Murine cytomegalovirus induces a Sjögren's syndrome-like disease in C57Bl/6-lpr/lpr mice

ARTHRlTlS & RHEUMATISM Vol. 41, No. 12, December 1998, pp 2175-2184 0 1998, American College of Rheumatology 2175

MURINE CYTOMEGALOVIRUS INDUCES A SJOGREN'S SYNDROME-LIKE DISEASE IN C57B1/6-lpr/lpr MICE

MARTIN FLECK, EARL R. KERN, TONG ZHOU, BERNHARD LANG, and JOHN D. MOUNTZ

Objective. To analyze Fas and tumor necrosis factor receptor I (TNFRI) apoptosis pathways in salivary gland inflammatory disease induced by murine cytomegaloviru s (MCMV) infection.

Methods. Four different strains of mice (C57B1/6 [B6] - +/ + , Fas-deficient B6-lprllpr, TNFRI-deficient B6- tnfrl"", and B6-tnfrlo"-lpr/lpr mice) were infected intraperitoneally with the Smith strain of MCMV (1 X lo5 plaque-forming units). Viral load was determined by a plaque assay, inflammation and apoptosis by immunohistochemistry and staining with terminal dUTP nick- end labeling, and autoantibodies by enzyme-linked immunosorbent assay.

Results. Infectious MCMV was not detectable by day 100. Although all MCMV-infected mice developed acute sialadenitis by day 28, a chronic (>lo0 days), severe salivary gland inflammation and anti-Ro and anti-La antibodies developed only in the B6-Zprllpr mice. Apoptotic cells were detected during the acute, but not the chronic, phase of inflammation.

Conclusion. Both Fas- and TNFRI-mediated apoptosis contribute to the clearance of MCMV-infected cells in the salivary glands. However, because Fas-

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Dr. Fleck's work was supported by a grant from the Deutschc Forschungs Gcmcinschaft. Dr. Kern's work was supported by Health Service contract no. N01-AI-65290 from the National Institute of Allergy and Infectious Diseases, NIH. and a grant from Sankyo. Inc. Dr. Zhou is the recipient of an Arthritis Foundation Investigator Award. Dr. Mountz's work was supported in part by a Veterans Administration Career Development and Mcrit Review Award and NIH grants N01-AR-6-2224 and R01-AR-42547 from the National Institute of Arthritis and Musculoskeletal Diseases.

Martin Fleck; MD: The University of Regensburg, Regens- burg, Germany. and The University of Alabama at Birmingham; Earl R. Kern, PhD, Tong Zhou, MD: The University of Alabama at Birmingham; Bernhard Lang, MD: The Univcrsity of Regensburg, Regensburg, Gcrmanv; John D. Mountz, MD, PhD: The Univcrsity of Alabama at Birmingham, and Veterans Administration Medical Cen- ter, Birmingham, Alabama.

Address reprint requcsts to John D. Mountz, MD, PhD, Department of Medicine, The University of Alabama at Birmingham, 701 South 19th Street, LHRB 473, Birmingham, AL 35294.

Submitted for publication February 17, 1998; acceptcd in revised form May 26, 1998.

mediated apoptosis is necessary for the down- modulation of the immune response, a defect in this process can lead to a postinfection, chronic inflammatory response that resembles Sjogren's syndrome.

Sialadenitis is a common feature of several disease states, including primary Sjiigren's syndrome (SS), acute inflammation due to viral infections, and a sec- ondary syndrome associated with various autoimmune diseases (1,2). SS is an autoimmune disease that is characterized by infiltration of the exocrine glands with mononuclear cells, predominantly T cells, which, due to acinar and ductal gland destruction, leads to a sicca syndrome (1-3). Activation of autoreactive B cells, as indicated by the production of autoantibodies against the Ro/SSA and La/SSB autoantigens, i s also typical in SS (1-3). Recent evidence indicates that apoptosis mediated by Fas (CD95iApo-1) may contribute to the pathogenesis of SS, since increased Fas-mediated apoptosis in acinar cpithelial cells and decreased apoptosis of infiltrating T cells has been reported by several investigators (4-6).

Viral infections may be involved in SS. Several studies have suggested an association between SS and, mainly, hepatitis C virus (7-9) and viruses of the herpes family, including Epstein-Barr virus (EBV) (10-15) and cytomegalovirus (CMV) (1 6,17). Infection of immuno- compromised patients with human CMV leads to wide- spread disease, including sialadenitis ( 3 8,19). Murine CMV (MCMV) has homology with certain regions of human CMV (20), and MCMV infection in mice exhib- its a course of infection similar to that in humans with regard to the establishment of acute and chronic infection, viral persistency and latency, and the host immune response (21,22).

Although all strains of mice that are homozygous for the Ipr gene develop autoimmune disease, the disease phenotype and its severity also depend on the genetic background and environmental factors (23-26). Aged MRL-Zpr/Zpr mice spontaneously develop an auto-

2176 FLECK ET AL

immune disease with features resembling those of SS. Circulating autoantibodies, including anti-Ro and anti- La, are present and the salivary glands are infiltrated with CD4+ and CD3+ B220- T cells (27,28). These infiltrating 1 cells show reduced apoptotic function, thus suggesting that de€ective Fas-mediated apoptosis might contribute to the inflammation of the salivary glands in MRL-lpr/lpr mice (29). Expression of the Ipr gene on the C57BV6 (B6) background results in the development of a milder autoimmune disease of later onsct. Although they exhibit glomerulonephritis after -6-8 months of age, Bfi-lprilpr mice do not spontaneously develop significant salivary gland disease (23-26).

Apoptosis can be mediated by either tumor necrosis factor receptor I (TNFRI) or Fas. These receptors are homologous, especially in the intracellular death domains, where some of the same cytoplasmic signaling proteins are utilized (30,31). In contrast to Fas-dcficient B6 mice, TNFRT-deficient mice do not develop lymphoproliferation or autoimmune disease. Mice that are deficient in both Fas and TNFRI, however, exhibit higher mortality, accelerated lymphoproliferation, and exacerbated autoimmune disease in comparison with B6-Ipr/Ipr mice. Thus, the Fas and TNFRI signaling and apoptosis pathways may compensate for each other (25).

To determine the significance of apoptosis mediated by Fas and TNFRI in viral-induced salivary gland disease, B6-+/+ mice, B6-lpr/Zpr mice, TNFRI-knockout (B6-tnfr‘”l’) mice, and Fas-deficient TNFR1-knockout (B6-tnj~””-lpr/lpr) mice were inoculated intraperitoneally with MCMV. The present results indicate that apoptosis mediated by either Fas or TNFRI can contribute to the clearance of MCMV-infected cells from salivary gland tissue. However, apoptosis mediated by Fas, but not by TNFRI, is critical for down-modulation of the immune response, so that defective expression of Fas will lead to a chronic, inflammatory SS-like disease following MCMV infection, despite the apparent absence of infectious virus.

MATERIALS AND METHODS

Animals. Female B6--/+ and B6-lprllpr mice, ranging in age from 10 weeks to 14 weeks, were obtained from Jackson Laboratories (Bar Harbor, ME). B6-tnfi/‘lo and Bh-tnfkO’*-lpr/ lpr mice were generated as described previously (25) and were bred and maintained in our own animal facility at The Uni- versity of Alabama at Birmingham, under pathogen-free conditions. At least 5 mice were analyzed at each time point.

Virus and virus titration. Analysis of viral clearance was carried out using 5 mice per group for each of the 4 mouse strains (B6-+/+, B6-lpr/lpr, B6-tnfrl0’”, and B6-tnfvlo‘o-lpr/lpr). Samples were obtained at 5 time points (7, 14, 28, 80, and 100

days after viral infection). The MCMV stock-virus pool (3 X 10’ plaque-forming units [PFU]/ml) was prepared by intraperitoneal inoculation of femalc BALB/c mice with the Smith strain of MCMV, which was obtained from Amcrican Typc Culture Collection (Rockvillc, MD). The salivary glands were collected 12 days later and homogcnizcd in minimal essential medium (Gibco, Grand Island, NY) containing 10% fetal bovine serum, using a mechanical grinder. The suspension was then centrifugcd at 1,500 revolutions per minute for 15 minutes at 4°C to remove ccll debris. The supernatant was dispensed into aliquots. which were stored at -80°C until used. For determination of the virus titer in salivary gland tissucs of experimenlal mice, salivary glands were removed and homog- enized as 10% (weightlvolume) suspensions in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with L-glutaminc, 10% fetal calf serum, penicillinlstreptomycin, and amphotcricin B. The homogenates were titrated in duplicate in log,,, dilutions on subconfluent primary murine embryo fibroblasts in 12-well plates. Scvcn days later, monolayers were stained with neutral red and the number of plaques was counted. To verify that the virus titcr was 0 at days 80 and 100 after MCMV infection in the B6-+/+, B6-lpr/lpr, and B6- tnfvl0” mice, duplicatc analyscs were carried out on separate occasions using differcnt platcs of indicator cells.

Histologic assessment and immunophenotyping. His- tologic analysis was carried out on thc salivary glands fi-om the 4 strains of mice (5 miceigroup) on days 0, 7, 28, and 100. Salivary glands were removed and fixed in 10% phosphate buffered formalin. Aftcr paraffin embedding, tissue sections were cut (5 pm) and staincd with hcmatoxylin and eosin for histologic evaluation. Histopathologic scoring was performed by rating the severity of lesions and infiltration on a scale from 0 to 5. The following criteria were applied for the grading scale: 0 = normal histologic appearance, 1 = minimal mononuclear cell infiltration, with or without minimal cell destruction, 2 = modest mononuclear cell infiltration with 1-2 clear foci per lobe, with focal cellular destruction, 3 = numerous aggregates (3-5) of mononuclear cells per lobe, with destruction of acinar or ductal cells at several locations, 4 = severc infiltration with mononuclear cells in multiple foci per lobe, with destruction of acinar and ductal cells, and 5 = diffuse infiltration with mononuclear cells and the most severe cellular destruction. The histologic score was assessed in a blinded manner by 3 different examiners. Each observer assigned a histologic score after examining at least 10 different fields of vicw in tissuc sections from each strain of mice at each time point. Thc histologic score for each strain was determined by calculating the mean iz SEM of the 3 observers’ scores.

For immunohistochemistry studies, slides were first incubatcd with a peroxidasc-conjugated monoclonal antibody specific for T cell receptoriCD3 (Dako, Glostrup, Denmark) or a biotin-conjugated monoclonal antibody specific for B220 (PharMingen, San Diego. CA). The biotinylated antibody specific for B220 was detected by application of peroxidase- conjugated streptavidin (Sigma, St. Louis, MO). Positive reactions were revealed using diaminobenzidene substrate (Sig- ma), and methyl green was used for counterstaining.

Terminal deoxynucleotidyl transferase (TDT)- mediated dUTP nick-end labeling (TUNEL) analysis. The extent of apoptosis was analyzed by the TUNEL method, which was modified slightly from that described prcviously

MCMV-INDUCED DISEASE IN C57B1/6-@/lpr MICE 2177

(32). Briefly, formalin-fixed and paraffin-embedded lissue sections were deparaffinized and rehydrated. After thorough washing with deionized water, the tissue scctions wcrc incubated with 10 p,g/ml proteinase K (Sigma) at room temperature for 15 minutes, and then incubated at 37°C for 60 minutes with freshly prepared TDT reaction mix (Boehringer Mann- heim, Indianapolis, IN) containing 0.4 unitsip1 TDT, 10 nM digoxigenin-modified dUTP, and TDT buffer. The incorpo- rated digoxigenin-dUTP was detected by incubation with alkaline phosphatase-conjugated antidigoxigenin antibody at room ternpcrature for 60 minutes, and positive reactions were revealed using nitroblue tetrazolium!BCTP substrate. Methyl grccn was used for counterstaining. The percentage of apoptosis at day 28 and day 100 after infection was determined using 5 miceigroup at each time point. At least 10 different fields of view in cach strain of mice wcre examincd at each time point. In each field of view, the total number of inflammatory cells and the total number of acinar and epithelial cells was determined, and the percentage of apoptosis in each population was determined by dividing the number of apoptotic cells by the total number of each cell type, respectively. The percentage of apoptosis in each cell type was determined for each mouse individually, and then the mean i SEM was calculated.

Quantitation of autoantibudies. Serum levels of anti- double-stranded DNA (anti-dsDNA) autoantibodies and rheumatoid factor (RF) were dctcrmined by sandwich cnzyme- linked immunosorbent assay (ELISA) as described previously (33). Bricfly, for quantitation of anti-dsDNA antibodies, 96- well microtiter plates were precoated with 10 p,g/ml of poly-L- lysine, followed by coating with 10 pdml of dsDNA or 10 p,g/ml of poly-L-glutamic acid as background control. For detection of RF, 96-well microtiter plates were coated with 4 p,g/ml of affinity-purified rabbit IgG (all reagents were obtained from Sigma). For detection of antibodies specific for RoiSSA or LalSSB, standard ELISA plates precoatcd with autoantigen wcrc used (Upjohn Pharmacia, Minneapolis, MN). The sera wcre diluted at 1:100 and incubated at room temperature for 4 hours. Bound anti-dsDNA antibodies or RF were detected by isotype-specific alkaline phosphatase- conjugated goat anti-mouse Ig (PharMingen), and p-nitrophenylphosphatc (Sigma) was used as substrate. Color development was measured at 405 nm using an Emax micro- plate reader (Molecular Devices, Mcnlo Park, CA).

Statistical analysis. Viral titers, histologic scores, percentage of apoptotic cells, and autoantibody levels in each group of mice were compared at the same time point. Student's 2-tailed t-test was used for statistical analysis when 2 different groups of samples were compared. One-way analysis of vari- ance was used when more than 2 groups of samplcs were compared. A P value of less than 0.05 was considered statistically significant. Values arc expressed as the mean t SEM.

RESULTS

Combined defects in Fas- and TNFRI-mediated apoptosis leading to delayed clearance of infectious MCMV. B6- +I+, B6-lpr/lpr, B6-fr~fr*'~, and B6-tnfro'O-lprI lpr mice were infected intraperitoneally with the Smith strain of MCMV (1 X lo5 PFU). Salivary glands (5 mice

6 T

0 25 50 75 100 1

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Figure 1. Delayed clearance of infectious murine cytomegalovirus (MCMV) in Fas and tumor necrosis factor receptor 1 double-deficient mice. B6-+/+ (O), B6-lpr/lJ~r ( 0 ) , B6-ln[P'" (O), and B6-tr$ifl'o-Zpr/lpr (A) mice were inoculated intraperitoneally with MCMV (1 X 105 plaque-forming units [PFU]). Quantit ation of infectious MCMV in the salivary glands was performed in at least 5 individual mice at each of the indicated time points using a plaque assay. Bars show the mean 2 SEM. * = P < 0.05 compared with the other groups of mice at these time points.

per group) were obtained at 5 different time points after virus infection, and the titers of MCMV in the salivary gland tissue were determined separately by a plaque assay using mouse embryo fibroblasts (Figure 1). High titers of infectious MCMV were detected in the salivary glands of all mouse types within 7 days of viral infection. By day 14, viral clearance in B(i-+/+ mice had been initiated (P < 0.05) , whereas the infectious titers of MCMV were at their peak in the other types of mice. Progressive clearance of infectious MCMV from B6- +/+, B6-@/@, and B6-tnpo'0 mice occurred after day 14, and by days 80 and 100, infectious virus was not detectable in any of these mice. In contrast, the viral titers in B6-tn$-o'0-lpr/&r mice were highest at days 7 and 14 after infection, and the infection persisted with high levels of viral replication up to day 100 (P < 0.05). These results indicate that deficiencies in either Fas or TKFRI do not affcct the clearance of MCMV in B(i-Zpr/lpr and B6-tnfr'"' mice compared with B6-+/+ mice. However, combined deficiencies in both Fas and TNFRI will result in delayed clearance of infectious MCMV from the salivary glands.

2178 FLECK ET AL

Day 28 Day 100

Figure 2. MCMV-induccd chronic disease in B6-lprilpv mice. B6-+/+, B6-tnfS", Bfj-&r/&r, and B6-tr@'*-&r/ilpr mice were inoculated intraperitoneally with MCMV (1 X 10' PFU). Histologic evaluation was performed at different time points after infection. Representative sections from the 4 mouse strains are depicted. A, B6-+/+ infected with MChW, on day 28; B, B6-+/+ on day 100; C, B6-bzfp'" infected with MCMV, on day 28; D, B6-tnfi'J'' on day 100; E, Bb-@Llpr infected with MCMV, on day 28; F, B6-lpr/lpr on day 100; G, B6-tnfiulo-&r/&r infected with MCMV, o n day 2.8; H, uninfected B6-/pr/&r control. (Original magnification X 320.) See Figure 1 for definitions.

MCMV-INDUCED DISEASE IN C57B1/6-lpp./lpr MICE 2179

MCMV-induced, SS-like chronic sialadenitis in Fas-mutant lprllpr mice. Extensive inflammatory cell Salivarv Gland infiltration was observed in the salivary glands of B6-

tion, a time point at which infectious MCMV was still detectable in these mice (Figures 2A, C, and E). Inflam- mation was not observed in the salivary glands of B6-+/+ and B6-tnfr0'0 mice on day 100 postinfection, at which time infectious MCMV was cleared from the salivary glands in these mouse types (Figures 2B and D). In contrast, a persistent inflammatory disease maintained by foci of infiltrating mononuclear cells in the salivary glands of BG-lpr/lpr mice was observed on day 100 postinfection, despite the absence of detectable virus in the salivary gland tissue of B6-lprilpr mice at this late time point (Figure 2F). There was nearly equivalent inflammation in MCMV-infected B6-tnfrl""-lpr/lpr mice on day 28 (Figure 2G) as there was in B6-&r/Zpr mice on day 28 (Figure 2E). This inflammation required the initial infectious trigger provided by MCMV, since no inflammation could be observed in the salivary glands of uninfected, sex- and age-matched B6-lprilpr mice (Fig- urc 2H).

Infiltration of thc salivary glands as well as acinar and ductal cell destruction were assessed by histopatho-

+/+, B6-tnfrD", and B6-lpr/lpr mice at day 28 postinfec- 66-/pr/ ' /p~( Day 1 00)

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day Figure 4. Tmmunophenotype o f infiltrating cells. A, Infiltrating mononuclear cells in salivary glands of €36-@r/@r mice I00 days

Figure 3. Histologic scoring of tissue sections. Salivary gland tissue sections from B6-+/+ (U), B6-tnfP'" (W), Bb-lpr/lpr (H), and Bh-fnJP"-lpr/@r (W) mice were assessed for scvcrity of inflammation and tissue damage on a relativc scale ranging from 0 (not present) to 5 (most severe), by 3 blinded observers. * = P < 0.05 versus controls, by Student's t-test.

following murinc cytomcgalovirus (MCMV) infcction (stained with hematoxylin and eosin). B, Infiltrating CD3+ T cclls during the chronic phase of MCMV-induced sialadenitis in B&bir/@r mice at day 100. C, Expression or B220 antigen in lymphocytes 100 days after MCMV infection in salivary glands of Bh-lpr//pr mice. (Original magnification X 320.)

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FLECK ET AL

Iprllpr

Figure 5. Apoptotic cells in situ. Apoptotic cells were identified by labeling the characteristic DNA strand breaks using the terminal dUTP nick-end labeling reaction in salivary gland tissue from murine cytomegalovirus- infected B6-+/+ (A and C) and B&lpr/lpr (B and D) mice at days 28 and 100, respectively. Small arrows indicate apoptosis among infiltrating cells. Large arrows indicate epithclial cclls undcrgoing apoptosis. (Original magnification X 320.)

logic analysis. Three observers graded thc histologic appearance of each sample on a relative scale ranging from 0 (normal) to 5 (most severe), as defined in Materials and Methods. Figure 3 indicates a statistically significant increase in the histologic score for inflammation at day 100 in the Bb-lpr/lpr mice and B6-tnfrlolo-lpri Zpr mice compared with the control mice, as determined by Student's t-test (P < 0.05).

Immunophenotype of infiltrating cells. Previous studies have demonstrated that MCMV infection induces infiltration of the salivary glands with inflammatory cells, including T cells, natural killer cells, and macrophages (34-36). To characterize the cell population associated with the development of chronic sialadenitis, salivary glands were obtained from B6-lpr/@r mice at different time points following MCMV infection and analyzed by immunohistochemistry. There were substan- tial numbers of CD3+ T cells that were B220 negative (Figure 4B). As a positive control, lymph nodes were evaluated on each section that were CD3du" and B220

positive (data not shown). Thus, the phenotype of infiltrating cells in the MCMV-induced sialadenitis in H6-Zprilpr mice was similar to that observed in the spontaneous sialadenitis that develops in aged MRL-lprl $r mice (27,28).

Apoptotic cells in situ in salivary gland tissue after infection with MCMV. To analyze apoptosis of acinar epithelial cells and inflammatory cells, salivary gland tissue from B6-+/+ and B6-Zprllpr mice was examined both during acute MCMV-induced sialadenitis associated with high viral replication (day 28) and during late-stage, chronic inflammatory disease (day 100) in B6-lpriZpr mice. Apoptosis was observed in acinar and epithelial cells and in high numbers of infiltrating cells on day 28 in both B6+/+ and B6-lpr/Zpr mice (Figure 5) . There was a statistically significant increase in apoptosis of inflammatory cells in the B6-+/+ mice compared with the Bfi-ijdZpr mice ( P < 0.01 ) (Figure 6). On day 100, there were no apoptotic inflammatory cells detected in the B6-+/+ mice, and only 1.2% of the

MCMV-INDUCED DTSEASE IN C57B1/6-@r/@r MICE 2181

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Figure 6. Apoptosis of inflammatory and glandular cells in Bb-lyr/@r mice compared with B6-+/+ mice. Quaiititation of apoptosis of inflammatory and glandular cells was performed on tissue sections from at least 5 individual mice at the indicated time points (day [D] 28 and day 100 after infection). Bars show the iiiean and SEM. * = P < 0.01; * * = P < 0.05.

inflammatory cells were undergoing apoptosis in the B6-lprilpr mice. There was no significant difference in the percentage of apoptotic glandular cells betwcen these 2 mouse strains on day 28, but there was an increase in the percentage of apoptosis of glandular cells on day 100 in the B6-lprilpr mice compared with the B6-+i+ mice (P < 0.05). These results indicate that during the early stage of inflammatory disease, defective Fas-mediated apoptosis is associated with a persistent inflammatory infiltrate, and during later phases of the disease, increased apoptosis of glandular cells occurs in B6-lpr/lpr mice compared with B6-ti+ mice.

Enhanced autoantibody production in MCMV- infected B6-lpr/lpr mice. The levels of RF, anti-dsDNA, anti-Ro/SSA, or anti-LaiSSB autoantibodies in B6- +/+ mice 100 days after infection with MCMV were no higher than those in uninfected B6-+i+ mice (Figure 7). Higher expression of RF and anti-dsDNA antibodies was exhibited by uninfected Bb-lprilpr mice compared with B6-+i+ mice in the absencc of highcr expression of anti-Ro or anti-La antibodies. MCMV infection resulted in a significantly higher level of RF in B6-lyrilpr mice (P < 0.0s) (Figure 7). Furthermore, significantly higher expression of anti-Ro and anti-La autoantibodies was observed in the B6-lprilpr mice 100 days after MCMV infection, but this was not observed in uninfected B6-lpri lpr mice or in infected or uninfected B6-+/+ mice.

DISCUSSION

The contribution of viral infections to the pathogenesis of SS is controversial. The chronic sialadenitis of

human SS has been associated with several different viruses, including CMV (16,17), EBV (10,14), hepatitis C virus (&lo), and human herpes virus 6 (37), as determined by the presence of virus-specific DNA or RNA sequences in tissue samples. Other investigators, however, were not able to demonstratc a link between SS and infections with EBV (11,12) or CMV (11). One of the major problems in the interpretation of these findings is that biopsy samples were obtained at different time points after the onset of SS or chronic sialadenitis. Actively infected cells and viral antigens are cleared at different rates in different individuals. Moreover, whereas EBV is known to have the capacity to establish a latent infection in B cells, the cell population that eventually becomes latently infected during the course of CMV as well as MCMV infection is not clearly defined, and the mechanisms by which latency is established are poorly understood (38,39). Evidence indicates that blood cell progenitor cells in the bone marrow may be a source of latent CMV infection (40,41), and endothelial cells appear to be a potential site of latent MCMV infection (42). Furthermore, MCMV DNA has

Rheumatoid Factor (IgG)

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Figure 7. Enhanced autoantibody production in murine cytomegalovirus (MCMV)-infected Ipri!pr mice. Serum levels of rheumatoid factor as well as anti-double-stranded DNA (&DNA), anti-Ro (SS-A), and anti-La (SS-€3) autoantibodies were evaluated 100 days following MCMV infection in Bb-+i+ (0) and B6-lprilpr (A) mice, and in uninfected control B6-+/+ (0) and Bb-lprilpr (0) mice, using specific enzyme-linked immunosorbent assays. The optical density (O.D.) at 405 nm was plotted for each mouse separately. Bars show the mean -t SEM. * = P < 0.05 compared with the other groups of mice.

2182 FLECK ET AL

been detected in several types of tissue, including salivary glands, in the absence of infectious virus, which again suggests the occurrence of latent MCMV infection (43,44). In these experiments, persistence of replicating MCMV in tissues other than the salivary gland did not occur, and the virus was rapidly cleared from the liver, lung. and kidney in both B6-+/+ and B6-Zpr/Zpr mice within 2 weeks of infection.

The present results demonstrating that MCMV induces a chronic, postviral sialadenitis in I36-Zprilyr mice do not indicate that human SS is due to MCMV or requires an underlying defect in Fas-mediated apoptosis. Further analysis in humans who are preferably at the early stages of S S will be required to determine the role of virus infections as an initial trigger in human SS.

The present report is the first study of inflammatory salivary gland disease induced by infection with MCMV in B6-+/+, B6-lpr/lpr, and B6-tnfiDf0 mice. These results demonstrate that intraperitoneal administration of MCMV leads to a persistent infection in the salivary glands. Actively infected cells were cleared between 28 days and 80 days following infection in all 3 strains of mice, which is consistent with previous studies that have analyzed MCMV replication in the salivary glands of B6-+/+ mice (39,44). However, a chronic sialadenitis was established only in the salivary glands of Bb-lpr/lpr mice, despite the absence of detectable infectious MCMV by day 100. This chronic inflammation was mainly promotcd by T cells, since numerous CD3- expressing cells could bc identified by immunohistochemistry. Because the T cells did not coexpress B220, they were not identical to the population of B220- positive T cells that are predominant in the enlarged lymph nodes characteristic of BO-lpr/Zpr mice (27-29). These results suggest thal a viral infection can initiate a chronic inflammatory disease, but thc presence of infectious virus is not required to maintain the inflammation. However, the possibility of a persistent infection at a very low level or a latent infection without replication cannot be excluded. These results imply that the detection of viruses in salivary gland biopsy tissue from patients with chronic sialadenitis would depend on the timing of the biopsy with regard to thc initial infection. This may, in part, explain why some investigators find an association between chronic sialadenitis and viral infections whereas others do not.

Lymphocytic infiltration of the salivary glands may occur in response to the presentation of viral and self antigens by antigen-presenting cells. Wittingham et a1 (14) noted that in EBV-induced sialadenitis, an association between viral RNA and the La nucleopro-

\ B6-lnfr o'o-lpr/lpr - TNFa + TNFRI FasL Fas

Infection Infection

(3 Normal

I Chronic I Inflammation Chronic I Inflammation

Figure 8. Different clearance of virus and inflammation in B6-++, B6-1pr/lpr> and B6-tnfiI"o~lyr/lpr mice. Murine cytomegalovirus (MCMV)-inlected host cells elicit an immune response that results in clearance of replicating virus by day 80 in the salivary glands. Viral clearance requires the presence o f either tumor necrosis factor receptor 1 (TNFRT) or Faa, since clearance occurs in mice deficient of either of these 2 receptors. hut not in doublc-deficient B6-trzfrlo'"-lpr/lpr mice. Therc is cfficicnt clearance of the inflammatory response to the virus in BO-++ mice, but not in B6-lyrilpr mice, which develop a chronic inflammation in the salivary glands and demonstrate autoantibody production of anti-Ro and anti-La. This inflammation is not associated with the replicating virus. which is cleared by day 80. B6-fr~/rl~'~-lpr/lpr mice also develop a chronic inflammation in the salivary glands, but in contrast to B6-49417 micc. this is associated with low clearance of MCMV from the salivary glands and chronic infection of these mice at 100 days postinfection. FasL = Fas ligand.

tein resulted in a break in the immunologic tolerance of T cells, with induction of anti-La autoantibodies by polyclonal activation of B cells; this process may lead to autoimmune sialadenitis (14). The most frequently rec- ognized linear epitopes of the LaiSSB autoantigen exhibit sequence similarities with proteins encoded by a wide range of human viruses, particularly those of the herpes virus group (45). Other foreign or self antigens, such as a-fodrin, have been proposed as an initiating factor (46). The present experiments demonstrate a significant increase in the levels of anti-La/SSB and anti-Ro/SSA autoantibodies in the sera of B6-lpr/lyr mice infected with MCMV compared with uninfected B6-lprilpr control mice or with B6-+/+ mice after MCMV infection. Thercfore, we propose that the ini- tially increased MCMV replication in the salivary glands combined with the defect in Fas-mediated apoptosis in inflammatory lymphocytic cells lcads to a tolerance loss in B6-Zpr/lpr mice, with generation of T cells that pro- mote production of anti-Ro and anti-La autoantibodics.

MCMV-INDUCED DISEASE IN C57B1/6-lpr/lpr MICE 21 83

Altered apoptosis has been reported in salivary gland ductal and acinar cells as well as in lymphocytic infiltrating cells during chronic sialadenitis. Increased Bcl-2 expression in infiltrating lymphocytes associated with decreased sensitivity to Fas-mediated apoptosis has been described in patients with SS (6,47,48). Our findings of decreased apoptosis and the persistence of infiltrating lymphocytes in the salivary glands of Fas- deficient B6-@r/lpr mice are consistent with this. Other studies indicate that ductal and acinar cells may be more susceptible to apoptosis, and that T cells may produce Fas ligand, thus inducing apoptosis of the acinar cells and salivary gland destruction (5,49). In the present study, our observation of increased apoptosis of glandular cells at day 100 in B6-lpriZpr mice is consistent with these findings, except that apoptosis pathways other than Fas were likely mediating the apoptosis in Bb-lpdlpr mice. Other apoptosis pathways leading to the destruction of acinar cells have also been described, including cell death in cell lines treated with interferon-? and tumor necrosis factor a (TNFa) (50,Sl). This is supported by the finding of elevated levels of TNFa in the sera of MCMV-infected B6-lpr/lpr mice (Fleck M et al: unpublished observations). These results suggest that multiple pathways of apoptosis may be operative in infiltrating lymphocytes and in ductal and acinar cells, and that dysregulation of apoptosis may occur in patients with SS.

The present data suggest that clearance of MCMV infection requires the presence of either Fas or TNFRI, since viral clearance does not occur in B6-lprl lpr-tnfp”” mice. Fas signaling is required for the subse- quent clearance of inflammatory cells, since chronic inflammation occurs in Fas-deficient B6-EprlEpr mice but not in B6-tnfP’” mice (Figure 8). These results suggest that Fas-mediated apoptosis after MCMV infection is important for down-modulation of the immune response to the virus, and plays a role in prevention of chronic inflammation and autoantibody production.

In summary, we have shown that an infectious agent (MCMV) and a genetic defect (the Fas mutation) can induce an SS-like disease that persists after clearance of the virus, and continues despite high apoptosis of infiltrating cells. Thus, features of the chronic disease state do not necessarily reflect the genetic or environmental factors that caused the disease. This model is consistent with the concept that autoimmune disease develops in genetically predisposed individuals after an exposure to an environmental stimulus. The clinical features of an autoimmune disease triggered by an infectious agent, as in the current model, would most

likely depend on the tropism of the infectious agent and on the genetic susceptibility of the individual, leading to persistent inflammation after clearance of the infection.

ACKNOWLEDGMENTS

The authors wish to thank Joyce Palmer and Rachel Rybak for their excellent asqistance during the studies, Fiona Hunter and Judy White for their expert review and preparation of the manuscript, and Mike Amos, PhD, from Pharmacia Upjohn, for providing the ELISA kits for detection of the autoantibodies.

REFERENCES

1. Tala1 N. Sjogren’s syndrome and connective tissue diseases associated with other immunologic disordcrs. In: McCarty DJ, Koop- man WJ, editors. Arthritis and allied conditions. Philadclphia: Lea & Febiger; 1993. p. 1343-56,

2. Fox RI, Maruyama T. Pathogenesis and treatment of Sjogren’s syndrome. Curr Opin Rheumatol 1997:9:393-9.

3 . Fox RI. Clinical features, pathogenesis, and treatment of Sjogren’s syndrome. Curr Opin Rheumatol 1996;8:438-45.

4. Kong L, Ogawa N, Nakabayashi T, Liu GT, D’Souza E, McGuff HS, et al. Fas and Fas ligand expression in the salivary glands of patients with primary Sjogren’s syndrome. Arthritis Rheum 1997;

5. Sumida T, Matsumoto I, Murata H, Namekawa T, Matsumura R, Tomoika H, et al. TCR in Fas-sensitive T cells from labial salivary glands of patients with Sjogren’s syndrome. J lmmunol 1997;158: 1020-5.

6, Ichikawa Y, Arimori K, Yoshida M, Horiki T; Hoshina Y, Morita K, et al. Abnormal expression of apoptosis-related antigens. Fas and bcl-2, on circulating T lymphocyte subsets in primary Sjogren’s syndrome. Clin Exp Rheumatol 1995;13:307-13.

7. Koike K, Moryia K, Ishibashi K, Yotsuyanagi H, Shintani Y, Fujie H, et al. Sialadenitis histologically resembling Sjogren’s syndrome in mice transgenic for hepatitis C virus envelope genes. Proc Natl Acad Sci U S A 1997;94:233-6.

8. Scott CA, Avellini C, Desinan L, Pirisi M, Ferraccioli GF, Bardus P, et al. Chronic lymphocytic sialadenitis in HCV-related chronic liver disease: comparison of Sjogren’s syndrome. Histopathology

9. Haddad J, Deny P, Munz-Gotheil C, Ambrosini JC, Trinchet JC, Pateron D, et al. Lymphocytic sialadenitis of Sjiigren’s syndrome associated with chronic hepatitis C virus liver disease. Lancet

10. Wen S, Shimizu N, Yoshiyama H, Mizugaki Y, Shinozaki F, Takada K. Association of Epstein-Barr virus (EBV) with Sjogren’s syndrome: differential EBV expression patterns between epithelial cells and lymphocytes in salivary glands. Am J Pathol 1996;149:

11. Maitland N, Flint S, Scully C, Crean SJ. Detection of cytomegalovirus and Epstein-3arr virus in labial salivary glands in Sjogren’s syndrome and non-specific sialadenitis. J Oral Pathol Med 1995;

12. Syrjanen S, Karja V, Chang FJ, Johansson B: Syrjanen K. Epstein- Barr virus involvement in salivary gland lesions associated with Sjogren’s syndrome. ORL J Otorhinolaryngol Relat Spec 1990;52:

13. Clark DA, Lamey PJ. Jarett RF, Onions DE. A model to study viral and cytokine involvement in Sjogren’s syndrome. Autoimmu- nity 1994;18:7-14.

40:87-97.

1997;30:41-8.

19923391321-3,

1511-7.

24:293-8.

254-9.

2184 FLECK ET AL

14. Wittingham S, McNeilagc J, Mackay IR. Primary Sjogren’s syndrome after mononucleosis. Ann Intern Med 1985;102:490-3.

15. Newhrk MM, Shiroky JB, Johnson N, Danoff I), lscnberg DA, Shustik C, et al. Rheumatic disease patients, prone to Sjogren’s syndrome and/or lymphoma, mount an antibody responsc to BHRF1, the Epstein-Barr viral homologue of BCL-2. Br J Rheu- matol 1996;35:1075-81.

16. Thorn JJ, Oxholm P, Andersen HK. High levels of complement fixing antibodics against cytomegalovirus in patients with primary Sjiigren’s syndrome. Clin Exp Rheumatol 1988;6:71-4.

17. Wax TD, Layfield LJ, Zaleski S, Bhargara V, Cohen M, Lyerly HK, et al. Cytomegalovirus sialadenitis in patients with the ac- quired immunodeficiency syndrome: a potential diagnostic pitfall with fine-needle aspiration cytology. Diagn Cytopathol 1994;lO:

18. Schiodt M, Greenspan D, Daniels TE, Nelson J, Legott PJ, Wara DW, et al. Parotid gland cnlargcmcnt and xcrostomia associated with labial sialadenitis in HIV-infected patients. J Autoimmun

19. Smith NA, Leen EJ, Derias NW, Nicholson F; Bingham JS. Massive salivary gland swelling due to primary cytomegalovirus infection in an AIDS patient. Int J STD AIDS 1997;8:528-9.

20. Messerle M, Keil GM, Schncidcr K, Koszinowski UH. Character- ization of the murine cytomegalovirus genes encoding the major DNA binding protein and the ICP18.5 homolog. Virology 1992;

21. Hudson JB. The murine cytomegalovirus as a model for the study of viral pathogenesis and pcrsistcnt infections. Arch Virol 1979;

22. Tanaka K, Koga Y, I,u YY, Zhang XY, Wang Y. Kimura G, et al. Murine cytomegalovirus-associated pneumonitis in the lungs free of the virus. J Clin Invest 1994;94:1019-25.

23. Cohen PL, Eisenberg FL4. Autoimmune Ipr and gld mice. Annu Rev Immunol 1992;9:243-65.

24. Gilkeson GS, Ruiz P, Pritchard AJ, Pisetsky DS. Genetic control of inflammatory arthritis and glomcruloncphritis in congenic lpr mice and their F1 hybrids. J Autoimmun 1991;4:595-609.

25. Zhou T, Edwards CK 111, Yang P, Wang Z, Bluethniann H, Mountz JI). Greatly accelerated lymphadenopathy and autoimmune disease in Ipr mice lacking tumor necrosis factor receptor 1. J Immunol lY96;156:2661-5.

26. Vaishnaw AK, McNally JD, Elkon KB. Apoptosis in the rheumatic diseases. Arthritis Rheum 1997;40:1917-27.

27. Theofilopoulos AT, Dixon FJ. Murine models of systemic lupus erythematosus. Adv Immunol 1987;37:269-380.

28. Mountz JD, Gause WC. Murine models of autoimmune disease and Sjogren’s syndrome. Curr Opin Kheumatol 1993;5:557-69.

29. Skarstein K, Nerland AH, Eidsheim M, Mountz JD, Jonsson R. Lymphoid cell accumulation in salivary glands of autoimmune MRL mice can be due to impaired apoptosis. Scand J Immunol 1997;46:373-8.

169-72.

1989;2:415-25.

191 35 -67 ,

62A-29.

30. Nagata S. Apoptosis by death factor. Cell 1997:88:355-65. 31. Wallach D. Placing death under control. Naturc 1997;388:124-6. 32. Gavrieli Y, Sherman Y. Ben-Sasson SA. Identification of pro-

grammed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1991-;119:4Y2-501.

33. Zhou T, Bluethman H, Eldridge J, Berry K, Mountz JD. Origin of CD4-CD8-B22Ot T cells in MRL-lprilpr mice. J Immunol 1993; 150:3651-67.

34. Quinnan GV, Manischewitz JE. Ennis FA. Cytotoxic T lymphocyte response to murine cytomegalovirus infection. Naturc 1978;273: 541-3.

35. Heise MT, Virgin HW. The T cell independent role of IFN- gamma and TNF-alpha in macrophage activation during murine cytomegalovirus and herpes simplex virus infection. J Virol 1995; 69:904-9.

36. Salazar-Mathcr TP, Orange JS, Biron CA. Early murine cytomegalovirus (MCMV) infection induces liver natural killer (NK) cell inflammation and protection through macrophage inflammatory protein la (MTP-la)-dependent pathways. J Exp Med 1998;187:

37. Ranger-Rogez S, Vidal E, Labrousse F, Riche A, Vidal J, Col- lincau M, et al. Large-scale study suggests no direct link between human herpesvirus-6 and primary Sjogrcn’s syndrome. J Med Virol 1995;47:198-203.

38. Toro AI, Ossa J. PCR activity of CMV in healthy CMV- seropositive individuals: does latency need redefinition? Res Virol

39. Reddehase MJ. Baithcscn M, Rapp M, Jonjic S, Pavic I, Koszi- nowski UH. The conditions of primary infection define the load of latent viral genome in organs and the risk of rccurrent qtomega- lovirus disease. J Exp Med 1994;179:185-93.

40. Kondo K, Kaneshima H. Mocarski ES. Human cytomegalovirus latent infection of granulocyte-macrophage progenitors. Proc Natl Acad Sci U S A 1994;91:11879-83.

41. Kondo K, Mocarski ES. Cytomegalovirus latency and latency- specific transcription in hematopoietic progenitors. Scand J Infect Dis 1995;99:63-7.

42. Koffron AJ, Mueller KH, Kaufman DB? Stuart FP, PaLterson B, Abecassis MI. Direct evidence using in situ polymerase chain reaction that the endothelial cell and T-lymphocyte harbor latent murine cytomegalovirus. Scand J Infect Dis lY95;99:61-2.

43. Schmader K, Henry SC, Rahija RJ, Yu Y, Daley GG. Hamilton JD. Mouse cytomegalovirus reactivation in severe combined immune deficient mice after implantation of latently infected salivary gland. J Infect Dis 1995;172:531-4.

44. Kurz S, Steffens HP, Mayer A, Harris JR, Reddehase MJ. Latency versus persistence or intermittent recurrences: evidence for a latent state of murine cytomegalovirus in the lungs. J Virol

45. Haaheim LR, Hake AK, Kvakestad R, Stern B, Normann 0, Jonsson R. Scrum antibodies from patients with primary Sjiigren’s syndrome and systemic lupus crythcmatosus rccognize multiple epitopes on the La (SS-B) autoantigen resembling viral protein sequences. Scand J Immunol 1996:43:115-21.

46. Haneji N , Nakamura T, Taho K. Yanagi K, Higashiyama H, Saito 1, et al. ldcntification of alpha-fodrin as a candidate autoantigen in primary Sjogren’s syndrome. Science 1997;276:604-7.

47. Fox RI. Sjiigren’s syndrome: controversies and progress. Clin Lab

48. Ogawa N, Dang H, Kong L, Anaya J-M, Liu GT, Tala1 N. Lymphocyte apoptosis and apoptosis-associated gene expression in Sjogren’s syndrome. Arthritis Rhcum 1996;39:1875-85.

49. Manganelli P, Quaini F, Andreoli AM, Lagrasta C. Pilato FP, Zuccarelli A, et al. Quantitative analysis of apoptosis and bcl-2 in Sjiigren’s syndrome. J Rheumatol 1997;24:1552-7.

SO. Azuma M, Motegi K, Aota K? Hayashi Y, Sato M. Role of cytokines in the destruction of acinar structure in Sjiigren’s syndrome salivary glands. Lab Invest 1997;77:269-80.

51. Wu AJ. Chcn ZJ, Tsokos M, O’Conncll BC, Ainbudkar IS, Baum BJ. Interferon-gamma induced ccll dcath in a culturcd human salivary gland cell line. J Cell Physiol 1996;167:297-304.

1-14.

1996;147:233-8.

1997;71:2980-7.

Med 1997;17:431-44.

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Murine cytomegalovirus induces a Sjögren's syndrome-like disease in C57Bl/6-lpr/lpr mice