role in the pathogenesis of the choroiditis and scleritis in this strain. Invest Ophthalmol ... seen in these patients are scleritis, present in patients with rheumatoid ...
Investigative Ophthalmology & Visual Science, Vol. 32, No. 10, September 1991 Copyright © Association for Research in Vision and Ophthalmology
Autoimmune Ocular Disease in MRL/Mp-lpr/lpr Mice Is Suppressed by Anti-CD4 Antibody Douglas A. Jobs and Robert A. Prendergasr MKL/Mv-lpr/lpr (MRL/lpr) mice spontaneously have a systemic autoimmune disease, characterized by vasculitis, lymphadenopathy, glomerulonephritis, and autoantibody formation. Among the many autoimmune lesions present are focal ocular inflammatory infiltrates, involving the choroid and sclera. These lesions appear to be related to the vasculitis seen in MRL/lpr mice and are mediated by L3T4-positive helper T-cells (CD4-positive T-cells). Systemic treatment of MRL/lpr mice with a monoclonal anti-L3T4 antibody (anti-CD4) resulted in a dramatic reduction of both the frequency and severity of the ocular disease, supporting the hypothesis that the CD4-positive T-cells play an essential role in the pathogenesis of the choroiditis and scleritis in this strain. Invest Ophthalmol Vis Sci 32:2718-2722,1991
Ocular involvement is common in systemic autoimmune diseases in humans. Among the lesions seen in these patients are scleritis, present in patients with rheumatoid arthritis and vasculitis;12 secondary Sjogren's syndrome in patients with rheumatoid arthritis and systemic lupus erythematosus (SLE);2 and uveitis in patients with spondyloarthopathies.2 Choroidal involvement is less frequent, but it was described in patients with SLE and vasculitis.2 There are several strains of autoimmune mice, including MRL/ Up-lpr/lpr (MRL/lpr), MRL/Mp-+/+ (MRL/+), (NZBXNZW) Fl hybrid (NZB/W), BxSB, and Palmerston-North strains.3"6 Among the autoimmune lesions seen in MRL/lpr mice are focal ocular inflammatory infiltrates involving the choroid and sclera.7'8 These lesions are present in MRL/lpr mice, but not in congenic MRL/+ mice or in NZB/W mice.8 The choroidal and scleral lesions in these mice appear to be related to the vasculitis commonly seen in MRL/lpr mice,8 and the lesions consist largely of CD4-positive helper T-cells. We found that treatment of these mice with systemic, monoclonal anti-CD4 antibody results in marked suppression of the ocular autoimmune pathology.
From the Wilmer Ophthalmological Institute, the Department of Ophthalmology, the Johns Hopkins University School of Medicine, Baltimore, Maryland. Supported by grants EY05912 and EY03521 from the National Institutes of Health, Bethesda, Maryland, and a grant from Research to Prevent Blindness, New York, NY. Submitted for publication: November 21, 1990; accepted April 25, 1991. Reprint requests: Douglas A. Jabs, MD, Wilmer Ophthalmological Institute, 550 North Broadway, Suite 700, Baltimore, MD 21205.
Materials and Methods Monoclonal Antibodies Monoclonal antibodies were prepared as previously described.910 Hybridoma cells secreting a monoclonal antibody (GK 1.5) to the murine CD4 antigen L3T4 were obtained from the American Type Culture Collection (Rockville, MD).11'12 One million cells were injected into the peritoneal cavity of mice with severe combined immunodeficiency (SCID) obtained from the Jackson Laboratories (Bar Harbor, ME). These mice lack both T- and B-cell function; therefore, all immunoglobulin recovered from the peritoneal cavity would be monoclonal antibody secreted by the hybridoma cells. Antibody was harvested from the SCID peritoneal exudate using daily paracentesis starting 8 days after cell transfer. The immunoglobulin was purified partially using ammonium sulfate precipitation and dialysis against phosphate-buffered saline. The partially purified antibody then was standardized for protein content using spectrophotometric absorbance at 280 nm. Treatment Protocol
The MRL/lpr mice were obtained from the Jackson Laboratories and kept under standard conditions in the animal facilities of the Woods Research Building of the Johns Hopkins Hospital. The animals were treated starting at 1 month of age with one initial intravenous injection and weekly intraperitoneal injections of 2 mg of monoclonal anti-L3T4. Two control groups were used; one was given injections of normal saline, and a second was given injections of normal rat serum (NRS). The normal rat serum used in these experiments underwent ammonium sulfate precipitation and dialysis identical to that used for the prepara-
TREATMENT OF MICE WITH ANTI-L3T4 / Jobs and Prendergosr
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Table 1. Treatment of MRL/lpr mice Treatment group Saline Normal rat serum Anti-L3T4
Any ocular inflammation*
Anterior episcleritis
8/9 9/9 2/9
6/9 8/9
m
Choroiditis
Scleritis
5/9 9/9 1/9
4/9 2/9 0/9
Number of animals with lesions/number of animals analyzed.
>~
tion of the monoclonal anti-L3T4. At 5 months of age, the animals were killed by exsanguination, and both their eyes were removed. Both eyes were fixed in 4% buffered formaldehyde, embedded in paraffin, sectioned at 5 /im, and stained with hematoxylin and eosin. These experiments conformed to the ARVO Resolution on the Use of Animals in Research. Results
->
The results of treatment are shown in Table 1. In the two control groups, saline and NRS, ocular inflammatory disease was common. Eight of nine salinetreated animals had some type of ocular inflammation, and all nine NRS-treated animals had ocular disease. By contrast, only two of the nine animals treated with monoclonal anti-L3T4 had ocular disease (P < 0.001, by Fisher's exact test). The ocular inflammation could be divided into three types of lesions. The most common one was an anterior episcleritis; the second, a choroidal infiltrate; and the third, posterior
Fig. 1. Anterior episcleritis in a saline-treated MRL/lpr mouse (H & E, X525).
scleritis. Anterior episcleritis was present in six of nine saline-treated and eight of nine NRS-treated animals but only in two of nine anti-L3T4-treated animals (P < 0.001). In general, the episcleral lesions consisted of a mononuclear inflammatory cell infiltrate surrounding small vessels in the episcleral region at the angle of the eye (Fig. 1). Anti-L3T4-treated animals generally had no such lesions (Fig. 2). Similarly, choroiditis was present in five of nine saline-treated and nine of nine NRS-treated mice but only one of nine anti-L3T4treated mice (P < 0.001). In the control mice, the prominent cell infiltrate was mononuclear in nature (Fig. 3), and anti-L3T4-treated mice had a normal-appearing choroid (Fig. 4) with the single exception noted. Scleritis was seen in four of nine saline-treated, two of nine NRS-treated, and none of the anti-L3T4treated mice (P = 0.07). The primary lesion in the sclera was centered around small vessels where a mononuclear inflammatory reaction of the same general character was present. Other ocular lesions included orbital vasculitis in one saline-treated mouse
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Fig, 2. Anti-L3T4-treated MRL/lpr mouse showing absence of inflammation in the anterior episcleral region (H & E, X525).
and optic perineuritis in one NRS-treated mouse. No anti-L3T4-treated mice had other ocular lesions These results show a significant reduction in both the frequency and severity of the ocular lesions by treatment with anti-L3T4. Two additional animals were killed at 3 months of age for fluorescein-activated cell sorted (FACS) analysis of the lymphocyte populations of their spleens. This analysis showed virtually com-
plete elimination of the L3T4-positive cell population from the spleens of the anti-L3T4-treated MRL/lpr mice, with a reduction of the L3T4-positive T-cells from 18.1% in the saline-treated group and 20.8% in the NRS-treated group to 0.6% in the anti-L3T4treated group. In addition to the beneficial effect of treatment with monoclonal anti-L3T4 on the ocular lesions, such
Fig. 3. Choroiditis in a salinetreated MRL/lpr mouse (H & E, X525).
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TREATMENT OF MICE WITH ANTI-L3T4 / Jobs ond Prendergosr
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Fig. 4. Choroid of an anti-L3T4treated MRL/lpr mouse, showing absence of inflammation (H & E, X500).
treatment also had a similar effect on the systemic disease. The adenopathy markedly improved, and lymph node weights were reduced 60%. Double-negative T-cells were still present in the lymph nodes, but the size of nodes markedly improved. The vasculitis similarly was eliminated; renal vasculitis was detected in six saline-treated animals, seven NRS-treated animals, and no anti-L3T4-treated animals. Anti-nDNA antibodies were eliminated by treatment and the elevated immunoglobulin levels seen in control mice were not present in anti-L3T4-treated animals (manuscript in preparation). Thus, anti-L3T4 therapy had a beneficial effect on the systemic autoimmune disease, in particular the vasculitis, compared with the control groups in this study. Discussion MRL/lpr mice are congenic with MRL/+ mice and differ from them only by the presence of the autosomal recessive gene, the Ipr gene. This gene accelerates the autoimmune disease in MRL mice and causes a massive lymphadenopathy.3"6 The primary immunologic disorder in MRL/lpr mice appears to be T-cell mediated. Neonatal thymectomy of MRL/lpr mice1314 or treatment with monoclonal anti-T-cell antibody9 results in the amelioration of the autoimmune disease in this strain. Similarly, the polyclonal B-cell activation, hypergammaglobulinemia, and autoantibody formation in MRL/lpr mice appear to be T-cell driven because lymphocytes of MRL/lpr mice spontaneously secrete a B-cell differentiation factor in
vitro.1516 Among the many systemic lesions seen in MRL/lpr mice is vasculitis.17"19 This vasculitis appears to be largely T-cell mediated19 with most cells being L3T4-positive helper T-cells. L3T4 is the murine CD4 antigen. Because the ocular lesions in MRL/ Ipr mice appear to be related to the vasculitis8 and because this process is largely L3T4-positive helper T-cell mediated,19 we hypothesized that treatment with anti-L3T4 would result in amelioration of the disease in MRL/lpr mice. A dramatic reduction in both the incidence and severity of the ocular lesions was seen in the group treated with anti-L3T4; this was consistent with our hypothesis that the ocular disease was mediated by CD4-positive (L3T4-positive) helper T-cells. MRL/lpr mice also develop massive lymphadenopathy not seen in the congenic MRL/+ substrain. These massively enlarged lymph nodes are composed primarily of double-negative T-cells.20"22 These cells are Thy 1.2 positive but L3T4 and Lyt 2 negative. The exact nature of these double-negative T-cells and their relationship to the other autoimmune lesions seen in MRL/lpr mice are unknown. However, the predominance of the L3T4-positive helper T-cells in the target organ lesions of MRL/lpr mice19 and the elimination of the ocular inflammatory lesions by anti-L3T4 therapy suggest that these double-negative T-cells are not involved directly in the autoimmune process in the target organ lesions. In the past, many of the ocular inflammatory lesions in humans, such as scleritis and uveitis, were
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believed to be caused by circulating immune complexes and immune complex deposition disease. More recently there has been evidence that ocular inflammatory lesions may be T-cell mediated. Experimental allergic uveal retinitis (EAU) can be transferred by T-cells,23 and others24 showed that treatment of EAU with monoclonal anti-CD4 antibodies can ameliorate this disease in rats. Our results in treating scleritis and choroiditis in the MRL/lpr animal model suggest that these lesions are also helper T-cell mediated. The successful treatment of one patient with scleritis with cyclosporine,25 a drug that is selectively active against T-cells,26'27 suggests that some cases of scleritis in humans also may be helper T-cell mediated. Key words: autoimmune disease, choroiditis, monoclonal antibody therapy, MRL/Mp-lpr/lpr mice, scleritis, vasculitis
Acknowledgments The authors thank Bella Lee for technical assistance and Amy KJemm for preparation of the manuscript.
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antibody GK-1.5: Similarity of L3T4 to the human Leu-3/T4 molecule. J Immunol 131:2445, 1983. 12. Dialynas DP, Wilde DB, Marrack P, Pierres A, Wall KA, Havran W, Hen GO, Loken MR, Pierres M, Kappler J, and Fitch FW: Characterization of the murine antigenic determinant, designated L3T4 A, recognized by monoclonal antibody GK-1.5: Expression of L3T4 A by functional T cell clones appears to correlate primarily with class IIMHC antigen reactivity. Immunol Rev 74:29, 1983. 13. Steinberg AD, Roths JB, Murphy ED, Steinberg RT, and Raveche ES: Effects of thymectomy or androgen administration upon the autoimmune disease of MRL/Mp-lpr/lpr mice. J Immunol 125:871, 1980. 14. Hang L, Theofilopoulos AN, Balderas RS, Francis SJ, and Dixon FJ: The effect of thymectomy on lupus-prone mice. J Immunol 132:1809, 1984. 15. Prud'homme GJ, Park CL, Fieser TM, Kofler R, Dixon FJ, and Theofilopoulos AN: Identification of a B cell differentiation factor(s) spontaneously produced by proliferating T cells in murine lupus strains of the Ipr/lpr genotype. J Exp Med 157:730, 1983. 16. Prud'homme GJ, Balderas RS, Dixon FJ, and Theofilopoulos AN: B cell dependence on and response to accessory signals in murine lupus strains. J Exp Med 157:1815, 1983. 17. Berden JHM, Hang L, McConahey PJ, and Dixon FJ: Analysis of vascular lesions in murine SLE: I. Association with serologic abnormalities. J Immunol 130:1699, 1983. 18. Alexander EL, Moyer C, Travlos GS, Roths JB, and Murphy ED: Two histopathologic types of inflammatory vascular disease in MRL/Mp autoimmune mice: Model for human vasculitis in connective tissue disease. Arthritis Rheum 28:1146, 1985. 19. Jabs DA and Prendergast RA: Reactive lymphocytes in lacrimal gland and renal vasculitic lesions of autoimmune MRL/ Ipr mice express L3T4. J Exp Med 166:1198, 1987. 20. Theofilopoulos AN, Eisenberg RA, Bourdon M, Crowel JS Jr, and Dixon FJ: Distribution of lymphocytes identified by surface markers in murine strains with systemic lupus erythematosus-like syndromes. J Exp Med 149:516, 1979. 21. Lewis DE, Giorgi JV, and Warner NL: Flow cytometry analysis of T cells and continuous T-cell lines from autoimmune MRL/lpr mice. Nature 289:298, 1981. 22. Wofsy D, Hardy RR, and Seaman WE: The proliferating cells in autoimmune MRL/lpr mice lack L3T4, an antigen on "helper" T cells that is involved in the response to class II major histocompatibility antigens. J Immunol 132:2686, 1984. 23. Mochizuki M, Kuwabara T, McAllister C, Nussenblatt RB, and Gery I: Adoptive transfer of experimental autoimmune uveoretinitis in rats: Immunopathogenic mechanisms and histologic features. Invest Ophthalmol Vis Sci 26:1, 1985. 24. Atalla L, Linker-Israeli M, Steinman L, and Rao NA: Inhibition of autoimmune uveitis by anti-CD4 antibody. Invest Ophthalmol Vis Sci 31:7, 1990. 25. Nussenblatt RB and Palestine AG: Cyclosporine: Immunology, pharmacology, and therapeutic uses. Surv Ophthalmol 31:159, 1986. 26. Hess AD, Tutschka PJ, and Santos GW: Cyclosporine induced tolerance-mechanism of maintaining specific immunologic unresponsiveness. Transplant Proc 15:539, 1983. 27. Wagner H: Cyclosporin A: Mechanism of action. Transplant Proc 15:523, 1983.
