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. 2004 May;136(2):200–206. doi: 10.1111/j.1365-2249.2004.02457.x

Corneal stromal cells selectively inhibit production of anti-inflammatory cytokines by activated T cells

V HOLÁŇ *,†, A VÍTOVÁ *,‡, J PINDJÁKOVÁ *,†, M KRULOVÁ *,†, A ZAJÍCOVÁ *,†, M FILIPEC ‡
PMCID: PMC1809025  PMID: 15086381

Abstract

The eye has been described as an immunologically privileged site where immunity is purely expressed. It has been demonstrated that administration of antigen into the eye induces only a weak immune response. However, the anterior part of the eye represents an important protective barrier against pathogens and other harmful invaders from the outer environment. Therefore, effective immune mechanisms, which operate locally, need to be present there. Because the cornea has been shown to be a potent producer of various cytokines and other molecules with immunomodulatory properties, we investigated a possible regulatory role for the individual corneal cell types on cytokine production by activated T cells. Mouse spleen cells were stimulated with the T cell mitogen concanavalin A in the presence of either corneal explants or cells of corneal epithelial or endothelial cell lines and the production of T helper 1 (Th1) or Th2 cytokines was determined by enzyme-linked immunosorbent assay (ELISA) or reverse transcription–polymerase chain reaction (RT-PCR). We found that the cornea possesses the ability to inhibit, in a dose-dependent manner, production of the inhibitory and anti-inflammatory cytokines interleukin (IL)-4 and IL-10 by activated T cells. The production of cytokines associated with protective immunity [IL-2, IL-1β, interferon (IFN)-γ] was not inhibited under the same conditions. Corneal explants deprived of epithelial and endothelial cells retained the ability to suppress production of anti-inflammatory cytokines. This suppression was mediated by a factor produced by corneal stromal cells and occurred at the level of cytokine gene expression. We suggest that by this mechanism the cornea can potentiate a local expression of protective immune reactions in the anterior segment of the eye.

Keywords: eye, cornea, cytokines, immunoregulation, inflammation

INTRODUCTION

The unique features of the ocular immune response have been well documented. They have been described as ‘immune privilege’, referring to a decrease in the expression of immunity in the eye [1–3] or as an anterior chamber-associated immune deviation (ACAID) characterized by a suppression of some forms of systemic immunity after the administration of antigen into the eye [4,5]. The weaker expression of immunity in the eye has been explained by the low vascularization, especially in the anterior part of the eye, by the expression of Fas ligand and TRAIL molecules on corneal cells, by the activity of γ/δ regulatory T cells, and by the presence of various inhibitory substances present in the aqueous humour [6–10]. As a consequence, corneal allografts survive significantly longer than allografts at conventional sites of the body [11–13].

The eye is protected from the outer environment by the tear film and by the cornea. The cornea is a thin tissue composed of an outer layer of epithelial cells, the central stroma containing some modified fibroblasts (keratocytes) and an inner layer of endothelial cells. The number of each type of corneal cell in the mouse does not exceed a few thousand. The cornea is normally in contact with the outside environment and thus immunological protective mechanisms, similar to those in mucosal tissues exposed to a ‘danger’ from outside, have to develop and operate efficiently here. It has been shown that the cornea is a potent producer of nitric oxide (NO) [14,15], a molecule which is toxic to various pathogens and possesses immunomodulatory properties [16]. The production of various cytokines, including interleukin (IL)-1, IL-6, IL-8, IL-18, interferon (IFN)-γ and TGF-β, has also been demonstrated in cultured corneal cells in vitro[17–21].

The high production by the cornea of molecules with immunomodulatory properties led to the assumption that the cornea could be an important tissue regulating immunity in the anterior part of the eye. To test this hypothesis, we removed corneas from healthy mice and co-cultivated these explants with syngeneic spleen cells stimulated with the T cell mitogen concanavalin A (Con A). We found that corneal stromal cells selectively inhibited production of the anti-inflammatory cytokines IL-4 and IL-10 but did not affect production of the cytokines (IL-2, IL-1β, IFN-γ) contributing to inflammatory reactions. We suggest that through this mechanism the cornea ensures the expression of protective inflammatory reactions in the anterior segment of the eye.

MATERIALS AND METHODS

Animals

Mice of both sexes of the inbred strain BALB/c aged 7–9 weeks were used in the experiments. The mice were from the breeding unit of the Institute of Molecular Genetics, Prague. All experiments were approved by the local Animal Ethics Commitee.

Corneas and corneal cell lines

Corneal buttons (2 mm in diameter) were removed aseptically from the eyes of normal BALB/c mice and were added to cultures of Con A-stimulated (1·5 µg/ml, Sigma-Aldrich, St Louis, MO, USA) syngeneic spleen cells or bacterial lipopolysaccharide (LPS)-stimulated (1·5 µg/ml, Difco Laboratories, Detroit, MI, USA) peritoneal exudates (PE) cells. In some experiments, layers of epithelial or endothelial cells were removed surgically before adding the corneal explants to the cultures. To prepare supernatants from corneal cells, four corneas were cultivated without any stimulation for 48 h in 500 µl of complete RPMI-1640 medium (Sigma) containing 10% fetal calf serum (FCS, Sigma), 10 mm HEPES buffer, antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin) and 5 × 10−5m 2-ME. Permanent lines of immortalized mouse corneal epithelial and endothelial cells [22] were grown in vitro.

Cytokine production and determination

Spleen cells at a concentration 0·6 × 106 cells/ml were cultivated in 48-well tissue culture plates (Nunclon, Roskilde, Denmark) in a volume of 500 µl of complete RPMI-1640 medium either unstimulated or stimulated with Con A (1·5 µg/ml). The supernatants were harvested after a 24-h (IL-2 detection) or 48-h (IL-4, IL-10, IFN-γ) incubation period. Peritoneal exudate cells obtained by washing the peritoneal cavity of normal BALB/c mice were cultivated at a concentration of 1 × 106 cells/ml in a volume of 500 µl of complete RPMI-1640 medium either unstimulated or stimulated with LPS (1·5 µg/ml) and rmIFN-γ (400 U/ml). The supernatants were harvested after 48 h for IL-1β detection. To test the effects of corneal cells on cytokine production, corneal explants (two corneas per 500 µl of culture medium, or one cornea per 250 µl of medium, if not indicated otherwise), or cells of corneal epithelial or endothelial cell lines (104 cells/well), were added to the cultures of stimulated spleen or PE cells. In some experiments, selective inhibitors of inducible NO synthase (iNOS) 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT, Sigma) or L-N(6)-(1-iminoethyl)lysine (L-NIL, Sigma) at a final concentration of 1 µg/ml, or 20 µg/ml of neutralizing monoclonal antibody (MoAb) anti-IFN-γ (clone H22·1, Santa Cruz Biotechnology, Santa Cruz, CA, USA), were added to the cultures containing corneas. For detection of suppressive activity in cell-free supernatants, the supernatants obtained after cultivation of corneas were added to the cultures of Con A-stimulated spleen cells to make 60, 30 or 15% of the final volume.

The production of cytokines was detected in the supernatants by enzyme-linked immunosorbent assay (ELISA) using cytokine specific capture and detection antibodies purchased from PharMingen (San Diego, CA, USA) (IL-2, IL-4, IL-10 and IFN-γ detection) or R&D Systems (Minneapolis, MN, USA) (IL-1β determination). Cytokine standards (recombinant cytokines of known concentrations) were added to all cytokine determinations to quantify the reaction.

Reverse transcription–polymerase chain reaction (RT-PCR)

The expression of genes for IL-4 and IFN-γ was detected using RT-PCR. Spleen cells (0·3 × 106 cells in 500 µl of cultivation medium) were cultivated either unstimulated or were stimulated with Con A (1·5 µg/ml) in the absence or presence of corneal explants. After 48 h of incubation, the corneal explants were removed and total RNA was isolated from the spleen cells using TRI Reagent (MRC Inc., Cincinnati, OH, USA). Two µg of total RNA were reverse transcripted into cDNA in a 20 µl reaction mixture, as described previously [23]. The cDNA samples were first normalized to yield equal amounts of β-actin. Consequently, the samples were hybridized with 5′- and 3′ primers for IL-4 (sense, 5′-ACGGAGATGGATGTGCCAAACGTC-3′; antisense, 5′-CGAGTAATCCATTTGCATGATGC-3′) and IFN-γ (sense, 5′-TACTGCCACGGCACAGTCATTGAA-3′; antisense, 5′-GCAGCGACTCCTTTTCCGCTTCCT-3′). The PCR products were electrophoresed on an ethidium bromide-stained agarose gel.

Statistics

All results are expressed as means ± s.e.m. The statistical significance of differences between the means of individual groups was calculated using the Student's t-test.

RESULTS

Effects of corneal cells on the production of pro- and anti-inflammatory cytokines

To test the effects of corneal cells on the production of cytokines by activated T cells, spleen cells were stimulated with Con A in the absence or presence of corneal explants (1, 2 or 3 explants per culture) and the level of cytokines in the culture supernatants was determined by ELISA. A significant and profound suppression of IL-4 (Fig. 1a) and IL-10 (Fig. 1b) production was observed in cultures containing corneas, while the production of Th1 cytokines IL-2 and IFN-γ was unchanged in the same cultures (Figs 1c, d). Similar to the production of IL-2 and IFN-γ, production of IL-1β, which is a principal inflammatory cytokine, was also not inhibited in cultures of PE cells stimulated with LPS in the presence of corneal explants (Fig. 1e). To exclude the possibility that the low levels of IL-4 in cultures containing corneas were due to the absorption of cytokines by corneal cells, we measured expression of genes for IL-4 and IFN-γ in cultures of spleen cells stimulated with Con A in the presence of corneal explants. As demonstrated in Fig. 2, the expression of the gene for IL-4, but not for IFN-γ, was markedly suppressed in the cultures containing corneas.

Fig. 1.

Fig. 1

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Selective suppression of anti-inflammatory cytokine production by corneal cells. Spleen cells or peritoneal exudate cells were stimulated with mitogens in the absence or presence of corneal explants (1, 2 or 3 explants per culture) and production of IL-4 (a), IL-10 (b), IL-2 (c), IFN-γ (d) and IL-1β(e) was determined by ELISA. Data are displayed as mean ± s.e.m. and are representative of three comparable experiments. *P < 0·05, **P < 0·001.

Fig. 2.

Fig. 2

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Corneal cells selectively inhibit expression of cytokine genes. Spleen cells were cultivated unstimulated (lane 1) or were stimulated with Con A in the absence (lane 2) or presence (lane 3) of corneal explants (two explants per culture) and actin, IL-4 and IFN-γ gene expression was determined in spleen cells by RT-PCR.

Identification of corneal cell type mediating suppression of anti-inflammatory cytokine production

To identify the corneal cell type responsible for the suppressive activity, epithelial and/or endothelial cells were surgically removed from the corneas and such modified corneal explants without epithelial or endothelial cells were tested for suppression of IL-4 production. Similar to the intact corneas, which strongly inhibited IL-4 production, corneal explants without epithelial or endothelial cells retained suppressive activity (Fig. 3a). The activity was maintained even in corneal stroma obtained after removing both epithelial and endothelial cells (Fig. 3a). The suppressive activity of corneal explants was attenuated by irradiation (3000 R) of the explants before their cocultivation with spleen cells (data not shown). This observation suggests that the inhibition of cytokine production is due to an active suppression by corneal stromal cells rather than by a substance released from the stromal matrix. Figure 3b shows the selectivity of suppression: production of IFN-γ was not inhibited in cultures containing intact or modified corneas or corneal stroma. Separated epithelial or endothelial cells, or both together, when added to the cultures of Con A-stimulated spleen cells, did not suppress IL-4 production (Fig. 3a). To confirm these results, permanent lines of immortalized mouse corneal epithelial or endothelial cells were tested for their ability to inhibit cytokine production. No selective suppression of IL-4 production was found (Fig. 3a).

Fig. 3.

Fig. 3

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Identification of the corneal cell type mediating selective suppression of IL-4 production. (a) Production of IL-4. Spleen cells were stimulated with Con A in the absence or presence of intact corneal explants (C), corneal explants deprived of either epithelial (S + En) or endothelial (S + Ep) cells, corneal stroma (S), fresh corneal epithelial (Ep) or endothelial (En) cells or both cell types (Ep + En), or in the presence of cells of the immortalized mouse corneal epithelial (Ep line) or endothelial (En line) cell lines. (b) Production of IFN-γ in the same cultures as described in Fig. 3a. Production of IL-4 and IFN-γ was determined in the supernatants by ELISA. Data are representative of three comparable experiments. *P < 0·001.

The suppression of IL-4 production is mediated by a soluble factor produced by corneal cells

The suppression of anti-inflammatory cytokine production observed in cultures containing corneas can result from the contact between corneal and spleen cells or may be mediated by a soluble factor produced by the cornea. To test these two possibilities, corneas from healthy mice were cultivated for 48 h in culture medium and the obtained cell-free supernatants were added to cultures of spleen cells stimulated with Con A. As demonstrated in Fig. 4, the supernatants inhibited, in a dose-dependent manner, the production of IL-4 (Fig. 4a) but not of IFN-γ (Fig. 4b), suggesting the existence of a suppressive molecule secreted by the corneal cells. As the cornea produces a high level of NO and the immunosuppressive effects of NO have been described, we tested whether NO could be the suppressive molecule in our model. We used selective inhibitors of iNOS at concentrations at which they completely inhibit NO production by stimulated macrophages or corneas (data not shown). However, neither AMT nor L-NIL added to the cultures of spleen cells stimulated with Con A in the presence of corneas abrogated suppression of IL-4 production (Fig. 4c). The only known cytokine which binds preferentially to Th2 cells and which could selectively inhibit Th2 cytokine production is IFN-γ. We therefore tested whether neutralizing anti-IFN-γ antibodies added to the cultures containing corneas can abrogate suppression of IL-4 production. It was found that anti-IFN-γantibodies did not affect the suppression of IL-4 production in cultures containing corneal explants (Fig. 4d).

Fig. 4.

Fig. 4

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Suppression of IL-4 production is mediated by a factor produced by cornea. The supernatants obtained after cultivation of corneal explants were added (60, 30 or 15% of the culture volume) to the cultures of spleen cells stimulated with Con A. The production of IL-4 (a) and IFN-γ (b) was measured by ELISA. To characterize the molecule responsible for the suppression of IL-4 production, specific iNOS inhibitors AMT or L-NIL (c) or neutralizing anti-IFN-γ antibody (d) were added into the cultures of spleen cells stimulated with Con A in the presence of corneal explants (C). Each experiment was repeated at least three times with comparable results. *P < 0·05, **P < 0·001.

DISCUSSION

Immunological privilege of the eye and the unique features of immunity after the administration of an antigen into the eye have been well documented [1–5]. However, specific immunity or tolerance can be expressed effectively in the eye, as has been demonstrated by a second-set rejection or acceptance of corneal allografts in systemically sensitized or tolerized recipients [13,24]. Therefore, the anatomic barrier separating the anterior part of the eye from the immune system is not so fully effective and local immunoregulatory mechanisms must operate efficiently here and be responsible for the alterations of immunity observed after antigen administration into the eye. These mechanisms must protect the eye from self-damaging strong inflammatory reactions but must also allow protection of the eye against harmful invaders from outside [3].

We have demonstrated here a role for the cornea in ensuring protective inflammatory reactions in the anterior part of the eye. We found that the cornea inhibits, in a cornea mass-dependent manner, production of the anti-inflammatory cytokines IL-4 and IL-10 but did not affect production of cytokines contributing to the inflammatory reactions. Corneal stromal cells have been identified as a source of a molecule with a selective suppressive activity. No suppression of IL-4 production was found in cultures containing activated T cells and separated fresh corneal epithelial or endothelial cells or permanent lines of mouse corneal epithelial or endothelial cells. Using a different system, involving cultured rat corneal endothelial cells and mouse T hybridoma cells or T cell lines, Mi et al. [25] observed suppression of both IL-2 and IL-4 production. The difference may be due to a xenogeneic system (rat corneal endothelial cells and mouse permanent lines of T cells) in the experiments of Mi et al. and a syngeneic system of fresh mouse corneal endothelial and spleen cells in our model.

Using specific inhibitors of iNOS we excluded the possibility that suppression in our model was mediated by NO, a molecule which has been shown to have immunosuppressive properties [26,27] and which can be produced in relatively large quantities by corneal cells [14,15]. The suppressive molecule also appears to be distinct from IFN-γ, a cytokine which influences the production of Th2 cytokines [28]. Although the cornea produces a number of neuropeptides and other biologically active molecules [10,29], none of these substances has been shown to have a property which selectively inhibits production of Th2 cytokines. We therefore suggest that the selective suppression of anti-inflammatory cytokine production is mediated by an as yet uncharacterized molecule produced by corneal stromal cells. This molecule inhibits Th2 cytokine production at the level of cytokine gene expression. We propose that by production of this molecule the cornea can potentiate expression of the protective inflammatory reactions in the anterior part of the eye, and thus the cornea may contribute to the protection of the eye against invaders from outside.

So far, mainly only those mechanisms contributing to the suppression of immunity in the eye have been proposed and characterized [6, 7, 8, 9, 10,30]. Nevertheless, reports have appeared showing production of proinflammatory cytokines by the cells of the anterior segment of the eye. It has been shown that human corneal cells produce IL-1, which is a principal inflammatory cytokine [17]. Corneal epithelial cells are also a potent source of IL-18, which may play an important role in initiating IFN-γ-mediated inflammatory responses in the cornea [21]. A large number of various cytokines and chemokines are also produced by corneal endothelial cells [19]. In addition, human ocular cells produce macrophage migration inhibitory factor which has the capacity to enhance Th1 cytokine production and thus could play a role in inflammatory reactions in the eye [31]. It thus appears that, to ensure protective inflammatory reactions in the anterior part of the eye, corneal epithelial and endothelial cells produce cytokines which can mediate or contribute to the inflammatory reactions.

Our results extend those reports demonstrating production of proinflammatory cytokines by corneal cells and show the ability of corneal stromal cells to potentiate inflammatory reactions by the selective inhibition of anti-inflammatory cytokine production. Corneal stromal cells have been identified as the principal source of the molecule, which selectively inhibits the production of anti-inflammatory cytokines. It has already been demonstrated that corneal stromal keratocytes may modulate immune responses as antigen-presenting cells [32]. In the environment of a local depression of IL-4 production, keratocytes can direct development of the immune response preferentially to a Th1 type which is associated with inflammatory reactions. It has been shown recently that inflammatory reactions can be induced in the environment of immune privilege and that immune privilege persists in the eye with extreme inflammation [33]. The importance of the balance of individual cytokines for the optimal expression of inflammatory reactions in the cornea and for the protection of the eye has been suggested [34–37].

Our results thus demonstrate that the cornea is not only a passive barrier, protecting the eye from the outer environment or a site where proinflammatory cytokines are produced. The corneal stromal cells inhibit selectively, by the secretion of an as yet uncharacterized suppressive molecule, production of anti-inflammatory cytokines but did not affect the production of cytokines contributing to inflammatory reactions. The results thus show the existence of a local regulation of protective immunological mechanisms in the anterior segment of the eye.

Acknowledgments

This work was supported by the Wellcome Trust Grant 061367/Z/000/Z, grants NI/6659–3 and NI/7531–3 from the Grant Agency of the Ministry of Health of the Czech Republic, grant 310/02/D162 from the Grant Agency of the Czech Republic and projects LN00A026 and MSM 113100003 from the Ministry of Education of the Czech Republic. The authors thank J. Y. Niederkorn for providing permanent lines of corneal epithelial and endothelial cells.

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