Elsevier

Experimental Eye Research

Volume 90, Issue 2, February 2010, Pages 223-237
Experimental Eye Research

Lymphocytic infiltration leads to degradation of lacrimal gland extracellular matrix structures in NOD mice exhibiting a Sjögren's syndrome-like exocrinopathy

https://doi.org/10.1016/j.exer.2009.10.008Get rights and content

Abstract

We previously reported that lacrimal glands (LGs) of male non-obese diabetic (NOD) mice, an established mouse model of autoimmune inflammatory LG disease that displays many features of human LGs in patients afflicted with Sjögren's syndrome (SjS), exhibit significant degradation of extracellular matrix (ECM) structures as well as increased expression of matrix metalloproteinases (MMPs). The purpose of the current study was to expand the spectrum of proteases identified, to clarify their probable origin as well as to identify the contribution of these changes to disease pathogenesis. We explored in depth the changes in ECM structures and ECM protease expression at the onset of disease (6 weeks) versus late stage disease (18 weeks) in male NOD mouse LGs, relative to LGs of age-matched male NODscid, a severely immunocompromised congenic strain, and healthy BALB/c mice. LG tissues were examined using routine histological, immunohistochemical, Western Blot and gene expression analyses novel multiphoton imaging technologies. We further characterized the profile of infiltrating immune cells under each condition using flow cytometry. Our results show that the initial infiltrating cells at 6 weeks of age are responsible for increased MMP and cathepsin H expression and therefore initiate the LG ECM degradation in NOD mice. More importantly, NODscid mice exhibited normal LG ECM structures, indicating the lymphocytes seen in the LGs of NOD mice are responsible for the degradation of the LG ECM. The disease-related remodeling of LG ECM structures may play a crucial role in altering the acinar signaling environment, disrupting the signaling scaffolds within the cells, which are required to mobilize the exocytotic trafficking machinery, ultimately leading to a loss of LG function in patients afflicted with SjS.

Introduction

Proteases such as matrix metalloproteinases (MMPs) and lysosomal cysteine proteinases (cathepsins) play important roles during normal embryonic development, including the morphological development of glandular structures (Thompson and Spooner, 1983, de la Cuadra-Blanco et al., 2006, Patel et al., 2006). Their degradation of extracellular matrix (ECM) proteins alters the tissue microenvironment, allowing cellular migration and differentiation (Thompson and Spooner, 1983, Patel et al., 2006); however, numerous studies have demonstrated their close association with various diseases, including rheumatoid arthritis and osteoarthritis (Xue et al., 2007, Takaishi et al., 2008). Increased levels of MMPs have also been implicated in the progression of the autoimmune disease Sjögren's syndrome (SjS) in which lymphocytes infiltrate lacrimal (LGs) and salivary glands (SGs) (Perez et al., 2000; Goicovich et al., 2004, Molina et al., 2006, Schenke-Layland et al., 2008). These events lead to atrophy and ultimately destruction of these tissues, resulting in severe dry eye and dry mouth symptoms which are characteristic of this disease state (Fox et al., 1983, Fox et al., 1986, Matsumoto et al., 1996). Because the LGs are seriously affected, patients cannot produce reflex tears, resulting in severe damage to the ocular surface including squamous metaplasia of the ocular surface epithelium, pain, corneal scarring and in severe cases, blindness (Tsubota et al., 1996).

Although a number of theories have been proposed to explain the onset and progression of lymphocytic infiltration into the LG and SG, the disease is still very poorly understood. The use of mouse models has been important in advancing our knowledge of the etiology of SjS. Perhaps one of the best known models is the male non-obese diabetic (NOD) mouse (Robinson et al., 1997, Yamachika et al., 1998, Humphreys-Beher and Peck, 1999, da Costa et al., 2006). This strain spontaneously develops diabetes as well as lymphocytic infiltration in SGs (sialoadenitis) and LGs (dacryoadenitis), an effect that is associated with reduced secretory flow (van Blokland and Versnel, 2002, Barabino and Dana, 2004, da Costa et al., 2006). The two autoimmune processes of diabetes and SjS occur independently in the same animal. For reasons not yet understood, male NOD mice are more susceptible to dacryoadenitis than female mice, whereas SjS is more prevalent in female patients. Lymphocytic infiltration into the LGs, which is accompanied by decreased production of lacrimal fluid, is typically detected in male mice from ∼6 to 10 weeks. The severely immunocompromised congenic strain (NODscid) also exhibits abnormalities in SG and LG function (Yamachika et al., 2000, van Blokland et al., 2003, Robinson et al., 1997, Kong et al., 1998), suggesting that the disease process exhibited in this model includes both exocrine tissue-dependent as well as inflammatory cell-dependent events. The comparison between the LGs of NOD and the NODscid strains provides a powerful tool for distinguishing processes initiated within the exocrine tissue versus processes initiated by the immune system.

It is clear from different studies of mouse models of SjS that abnormalities in protease activity participate in disease development and progression. For instance, prior to the appearance of glandular infiltrating lymphocytes, aberrant expression of specific apoptotic proteases (caspases) is detectable in the salivary gland lysates of NOD mice (Robinson et al., 1997, Yamachika et al., 1998, Yamachika et al., 2000). Another study in a different mouse model of SjS implicated aberrant cathepsin S activation sialoadenitis, showing that inhibition of cathepsin S was able to prevent SG-specific autoimmunity (Saegusa et al., 2002). Each of these previous studies has linked the enhanced expression of proteases to changes in the acinar cells rather than to immune cell infiltrates. Recently, we were able to demonstrate in the LG of male NOD mice with established disease (e.g., 18 weeks of age) that they exhibited significant degradation of ECM structures as well as increased expression of MMPs (Schenke-Layland et al., 2008). In order to understand the origin of the increased protease levels and the contribution of these changes to disease pathogenesis, we have here explored in depth the changes in ECM structures and ECM protease expression at the onset of disease (6 weeks) versus later stage disease (18 weeks) in male NOD mouse LGs, relative to NODscid and BALB/c mice. We have also characterized the profile of the infiltrating immune cells under each condition. Our results show that the initial infiltrating cells at 6 weeks of age, which are responsible for the increased MMP and cathepsin H expression, initiate the ECM degradation.

Section snippets

Animals

This study was conducted using LGs isolated from male BALB/c (Jackson Labs, Bar Harbor/ME, USA), NOD and NODscid (both Taconic Farms, Germantown/NY, USA) mice at the ages of 6 and 18 weeks as previously described (Schenke-Layland et al., 2008). All procedures conformed to the standards and procedures for the proper care and use of animals in accordance with the institutional guidelines, and as described in the Declaration of Helsinki and the Guiding Principles in the Care and Use of Animals

Histology and immunohistostaining reveal significant changes in ECM protein expression patterns in NOD LGs

No evidence of inflammation was observed within LG tissues isolated from BALB/c (Supplmentary Fig. 1A and D) or the lymphocyte-deficient NODscid (Supplementary Fig. 1C and F) mice. In contrast, and similar to our previously reported results (Schenke-Layland et al., 2008), routine histological analysis demonstrated an infiltration of mononuclear inflammatory cells into the glandular parenchyma of NOD LGs, predominantly around acini and ducts, starting at the age of 6 weeks, which was further

Discussion

In our previous study, we provided a detailed analysis of the LG matrix structure in combination with a qualitative matrix protein evaluation (Schenke-Layland et al., 2008), representing the first comprehensive identification and characterization of the composition of LG ECM. Using routine histology, biochemistry, immunohistochemistry and gene expression analyses in combination with novel multiphoton imaging, we could demonstrate that healthy murine LGs are composed of collagen types I, III,

Acknowledgments

The authors would like to thank Yekaterina Butylkova for her excellent technical assistance and Dr. Ali Nsair for his helpful comments. This work was supported by the NIH (5T32HL007895-10 to K.S-L.; EY-11386 to S.H-A.) and by the Canadian Institutes of Health Research (MOP-68836 to D.P.R.).

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