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(Investigative Ophthalmology and Visual Science. 2001;42:399-401.)
© 2001 by The Association for Research in Vision and Ophthalmology, Inc.

The Role of Fas-Fas Ligand–Mediated Apoptosis in Autoimmune Lacrimal Gland Disease in MRL/MpJ Mice

Douglas A. Jabs1,2, Bella Lee1, Judith Whittum-Hudson3 and Robert A. Prendergast1

1 From the Departments of Ophthalmology and 2 Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland; and the 3 Department of Medicine, Wayne State University School of Medicine, Detroit, Michigan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. MRL/MpJ mice spontaneously develop lacrimal gland inflammation and are a model for the human disorder Sjögren’s syndrome. MRL/MpJ-lpr/lpr (MRL/lpr) and MRL/Mp-+/+ (MRL/+) mice are congenic substrains, which differ only by a single autosomal recessive gene, the lpr mutation. This mutation results in defective Fas protein, defective lymphocytic apoptosis, and accelerated autoimmune lacrimal gland disease in MRL/lpr mice. We evaluated apoptosis in the lacrimal glands of MRL/lpr and MRL/+ mice.

METHODS. Inflammatory cells in the lacrimal glands of MRL/lpr and MRL/+ mice were evaluated for apoptosis with TUNEL staining and Fas and Fas ligand expression with immunohistochemistry.

RESULTS. MRL/lpr mice had a greater percentage of the lacrimal gland replaced by inflammatory infiltrate (30.3% ± 7.0%) than did MRL/+ mice (13.0% ± 3.0%, P = 0.02). However, similar amounts of lymphocytic apoptosis were present in the lacrimal glands of MRL/lpr and MRL/+ mice. The mean number of apoptotic cells per unit area of inflammation was 23.8 ± 2.4 in MRL/lpr mice and 24.6 ± 6.0 in MRL/+ mice (P = 0.91). Fas expression was absent on lymphocytes in MRL/lpr mice but was present on lymphocytes in MRL/+ mice. Fas ligand expression was present on epithelial structures in both substrains.

CONCLUSIONS. The accelerated lacrimal gland disease inflammation in MRL/lpr mice does not appear to be due to decreased apoptosis in the microenvironment of the lacrimal gland of MRL/lpr mice. It appears that in MRL/lpr mice there is defective extrathymic lymphoid apoptosis, permitting a relatively greater expansion of autoreactive T cells, which subsequently invade the lacrimal gland.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MRL/MpJ mice are autoimmune mice that spontaneously develop lacrimal gland inflammation and are a model for the human disorder Sjögren’s syndrome.1 2 3 4 The lacrimal gland inflammation in MRL/MpJ mice is similar to that seen in humans with Sjögren’s syndrome in that the majority of cells infiltrating the lacrimal glands are CD4+ T cells with lesser numbers of CD8+ T cells, B cells, and macrophages.2 4 MRL/MpJ mice exist as two congenic substrains, MRL/MpJ-lpr/lpr (MRL/lpr) and MRL/MpJ-+/+ (MRL/+) mice. The lpr gene is an autosomal recessive mutation that results in a defective Fas protein (CD95), the consequences of which are defective extrathymic lymphocytic apoptosis and accelerated autoimmune disease.5 6 7 MRL/lpr mice develop accelerated lacrimal gland inflammation, when compared to MRL/+ mice, in that the disease has an earlier onset and, at comparable ages, greater severity.2 Two possible mechanisms may be considered by which the defective apoptosis caused by the lpr mutation could accelerate lacrimal gland disease in MRL/MpJ mice: defective apoptosis in the microenvironment of the lacrimal gland permits the infiltrating inflammatory cells to accumulate and expand; or defective apoptosis in the peripheral lymphoid tissues permits the accumulation of autoreactive lymphocytes, which then invade the lacrimal gland. To distinguish between these possibilities, we evaluated apoptosis in lacrimal gland inflammation in MRL/lpr and MRL/+ mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
One-month-old MRL/lpr and MRL/+ mice were obtained from the Jackson Laboratories (Bar Harbor, ME) and kept under standard conditions until killed. Eleven 3- to 6-month-old mice of each strain were killed by anesthesia overdose followed by exsanguination, and both lacrimal glands were removed. Lacrimal glands were embedded in OCT compound (Miles, Inc., Elkhart, IN), frozen in liquid nitrogen, sectioned at 6 µm, and processed either for TUNEL staining or for immunohistochemistry for Fas and Fas ligand expression. In addition, lacrimal glands from three BALB/c mice were used for TUNEL staining and for immunohistochemistry as appropriate controls. These experiments adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the institutional Animal Care and Use Committee.

TUNEL Staining
Slides were fixed in acetone for 5 seconds, washed once in distilled water, and then dried at 42°C. Tissue sections were immersed in 10 mM Tris-HCl at pH 8.0 for 5 minutes, incubated with 20 µg/ml proteinase K in 10 mM Tris-HCl for 15 minutes at room temperature, and washed in double-distilled water (DDW) four times for 2 minutes each. To accomplish nuclear stripping, endogenous peroxidase was inactivated by covering the sections with 100 to 200 µl 3% hydrogen peroxide for 5 minutes at room temperature and rinsing in DDW. Sections were preincubated in TdT buffer (1X: 30 mM tris base, 140 mM sodium cacodylate, pH 7.2, 1 mM cobalt chloride). Each slide was incubated with 75 µl reaction mixture coverslips in a humid atmosphere for 60 minutes at 37°C. The reaction mixture consisted of TdT-buffered biotinylated-dUTP stock, and TdT at 0.3 enzyme units/µl, final solution, in DDW. The reaction was terminated by transferring slides to a bath of 300 mM NaC1 and 30 mM sodium citrate for 15 minutes at room temperature. The slides were washed for 15 minutes at room temperature with phosphate-buffered saline (PBS), blocked with 2% bovine serum albumin for 10 minutes, and washed with PBS for 5 minutes. The sections were incubated with avidin-conjugated peroxidase in PBS at 37°C for 30 minutes and stained with the 3-amino-9-ethylcarbazole (AEC) reagent for 30 minutes at 37°C. Sections were then washed in PBS. Light counterstaining with hematoxylin was performed, and slides were mounted in glycerol-gelatin and covered.8 Because of the loss of two sections from slides with proteolytic treatment, a second series of experiments was performed omitting proteinase K treatment. Before terminal transferase labeling the following steps were used in this series. Slides were immersed in 1% hydrogen peroxide/methanol for 30 minutes, washed twice with PBS, blocked with the avidin-biotin blocking kit (Vector Laboratories, Burlingame, CA) for 10 minutes, rinsed twice more with PBS, and labeled with TdT buffer as above. No substantive differences were seen between the two methods, and a total of nine slides for each strain was available for analysis. The proportion of lymphocytes undergoing apoptosis was counted with a net micrometer disc, which covered an area of 0.35 mm2, and the number of cells staining positively per unit area of inflammation was calculated. This area was estimated to encompass an average of 59,600 cells. All foci of inflammation in one histologic section of each lacrimal gland were counted.

Immunohistochemistry
Lacrimal gland sections were fixed in chilled (4°C) acetone, air-dried, rehydrated in PBS, and incubated with the appropriate blocking agent (Vector Laboratories) for 20 minutes. Monoclonal antibodies to Fas (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or Fas ligand (Boehringer Mannheim, Indianapolis, IN)9 10 were applied, and the slides were incubated for 60 minutes. Slides were washed in PBS and incubated with a biotinylated antibody for 30 minutes, rinsed in PBS, incubated with the ABC reagent for 45 minutes, and washed again in PBS, and the reaction product was developed with hydrogen peroxide in AEC containing acetate buffer and counterstained with Harris hematoxylin or methyl green (Sigma, St. Louis, MO).2 3 11 For each run and for each antibody, appropriate positive controls (e.g., ocular, thymus, or spleen sections from appropriate animals) and negative controls (tissues omitting the primary antibody) were run to ensure quality control. A semiquantitative scoring system was used for Fas staining as follows: 0, no staining; 1+, <25% cells positive; 2+, 25% to 50% cells positive; 3+, 51% to 75% cells positive; and 4+, >75% cells positive.

Lacrimal Gland Inflammation
The percentage of the lacrimal gland replaced by inflammation was estimated with the net micrometer disc.

Statistics
Comparisons of the percentage of the lacrimal gland replaced by inflammation and of the number of cells per unit area undergoing apoptosis were performed with the two-sample t-test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The mean ages of the MRL/lpr and MRL/+ mice were 4.8 ± 0.1 and 4.6 ± 0.3 months, respectively. At this age, MRL/lpr mice had a greater percentage of the lacrimal gland replaced by the inflammatory infiltrate than did MRL/+ mice (Table 1 and Fig. 1 ). The mean percentage of the lacrimal gland occupied by the inflammatory infiltrate was 30.3% ± 7.0% in MRL/lpr mice and 13.0% ± 3.0% in MRL/+ mice (P = 0.02). TUNEL staining demonstrated nuclear changes from apoptosis in both MRL/lpr and MRL/+ mice (Fig. 2) . The degree of apoptosis among the mononuclear inflammatory cells infiltrating the lacrimal gland was similar between MRL/lpr mice and MRL/+ mice (Table 1) . The mean number of apoptotic cells per unit area of infiltrate was 23.8 ± 2.4 in MRL/lpr mice and 24.6 ± 6.0 in MRL/+ mice (P = 0.91). Immunohistochemistry revealed staining for Fas on 50% to 75% (score, 3+) of the infiltrating lymphocytes in MRL/+ mice (Fig. 3) , but no staining for Fas could be seen in MRL/lpr mice. Diffuse positive staining for Fas ligand was seen on epithelial structures in both MRL/lpr and MRL/+ mice. No staining for Fas ligand was seen in control sections omitting the primary antibody. In BALB/c mice there were no infiltrates; therefore, there were no lymphocytic apoptosis (TUNEL staining) and no lymphocytes for analysis of lymphocytic Fas expression. As in MRL/MpJ mice, in BALB/C mice there was diffuse staining for Fas ligand on epithelial structures.


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  		Table 1. Apoptosis in MRL/MpJ Mouse Lacrimal Glands

 


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  		Figure 1. Lacrimal gland inflammation in (A) a 6-month-old MRL/lpr mouse and (B) a 6-month-old MRL/+ mouse. The MRL/lpr mouse has extensive inflammation throughout the lacrimal gland, whereas the MRL/+ mouse has small focal infiltrates (hematoxylin and eosin; original magnification, x50).

 


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  		Figure 2. Apoptosis in lymphocytes infiltrating the lacrimal glands of MRL/MpJ mice, showing similar amounts of apoptosis in (A) MRL/lpr mice and (B) MRL/+ mice. Apoptotic cells are stained dark black (original magnification, x160).

 


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  		Figure 3. Immunohistochemistry for Fas antigen, showing absence of staining in (A) MRL/lpr mice and substantial staining in (B) MRL/+ mice. Positive staining of lymphocytes is detected by the dark reaction product on the cell surface (original magnification, x200).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Apoptosis is programmed cell death, which occurs after an appropriate triggering event. Apoptosis is one mechanism by which both normal development and the immune response are regulated. After an offending organism has been eliminated, the immune response must be downregulated to prevent ongoing tissue destruction, and the Fas–Fas ligand system is involved in the regulation of the immune response. Defects in either Fas or Fas ligand result in autoimmune disease in both mice and in humans.12 13 The lpr mutation results in a defective Fas protein, whereas the gld mutation results in a defective Fas ligand, and both mutations result in accelerated autoimmune disease in MRL/MpJ mice.12 In humans, defects in Fas result in the autoimmune lymphoproliferative syndrome, characterized by lymphoproliferation and systemic autoimmune disease.13

MRL/lpr mice have an accelerated course of autoimmune lacrimal gland inflammatory disease compared with MRL/+ mice. Specifically, the onset of diseases is earlier in MRL/lpr mice and the amount of inflammation is greater than in MRL/+ mice at comparable ages.3 Our data on the percentage of lacrimal gland occupied by the inflammatory infiltrate confirm these differences, because MRL/lpr mice had a significantly greater amount of disease. Because MRL/lpr and MRL/+ are congenic and differ only by the lpr gene, these experiments were performed to evaluate hypotheses as to how the defective Fas in MRL/lpr mice might accelerate the autoimmune lacrimal gland disease. Although it was possible that failure of apoptosis in the microenvironment of lacrimal gland in MRL/lpr mice permitted the inflammatory cells invading the lacrimal disease to accumulate at the site, our data do not provide support for this hypothesis. Instead, the amount of lymphocyte apoptosis was similar between MRL/lpr and MRL/+ mice. Apoptotic cells appeared to be haphazardly scattered throughout the inflammatory infiltrate, and no specific pattern could be discerned. MRL/+ mice appeared to have a greater variability in the number of apoptotic cells per unit area because of the low numbers of apoptotic cells present, the smaller size of the inflammatory infiltrates in MRL/+ mice, and the haphazard distribution of apoptotic cells, producing greater sampling variability in MRL/+ mice. However, because of the overall similarity in the amount of lymphocyte apoptosis in the lacrimal gland between the two substrains, the effect of the defective lymphocyte apoptosis in MRL/lpr mice appears to occur outside the lacrimal gland.

As anticipated, Fas staining was seen on the lymphocytes in the lacrimal gland in MRL/+ mice but not in MRL/lpr mice, and Fas ligand staining was present in both strains. Fas ligand expression on lacrimal gland epithelial structures has been reported in other murine models of Sjögren’s syndrome.10 Although MRL/+ mice may use the Fas ligand system to initiate lymphocyte apoptosis, MRL/lpr mice cannot do so and must use alternative pathway(s) to initiate the apoptosis seen in the lacrimal glands; these pathways could include the tumor necrosis factor (TNF) receptor or death receptor 3, 4, or 5.14 Of note, MRL/lpr mice have been reported to have elevated levels of TNF{alpha}.15 In the human disease Sjögren’s syndrome, Fas is expressed on the infiltrating lymphocytes in the glands, and Fas ligand on the acinar epithelial cells; however, apoptosis appears to be blocked, possibly by bcl-2 expression.16 It is possible that, as in human disease, lymphocytic apoptosis in the lacrimal gland is blocked in both MRL/+ and MRL/lpr mice, resulting in reduced apoptosis in both substrains; however, our data suggest that apoptosis is not blocked differentially between the two substrains in the microenvironment of the lacrimal gland.

Previous work has shown that MRL/lpr mice have defective lymphocyte apoptosis in peripheral (extrathymic) lymphoid organs.6 7 17 Our data demonstrate that the extent of apoptosis within the lacrimal gland inflammation is similar between MRL/lpr and MRL/+ mice. They also suggest that the accumulation of autoreactive lymphocytes, which is a consequence of the defective apoptosis in peripheral lymphoid organs, permits the expansion of that population of lymphocytes, which then invade the lacrimal gland in greater numbers in MRL/lpr than in MRL/+ mice and that this phenomenon is responsible for the accelerated lacrimal gland disease seen in MRL/lpr mice, when compared to its congenic substrain MRL/+.


   Acknowledgements
 
The authors thank Donald P. Zack, MD, PhD, whose laboratory provided technical assistance with the TUNEL staining and John H. Kempen, MD, MPH, MHS, who provided the statistical analysis.


   Footnotes
 
Supported by National Eye Institute Grant EY-05912 (DAJ) and National Institute of Allergy and Infectious Disease Grant AI-44493 (JW-H), National Institutes of Health, Bethesda, Maryland.

Submitted for publication May 19, 2000; revised September 7, 2000; accepted October 16, 2000.

Commercial relationships policy: N.

Corresponding author: Douglas A. Jabs, The Wilmer Eye Institute, The Johns Hopkins University School of Medicine, 550 North Broadway, Suite 700, Baltimore, MD 21205. djabs{at}jhmi.edu


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Jabs, DA, Alexander, EL, Green, WR (1985) Ocular inflammation in autoimmune MRL/Mp mice Invest Ophthalmol Vis Sci 26,1223-1229[Abstract/Free Full Text]
  2. Jabs, DA, Prendergast, RA (1988) Murine models of Sjögren’s syndrome: immunohistologic analysis of different strains Invest Ophthalmol Vis Sci 29,1427-1443
  3. Jabs, DA, Enger, C, Prendergast, RA (1991) Murine models of Sjögren’s syndrome: evolution of lacrimal gland inflammatory lesions Invest Ophthalmol Vis Sci 32,371-380[Abstract/Free Full Text]
  4. Jabs, DA, Prendergast, RA (1987) Reactive lymphocytes in lacrimal gland and renal vasculitic lesions of MRL/lpr mice express L3T4 J Exp Med 166,1198-1203[Abstract/Free Full Text]
  5. Watanabe-Fukunaga, R, Brannan, CI, Copeland, NG, Jenkins, A, Nagata, S. (1992) Lymphoproliferative disorder in mice explained by defects in Fas antigen that mediates apoptosis Nature 356,314-317[Medline][Order article via Infotrieve]
  6. Singer, GG, Abbas, AK (1994) The Fas antigen is involved in peripheral but not thymic deletion of T lymphocytes in T cell receptor transgenic mice Immunity 1,365-371[Medline][Order article via Infotrieve]
  7. Cui, H, Ju, ST, Sherr, D. (1996) Functional expression of Fas (CD95) protein in autoimmune lpr mice Cell Immunol 174,3335-3341
  8. Ben-Sasson, SA, Sherman, Y, Gavrieli, Y. (1995) Identification of dying cells—in situ staining Schwartz, L Osborne, B eds. Methods in Cell Biology 46,29-39 Academic Press, Inc San Diego. [Medline][Order article via Infotrieve]
  9. Griffith, TS, Brunner, T, Fletcher, SM, Green, DR, Ferguson, TA (1995) Fas ligand-induced apoptosis as a mechanism of immune privilege Science 270,1189-1192[Abstract/Free Full Text]
  10. Kong, L, Robinson, CP, Peck, AB, et al (1998) Inappropriate apoptosis of salivary and lacrimal gland epithelium of immunodeficient NOD-scid mice Clin Exp Rheum 16,675-681[Medline][Order article via Infotrieve]
  11. Hsu, S, Raine, L, Fanger, H. (1981) Use of avidin-biotin-peroxidase (ABC) techniques: a comparison between ABC and unlabeled antibody (PAP) procedures J Histochem Cytochem 29,577-580[Abstract]
  12. Ito, MR, Terasaki, S, Itoh, J, Katoh, H, Yonehar, S, Mose, N. (1997) Rheumatic diseases in an MRL strain of mice with a deficit in the functional Fas ligand Arthritis Rheum 40,1054-1063[Medline][Order article via Infotrieve]
  13. Straus, SE, Sneller, M, Lenardo, MJ, Puck, JM, Strober, W. (1999) An inherited disorder of lymphocyte apoptosis: the autoimmune lymphoproliferative syndrome Ann Intern Med 130,591-601[Abstract/Free Full Text]
  14. Ashkenazi, A, Dixit, VM (1998) Death receptors: signaling and modulation Science 281,1305-1308[Abstract/Free Full Text]
  15. Theofilopoulos, AN, Lawson, BR (1999) Tumor necrosis factor and other cytokines in murine lupus Ann Rheum Dis 58(suppl I),149-155
  16. Kong, L, Ogawa, N, Nakabayashi, T, et al (1997) Fas and Fas ligand expression in the salivary glands of patients with primary Sjögren’s syndrome Arthritis Rheum 40,87-97[Medline][Order article via Infotrieve]
  17. Rathmell, JC, Goodnow, CC (1994) Effects of the lpr mutation on elimination and inactivation of self-reactive B cells J Immunol 153,2831-2842[Abstract]



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