patients include scleritis, found in patients who had rheumatoid arthritis or vasculitis [reviewed in 1]; sec- ondary Sjogren's syndrome, seen in patients who had.
Investigative Ophthalmology & Visual Science, Vol. 32, No. 6, May 1991 Copyright © Association for Research in Vision and Ophthalmology
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Ocular Inflammation in MRL/Mp-lpr/lpr Mice Douglas A. Jobs and Robert A. Prendergasr A systemic autoimmune disease spontaneously developed in MRL/Mp-lpr/lpr (MRL/lpr) mice. The disease was characterized by vasculitis, lymphadenopathy, glomerulonephritis, and autoantibody formation. Among the many autoimmune lesions are focal ocular inflammatory infiltrates involving the choroid and sclera. Of 104 mice examined histologically, the sclera was most often involved, with 30% of mice 5 months of age or older having scleral lesions that were often centered around small arteries. The choroid was the second most frequently involved tissue, with focal inflammation developing in 16% of these mice. Immunohistologic analysis of the ocular infiltrates showed that most of the mononuclear cells seen were L3T4 + T cells (CD4+ helper T cells), suggesting that the process was largely T cell-mediated. Smaller numbers of B cells and Lyt 2+ T suppressor/cytotoxic cells were seen. No such lesions were seen in congenic MRL/Mp- + / + , (NZBxNZW) Fl hybrid mice, and control BALB/c mice; vasculitis did not develop in all of the mice. These results suggest that the ocular lesions in MRL/lpr mice may be a model for ocular involvement in patients who have systemic necrotizing vasculitis. Invest Ophthalmol Vis Sci 32:1944-1947,1991
In humans, ocular involvement is common in systemic autoimmune diseases. The lesions seen in these patients include scleritis, found in patients who had rheumatoid arthritis or vasculitis [reviewed in 1]; secondary Sjogren's syndrome, seen in patients who had rheumatoid arthritis or systemic lupus erythematosus (SLE);1 and uveitis seen in patients who had spondyloarthropathies.' Choroidal involvement is less frequent, but has been described in patients who had SLE and vasculitis.1 Several strains of autoimmune mice have been described [reviewed in 2], including the MRL/Mp-lpr/lpr (MRL/lpr), the MRL/Mp- +/+ (MRL/+), (NZBxNZW) Fl hybrid (NZB/W), BxSB, and Palmerston-North (PN) strains. Focal lacrimal gland inflammatory infiltrates develop in all of the MRL/lpr, MRL/+, and NZB/W mice and this is a model for the human disorder, Sjogren's syndrome.3"5 We previously reported ourfindingsof analyzing these three strains for lacrimal gland inflammation and here, report our results of histologic analysis of the eyes of these mice for ocular inflammation.
Materials and Methods. Animals: MRL/lpr, MRL/ +, NZB/W, and BALB/c mice were obtained commercially (Jackson Laboratory, Bar Harbour, Maine) and kept under standard conditions in the animal facilities of the Woods Research Building of the Johns Hopkins Hospital. Animals were killed by exsanguination and both eyes were removed. One eye was fixed in 4% buffered formaldehyde, embedded in paraffin, sectioned at 5 n, and stained with hematoxylin and eosin. The other eye was embedded in OCT, frozen in liquid nitrogen, sectioned at 6 ^ on a cryostat, and stained as described below. Approximately 10-20 mice, in groups of approximately equal numbers of males and females, were killed at various time points during the course of their autoimmune disease. For MRL/lpr mice, death occured at 1,2, 3, 4, 5, 6, and occasionally 7 or more months of age; for MRL/+ mice, animals were killed at 1, 3, 6, 9, 12, 18 months, and 2 yr of age. For NZB/W mice, animals were killed at 1, 3, 6,9 months, and 1 yr of age; for BALB/c mice, animals were killed at 1,2,3,4, 5,6,9,12,18 months, and 2 yr of age. In addition to these ocular structures, lacrimal gland and other tissues were removed and analyzed. These results have been previously reported.57 These investigations adhered to the ARVO resolution on the use of animals in research. Immunohistologic analysis: Analysis of frozen sections of eyes was performed as previously described,57 using a panel of monoclonal antibodies to cell surface markers and the avidin-biotin-peroxidase (ABC) technique.8 Monoclonal antibodies used were: rat antiThy 1.2 (Becton Dickinson, Mountain View, CA) for T cells; rat anti-L3T4 (GK 1.5, American Tissue Culture Collection [ATCC], Rockville, MD) for helper T cells (CD4); rat anti-Lyt 2 (Becton Dickinson) for suppressor/cytotoxic T cells (CD8); rat anti-Mac 3 (M3/ 84.6.34, ATCC) for macrophages; goat F (ab')2 antimouse IgG + IgM (Tago, Inc., Burlingame, CA) for surface immunoglobulin on B cells (slg), or a pan-B cell (pre-B, B cells, and plasma) monoclonal antibody (RA3-LC2/1, ATCC), for B cells. Frozen sections were fixed in 4°C acetone, air-dried, rehydrated, and phosphate buffered saline (PBS), and incubated with
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Fig. 1. Anterior episcleral inflammation in a six-month-old female MRL/lpr mouse eye. There is a mononuclear inflammatory cell infiltrate at the anterior edge of the sclera just beneath the conjunctiva (hematoxylin and eosin, magnification X400).
Fig. 3. Mononuclear inflammatory infiltrate in the choroid and sclera of a 7-month-old male MRL/lpr mouse. There is a scleral inflammation with overlying choroidal inflammation (hematoxylin and eosin, magnification X280).
the appropriate blocking agent (Vector Laboratories, Burlingame, CA) for 20 min. The primary monoclonal antibody was applied and the slides were incubated for 16 min. Slides were then washed in PBS, incubated with a bibtinylated second antibody for 30 min, rinsed in PBS, incubated with the ABC reagent for 45 min, washed in PBS, developed with 0.1% hydrogen peroxide and 3-amino-9-ethyl-carbazole containing acetate buffer, and counterstained with Harris' hematoxylin. The percentage of mononuclear inflammatory cells staining positively with a given monoclonal antibody was enumerated using a 10 X 10 grid net micronometer disk covering an area of 0.16 mm 2 with a 25 X objective. Because limited tissue was available, not all infiltrates were analyzed for
all markers. The number of sections analyzed for Thy 1.2 was seven; for L3T4, ten; for Lyt 2, seven; for B cells, five; and for macrophages, five. Results. MRL/lpr mice: Histologic analysis was performed on 104 MRL/lpr mice. Only one of the 43 MRL/lpr mice, who were less than 5 months of age (2%), had any ocular inflammatory lesions, an animal with an anterior episcleritis. Of the older animals studied, 5 of 20 (25%) animals who were 5 months of age3 11 of 23 (48%) animals who were 6 months of age, and 12 of 18 (67%) animals who were more than 6 months of age had ocular inflammatory lesions. There was a significant trend for an increasing frequency of ocular inflammation with increasing age (P = 0.001, x 2 for trend). Of the 61 animals who were 5 months of age or older, 30% had scleral disease (Fig. 1), 16% choroidal infiltrates (Fig. 2), and 11% other ocular inflammatory lesions, including extraocular muscle myositis and keratitis. Overall, 46% of the mice who were five months or older had abnormal findings. The choroidal disease was not necessarily related to the scleral disease; half of the mice who had choroiditis also had scleritis (Fig. 3) and half did not. Ten animals had evidence of a more severe inflammatory process^ and in all cases, evidence of ocular vasculitis was seen. This more severe process generally consisted of a posterior scleral inflammation with vasculitis (Fig. 4), and occasionally produced severe ocular destruction. Seventy-five percent of the animals had bilateral ocular lesions. MRL/+, NZB/W, and BALB/c mice: No ocular inflammatory lesions were found in any of the NZB/ W mice analyzed at any age (57 mice) or any of the control BALB/c mice analyzed at any age (212 mice). The only abnormal finding in the 63 MRL/4- mice
Fig. 2. Mononuclear choroidal inflammatory infiltrate in a 6month-old female MRL/lpr mouse (hematoxylin and eosin, magnification X280).
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INVESTIGATIVE OPHTHALMOLOGY G VISUAL 5CIENCE / May 1991
Fig. 4. Posterior segment scleral vasculitis in a 5-month-old female MRL/lpr mouse eye, demonstrating fibrinoid necrosis and a mononuclear cell inflammatory infiltrate (hematoxylin and eosin, magnification X380).
analyzed was extraocular muscle myositis in an 18month-old mouse. Immunohistology: Immunohistologic analysis was performed on MRL/lpr mice with evidence of ocular disease and on age and sex-matched control BALB/c mice. In BALB/c mice, the nerve fiber layer of the retina stained for Thy 1.2, a marker shared by T cells and neurologic tissue. No staining for L3T4 (CD4), Lyt 2 (CD8), or B cells could be detected in the retina, choroid, or sclera. In contrast, MRL/lpr mice often showed focal collections of mononuclear inflammatory cells. The most common finding was that of scattered Thy 1,2+, L3T4+, Lyt 2- T cells in the anterior episcleral region. Analysis of 10 larger focal collections in the choroid or sclera showed that 78% of the cells were Thy 1.2+; 49% were L3T4+; 14% Lyt 2+; 11% were B cells; and 3% were macrophages (Table 1). No differences in the cellular profile were seen between scleral and choroidal infiltrate. One animal with extraocular muscle myositis showed an unusual finding in which one extraocular muscle was largely infiltrated with L3T4+ T cells, whereas in another extraocular muscle, the infiltrate was predominantly composed of Mac 3+ macrophages. Discussion. The three, autoimmune mice analyzed above have been proposed as models for a variety of autoimmune disorders. NZB/W mice and MRL/lpr mice have been proposed as animal models for lupus2 and/or Sjogren's syndrome.3"5 In addition, MRL/lpr mice have been proposed as a model for rheumatoid arthritis and for vasculitis,2'7'9 whereas MRL/+ mice have been proposed as a model for Sjogren's syndrome.3"5 The results reported here confirm and extend our previous observation of ocular inflamma-
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tion in a small number of MRL/lpr mice.3 Furthermore, the relationship of pathologic features to age has been shown and the immunohistologic profiles of the infiltrates analyzed. MRL/lpr and MRL/+ mice are congenic substrains that differ only by a single autosomal recessive gene, the Ipr gene. This gene causes a massive, but benign lymphadenopathy in MRL/lpr mice and markedly accelerates the autoimmune disease seen in the MRL/Mp strain. MRL/+ mice live until 2 yr of age, whereas MRL/lpr mice die at 6-7 months of age; a more aggressive glomerulonephritis develops in MRL/lpr mice than in MRL/+ mice; and a systemic necrotizing vasculitis develops in MRL/lpr mice that is not seen in MRL/+ mice. Although lacrimal gland inflammatory infiltrates similar to that seen in the human disorder Sjogren's syndrome develop in both substrains, ocular inflammatory lesions, such as choroiditis and scleritis, developed only in MRL/lpr mice. The finding of these lesions only in MRL/lpr mice and the demonstration that the more severe lesions appear to be related to vascular inflammation, suggest that the ocular lesions seen in MRL/lpr mice are indeed related to the vasculitis. Vasculitis is not seen in either NZB/W mice or in control strains, such as BALB/c mice; and inflammatory ocular lesions did not develop in either of these strains. Scleritis is a common feature of patients who had either rheumatoid arthritis or systemic necrotizing vasculitis, such as Wegener's granulomatosis.1 In patients who have rheumatoid arthritis, scleritis is believed to be a marker for the development of rheumatoid vasculitis.1 Overall, scleral disease occurs in 1% percent of patients who have rheumatoid arthritis; whereas it occurs in 14% of patients who have rheumatoid vasculitis.1 In Wegener's granulomatosis, scleral disease has occured in up to half of the patients.1 The finding of ocular inflammation in MRL/ Ipr mice which have been proposed as a model for rheumatoid arthritis or for vasculitis,2'7'9 is consistent with the observation in human disease. In rheumatoid arthritis, scleritis is most often seen in patients
Table 1. Immunohistologic analysis of MRL/lpr ocular lesions Mean ± standard deviation (%)
Range (%)
Thy 1.2 L3T4
78 ± 2 49 ± 17
75-80 23-65
Lyt 2 Pan-B/sIg* Mac 3
14 ± 10 11 ± 11 3± 3
0-24 0-23
Ceil type T-cells Helper T-cells
