Quantitative analysis of lacrimal gland function, apoptotic figures, Fas and Fas ligand expression of lacrimal glands in dry eye patients

doi:10.1016/S0014-4835(02)00279-8

Experimental Eye Research

Volume 76, Issue 2, 1 February 2003, Pages 233-240

https://doi.org/10.1016/S0014-4835(02)00279-8 Get rights and content

Abstract

The purpose of our study was to examine the correlation between the lacrimal gland function and apoptotic figure, Fas and Fas ligand (FasL) expression in the lacrimal gland. A total of 15 dry eye patients (nine Sjögren's syndrome and six non-Sjögren's syndrome-type dry eye) were recruited for the study. Lacrimal function was evaluated by Schirmer tests 1 and 2. Lacrimal gland biopsies were performed and sections were analyzed by immunohistochemistry using APO2·7, an antibody to Fas and FasL. Quantitative analysis of fluorescein staining was performed by a scanning laser microscopy. Schirmer test 2 results were lower in Sjögren's syndrome-type dry eye and were associated with positive staining of acinar cells with APO2·7 and of infiltrating lymphocytes with FasL. There was a good correlation between the results of Schirmer test 2 and APO2·7 and FasL staining. Lacrimal gland dysfunction is related to the apoptotic figure of acinar cells possibly induced by FasL on the infiltrating lymphocytes.

Introduction

Various disease mechanisms may account for dry eye syndrome, including decreased blink rate, shortened tear break-up time or delayed tear clearance; all of which are not directly related to lacrimal gland dysfunction (Tseng and Tsubota, 1997). However, in Sjögren's syndrome (SS), severe lacrimal gland dysfunction is the key mechanism (Pflugfelder et al., 1993, Tsubota et al., 1996, Xu et al., 1996). We have previously reported that these types of dry eye have lymphocytic infiltration into the lacrimal gland accompanied by some acinar cell destruction, resulting in dramatic dysfunction of the lacrimal gland (Xu et al., 1996). Reflex tear production is lost, even with strong stimulation such as nasal stimulation, as is basic secretion (Tsubota et al., 1996, Tsubota et al., 1999a). In this situation, essential tear components cannot reach the ocular surface epithelium, and the epithelium may develop squamous metaplasia (Tseng, 1985, Tsubota, 1998, Tsubota et al., 1999b).

A mysterious feature of the lacrimal gland in SS is the presence of some intact-looking acinar cells remaining in the lacrimal gland, even though lacrimal gland function is almost totally lost. We have proposed that these normal-looking acinar cells in SS are in the early phase of apoptosis and cannot function (Fujihara et al., 1999, Tsubota et al., 2000). This concept can explain the difference in lacrimal gland function between SS and SS with enlarged exocrine glands; both of which have massive lymphocyte infiltration. Whereas, SS shows a lot of apoptotic figures in the lacrimal gland, there are almost no apoptotic figures in the lacrimal gland in patients with SS with enlarged exocrine glands and non-Sjögren's syndrome-type dry eye (Fujihara et al., 1999, Tsubota et al., 2000). In the animal model of SS, we also showed the importance of apoptosis in acinar cells for the destruction of exocrine glands (Saito et al., 1999). Thus, we now are able to hypothesize that lacrimal gland function is related to the apoptotic process in lacrimal gland acinar cells. Furthermore, apoptosis may be related to the Fas and Fas ligand (FasL) interaction. In this paper, we have quantitatively compared lacrimal gland function (Schirmer test result) and apoptosis, and Fas and FasL expression of lacrimal gland acinar cells. As expected, there was a correlation between function and the number of apoptotic figures in the lacrimal gland, and with FasL expression in infiltrating lymphocytes.

Section snippets

Diagnosis of Sjögren's syndrome and dry eye

The diagnosis of SS was made by a modification of proposed criteria of Fox et al. (1986); (1) objective evidence of keratoconjunctivitis sicca, as documented by Rose Bengal or fluorescein dye staining, (2) objective evidence of diminished salivary gland flow, (3) minor salivary gland biopsy, obtained through normal mucosa, where the specimen contains at least four evaluative salivary gland lobules, and having an average of at least 2 foci/4 mm² or positive lymphocyte infiltration of lacrimal

Results

In SS lacrimal glands, some acinar cells were clearly stained by APO2·7 antibody indicating the early phase of apoptosis (Fig. 1(a) and (b)). This antibody reacts with the mitochondria protein induced in the early phase of apoptosis (Zhang et al., 1996), and was shown to stain identical cells, which were stained by the TUNEL method (Fujihara et al., 1999). It was shown that this antibody is a useful and early marker for apoptosis, not only in hemopoietic cells but also in epithelial cells (

Discussion

This paper shows a correlation between the presence of apoptotic figures in lacrimal glands and gland function. There was a further correlation between lacrimal gland function and FasL expression, which suggests that the Fas–FasL interaction is an important mechanism in lacrimal gland dysfunction, which is probably mediated via infiltrating lymphocytes.

Tear function was correlated with lacrimal gland apoptosis. This result suggests that normal-looking acinar cells in SS are already in an early

Acknowledgements

A grant from the High Tech Research Center of Tokyo Dental College, Chiba, Japan and an award from the Medical School Faculty and Alumni Grants of Keio University Medical Science Fund, Tokyo, Japan, supported this study.

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Activated CD4+ T cells can trigger CD8+ T cell differentiation which could lead to lacrimal epithelial cell death through Fas–FasL system or by releasing perforin-granzyme. Expression of FasL and its receptor Fas, have been observed in the lacrimal (Tsubota et al., 2003) and salivary glands of SS patients (Matsumura et al., 1996) and in the salivary glands of the NOD mice (Van Blokland et al., 2003). The interaction of Fas and FasL initiates a chain of biochemical events which leads to cleavage and activation of caspase-3 with subsequent substrate cleavage and apoptosis (Manganelli and Fietta, 2003; Kulkarni et al., 2006; O’brien and Kirby, 2008).
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Quantitative analysis of lacrimal gland function, apoptotic figures, Fas and Fas ligand expression of lacrimal glands in dry eye patients

Abstract

Introduction

Section snippets

Diagnosis of Sjögren's syndrome and dry eye

Results

Discussion

Acknowledgements

Cell

Oral Surg. Oral Med. Oral Pathol.

Cell

Ophthalmology

Ophthalmology

Am. J. Ophthalmol.

Progr. Reti. Eye Res.

Apoptosis and expression of inflammation-related proteins following IFN g treatment of epithelial conjunctival cells in vitro

Invest. Ophthalmol. Vis. Sci.

Involvement of the CD95 (APO-1/Fas) receptor/ligand system in multiple sclerosis brain

J. Exp. Med.

Involvement of an ICE-like protease in Fas-mediated apoptosis

Nature