157 SJOGREN’S SYNDROME IN MRL/1 AND M R L h MICE ROBERT W. HOFFMAN, MARGARET A. ALSPAUGH, KIM S. WAGGIE, JAMES B. DURHAM, and SARA ELLEN WALKER Six autoimmune murine models (MRLh, MRL/n, NZB, NZB/NZW, PN, C57BL/6J-lpr/lpr) were compared with normal control C57BL/6J and DBM2 mice to determine if spontaneous autoimmune disease was associated with evidence of Sjogren’s syndrome. Schirmer tests documented dry eyes in NZB/NZW and PN mice; other autoimmune strains and controls had normal tear formation. All autoimmune mice had conjunctivitis, but this abnormality was most severe in the PN strain. Ninety-eight percent of MRL/I and MRL/n mice had mononuclear cell infiltrates in lacrimal glands, and salivary glands were involved to a lesser degree. New Zealand mice and PN mice had smaller gland lesions. The extensive gland destruction in MRL/I and MRLln mice suggested that these substrains merit further studies as animal models of Sjogren’s syndrome. ____ From the Veterans Administration Medical Center, Columbia, Missouri and the University of MissouriXolumbia. Supported by the Medical Research Service of the Veterans Administration and grant 2 R 0 1 AM 25868 from the National Institutes of Health, U.S. Public Health Service. Dr. Waggie’s fellowship was supported by DHHS grants RR00471 and RROSO16. Robert W. Hoffman, DO: Resident, Department of Medicine, University of Missouri-Columbia; Margaret A. Alspaugh, PhD: Associate Professor of Medicine and Pathology, University of Missouri-Columbia; Kim S. Waggie, DVM: Research Associate, Department of Veterinary Pathology, University of Missouri School of Veterinary Medicine; James B. Durham, MD: Assistant Professor of Pathology, University of Missouri-Columbia; Sara Ellen Walker, MD: Chief, Rheumatology Section, Harry S. Truman Memorial Veterans Hospital and Associate Professor of Medicine, University of Missouri-Columbia. Address reprint requests to Sara Ellen Walker, MD, 1 IIF, Harry S. Truman Memorial Veterans Hospital, 800 Stadium Road, Columbia, MO 65201. Submitted for publication March 21, 1983; accepted in revised form August 22, 1983. Arthritis and Rheumatism, Vol. 27, No. 2 (February 1984) Sjogren’s syndrome was defined originally as keratoconjunctivitis sicca and xerostomia associated with a connective tissue disease (1). Although carefully tabulated data concerning the true incidence of Sjogren’s syndrome are not available, several authors have suggested that it is one of the most common rheumatic diseases (2,3). Sjogren’s syndrome may occur with a variety of connective tissue diseases, including rheumatoid arthritis and systemic lupus erythematosus (2). Primary Sjogren’s syndrome, or sicca syndrome, occurs in patients who have only ocular and oral involvement; this disease is of special interest because it is related to lymphoreticular malignancies (4) * Early attempts to induce Sjogren’s syndrome in experimental animals were not consistently successful. Chan ( 5 ) produced mild salivary gland inflammation in 50% of guinea pigs injected with submaxillary gland homogenate suspended in complete Freund’s adjuvant. Other investigators found that injections of salivary gland extract required 2 adjuvants (carbonyl iron and Bordetelfapertussis) to produce severe sialadenitis (6). The first spontaneous models of this disorder were reported by Kessler (7), who described mononuclear cell infiltration in lacrimal and salivary glands of autoimmune New Zealand black (NZB) and hybrid New Zealand blacWNew Zealand white (NZB/NZW) mice (8). These animals, which have been studied extensively as models of systemic lupus erythematosus, were considered to have coexisting Sjogren’s syndrome. Recently, new murine models of systemic lupus erythematosus have provided the opportunity to further examine relationships between murine lupus and HOFFMAN ET AL Sjogren’s syndrome. MRL/I mice and congenic MRL/Mplpr/lpr mice develop accelerated immune complex disease with features of lupus and rheumatoid arthritis. These animals have anti-Sm and anti-DNA antibodies, glomerulonephritis, vasculitis, IgM and IgG rheumatoid factors, and erosive arthritis (9,lO). The MRL/n substrain has mild autoimmune disease with low levels of anti-DNA antibodies and late-onset glomerulonephritis (9). Congenic C57BL/6J-lpr/lpr mice, derived by transferring the Ipr/lpr genome to standard inbred C57BL/6J mice, develop antinuclear antibodies and glomerulonephritis (1 1). Palmerston North (PN) mice have antibodies to DNA, immune complex glomerulonephritis, and vasculitis (12). In the current study tear formation, ocular tissues, and salivary and lacrimal glands were evaluated in autoimmune MRLII, MRL/n, NZB, NZB/NZW, and PN mice and results were compared with normal C57BL/6J and DBM2 control strains. All autoimmune strains had at least 1 manifestation of Sjogren’s syndrome but ocular dryness, conjunctivitis, and lacrimal and salivary gland inflammation varied in different murine models of lupus. MRL/I and MRL/n mice had pronounced, destructive mononuclear infiltrates in glandular tissue which resembled the lesions of Sjogren’s syndrome. MATERIALS AND METHODS Animals. MRLA mice, which are homozygous for the recessive Ipr (lymphoproliferative) gene, and a closely related line (MRL/n) without Ipr genes were obtained from Jackson Laboratories and colonies were established at the University of Missouri-Columbia in 1977. Animals from these colonies were donated to the study by Deborah Wilson, MD. In 1978, the IprApr genome was transferred to MRL/n to produce substrain MRL/Mp-lpr/lpr and MRL/n was renamed MRL/Mp-+I+ (9). MRLIMplprllpr and C57BL/6J-lpr/lpr mice were provided through the courtesy of E. D. Murphy, PhD and J. B. Roths of Jackson Laboratories and housed in the Research Service of the Harry S. Truman Memorial Veterans Hospital. New Zealand mice descended from breeding pairs obtained in 1969 (NZB) and 1975 (NZW) from the University of Otago in Dunedin, New Zealand. PN mice were offspring of animals donated in 1974 by Dr. Richard D. Wigley of Palmerston North, New Zealand. C57BL/6J and DBA/2 retired breeders were purchased from Jackson Laboratories. All mice were housed in plastic cages on hardwood bedding and fed Purina 5001 chow. Schirmer tests. Twenty to 37 male and female MRL/I (this group contained 4 MRL/Mplpr/lpr mice), MRL/n. NZB, NZBNZW, PN, C57BL/6J, and DBN2 mice aged 11 to 60 weeks were examined. Mice were anesthetized lightly with methoxyflurane, and a 0.5 x 3.0 mm strip of Whatman # 1 filter paper was placed under the lower lid of each eye near the medial canthus. After 2 minutes the strips were removed, soaked areas were marked, and the length of wetting was measured at l o x magnification using a micrometer on a dissecting microscope. Histologic studies. Necropsies were usually performed within 8 weeks after the Schirmer tests; in several instances, NZB and NZB/NZW males were autopsied 14 to 17 weeks after testing. Groups of 20 to 38 autoimmune and control mice of both sexes were bled from the right orbital vascular plexus and killed by cervical dislocation. A separate group of 20 C57BL/6J-lpr/lpr mice aged 38 weeks were autopsied, and lacrimal and salivary glands were evaluated for inflammation. Autoimmune mice were killed at ages when they were expected to have active disease. Mean ages at death were: MRLA males 28 weeks (range 16-52) (this group contained 2 MRL/Mp-IprApr males); MRL/I females 25 (range 16-30) (this group contained 2 MRL/Mplpr/lpr females); MRL/n males 47 (range 33-60); MRL/n females 43 (range 33-60); NZB males 47 (range 37-61); NZB females 54 (range 32-67); NZB/NZW males 33 (range 30-35); NZBNZW females 30 (range 20-36); PN males 35 (range 2750); PN females 37 (range 26-47). Control mice were killed when their ages were similar to autoimmune animals: C57BL/6J males at 35 weeks; C57BL16J females at 37 (range 35-40); DBA/2 males at 27 (range 30-36); DBA/2 females at 30. Serum was stored in plugged capillary tubes at -20°C. The left eye was dissected carefully from the orbit, fixed in one piece in 10% buffered formalin, and embedded in paraffin. Twenty to 100 serial lop sections were stained with hematoxylin and eosin. These tissue sections were in part the basis of an earlier study of band keratopathy and posterior uveitis in autoimmune and normal mice (13). The left lacrimal gland was removed separately and the parotid, submandibular, and sublingual salivary glands were taken en bloc. Complete autopsies were performed and samples of superficial cervical lymph nodes, lung, heart, liver, pancreas, spleen, and kidney were preserved in formalin. A section through each of these tissues was examined for infiltrates of mononuclear cells. In 139 animals, serial sections of ocular tissue contained samples of conjunctiva which were adequate for evaluation of conjunctivitis. Conjunctival inflammation was graded on a scale of 0-3: 0 = no inflammation; 1 = minimal inflammation; 2 = intermediate inflammation; 3 = diffuse infiltration of tissue with inflammatory cells. Cellular infiltrations in cross sections through lacrimal glands from 183 mice and salivary glands from 178 mice were graded on a scale of 0-4 based on a modification of the system of Chisholm and Mason (14). In this system, 0 = no inflammation; 1 = focal infiltration with mononuclear cells; 2 = one-fourth of gland replaced by mononuclear cells; 3 = one-third of gland replaced by mononuclear cells; 4 = more than half of gland replaced by mononuclear cells. To establish the reproducibility of both grading systems, representative slides of conjunctiva, lacrimal glands, and salivary glands were scored independently by 2 observers who were not aware of the strain being examined. Mean scores between both observers differed by less than 1 grade. Electron microscopy. Submandibular salivary glands 159 SJOGREN’S SYNDROME Table 1. Schirmer tests in autoimmune MRL/I, MRL/n, NZE), NZB/NZW, and PN mice, and in normal control C57BL/6J and DBAR mice Sex MRLA MRLh NZB NZBINZW PN C57BL/6J DBA/2 M F M F M F M F M F M F M F Number of mice examined Filter paper wetting (mm)+ 2.8 C 0.2 (1.0-4.7) 3.1 f 0.3 (1.1-6.8) 3.9 2 0.2 (2.0-5.5)$ 2.7 f 0.3 (1.0-5.4) 3.8 2 0.3 (1.7-6.5) 3.7 5 0.3 (1.2-6.3) 2.2 2 0.2 (1.2-4.1)§ 1.8 C 0.1 (1.1-3.1)§ 2.2 5 0.2 (1.1-4.9)§ 2.0 t 0.2 (0.6-3.8)s 2.8 5 0.2 (1.8-4.4) 3.3 -t 0.2 (2.0-4.6) 3.1 5 0.2 (2.2-4.6) 3.3 It 0.2 (2.1-5.6) Age (weeks)* 30 (29-30) 28 (21-31) 36 (35-36) 33 (31-37) 44 (36-.58) 48 (28-66) 25 ( 16-34) 26 ( I 1-34) 32 (28-49) 34 (25-47) 34 (34-35) 34 (34) 30 (29-30) 30 (30) 11 12 10 11 14 23 12 12 12 12 10 10 10 10 * Mean (range). t Mean f SEM (range). $ Mean wetting in MRLln males was significantly greater compared with mean wetting in females ( P < 0.025). 8 In NZBINZW males, NZBlNZW females, PN males, and PN females mean Schirmer test results were significantly smaller compared with control C57BL/6J and D B N 2 mice of the same sex. In each instance. P < 0.001. from 2 adult MRLh mice were cut into I-mm cubes in cold (4°C) 3% Millonigs phosphate buffered glutaraldehyde. After fixation for 3 hours at 4”C, the tissue was washed twice in cold Millonigs buffer and post-fixed for 1 hour in 1.5% cold Millonigs buffered osmic acid. The tissue was dehydrated in graded dilutions of ethanol, placed in 2 changes of propythene oxide, and embedded in Epon 812. The blocks were heated at 60°C for 16 hours, and thin sections stained with uranyl acetate and lead citrate were examined in a Philips EM 300 transmission electron microscope (15,16). Immunodiffusion tests. Terminal sera from 10 male and 10 female MRL/I, MRL/n, NZB, NZB/NZW, and PN mice were tested for precipitating antibodies to SS-A (Ro) and SS-B (La) by a modification of the Ouchterlony double diffusion method (17). The antigen for SS-A was extracted from human spleen and the antigen for SS-B was prepared from rabbit thymus (18). Prototype human sera known to possess SS-A or SS-B antibodies were used as reference sera at dilutions that gave optimal precipitin lines with antigen. Unpooled samples of serum from individual mice were placed in wells adjacent to antigen and to reference sera and examined for prccipitin lines of identity or nonidentity with the reference sera. Reactions were allowed to proceed at room temperature and plates were observed for precipitin lines at 24, 48, and 72 hours. Statistics. Student’s I-test was calculated using the method described by Snedecor and Cochran (19). RESULTS Schirmer tests. Table 1 lists mean values for Schirmer tests in autoimmune and control strains. In the MRL/n substrain, tear formation in males was significantly greater compared with females. In the other groups of mice, gender did not influence tear formation. Furthermore, severity of autoimmune dis- ease did not correlate with results of Schirmer tests. MRL/l mice, which develop early and severe immune complex disease, resembled the MRL/n substrain which has mild late-onset disease. Ocular wetting was diminished significantly in NZB/NZW and PN mice compared with control C57BL/6J and DBA/2 mice of the same sex. Conjunctivitis. Inflammation of the conjunctiva, a clinical and histologic manifestation of Sjogren’s syndrome in humans (20), was found in autoimmune mice (Figure 1). Conjunctivitis was most common in w rJl 0’ 000 ~ 0 0 0 0 goo00 00000 00000 00000 00000 pJ~~ 0 00 u u u u u u MRL/I MRL/n NZB NZB/NZW PN Control Figure 1. Conjunctivitis, a clinical and histologic manifestation of Sjogren’s syndrome, was graded on a scale of 0 to 3+. Inflammation of the conjunctiva was most common in MRLll mice; the most severe involvement was observed in PN mice. 0 = male; 0 = female. In the control column, solid symbols represent DBAl2 mice and open symbols represent CS7BL/6J mice. 160 HOFFMAN ET AL OJ ------MRL/I MRL/n NZB NZB/NZW PN C571pr Control Figure 3. Cellular infiltration in lacrimal glands was graded 0 to 4in a classification adapted from Chisholrn and Mason (14). All autoimmune strains had lacrimal gland inflammation. The most extensive lesions were found in MRLA, MRL/n, and NZB mice. See Figure 1 for explanations. Figure 2. Severe inflammatory changes in a section through the superior palpebral conjunctiva of a female MRL/n mouse aged 35 weeks. Conjunctival tissue contains diffuse infiltrates of mononuclear and polymorphonuclear cells. Overlying epithelium (arrow) is hyperplastic. On the left, the sebaceous glands are infiltrated with inflammatory cells (hematoxylin and eosin, original magnification x Lacrimal glands. Figure 3 illustrates grades of severity of inflammatory changes in lacrimal glands from autoimmune and control mice. The most extensive involvement was found in the MRL/l and MRLin substrains in which 100% and 95% of mice, respective- loo). the MRLA substrain, in which 85% of mice were affected. Conjunctival inflammation was generally characterized by scattered foci of mononuclear cells infiltrating the bulbar and palpebral conjunctiva. Occasional neutrophils were present with scattered cellular debris. MRL/I, MRL/n, NZB, and NZB/NZW mice commonly had extensive mononuclear cell infiltrates in the palpebral conjunctiva surrounding the tarsal plate. Typical conjunctivitis in a female MRWn mouse is illustrated in Figure 2. The most severe conjunctival involvement was observed in PN mice; 40% of these animals had grade 3 conjunctivitis. In the P N strain, infiltrates had a vasculitic component and mononuclear cells clustered around blood vessels and extended into surrounding tissue. Several PN mice with grade 3 conjunctivitis had extensive vessel wall destruction and lysis of infiltrating cells. The inflammatory process extended into adjacent extraocular muscles, where fiber necrosis and degeneration were observed. N o inflammation was found in the conjunctiva in normal control C57BL/6J and D B N 2 mice. Figure 4. Characteristic destructive changes in a lacrimal gland from a 16-week-old MRL/I male. Mononuclear cells infiltrate the adjacent parenchyma, resulting in focal destruction of secretory alveoli (hematoxylin and eosin, original magnification x 250). SJOGREN’S SYNDROME 161 4* 1 00 z 2 2*{ LL 003oc ouoo 0 CCJ 0 ocooo oconn w 0 J 01:c 00000 cioooo OOCIC~ DOODD CIJC 03 3 co000 00000 O??C2 ooxn 3cucc 30000 00030 oooou LIE ooooo oooor! ccc3c 0.88. mmmmm cooco coooo 0ocoo uoil ... oc030 000.. a .. ------nooon ccooc! 000 0 MRL/I MRL/n NZB NZWNAN PN C571pr 00 llOUO. Control Figure 5. Inflammation in submandibular salivary glands was graded 0 to 4 + . Sialadenitis was most severe in NZB/NZW mice. Foci of inflammation were present in 38% of control mice. See Figure 1 for explanations. ly, had lacrimal gland infiltrates. Lacrimal glands from the MRL substrains typically contained large multifocal confluent infiltrates of lymphocytes, with occasional histiocytcs and plasma cells. These lesions were characterized by perivascular and periductal aggregates of cells that infiltrated the adjacent parenchyma and resulted in focal destruction of secretory alveoli. Fibrosis was present in areas diffusely infiltrated with inflammatory cells. A lesion in an MRL/I male is illustrated in Figure 4. Lacrimal gland infiltrates were found in 85% of NZB, 57% of NZB/NZW, and 75% of PN mice. Inflammation in these animals was less severe compared with MRL mice. In congenic C57BL/6J-lpr/lpr mice, 28% of animals had grade 1 and 2 infiltrates. No control mice had a histologic score greater than grade 1, and most controls had no evidence of inflammation. Submandibular glands Grading of inflammation in the submandibular salivary gland is shown in Figure 5. Eighty-six percent of MRL/l mice and 94% of MRL/n mice had grade I and 2 infiltrates. Sialadenitis in the submandibular glands resembled lacrimal gland involvement, with collections of mononuclear cells around vessels and ducts. Lymphocytes and plasma cells extended into and destroyed adjacent glandular tissue. Figure 6 illustrates a characteristic lesion resulting in destruction of a duct and acinar tissue. Moderately severe involvement was found in 2 NZB/NZW mice, which had grade 3 lesions; the incidence of submandibular gland inflammation in this group was 40%. Minimal inflammation was found in submandibular glands from NZB and PN mice, and C57BL/6J-lpr/lpr mice had normal submandibular glands. Focal grade 1 infiltrates were found in 38% of C57BL/6J and DBAR mice. Parotid glands. Inflammation of grade 1 and 2 severity involved 23% of MRL/I and 26% of MRL/n parotid glands. Eighteen percent of NZB mice, 12% of NZB/NZW mice, and 10% of PN mice had grade 1 infiltrates. Mononuclear cell infiltrates were not found in parotid glands from C57BL/6J-lpr/lpr. C57BL/6J, and DBAR mice. Sublingual glands. Focal grade 1 sublingual gland lesions occurred in 4-19% of autoimmune MRL/l, MRL/n, NZB, NZB/NZW, and PN mice; C57BL/6J-lpr/lpr mice had no inflammatory changes. Unexpectedly, 4 female control C57BL/6J mice had infiltrates in sublingual glands. Two glands were classified as grade 1 , and 2 glands were grade 2. The overall incidence of sublingual inflammation in controls was 13%. Examination of other tissues. As expected from our experience and the work of others (8-12), glomerulonephritis and vasculitis were common in renal Figure 6 . Submandibular gland infiltrated with mononuclear cells, 35-week-old MRL/I female. A duct and acini are invaded by inflammatory cells (hematoxylin and eosin, original magnification x 250) 162 tissue from autoimmune mice. Variable numbers of lymphocytes and plasma cells surrounded the renal pelvis and renal arteries in the autoimmune strains; in some instances focal collections of mononuclear cells were scattered throughout the renal parenchyma. Hyperplasia of lymphoid tissue was observed frequently in the lupus strains. Several animals had lymphomas and in 2 instances, metastases to salivary glands were identified. These lesions were clearly distinct from the cellular infiltrates that resembled Sjogren’s syndrome. Lymphocytic infiltrates were found commonly around bronchioles and pulmonary arteries in the autoimmune strains. These lesions were most severe in the MKL/l substrain. Pulmonary and renal infiltrates were not present in the normal strains. The heart, liver, and pancreas were not infiltrated with lymphocytes in autoimmune or control mice. Electron microscopy. In 2 MRL/n females, cellular infiltrates in lacrimal glands were composed of small and medium lymphocytes. These cells were observed infiltrating acini and ductal epithelium (Figure 7). Immunodiffusion tests. Antibodies to SS-A and SS-B were not detected by immunodiffusion in sera from MRL/I, MRL/n, NZB, NZB/NZW, and PN mice. Figure 7. Electron micrograph of lacrimal gland tissue from a female MRLh mouse. Architecture of the gland has been destroyed by infiltration of lymphocytes. Representative lymphocytes are labeled (L). Prominent endoplasmic reticulum, prominent Golgi apparatus, and cytoplasmic extrusion identify these cells as activated lymphocytes. Two fibroblasts (F) appear to be laying down bundles of collagen (arrowheads) (transmission electron microscopy, original magnification x 3,300). HOFFMAN ET AL DISCUSSION The first models of spontaneous Sjogren’s syndrome were described by Kessler (7,21), who reported corneal changes in NZB/NZW mice and periductal and periarteriolar infiltrates in salivary and lacrimal glands from NZB and NZB/NZW mice. Keyes and associates (22) confirmed the finding of histopathologic abnormalities in NZB/NZW hybrids and reported epimyoepithelial-like structures in glandular tissue. Examination of parotid and submandibular salivary glands of NZB/NZW mice by electron microscopy showed that the inflammatory lesions consisted of lymphocytes surrounding vessels and extending into adjacent glandular parenchyma. Ductal cell proliferation and epimyoepithelial island formation were not found (23,24). In the current study, we compared 6 murine models of lupus with 2 normal strains of mice that do not develop autoimmune disease. Modified Schirmer tests, histologic evaluation of conjuctivae and lacrimal and salivary glands, and serologic assays for antiSS-A and anti-SS-B antibodies were utilized to determine if lupus-like disease was invariably associated with manifestations of Sjogren’s syndrome. We are aware of only one earlier study which examined tear production in animal models of autoimmune disease. Kessler (21) performed Schirmer tests in 6 NZB/NZW mice. Mean wetting was 3.1 mm, compared with 4.8 mm in NZW mice used as controls. Our studies demonstrated significant ocular dryness in NZB/NZW and PN mice, which had moderately severe lesions in the lacrimal glands. In contrast, MRL/l and MRL/n mice did not have functionally dry eyes, although both substrains had extensive inflammation and destruction of lacrimal glands. Ocular wetting appeared to correlate with body weight in the autoimmune and normal strains of mice tested, and normal tear production in the large MRL animals may have overestimated lacrimal gland function. Nevertheless, the unexpected discrepancy between results of Schirmer tests and lacrimal gland pathology may simply reflect the inadequacy of this test as a sensitive measure of ocular wetting in small animals. In the current study we found varying degrees of inflammation of the conjunctiva in autoimmune strains. Conjunctivitis was most severe in PN mice, which exhibited focal and diffuse inflammation with perivascular infiltrates of lymphocytes and polymorphonuclear leukocytes. The other autoimmune strains had less severe involvement of conjunctivae, and conjunctivitis was not found in C57BL/6J and DBA/2 controls. These findings suggested that individual mu- SJOGREN’S SYNDROME rine models of lupus exhibited selective manifestations of ocular pathology and did not have diffuse inflammatory eye disease. A unique feature of this study was the use of a grading system to compare severity of inflammation in glands from a series of autoimmune models. In autoimmune mice, the most severe inflammatory changes were found in MRL/I and M R L h lacrimal glands. In 25% of MRL mice, at least one-third of the area in lacrimal gland cross sections was replaced by mononuclear cell infiltrates. The most severely involved salivary glands were submaxillary glands. Parotid glands were involved to a mild degree, and sublingual salivary glands resembled controls. Although infiltrates of grade 2+ severity were observed in lacrimal glands from 3 of 18 C57BL/6J-lpr/lpr mice, salivary glands from this strain were normal. It was concluded that the lpr/lpr genome, which accelerates autoimmunity in mice with the MRL genetic background, was not linked with severe or consistent inflammatory lesions in lacrimal and salivary glands. Although severity of inflammation differed in various strains of autoimmune mice, the pattern of mononuclear cell infiltration was the same. Characteristically, lymphocytes and plasma cells surrounded arterioles and ducts, and cells invaded and destroyed acinar tissue. The distribution of inflammatory cells in these animals resembled the abnormalities in lacrimal and salivary glands of NZB and NZB/NZW mice which were described by other investigators (7,23,24). MRL/l mice have severe, early-onset immune complex disease which possesses many characteristics of systemic lupus erythematosus (9) and rheumatoid arthritis (10) A 60% incidence of periductal mononuclear cell infiltrates in salivary glands from 5- to 6month old MRL/I females has been reported by Hang et al(10). Because sialadenitis was not found preferentially in mice affected with destructive rheumatoid-like arthritis, the gland infiltrates were not considered to be related to a specific autoimmune process. Several features of our report offer new perspectives in interpreting lacrimal and salivary inflammation in the MRLA model of lupus. By using a detailed grading system to classify severity of inflammation and studying large numbers of mature mice of both sexes, we were able to reliably demonstrate extensive infiltrates resembling Sjogren’s syndrome in a large percentage of MRL/I and MRL/n mice. Smaller infiltrates were observed in the other autoimmune strains and in a small number of control mice. Mononuclear infiltration followed a strict order of involve- 163 ment in glandular tissue. In almost every instance infiltrates were most extensive in lacrimal glands, and decreasing grades of severity of inflammation were assigned to submandibular, parotid, and sublingual salivary glands, respectively. Furthermore, severity of lesions in these glands did not correlate with the extent of pulmonary or renal infiltration in individual animals. These patterns of involvement support our conclusion that the mononuclear infiltrates reflect a specific, destructive inflammatory process in lacrimal and salivary glands. Examination at the ultrastructural level confirmed the invasive nature of lymphocytic infiltrates in lacrimal glands from 2 MRL/n mice. Electron microscopy was also used to examine lacrimal gland tissue for structures resembling viruses. Type C viruses have been implicated in the pathogenesis of murine lupus (25), and infections with Epstein-Barr virus (26) and cytomegalovirus (27) have been associated with rheumatoid arthritis and Sjogren’s syndrome. Viral particles were not found in the limited number of animals we examined. We also did not find tubuloreticular structures, which have been observed in humans with Sjogren’s syndrome (28). Nevertheless, the absence of viral structures did not exclude the possibility that viral infection contributed to inflammatory lesions in lacrimal and salivary glands of autoimmune mice. Although the precise roles for antibodies to SSA and SS-B have not been defined, they are important markers for Sjogren’s syndrome and systemic lupus erythematosus (2). In some instances, these antibody systems have been implicated as participants in immune complex-mediated tissue injury (2,29-3 I). Although we could not detect anti-SS-A or anti-SS-B using the relatively insensitive Ouchterlony technique, we believe that these antibodics should be sought in autoimmune mice by using more sensitive techniques. We are currently developing an enzyme-linked immunosorbent assay for antibodies to SS-A and SS-B. This sensitive method, which has been utilized to detect anti-SS-B antibodies in human serum (32), will allow sera from autoimmune mice to be reexamined for serologic markers of Sjogren’s syndrome. In humans with Sjogren’s syndrome, infiltrates in labial salivary glands consisted primarily of OKT4positive (T helper-inducer) lymphocytes (33). This finding was of interest in the context of the present study because MRL/I mice have excessive lymph node infiltration with theta antigen-positive cells which do not carry surface antigens Ly-I, Ly-2, or Ly-3. It has been postulated that excessive activity of these murine 164 HOFFMAN ET AL T helper cells contributes to disease in autoimmune mice b y stimulating B cells (34,35).Additional studies of lymphocyte surface markers on infiltrating cells in lacrimal and salivary glands from murine models of lupus will provide insight into the pathogenesis of Sjogren’s syndrome. Identification of subsets of lymphocytes in autoimmune mice may facilitate identification of a murine model that closely resembles Sjogren’s syndrome in humans. ACKNOWLEDGMENTS Harry Kessler, DDS, provided encouragement and helpful advice. The authors acknowledge the expert technical assistance of Julia Burge and Fortune Campbell. Electron microscopy was performed with the assistance of Wayland N. McKenzie, PhD. The Medical Media Service of the Harry S. Truman Memorial Veterans Hospital prepared the figures used in this manuscript. REFERENCES 1. 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