cyclosporine therapy suppresses ocular and...

Cyclosporine Therapy Suppresses Ocular and LacrimalGland Disease in MRL/Mp-lpr/lpr Mice

Douglas A. Jabs, Bella Lee, C. Lynne Burek, Alt M. Saboori, and Robert A. Prendergast

Purpose. MRL/Mp-lpr/lpr (MRL/lpr) mice spontaneously develop an autoimmune diseasecharacterized by lymphoproliferation, vasculitis, glomerulonephritis, autoantibody produc-tion, and ocular and lacrimal gland inflammation. Lacrimal gland lesions in MRL/lpr miceare a model for the human disorder Sjogren's syndrome. The target organ lesions in MRL/lpr mice, including those in the eye and lacrimal gland, are composed largely of CD4+ Tcells, with lesser numbers of CD8+ T cells and B cells. Cyclosporine tfierapy was evaluatedfor its effect on the autoimmune disease, particularly in the eye and lacrimal gland.

Methods. MRL/lpr mice were administered cyclosporine intraperitoneally at a dosage of 2 mgdaily from age 1 to 5 months. Animals were killed at 5 mondis and evaluated for the presenceof autoimmune disease. Control groups consisted of animals given daily injections with eithersaline or the cyclosporine diluent.

Results. Cyclosporine therapy was effective in reducing the ocular and lacrimal gland disease.Intraocular inflammation was present in 73% of control animals but in only 15% ofcyclosporine-treated animals (P < 0.003). Multifocal lacrimal gland inflammatory infiltrateswere present in 100% of controls but in only 23% of cyclosporine-treated animals (P <0.0001). Mean percent area involved by lacrimal gland inflammation was reduced from 19.7%to 4.7% by cyclosporine therapy (P = 0.0003). Systemic autoimmune disease manifestations,including lymphoproliferation, vasculitis, glomerulonephritis, and serologic abnormalities,also were improved.

Conclusions. Chronic cyclosporine therapy, started at an early age, is effective in controllingthe autoimmune disease in MRL/lpr mice, including die ocular and lacrimal gland lesions.Invest Ophthalmol Vis Sci. 1996;37:377-383.

Ucular inflammation, including scleritis and Sjo-gren's syndrome, are common in patients with sys-temic autoimmune disorders.1 MRL/Mp-lpr/lpr(MRL/lpr) mice spontaneously develop an autoim-mune disease characterized by lymphadenopathy, vas-culitis, glomerulonephritis, serologic abnormalities,and ocular and lacrimal gland inflammatory le-sions.2"8 MRL/lpr mice are congenic with MRL/Mp—+/+ (MRL/ + ) mice and differ only by a singleautosomal recessive mutation, the Z/wgene.2"4 The lprmutation results in a defective Fas protein and defec-

From the Department of Ophthalmology, The Johns Hopkins University School ofMedicine, Baltimore, Maryland.Supported by grants EY05912 and EY01765 from the. National Institutes of Healthand by an unrestricted grant from Research to Prevent Blindness.Submitted for publication May 17, 1995; revised September 29, 1995; accejHedOctober 4, 1995.Proprietary interest category: N.Reprint requests: Douglas A. Jabs, Wilmer Institute 550 N. Broadway, Suite 700,Baltimore, MD 21205.

tive lymphocytic apoptosis, causing massive lymphade-nopathy.9 Lymph nodes in MRL/lpr mice are com-posed largely of CD4- CD8- TCR «//?+ "doublenegative" T cells, whereas the target organ lesions,including those in the lacrimal gland and eye, arecomposed largely of CD4+ T cells with lesser numbersof CD8+ T cells and B cells.10"13

Therapies directed at T cells, including neonatalthymectomy, treatment with anti-T cell monoclonalantibody (mAb), and anti-CD4 mAb therapy, havebeen reported to improve systemic autoimmune andintraocular inflammatory lesions.14"19 However, the re-sponse of lacrimal gland inflammation to immunosup-pressive treatment may be very different from the re-sponse seen in systemic autoimmune disease.20 Treat-ment of MRL/lpr mice with anti-CD4 mAb largelyeliminated the systemic and ocular disease17'8 but itdid not decrease the lacrimal gland disease; insteadanti-CD4 mAb treatment resulted in lacrimal gland

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378 Investigative Ophthalmology & Visual Science, February 1996, Vol. 37, No. 2

lesions with an altered morphology and a lymphocyticinfiltrate composed largely of CD8+ T cells.20

Although cyclosporine therapy is effective in sup-pressing the systemic disease in MRL/lpr mice,21 aprevious study22 reported that cyclosporine therapyworsened the lacrimal gland disease. In this article,we present evidence to the contrary, namely that earlyand consistent cyclosporine therapy is effective in sup-pressing ocular and lacrimal gland lesions in MRL/lpr mice.

MATERIALS AND METHODS

Treatment Protocol

Female MRL/lpr mice were obtained from the Jack-son Laboratories (Bar Harbor, ME) and kept understandard conditions in the animal facilities of theWoods Research Building of the Johns Hopkins Hospi-tal. Treatment was begun at 1 month of age, and ani-mals were given daily intraperitoneal injections (7days/week) of 2 mg of cyclosporine (Sandoz Pharma-ceutical, East Hanover, NJ), in 0.5 ml diluted with5% Alkamuls (Rhone-Poulenc, Cranbury, NJ), a doseequivalent to 40 mg/kg body weight. Control animalswere given either daily intraperitoneal injections ofnormal saline or diluent. Animals were killed at 5months of age by exsanguination. Sera were collectedfor the studies outlined below. Submandibular, axil-lary, and inguinal lymph nodes were removed andweighed as a measure of lymphadenopathy. Spleenswere removed and weighed separately. Eyes, lacrimalglands, and kidneys were removed for histologic pro-cessing, fixed in 4% buffered formaldehyde, sectionedat 5 fim, and stained with hematoxylin and eosin. Tis-sue sections were evaluated for the presence of diseaseby a masked reader (RAP) unaware of the treatmentgroup for each animal. These experiments conformedto the ARVO Statement for the Use of Animals inOphthalmic and Vision Research.

Cyclosporine Levels

Plasma cyclosporine levels were measured in a subsetof animals at 3 months and 5 months of age usinghigh-performance liquid chromatography at SmithKline Beecham Clinical Laboratories (Owings Mills,MD).

Evaluation of Lacrimal Gland Histology

Lacrimal gland sections were graded using the pre-viously described, modified focus score scale.5"7 Usingthis system, lacrimal gland histologic sections werescored from 0 to 4 based on the presence or absenceof foci of 50 or more mononuclear inflammatory cells;grade 0 = no inflammatory cells; grade 1 = inflamma-tion without any focus; grade 2 = the presence of at

least one focus; grade 3 = multiple foci; and grade 4= multiple foci plus evidence of lacrimal gland de-struction (e.g., effacement of lobular architecture bymononuclear inflammatory cells, fibrosis, or other evi-dence of damage). In addition, the percentage of areain the lacrimal gland involved by inflammatory cellswas measured by planimetry, using a Zeiss (Ober-kochen, Germany) projection microscope.

Antinuclear Antibodies

Antinuclear antibodies were measured using indirectimmunofluorescence as previously described.23 Serawere screened at a 1:10 dilution, using 4-fim frozensections of rat liver as the substrate and a fluoresceinisothiocyanate-conjugated goat anti-mouse mAb as thesecond antibody. Antinuclear antibodies were read ina masked fashion and scored on a semiquantitativescale score of 0 to 4+, where 0 = negative, 1+ = weak,2+ = medium, 3+ = strong, and 4+ = very strongstaining.

Measurement of Anti-dsDNA Antibodies

Determination of anti-double-stranded DNA (anti-dsDNA) antibodies was performed using the methoddescribed by Pisetsky and Peters.24 Briefly, ELISAplates (Corning Laboratory Sciences, Corning, NY)were coated with 200 [A of salmon testes DNA (Sigma,St. Louis, MO) 5 /ig/ml in phosphate-buffered saline(PBS) in each well, and incubated at 37°C for 2 to 3hours. After decanting and washing the plates threetimes with PBS-Tween 20, the remaining sites in eachwell were blocked by incubation of 200 fA of 1 /^g/mlmethylated bovine serum albumin at 25°C for 1 hour.Plates were washed three times with PBS-Tween 20;then, serum diluted 1:2 with PBS-Tween 20 wasadded, and the plates were incubated at 25°C for 1hour. At the end of the incubation, plates were washedthree times, and 100 (j,\ of 1:1000 dilution of alkalinephosphatase-coupled goat anti-mouse (immunoglobu-lin G [IgG]) was added to each well. After 1 hour ofincubation at 25°C, each well was again washed threetimes, and then p-pheno\ phosphate was added as asubstrate. Reactions were stopped with the additionof 50 //I of 3 M NaOH in each well for 10 minutes.Serial dilutions were tested, beginning at 1:25. Eachserum also was tested on plates without coating withDNA. Plates were read at 410 nm. Results are ex-pressed as dilution (titer) giving optical density of<0.10. All determinations were performed in dupli-cate.

Determination of Total Immunoglobulin G

ELISA plates were coated with 100 /A of 5 //g/ml goatanti-mouse IgG (Capell, Cochranville, PA) in each wellat 4°C overnight. Plates were blocked with 1 % bovineserum albumin; after being washed three times with


Cyclosporine Therapy of MRL/lpr Mice 379

PBS-Tween 20, 100 fi\ of a 105 serum dilution wasadded to the individual wells and incubated at 25°Cfor 1 hour. Plates were read using optical density at410 nm. All autoantibody determinations and immu-noglobulin measurements were performed in amasked fashion.

Statistics

Group proportions were compared using either chi-square analysis or the Fisher's exact test. Group medi-ans were compared using the Wilcoxon rank sum test.

RESULTS

Cyclosporine Levels

Plasma cyclosporine levels were measured in two sa-line-treated and two Alkamuls-treated (cyclosporinediluent) mice at 3 months and another two from eachcontrol group at 5 months of age. In all eight controlmice, plasma levels were below the lower limit of de-tection (<30 /^g/1), that is, zero. Cyclosporine levelswere measured in three cyclosporine-treated mice at3 months and three other mice at 5 months of age.The mean level in the 3-month-old treated mice was875 /xg/1 (range, 334 to 1590 /xg/1), and in the 5-month-old treated mice, it was >1020 /Ltg/1 (range,904 to >1000 /ig/1). Despite the plasma levelsachieved, there was no evidence of wasting orcyclosporine toxicity in the cyclosporine-treatedgroup.

Lymphoproliferation

Lymphoproliferation was markedly improved by treat-ment with cyclosporine (Table 1). Median lymphnode weight was reduced 82%, and median spleenweight was reduced 50% by cyclosporine treatment (P= 0.0001 and P = 0.0002, respectively).

Ocular Pathology

Ocular disease was markedly suppressed by cy-closporine therapy (Table 2, Figure 1). The presenceof any ocular inflammation was decreased from 73%in control animals to 15% in cyclosporine-treated ani-mals (P < 0.003). This improvement was particularlyevident with the more severe ocular lesions, such aschoroiditis and scleritis, which collectively were pres-ent in 34% of control animals but in none of thecyclosporine-treated animals (P = 0.04). Other ocularinflammatory lesions seen in controls but notcyclosporine-treated animals included anterior uveitis(one mouse) and orbital vasculitis (one mouse).

Lacrimal Gland Pathology

Lacrimal gland inflammation was markedly sup-pressed by chronic cyclosporine therapy (Table 2).

One hundred percent of control animals had severelacrimal gland disease, grade 3 or 4, using the modi-fied focus score scale (Fig. 2). Only 23% ofcyclosporine-treated animals had grade 3 or greaterlacrimal gland disease (P< 0.0001). Furthermore, theevaluation of percentage area involved by inflamma-tion in the lacrimal gland revealed a 76% reductionin the mean amount of inflammation present, from19.7% in control animals to 4.7% in cyclosporine-treated animals (P = 0.0003).

Renal Pathology

Renal disease was decreased by cyclosporine therapy(Table 1). Glomerulonephritis was present in 100%of controls, and the median grade was 3+ using ourpreviously described 0 to 4+ scoring system.18'1"1 Glo-merulonephritis was detected in only 15% ofcyclosporine-treated animals (P< 0.00001). Vasculitisand interstitial nephritis were detected in 47% and60% of control animals, respectively, but vasculitis wasabsent and interstitial nephritis was present in only15% of cyclosporine-treated animals, respectively (P< 0.01 and P = 0.02, respectively).

Autoantibodies and Immunoglobulin Levels

Serologic abnormalities were decreased by cy-closporine therapy (Table 1). The median titer of anti-dsDNA was reduced from 1:600 in controls to 1:37.5in cyclosporine-treated animals (P = 0.004). Total IgGlevels were reduced from a median optical density of0.80 to 0.50 by cyclosporine therapy (P = 0.008), andthe median antinuclear antibody grade was decreasedfrom 3+ in controls to 1+ in cyclosporine-treatedmice (P= 0.002).

DISCUSSION

MRL/lpr mice differ from the congenic MRL/ + miceonly by a single autosomal recessive gene, the Ipr muta-tion.2"4 The Ipr mutation results in a defective Fasantigen and thereby defective lymphocytic apoptosis,which leads to the accumulation of double negativeT cells in the lymph nodes and hastens the develop-ment of autoreactive T cells.9 These autoreactive Tcells appear to drive the autoimmune disease in theseanimals. Both MRL/lpr and MRL/+ mice developlacrimal gland lesions and serologic abnormalities, butMRL/lpr mice develop a much more aggressive andrapidly fatal autoimmune disease than do MRL/ +mice.2"9 Furthermore, the lacrimal gland disease inMRL/lpr mice has a different immunocytochemicalprofile than that in MRL/+ mice. Both mice have apredominance of CD4+ T cells with few double nega-tive T cells present in the lacrimal glands, but MRL/Ipr mice have a significandy greater proportion of


380 Investigative Ophthalmology 8e Visual Science, February 1996, Vol. 37, No. 2

TABLE l. Systemic Autoimmune Disease in Control and Cyclosporine-treated MRL/lpr Mice

Treatment Saline Alkamuls* Controls] Cyclosporine P ValueX

Number of animals 7 8 15 13Lymphoproliferation

Median (mean ± SD) 2.5 (2.8 ± 1.6) 1.6 (2.1 ± 1.2) 2.2 (2.4 ± 1.4) 0.4 (0.5 ± 0.6)lymph node weight (g)

Median (mean ± SD) 0.6 (0.7 ± 0.4) 0.6 (0.6 ± 0.2) 0.6 (0.6 ± 0.3) 0.3 (0.3 ± 0.1)spleen weight (g)

Renal diseaseGlomerulonephritis (%)§ 100 100 100 15Median grade 3 2 3 0

glomerulonephritisVasculitis (%)§ 86 12 47 0Interstitial nephritis (%)§ 86 38 60 15

SerologyAnti-dsDNA|| 450 600 600 37.5Total IgUANA#

0.96 (0.89 ± 0.20) 0.69 (0.69 ± 0.14) 0.80 (0.79 ± 0.19) 0.50 (0.51 ± 0.20)3.5 3 3 1

0.0001

0.0002

<0.00001

<0.010.02

0.0040.0080.002

SD = standard deviation.* Alkamuls = diluent for cyclosporine.f Control group = saline- and alkamuls-treated groups combined.X P value for comparison of control vs. cyclosporine-treated groups. Group proportions were compared using chi-square of Fisher's exacttest, and group medians using the Wilcoxon rank sum test.§ Percent of animals with disease.|| Median titer (range 1:25 to 1:3200).\ Immunoglobulin, measured as median (mean ± SD) optical density.#Antinuclear antibodies, expressed as median grade (scale 0 to 4+).

CD4+ T cells and lower proportion of CD8+ T cellsthan do MRL/+ mice.6

Treatment directed toward T cells, such as neona-tal thymectomy1415 or anti-T cell mAb therapy,16 re-sults in marked improvement in the systemic autoim-mune disease in MRL/lpr mice. However, althoughtreatment with anti-CD4 mAb resulted in marked im-provement in the systemic autoimmune disease andintraocular disease,17"19 it did not improve the lacri-mal gland disease.20 Instead, the morphology of in-

flammatory lesions and immunocytochemical profilewere altered, but the extent of disease was not dimin-ished. These results demonstrate that the lacrimalgland and systemic autoimmune disease in MRL/lprmice may respond differendy to treatment.

Cyclosporine selectively inhibits T cells by the in-hibition of IL-2-IL-2 receptor (IL-2R) autocrine path-way.25'26 Because T cells are affected by cyclosporine,one might hypothesize that cyclosporine therapywould be effective for autoimmune disease in MRL/

TABLE 2. Autoimmune Ocular and Lacrimal Gland Disease in Controland Cyclosporine-treated MRL/lpr MiceTreatment

Number of animalsOcular disease

Any (%)§Episcleritis (%)Choroiditis (%)Posterior scleritis (%)

Lacrimal gland diseasePercent grade s=3+§Mean ± SD (median)percent area||

Saline

7

71291414

10022.5 ± 12.5 (20)

Alkamuls*

8

753838

0

10017.3 ± 17.3 (11.3)

Controls\

15

7333277

10019.7 ± 15.0 (15.9)

Cyclosporine

13

151500

234.7 ± 5.5 (2.7)

P ValueX

<0.0030.260.070.54

<0.00010.0003

* Alkamuls = diluent for cyclosporine.f Control group = saline- and alkamuls-treated groups combined.X Value for comparison of control vs. cyclosporine-treated groups. Group proportions were compared using chi-square or Fisher's exacttest, and group medians using the Wilcoxon rank sum test.§ Percent of animals with disease.|| Percent lacrimal gland area replaced by inflammatory cells.



,-• tf*.*.^.

FIGURE I. Choroidal lesions in MRL/lpr mice. (A) Choroidalinflammatory infiltrate in a saline-treated MRL/lpr mouse.(B) Chorioid of cyclosporine-treated MLR/lpr mouse show-ing absence of disease (hematoxylin and eosin; original mag-nification, X250).

lpr mice, a disease in which T-cell abnormalities ap-pear to be primary. However, adult MLR/lpr mice aredeficient in both the production of and the responseto IL-227; hence, cyclosporine therapy theoreticallycould accelerate autoimmune disease in MRL/lprmice. A previous study by Mountz et al21 reported thathigh-dose cyclosporine (40 mg/kg per day) was effec-tive in controlling the systemic autoimmune diseasein MRL/lpr mice, including the lymphadenopathy,but that autoantibody formation was not reduced. Inthat study, lacrimal glands were not evaluated. In theseexperiments low-dose cyclosporine therapy (10 mg/kg every other day) was considerably less effective thanhigh-dose dierapy.21

The dose of cyclosporine used in our experimentswas the high dose (40 mg/kg), and our results alsodemonstrate that cyclosporine therapy was effective incontrolling systemic autoimmune disease, includingglomerulonephritis and vasculitis. In contrast to the

results of Mountz et al,21 in our study both serologicabnormalities and total IgG were improved when com-pared to control animals.

In our study, ocular and lacrimal gland diseaseswere improved markedly by cyclosporine therapy. Thesuccess of cyclosporine therapy in suppressing the lac-rimal gland disease contrasts with the failure of anti-CD4 mAb therapy.20 Because IL-2 is produced byCD4+ T cells, the effects of cyclosporine and anti-CD4 mAb might have been predicted to be similar.However, because cyclosporine inhibits the genera-tion of cytotoxic T cells in vitro,26 it could affect bothCD4+ and CD8+ T cells and, hence, it could be effec-tive for the treatment of lacrimal gland disease inMRL/lpr mice, even though anti-CD4 mAb was not.Taken together, these two sets of results suggest thatboth CD4-I- and CD8+ T cells may be important inthe pathogenesis of lacrimal gland disease in MRL/lpr mice.

FIGURE 2. Lacrimal glands in MRL/lpr mice, (a) Lacrimalgland in a saline-treated MRL/lpr mouse showing extensivelacrimal gland inflammation, (b) Cyclosporine-treatedMRL/lpr mouse showing absence of lacrimal gland disease(hematoxylin and eosin; original magnification, X160).


382 Investigative Ophthalmology & Visual Science, February 1996, Vol. 37, No. 2

Our results stand in marked contrast to those ofSato and Sullivan,22 who reported that cyclosporinetherapy worsened lacrimal gland disease in MRL/lprmice. Several experimental differences may explainthe different results. Sato and Sullivan22 administeredcyclosporine to animals beginning at approximately3.5 months of age, a time at which autoimmune dis-ease already has begun, whereas in our experiments,cyclosporine therapy was begun at 1 month of age,before the onset of overt autoimmune disease. Previ-ous studies by us using anti-CD4 mAb therapy19 havedemonstrated that treatment begun "late" can havedifferent effects on the autoimmune disease in MRL/lpr mice than treatment begun "early." Furthermore,1-month-old MRL/lpr mice are relatively normal interms of the production of IL-2, whereas aged MRL/lpr mice have defects in both the production of andresponse to IL-2, at least when lymph node lympho-cytes are evaluated.27'28 Hence, cyclosporine may havedifferential effects on MRL/lpr mice depending onthe timing of the onset of therapy.

Furthermore, Sato and Sullivan22 used slow-re-lease pellets implanted subcutaneously instead of dailyintraperitoneal injections, and cyclosporine was ad-ministered only for a 21-day interval, after which ani-mals were evaluated. In our experiments, cyclosporinetherapy was given as daily intraperitoneal injections ofcyclosporine, which probably resulted in a muchhigher exposure to cyclosporine. Although the plasmalevels of cyclosporine were not measured in Sato andSullivan's experiments,22 the levels in our experimentswere substantial, generally in the 800 to 1000 yt/g/1range, and well above the therapeutic range for organtransplantation. Different doses of cyclosporine mayhave different effects on the immune system. In bonemarrow transplants, low-dose cyclosporine enhancesthe development of autologous graft-versus-host dis-ease, whereas high-dose cyclosporine is effective inpreventing and treating allogenic graft-versus-host dis-ease.29 Our data demonstrate that chronic daily, high-dose cyclosporine therapy begun at 1 month of agesuppresses lacrimal gland inflammation in MRL/lprmice. Further exploration of the differential effects ofthe dose and timing of onset of therapy using theintraperitoneal route may explain the differences be-tween our results and those of Sato and Sullivan.22

In conclusion, our study demonstrates thatchronic cyclosporine therapy of MRL/lpr mice is ef-fective in controlling the systemic autoimmune dis-ease, autoantibody formation, intraocular inflamma-tory lesions, and the lacrimal gland autoimmune dis-ease seen in this strain.

Key Words

autoimmune response, cyclosporine, immunopathology,lacrimal gland, Sjogren's syndrome

Acknowledgments

The authors thank Kevin G. McCarthy and Matthew Schi-nella for technical assistance.

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