosporine in castor oil vs castor oil alone) confirmed the results of their open trial. These clinical studies of topical cyclosporine in patients with refractory vernal keratoconjunctivitis are in accord with the experimental data in animals. No studies have been carried out to examine the effects of systemic or topical FK506 in allergic conditions of the human eyes.

5.3. Clinical Use of lmmunophilin Ligands in Allergic Conjunctivitis in Human

6. S U M M A R Y

Topical cyclosporine had been used in severe types of allergic conjunctivitis, e.g. vernal keratoconjunctivitis, before the current understanding of its mode of action on IgE receptor-mediated hypersensitivity. BenEzra et al. (1986) used 2°70 cyclosp0rine eye drops in olive oil in 12 children who had been suffering from severe vernal conjunctivitis. All patients had previously required short courses of systemic corticosteroids to relieve their symptoms which were refractory to local treatment with corticosteroid eye drops and cromolyn preparations. Eleven of the 12 children showed marked improvement after the first week of topical cyclosporine therapy. Nine patients demonstrated persistent improvement at the completion of the treatment schedule after 6 weeks. On longer follow-up, however, only 3 patients remained symptom-flee 2 months after

The immunophilin ligands (cyclosporine, FK506 and rapamycin) differ from conventional immunosuppressive agents (corticosteroids and cytotoxic agents) in many aspects; these include mode of action, efficacy and adverse side effects. Immunophilin ligands selectively inhibit T-cellmediated signal transduction that results in the transcription of interleukin 2 and certain other lymphokines. Experimental studies of these immunosuppressive agents indicate that immunophilin ligands are beneficial in suppressing T-cellmediated experimental autoimmune uveoretinitis in rats or monkeys, as well as allograft rejection of corneal transplantation in rabbits and rats. Clinical studies have shown that cyclosporine and FK506 administered systemically are beneficial in refractory uveitis with an endogenous etiology. In corneal transplantation in humans, systemical or

IMMUNOSUPPRESSANTSIN OCULAR DISEASES t o p i c a l c y c l o s p o r i n e has been d e m o n s t r a t e d to p r o l o n g the survival o f high-risk allografts. M o r e recently, i m m u n o p h i l i n ligands h a v e been s h o w n in v i t r o to inhibit I g E r e c e p t o r - m e d i a t e d signal t r a n s d u c t i o n t h a t results in exocytosis o f secretory granules f r o m b a s o p h i l s a n d m a s t cells. T h e effects o f F K5 0 6 o n an a n i m a l m o d e l f o r IgEm e d i a t e d o c u l a r disease was carried o u t in the rat. T o p i c a l F K5 0 6 was e f f e c ti v e in suppressing the intensity o f e x p e r i m e n t a l allergic c o n j u n c t i v i t i s a n d d e g r a n a t i o n o f m a s t cells in the c o n j u n c t i v a . Clinically, t o p i cal c y c l o s p o r i n e (eye drops) was e f fe ct i v e in i m p r o v i n g b o t h s y m p t o m s a n d signs o f patients with severe v e r n a l k e r a t o c o n j u n c t i v i t i s . A ll in all, the use o f these agents seems to h a v e a bright f u t u r e in the m a n a g e m e n t o f a wide r a n g e o f i m m u n o l o g i c a l diseases.

REFERENCES

ARAI, K., HOTOKEBUCH, T., MIYAHARA, H., ARITA, C., MOHTAI, M., SUGIOKA,Y. and KAIBARA,N. (1989) Limb allografts in rats immunosuppressed with FK506. I. Reversal of rejection and indefinite survival. Transplantation 48: 782- 786. ARMITAGE,J. M., KORMOS,R. L., FUNG,J. and STARZL,T. E. (1991) The clinical trial of FK506 as primary and rescue immunosuppression in adult cardiac transplantation. Transplant. Proc. 23: 3054- 3057. ATHENS, J. W., HAAB,O. P., RAAB,S. O., MAUER,A. M., ASHENBRUCKER, H., CARTWRIGHT, G.E. and WINTROBE,M. M. (1961) Leukokinetie studies. IV. The total blood, circulating and marginal granulocyte pools and the granulocyte turnover rate in normal subjects. J. Clin. Invest. 40: 989- 995. BELIN, M.W., BOUCHARD, C.S., FRANTZ, S. and CHMIELINSKA, J. (1989) Topical cyclosporine in highrisk corneal transplants. Ophthalmology 96: 1144- 1150. BELIN, M. W., BOUCHARD,C. S. and PHILLIPS, T. M. (1990) Update on topical cyclosporin A: background, immunology, and pharmacology. Cornea 9: 184- 195. BELL, T. A. G., EASTY,D. L. and McCULLAGH,K. G. (1982) A placebo-controlled blind trial of cyclosporin-A in prevention of corneal graft rejection in rabbits. Br. J. Ophthalmol. 66: 303- 308. BENEZRA, D., PE'ER, J., BRODSKY,M. and COHEN, E. (1986) Cyclosporine eyedrops for the treatment of severe vernal keratoconjunctivitis. Am. J. OphthalmoL 101: 278 - 282. BENEZRA, D., NUSSENBLATT,R. B. and TIMONEN,P. (1988) Optimal use of sandimmune in endogenous uveitis. In: Therapeutic Scheme f o r the Treatment o f SightThreatening Bilateral Endogenous Uveitis with Cyclosporine (CyA). Springer, Berlin. BERAUD, E., KOTAKE,S., CASPI, R. R., ODDO, S. M., CHAN,

C.-C., GERY,I. and NUSSENBLATT,R. B. (1992) Control

501

of experimental autoimmune uveoretinitis by low dose T-cell vaccination. Cell Immunol. 140:112 - 122. BIERER, B.E., MATTILA, P.S., STANDAERT, R.F., HERZENBERG, L. A., BURAKOFF,S. S., CRABTREE,G. and SCHREIBER, S.L. (1990a) Two distinct signal transmission pathways in T-lymphocytes are inhibited by complexes formed between an immunophilin and either FKS06 or repamycin. Proc. HatH. Acad. Sci. U.S.A. 87:9231-9235. BIERER, B. E., SOMERS,P. K., WANDLESS,T. J., BURAKOFF, S.J. and SCHREIBER, S.L. (1990b) Probing immunosuppressant action with a nonnatural immunophilin ligand. Science 250: 556- 559. BOREL, J. F., FEURER,C., GUBLER,H. U. and STAHELIN,H. (1976) Biological effects of cyclosporin A: a new antilymphocytic agent. Agents Actions 6:468 - 475. CALNE, R., COLLIER, D. and THIRU, S. (1987) Observation about FK506 in primates. Transplant. Proc. 19 (Suppl. 6): 63. CHAN, C.-C., MOCHIZUKI, M., NUSSENBLATT, R.B., PALESTINE, A.G., MCALLISTER, C., GERY, I. and BENEZRA, D. (1985a) T-lymphocyte subsets in experimental autoimmune uveitis. Clin. Immunol. Immunopathol. 35:103 - 110. CHAN, C.-C., MOCHlZUKI,M., PALESTINE,A. G., BENEZRA, D., GERY, I. and NUSSENBLATT,R. B. (1985b) Kinetics of T-lymphocyte subsets in the eyes of Lewis rats with experimental autoimmune uveitis. Cell. Immunol. 96: 430 - 434. CHAN, C.-C., NUSSENBLATT, R.B., FUJ1KAWA, L.S., PALESTINE, A.G., STEVENS, J.G., PARVER, L.M., LUCKENBACH, M.W. and KUWABARA, T. (1986) Sympathetic ophthalmia: immunopathological findings. Ophthalmology 93: 690-695. CHAN, C.-C., CASPI, R., MOCHIZUKI,M., DIAMANTSTEIN,T., GERY, I. and NUSSENBLATT,R. B. (1987) Cyclosporine and dexamethasone inhibit T-lymphocyte MHC class II antigens and IL-2 receptor expression in experimental autoimmune uveitis. Immunol. Invest. 16: 319- 331. CLAMAN, H. M. (1972) Corticosteroids and lymphoid cells. New EngL J. Med. 287: 388- 397. COHEN, I. J., DUKE, R. C., FADOK,V. A. and SELLINS,K. S. (1992) Apoptosis and programmed cell death in immunity. Ann. Rev. Immunol. 10: 267. COSTER, D.J., SHEPHERD, W. F. I., FOOK, T.C., RICE, N. S. C. and JONES,B. R. (1979) Prolonged survival of corneal allografts in rabbits treated with cyclosporin A. Lancet 29: 688- 689. CRONSTEIN, B. N., KIMMEL,S. C., LEVIN, R. I., MARTINIUK, F. and WEISSMANN,G. (1992). A mechanism for the antiinflammatory effects of corticosteroids: the glucocorticoid receptor regulates leukocyte adhesion to endothelial cells and expression of endothelial-leukocyte adhesion molecule 1 and intercellular adhesion molecule 1. Proc. HatH. Acad. Sci. U.S.A. 89:9991-9995. CuPPS, T.R., EDGAR, L.C. and FAUCI, A.S. (1982) Suppression of human B-lymphocyte function by cyclophosphamide. J. Immunol. 128: 2453. DALE, D. C., FAUCI, A. S., GUERRY,D. and WOLFF, S. M. (1975) Comparison of agents producing a neutrophilic leukocytosis in man. J. Clin. Invest. 56: 808- 813. DEL REY, A., BESEDOVSKY,H., SORKIN,E. and DINARELLO, C. A. (1987) Interleukin-1 and glucocorticoid hormones integrate an immunoregulatory feedback circuit. Ann. N.Y. Acad. Sci. 496: 85- 90.

502

M. MOCHIZUKI and M. DE SMET

DE PAULIS,A., CIRILLO, R., CICCARELLI,m., CONDORELL1,M. and MARONE, G. (1991) FK-506, a potent novel inhibitor of the release of proinflammatory mediators from human Fc e RI+ cells. J. lmmunol. 146: 2374- 2381. DE SMET, M. D., YAMAMOTO, J. H., MOCHIZUKI, M., GERY, I., SINGH, g . K . , SHINOHARA, T., WIGGERT, B., CHADER, G. J. and NUSSENBLATT,R. B. (1990) Cellular immune responses of patients with uveitis to retinal antigens and their fragments. Am. J. Ophthalmol. 110: 135 - 142. DE SMET, M. D., MAINIGI, S. and NUSSENBLATT,R. B. (1993) Immunologenicity and Immunopathogenicity of peptide determinants of human S-Ag in various rat strains. Invest. Ophthalmol. Vis. Sci. 34 (Suppl): 1143. ELLIOTT, J. F., L1N, Y., MIZEL, S. B., BLEACKLEY, R. C., HARNISH, D. G. and PARTKAU, V. (1984) Induction of interleukin 2 messenger RNA inhibited by cyclosporin A. Science 226: 1439- 1441. ERICZON, B. G., KUBOTA, K., GROTH, C. G., W1JNEN, R., TIEBOSCH, T., BUURMAN,W. and KOOTSTRA, G. (1990) Pancreaticoduodenal allotransplantation with FK506 in the cynomolgus monkey. Transplant. Proc. 22 (Suppl. 1): 72-73. FAUC'I, A . S . , DALE, D . C . and BALOW, J . E . (1976) Glucocorticoid therapy: mechanisms of action and clinical considerations. Ann. Intern. Med. 84: 304- 315. FOETS, B., M1SSOTTEN, L., VANDERVEEREN,P. and GOOSSENS, W. (1985) Prolonged survival of allogeneic corneal grafts in rabbits treated with topically applied cyclosporin A: systemic absorption and local immunosuppressive effect. Br. J. Opthhalmol. 69: 600 - 603. FUJIMURA, H., HIRAMATSU, Y., TAMURA, Y., YANAGIHARA, M., KODA, A., NAGAI, H., UDA, K., lso, T. and YAMAUCHI, H. (1980) Pharmacological studies of new sulfhydryl compound 2-mercapto-2-methylpropanoylL-cysteine (SA96). Folia Pharmacol. Jap. 76:117- 129. FUJINO, Y., OKUMURA,A., NUSSENBLATT,R. B., GERY, I. and MOCHIZUKI, M. (1988) Cyclosporine-induced specific unresponsiveness to retinal soluble antigen in experimental autoimmune uveoretinis. Clin. lmmunol. lmmunopathol. 46: 234-248. FUJINO, Y., MOCHIZUKI, M., CttAN, C.-C., RABER, J., KOTAKE, S., GERV, I. and NUSSENBLATT, R. B. (1991) FK506 treatment of S-antigen induced uveitis in primates. Curt. Eye. Res. 10: 679-690. FUNG, J., ABU-ELMAGD,K., JAIN, A., GORDON, R., TZAKIS, A., TODO, S., TAKAYA,S., ALESSIANI, M., DEMETRIS, A., BRONSTER,O., MARTIN, M., MIELES,L., SELBY, R., REYES, J., DOYLE, H., STIEBER,A., CASAVILEA,A. and STARZL, T. (1991) A randomized trial of primary liver transplantation under immunosuppression with FK506 vs cyclosporine. Trans. Proc. 23: 2977-2983. GASSMAN, A. E. and VAN FURTH, R. (1975) The effect of azathioprine (imunran) on the kinetics of monocytes and macrophages during the normal steady state and an acute inflammatory reaction. Blood 46: 51. GENG, J. G., BEVlLACQUA,M. P., MOORE, K. L., MCINTYRE, T. M., PRESCOTT, S. M., KIM, J . M . , BLISS, G. A., ZIMMERMAN, G. A. and McEVER, R. P. (1990) Rapid neutrophil adhesion to activated endothelium mediated by GMP-140. Nature 343: 757- 760.

GERY, I., MOCHIZUKI, M. and NtISSIiNBLATI, R. [3. (1990a) Retinal specific antigens and imnmnopathogenic processes they provoke. In: Progress' in Retinal Research (N. N. Osborne and G . J . Chader, eds) Vol. 5, pp. 7 5 - 109. Pergamon Press, Oxford. GERY, 1., WIGGERT, B., REDMOND, T. M., KUWAIL.XRA,1.. CRAWr:ORD, M. A., V~STICa, tt. P. and CtIAI)t R, G. J. (1990b) Uveoretinitis and pinealitis induced by immunization with interphotoreceptor retinoid-binding protein. Invest. Ophthalmol. Vis. Sci. 27: 1296- 1300. GILL, 1. T. J., KUNZ, H. W., MISRA, D. N. and CORTESI, H. A. L. (1987) The major histocompatibiti|y complex of the rat. Transplantation 43: 773- 785. GONZALO, J. A., GONZALEZ-GARCI~.,A., MARTINEZ-A,C. and KROEMER, G. (1993) Glucocorticoid-mediated control of the activation and clonal deletion of peripheral Tcells in vivo. J. exp. Med. 177: 1239- 1246. GUNTER, K. C., IRVING, S. G., ZU'FEI., P. F., SIEBENLIST, U. and KELLY, K. (1989) Cyclosporin A-mediated inhibition of mitogen-induced gene transcription is specific for the mitogenic stimulus and cell lype. J. lmmunol. 142:3286 3291. HANI)SCHUMACtIER, R. E., HARDING, M. W., RICE, J. and DRUG(iV, R. J. (1984) Cyclophilin: a specific cytosolic binding protein for cyclosporin A. Science 226: 544 - 547. HARDIN(;, M . W . and HANDSCHUMACHER, R.E. (1988) Cyclophilin, a primary molecular target for cyclosporine. Transplantation 46: 29S-35S. HARDING, C. V., LEYVA-COBIAN,F. and UNANUE,E. R. (1988) Mechanisms of antigen processing. Immunol. Rev. 106: 77 - 92. HERBORT, C. P., MATSUBARA,M., NISHI, M. and MOCHIZUK1, M. (1989) Penetrating keratoplasty in the rat: a model for the study of immunosuppressive treatment of graft rejection. Jap. Z Ophthalmol. 33: 212-220. HIKITA, N., LOPEZ, J. S., DE SMET, M. D., CHAN, C.-C., MOCHIZUKI, M. and NUSSENBEATT,R. B. (1992) Use of topical FK506 in the corneal graft rejection model in Lewis rats. Invest. Ophthalmol. Vis. Sci. 33: 832. HILL, J . C . (1989) The use of cyclosporine in high-risk keratoplasty. Am. J. Ophthalmol. 107: 506-510. HILL, J. C. and MASKE,R. (1988) An animal model for corneal graft rejection in high-risk keratoplasty. Transplantation 46: 2 6 - 30. HIROSE, S., TANAKA, T., NUSSENBLATT, R. B., PALESTINE, A. G., WIGGERT, B., REDMOND,T. M., CHADER, G. J. and GERY, I. (1988) Lymphocyte responses to retinalspecific antigens in uveitis patients and healthy subjects. Curr. Eye Res. 7: 393-402. HOFFMAN, A . L . , MAKOWKA, L., BANNER, B., CAI, X., fRAMER, D.V., PASCUALONE, A., TODO, S. and STARZL, T . E . (1990) The use of FK-506 for small intestine allotransplantation. Inhibition of acute rejection and prevention of fatal graft-versus-host disease. Transplantation 49:483 - 490. HOR1, S., OCHIAI, T., TAKAJIMA,K., NAGATA,M., SAKAMOTO, K., ENOMOTO, K., GOTO, T., KENMOCHI, T., NAKAGOHRI,T., ASANO,T. and ISONO, K. (1987) Effect of 15-deoxyspergualin on cardiac allotransplantation in the rat. Ishoku 22:291 -294. Hu, S. K., MITCHO, Y. L., ORONSKY, A. L. and KERWAR, S. S. (1988) Studies on the effect of methotrexate on macrophage function. J. Rheumatol. 15: 206-209.

IMMUNOSUPPRESSANTS IN OCULAR DISEASES

HULTSCH, T., RODRIGUEZ, J . L . , KALINER, M . A . and HOHMAN, R . J . (1990) Cyclosporin A inhibits degranulation of rat basophilic leukemia cells and human basophils: inhibition of mediator release without affecting PI hydrolysis of Ca 2+ fluxes. J. Immunol. 144: 2659 - 2664. HULTSCH, T., ALBERS, M.W., SCHREIBER, S.L. and HOHMAN, R . J . (1991) Immunophilin ligands demonstrate common features of signal transduction leading to exocytosis or transcription. Proc. HatH. Acad. Sci. U.S.A. 88: 6229-6233. HUNTER, P . A . , GARNER, A., WILHELMUS, K.R., RICE, N. S.C. and JONES, B . R . (1982) Corneal graft rejection: a new rabbit model and cyclosporin A. Br. J. Ophthalmol. 66: 2 9 2 - 302. HURD, E . R . and GIULIANO, V . J . (1975) The effect of cyclophosphamide on B- and T-lymphocytes in patients with connective tissue diseases. Arthritis Rheum. 18: 67. IMVENTARZA, O., TODO, S., LIRAS, G., UEDA, Y., FURUKAWA, H., WU, Y. M., ZHU, Y., OKS, A., DEMETRIS,J. and STARZL, T. E. (1990) Renal transplantation in baboons under FK506. Transplant. Proc. 22 (Suppl. 1): 64 - 65. INAMURA, N., NAKAHARA, K., KINO, T., GOTO, T., AOKI, H., YAMAGUCHI, I., KOHSAKA, M. and OCHIAI, T. (1988) Prolongation of skin allograft survival in rats by a novel immunosuppressive agent, FK506. Transplantation 45: 206 - 209. INOU, T., KUSABA, R., TAKAHASHI, I., SUGIMOTO, n., KUZUHARA, K., YAMADA, Y., YAMAGUCHI, J. and OTSUBO, O. (1980) Clinical trial of bredinin in renal transplantation. Transplant. Proc. 12: 526-528. IRSCHICK., E., MILLER, K., BERGER, M., SCHONITZER, D., KOLLER, J., WAGNER, H., GOTTINGER, W. and HUBER, C. (1989) Studies of the mechanism of tolerance induced by short-term immunosuppression with cyclosporine in high-risk corneal allograft recipients. Transplantation 48:986 - 990. IWAKI, Y., SAWA, K., FUJITO, S. and MOCHIZUK1, M. (1993) Effects of topical FK506 on experimental allergic conjunctivitis. Ocular lrnmunol. Inflamm. 1: 7 9 - 8 5 . KASWAN, R . L . (1988) Intraocular penetration of topically applied cyclosporine. Transplant. Proc. 20: 650-655. KAWASHIMA, H. and MOCHIZUKI, M. (1990b) Effects of a new immunosuppressive agent, FK506, on the efferect limb of the immune responses. ExplEye. Res. 51:565 - 572. KAWASHIMA, H., FUJINO, Y. and MOCHIZUKI, M. (1988) Effects of a new immunosuppressive agent, FK506, on experimental autoimmune uveoretinitis in rats. Invest. Ophthalmol. Vis. Sci. 29: 1265- 1271. KAWASHIMA,H., FUJINO, Y. and MOCH/ZUKI, M. (1990a) Antigen-specific suppressor cells induced by FK506 in experimental autoimmune uveoretinitis in the rat. Invest. Ophthalmol. Vis. Sci. 31: 2500-2507. KINO, T., HATANABE, H., HASH1MOTO, M., NISHIYAMA, M., GOTO, T., OKUHARA, M., KOHSAKA, M., AOKI, H. and IMANAKA,H. (1987a) FK506, a nobel immunosuppressant isolated from a streptomyces. I. Fermentation, isolation, and physio-chemical and biological characteristics. J. Antibiotics 40: 1249- 1255. KINO, Z., HATANAKA, H., MIYAYO, S., INAMURA, N., NISHIYAMA, M., YAJIMA, Z., GOTO, T., OKUHARA, M., KOHSAKA, M., AOKI, H. and OCHIAI, T. (1987b) FK506, a novel immunosuppressant isolated from a streptomyces. II. immunosuppressive effect of FK506 in vitro. J. Antibiotics 40: 1256- 1265.

5O3

KRONKE, M., LEONARD, W. J., DEPPER, J. M., ARYA, S. K., WONG-STAAL, F., GALLO, R. C., WALDMANN, T. A. and GREENE,W. C. (1984) Cyclosporin A inhibits T-cell growth factor gene expression at the level of mRNA transcription. Proc. HatH. Acad. Sci. U.S.A. 81: 5214-5218. LAGODZINSKI, Z., GORSKI, A. and WASlK, M. (1990) Effects of FK506 and cyclosporine on primary and secondary skin allograft survival in mice. Immunology 71: 148- 152. LARSON,R. S., HIBBS,M. L. and SPRINGER, T. A. (1990) The leukocyte intergrin LFA-1 reconstituted by cDNA transfection in a nonhematopoietic cell line is functionally active and not transiently regulated. Cell Regul. 1: 359-367. LIM, S . M . L . , THIRU, S. and WHITE, D . J . G . (1987) Heterotopic heart transplantation in the rat receiving FK506. Transplant. Proc. 19 (Suppl. 6): 6 8 - 70. LIU, E. Y., RAIZMAN, M. B., ROSNER, B., IHLEY, T. M. and FOSTER, C. S. (1989) Effects of blood transfusion and cyclosporin on rabbit corneal graft survival. Curr. Eye Res. 8: 523-531. MACPHEE, I. A. M., ANTONI, F. A. and MASON, D. W. (1989) Spontaneous recovery of rats from experimental allergic encephalomyelitis is dependent on regulation of the immune system by endogenous adrenal corticosteroids. J. exp. Med. 169:431-445. MAEDA, N., HOSOYA, H., IKEDA, T., WATANABE, K., OHHASHI, Y., KINOSmTA, S. and MANABE, R. (1992) Effect of peroral cyclosporine in high-risk corneal transplantation. Jap. J. Clin. Ophthalmol. 46: 1071 - 1076 (in Japanese). MASON, D., MACPHEE, I. and ANTONI, F. (1990) The role of the neuroendocrine system in determining genetic susceptibility to experimental allergic encephalomyelitis in the rat. Immunology 70:1 - 5 . MASUDA, K., NAKAJIMA, A., URAYAMA, A., NAKAE, K., KOGURE, M. and INABA, G. (1989) Double-masked trial of cyclosporin versus colchicine and long-term open study of cyclosporin in Behcet's disease. Lancet 8647: 1093 - 1095. MATSUMURA, N. and MIZUSHIMA, Y. (1975) Leucocyte movement and colchicine treatment in Behcet's disease. Lancet 2: 813. MILLER, K., HUBER, C., NIEDERWIESER, D. and GOTTINGER, W. (1988) Successful engraftment of high-risk corneal allografts with short-term immunosuppression with cyclosporine. Transplantation 4 5 : 6 5 1 - 6 5 3 . MOCHIZUKI, M., NUSSENBLATT, R . B . , KUWABARA, T. and GERY, I. (1983) Effects of cyclosporine on the efferent limb of the immune response. Transplant. Proc. 15 (Suppl. 1): 2364- 2366. MOCHIZUKI, M., NUSSENBLATT, R. B., KUWABARA, T. and GERY, I. (1984) Effects of cyclosporine and other immunosuppressive drugs on experimental autoimmune uveoretinitis in rats. Invest. Ophthalmol. Vis. Sci. 26: 226-232, 1985. MocmzuK1, M. and KAWASmMA, H. (1990) Effects of FK506, 15-deoxyspergualin, and cyclosporine on experimental autoirnmune uveoretinitis in the rat. Autoimmunity 8: 37 - 4 1 . MOCHIZUKI, M., MASUDA,K., SAKANE,T., INABA,G., ITO, K., KOGURE, M., SUGINO, N., UsuI, M., MIZUSHIMA, Y., OHNO, S., MIYANAGA,Y., HAYASAKA,S. and OHIZUMI, K. (1991) A multicenter clinical open trial of FK506 in

5O4

M. MOCHIZUKI a n d M. DE SMEq

refractory uveitis, including Behcet's disease. Transplant. Proc. 23: 3343- 3346. MOCH1ZUKI, M. (1992) Immunotherapy in ocular diseases. Acta Soc. Ophthalmol. Jap. 96: 1608-1634 (in Japanese). MOCHIZUKI, M., MASUDA, K., SAKANE, T., ITO, K., KOGURE, M., SUGINO, N., USUI, M., MIZUSHIMA, Y., OHNO, S., INABA, G., MIYANAGA,Y., HAYASAKA,S. and OHIZUMI, K. (1993) A clinical trial of FK506 in refractory uveitis. Am. J. Ophthalmol. 115: 763- 769. MONDEN, M., GOTOH, M., KANAI, T., VALDIVIA, K., UMESHITA, K., ENDOH, W., NAKANO, Y., KAWAI, M., OHZATO,H., UKEI,T., KONO, K., TONO, T., MURATA, M., WANG, K.S., OKAMURA, J., TANIMOTO, Y., HASHIMOTO, M. and MORI, T. (1990) A potent immunosuppressive effect of FKS06 in orthotopic liver transplantation in primates. Transplant. Proc. 22 (Suppl. 1): 6 6 - 7 1 . MORIVAMA, S, (1989) Experimental tracheal reconstruction by allotransplantation. Nippon Kyobu Geka Gakkai Zassi. 37: 218-216. MORRIS, R.E., Wu, J. and SHORTHOUSE, R. (1990) Comparative immunopharmacologic effects of FK506 and cya in in vivo models of organ transplantation. Transplant. Proc. 22 (Suppl. 1): 110-112. MOSTELLER, M. W., GEBHARDT, B. M., HAMILTON, A. M. and KAUFMAN, H . E . (1985) Penetration of topical cyclosporine into the rabbit cornea, aqueous humor, and serum. Archs Ophthalmol. 103:101 - 102. MURASE, N., TODO, S., LEE, P. H., LAI, H. S., CHAPMAN, F., NALESNtK, M . S . , MKOWKA, L. and STARZL, T . E . (1987) Heterotopic heart transplantation in the rat receiving FK-506 alone or with cyclosporine. Transplant. Proc. 19 (5 Suppl. 6): 71 -75. MURASE, N., KIM, D. G., TODA, S., CRAMER, D. V., FUNG, J . J . and STARZL, T . E . (1990a) Suppression of allograft rejection with FK506. I. Prolonged cardiac and liver survival in tars following short-course therapy. Transplantation 50:186 - 189. MURASE, N., KIM, D. G., TODA, S., CRAMER, D. V., FUNG, J. and STARZL, T. E. (1990b) FK506 suppression of heart and liver allograft rejection. II. The induction of graft acceptance in rats. Transplantation 50: 7 3 9 - 744. NAGATA, Y., KATABAMI,T., SEO, A., NUMAGA, J., HIJIKATA, K., TABUCHI, S., HAYASHI, K., IZAWA, Y., ANDO, K., KAWASHtMA, H. and MOCHIZUKI, M. (1992) Clinical efficacy of initial low-dose cyclosporin therapy in Behcet's disease. Folia Ophthalmol. Jap. 43: 6 - 12 (in Japanese). NI, M., CHAN, C.-C., NUSSENBLATT,R. B. and MOCHIZUKI, M. (1990) FR900506(FK506) and 15-seozyspergualin (15-DSG) modulate the kinetics of infiltrating vells in eyes with experimenitis. Autoimmunity 8: 4 3 - 51. NISHI, M., HERBORT, C. P., MATSUBARA,M., MORISHITA, Y., NISHIMURA, M., NIEDA, M., MORI, S. and MOCHIZUKI, M. Effects of the immunosuppressent FK506 on a penetrating keratoplasty rejection model in rat. Invest. Ophthalmol. Vis. Sci. (in press). NUSSENBLATT, R, B., GERY, I., BALLINTINE, E . J . and WACKER,W. B. (1980) Cellular immune responsiveness of uveitis patients to retinal S-antigen. Am. J. OphthalmoL 89:173 - 179. NUSSENBLATT, R.B., RODRIGUES, M.M., WACKER, W.., CEVARtO, S. J., SALINAS-CARMONA,M. C. and GERY, I.

(1981) Cyclosporin A: inhibition of experimental autoimmune uveitis in Lewis rats. J. Clin. Invest. 67: 1228 - 1231. NUSSENBLATT, R. B., MITTAL, K. K., RYAN, S. JR, GREIN, W . R . and MAUMENEE, A . E . (1982) Birdshot retionochoroidaopathy associated with HLA-A29 antigen and immune responsiveness to retinal S-antigen. Am. J. Ophthalmol. 94: 147-158. NUSSENBLATT, R. B., PALESTINE, A. G., ROOK, A. H., S( tl[_'R, I., WACKER, W. and GERY, 1. (1983) Treatment of intraocular inflammatory disease with cyclosporin A. Lancet 8344:235 - 238. NUSSENBLATT, R.B. and PALESTINE, A. G. (1989) Uveitis: fundamentals and clinical practice. Year Book Medical Publishers, Chicago. NUSSENBLATT, R. B,, CASPI, R., MAHDI, R., CHAN, C.-C., ROBERGE, F., LIDER, O. and WEINER, H. (1990) Inhibition of S-antigen induced experimental autoimmune umeroetinitis by oral induction of tolerance with S-antigen. J. Immunol. 144: 1689- 1695. OCHIAI, T., HORI, S., NAKAJIMA,K., NAGATA,M., ASANO, T., ISONO, K. and UMEZAWA,H. (1987a) Prolongation of rat heart allograft survival by 15-deoxyspergualin..L Antibiotics 40:249 - 250. OCHIAI, T., NAKAJIMA, K., NAGATA, M., HORI, S., ASANO, T. and ISONO, K. (1987b) Studies of the induction and maintenance of long-term graft acceptance by treatment with FK506 in heterotopic cardiac allotransplantation in rats. Transplantation 44: 734-738. OCHIAI, T., NAKAJIMA, K., NAGATA, M., SUZUKI, T., ASANO, T., UEMATSU, T., GOTO, T., HORI, S., KENMOCHI, T. and ISONO, K. (1987c) Effects of a new immunosuppressive agent, FK506, on hetrotopic cardiac allontransplantation in the rat. Transplant. Proc. 19: 1284. OCHIAI, T., NAGATA,M., NAKAJIMA,K., ISONO, K., INAMURA, N. and NAKAHARA, K. (1987d) Effects of FK-506 on xenotransplantation of the heart and skin in a mouse-rat combination. Transplant. Proc. 19 (Suppl. 6): 8 4 - 8 6 . OCHIAI, Y., NAGATA, M., NAKAJIMA, K., SUZUKI, T., SAKAMOTO, K., ENOMOTO, K., GUNJI, Y., UEMATSU,T. and HOR1, S. (1987e) Studies of the effects of FK506 on renal allografting in the beagle dog. Transplantation 44: 729 - 733. PARRtLLO, J. E. and FAUCl, A. S. (1978) Comparison of the effector cells in human spontaneous cellular cytotoxicity and antibody dependent cellular cytotoxicity: differential sensitivity of effector cells to in vivo and in vitro corticosteroids. Scancl. J. Immunol. 8: 9 9 - 107. PARRILLO, J . E . and FAUCI, A . S . (1979) Mechanisms of glucocorticoid action on immune processes. Ann. Rev. Pharmacol. Toxicol. 19: 179-201. PH1LIPS, S. M. and ZWE1MAN, B. (1973) Mechanisms in the suppression of delayed hypersensitivity in the guinea pig by 6-mecaptopurine. J. exp. Med. 137: 1494. REEM, G. H., COOK, L. A. and VILCEK, J. (1983) Gamma interferon synthesis by human thymocytes and Tlymphocytes inhibited by cyclosporin A. Science 221: 63 -65. ROBERGE,F. G., LORBERBOUM-GALSKI,H., LE HOANG, P., DE SMET, M., CHAN, C.-C., FITZGERALD, D. and PASTAN, I. (1989) Selective immunosuppression of activated Tcells with the chimeric toxin IL-2-PE40. Inhibition of experimental autoimmune uveoretinitis. J. lmmunol. 143:3498 - 3502.

IMMUNOSUPPRESSANTSIN OCULAR DISEASES ROBERGE, F. G., XU, D., CHAN, C.-C., DE SMET, M. D., NUSSENBLATT,R. B. and CHEN, H. (1993) Treatment of autoimmune uveoretinitis in the rat with rapamycin, an inhibitor of lymphocyte growth factor signal transduction. Curt. Eye Res. 12: 197- 203. ROBERTS, J. J., BRENT, T. P. and CRATHRON, A. R. (1971) Evidence for the inactivation and repair of the mammalian DNA template after alkylation by mustard gas and half mustard gas. Fur. J. Cancer 7: 515. SAITOH, Y., FUJISAWA, T., OGAWA, T., URABE, N., YAMAGUCHI, Y. and KIMIZUKA, G. (1990) Morphological rejection phases and cytotoxic activity in peripheral blood lymphocytes in canine lung allotransplantation. Jap. J. Surg. 20: 205- 211. SALISBURY,J. D. and GEBHARDT,B. M. (1981) Suppression of corneal allograft rejection by cyclosporin A. Archs Ophthalmol. 99: 1640-1643. SATO, K., YAMAGISHI, K., NAKAYAMA, Y., YOKOTA, K., UCHIDA, H., MASAKI,Y., WATANABE,K. and Aso, K. (1989) Pancreaticoduodenal allotransplantation with cyclosporine and FK506. Transplant. Proc. 21: 1074- 1075. SCHLESINGER,M. and ISRAEL,E. (1974) The effect of lectins on the migration of lymphocytes in vivo. Cell Immunol. 14:66 - 79. SCHREBER, S.L. "(1991) Chemistry and biology of the immunophilins and their immunosuppressive ligands. Science 251:283 - 287. SECCH1, A . G . , TOGNON, M.S. and LEONARDI, A. (1990) Topical use of cyclosporine in the treatment of vernal keratoconj unctivitis. Am. J. Ophthalmol. 110: 641 - 645. SEHGAL, S. R., BAKER, H. and VEZINA, C. (1975) Rapamycin (AY-22, 989), a new antifungal antibiotic. II. Fermentation, isolation and characterization. J. Antibiot. 28: 727-732. SEGAL, R., YARON, M. and TARTAKOVSKY, B. (1990) Methotrexate: mechanism of action in rheumatoid arthritis. Sere. Arthritis. Rheum. 20: 190-199. SELYE, H. (1953) On the mechanism through which hydrocortisone affects the resistance of tissues to injury. J. Am. reed. Assoc. 152: 1207-1213. SHAPIRO, R., JORDAN, M., SCANTLEBURY, V., FUNG, J., JENSEN, C., TZAKIS, A., MCCAULEY,J., CARROLL, P., RICORDI, C., DEMETRIS,A. J., MITCHELL,S., JAIN, A., IWAKI, Y., KOBAYASHI, M., REYES, J., TODO, S., HAKALA, T. R., SIMMONS, R. L. and STARZL, T. E. (1991) FK506 in clinical kidney transplantation. Transplant. Proc. 23: 3065- 3067. SHEPHERD, W. F. I., COSTER, D. J., CHIN FOOK, T., RICE, N. S. C. and JONES, B. R. (1980) Effect of cyclosporin A on the survival of corneal grafts in rabbits. Br. J. Ophthalmol. 64: 148-153. SIEKIERKA, J. J., HUNG, S. H. Y., POE, M., SHIRLEY, C. and SIGAL, N. H. (1989a) A cytosolic binding protein for the immunosuppressant FK506 has peptidyl-prolyl isomerase activity but is distinct from cyclophilin. Nature 341:755 - 757. SIEKIERKA,J. J., STARUCH,M. J., HUNG, S. n. Y. and SIGAL, N. H. (1989b) FK-506, a potent novel immunosuppressive agent, binds to a cytosolic protein which is distinct

5O5

from the cyclosporin A-binding protein, cyclophilin. J. lmmunol. 143: 1580-1583. SMITH, C. W., KISHIMOTO,T. K., ABBASS1,O., HUGHES, B., ROTHLEIN, R., MCINTIRE, L.V., BUTCHER, E. and ANDERSON, C. D. (1991) Chemotactic factors regulate lectin adhesion molecule 1 (LECAM-l)-dependent neutrophil adhesion to cytokine-stimulated endothelial cells in vitro. J. Clin. Invest. 87: 609-618. SPRINGER, T. A. (1990) Adhesion receptors of the immune system. Nature 346:425 - 434. STARZL, T. E., FUNG, J., VENKATARAMMAN,R., TODO, S., DEMETRIUS,A. J. and JAIN, A. (1989) FK506 for liver, kidney, and pancreas transplantation. Lancet 2: 1000- 1004. STERNBERG, E . M . , YOUNG III, W.S., BERNARDINI, R., CALOGERO,A. E., CHROUSOS,G. P., GOLD, P. W. and WILDER, R. L. (1989) A central nervous system defect in biosynthesis of corticotropin-releasing hormone is associated with susceptibility to streptococcal cell wallinduced arthritis in Lewis rat. Proc. natn. Acad. Sci. U.S.A. 86:4771-4775. STOOLMAN, L . M . (1989) Adhesion molecules controlling lymphocyte migration. Cell 56:907 - 910. TAKAHASHI,N., HAYANO,T. and SUZUKI,M. (1989) Peptidylprolyl cis-trans isomerase is the cyclosporin A-binding protein cyclophilin. Nature 337:473 - 475. TAMATANI,T., KOTANI, M., TANAKA,T. and MIYASAKA,M. (1991) Molecular mechanisms underlying lymphocyte recirculation. II. Differential regulation of LFA-1 in the interaction between lymphocytes and high endothelial cells. Eur. J. Immunol. 21: 855-888. TODO, S., DEMETRIS, A . J . , UEDA, Y., IMVENTARZE,O., OKUDA, K., CASAVILLA,A., CEMAJ, S., GHALAB, A., MAZZAFERRO, B., RHOE, B.S., TONGHUA, Y., MAKOWKA,L. and STARZL,T. E. (1987a) Canine kidney transplantation with FK-506 alone or in combination with cyclosporine and steroids. Transplant. Proc. 19 (Suppl. 6): 57-61. TODO, S., PODESTA, L., CHAPCHAP,P., KAHN, D., PAN, C.E., UEDA, Y., OKUDA, K., IMVENTARZA, O., CASAVILLA, A., DEMETRIS, A. J., MAKOWKA, t . and STARZL, T. E. (1987b) Orthotopic liver transplantation in dogs receiving FK-506. Transplant. Proc. 19 (Suppl. 6): 64-67. TODO, S., TZAKIS, A., REYES, J., ABU-ELMAGD, K., CASAVILLA,A., NOUR, B. M., SELBY, R., FUNG, J. J., VAN THIEL, D. and STARZL,T. E. (1991) Clinical small bowel or small bowel plus liver transplantation under FK506. Transplant. Proc. 103: 3093- 3098. TSUCHIMOTO,S., KUSUMOTO,K., NAKAJIMA,Y., KAKITA,A., UCHINO, J., NATORI,T., AIZAWA,M. (1989)Orthotopic liver transplantation in rats receiving FK506. Transplant. Proc. 21: 1064-1065. TZE, W. J., TAI, J., CHEUNG,S.-S. C., MURASE,N., RICORDI, C. and STARZL,T. E. (1991) Long-term survival of islet allografts in diabetic rats treated with FK506. Transplant. Proc. 103: 3208-3210. UCHIDA, H., YOKOTA,K., AKIYAMA,N., MASAKI,Y., ASO, K., OKUBO, M., OKUDAIRA,M., KATO, M. and KASIWAGI, N. (1979) Effectiveness of a new drug, bredinin, on

506

M. MOCHIZUKI and M. DE SMET

canine kidney allotransplant survival. Transplant. Proc. 11: 865-870. UNANUE, E . R . and CEROTTINI, J . C . (1989) Antigen presentation. F A S E B 3: 2496- 2502. VACCA, A., FELLI, M . P . , FARINA, S., MARTINOTTI, S., MARODER, M., SCREPANTI,1., MECO, D., PETRANGELI, E., FRETI, L. and GULINO, A. (1992) Glucocorticoid receptor-mediated suppression of the interleukin 2 gene expression through impairment of the cooperativity between nuclear factor of activated T-cells and AP-I enhancer elements. J. exp. Med. 175: 637. VEZ1NA, C., KUDELSKI, A. and SEHGAI_, S . N . (1975) Rapamycin (AY-22,989), a new antifungal antibiotic. Taxonomy of the producing streptomycete and isolation of the active principle. J. Antibiot. 28:721 - 726. VILLAS, P. A., DRONSFIELD, M. J. and BLACKENHORN, E. P. (1991) Experimental allergic encephalomyelitis and corticosterone studies in resistant and susceptible rat strains. Clin. Immunol. lmmunopathol. 61: 29-40. WAAGE, A., SLUPPHANG, G. and SHALABY, R. (1990) Glucocorticoids inhibit the production of IL-6 from monocytes endothelial cells and fibroblasts. Eur. J. Immunol. 20: 2439-2443. WACKER, W . B . , DONOSO, L . A . , KALSOW, C . M . , YANKEELOV, J. A. JR and ORGANISCIAK, D. T. (1977) Experimental allergic uveitis. Isolation, characterization, and localization of a soluble uveito-pathogenic antigen from bovine retina. .L lmmunol. 119: 1949- 1958.

WAI.Z, G., ARUFFO, A., KOLANUS,W., [~EVIIA( q~l /\, M. and SEED, B. (1990) Recognition by ELAM1 of the sialylLex determinant on myeloid and tumor cells. Sciem'c 250: 1132- 1135. WEIZIG, R., HOOKS, J. J., PERCOPO,C. M., NUSSINBt.AII, P,., CHAN, C.-C. and DETRI( K, B. (1988) Anti-la antibody diminishes ocular inflammation in experimental autoimmune uveitis. Curt. Eye Res. 7:809 -818. WIEDERHOLT, M., KOSSENDRUP,D., SCHULZ, W., I~IOFFMAN, F. (1986) Pharmacokinetics of topical cyclosporin A in the rabbit eye. Invest. Ophthal. Vi~. Sci. 27: 519- 524. WILLIAMS, K. A., ERICKSON, S. A. and COSTER, D. J. (1987) Topical steroid, cyclosporin A, and the outcome of rat corneal allografts. Br. J. Ophthalrnol. 71: 239-242. YASUNAMI, Y., RYU, S., KAMEI, T. (1990) FK506 as the sole immunosuppressive agent for prolongation of islet allograft survival in the rat. Transplantation 49: 682 - 686. Yu, D. T., CLr!MENTS,P. J., PETER, J. B., LEVY, J., PAULU%, H . E . and BARNETT, E.V. (1974) Lymphocyte characteristics in rheumatic patients and the effect of azathioprine therapy. Arthritis. Rheum. 17: 37. ZEMBOWICZ, A. and VANE, J. R. (1992) Induction of nitric oxide synthase activity by toxic shock syndrome toxin 1 in macrophage-monocyte cell lines. Proc. hath. Acad. Sci. U.S.A, 89: 2052-2055. ZUBIAGA, A. M., MUNOZ, E. and HU13ER, B. T. (1992) IL-4 and IL-2 selectively rescue Th cell subsets from glucocorticoid-induced apoptosis. J. ImmunoL 149: 1(17- 112.