Tear and mucus eotaxin-1 and eotaxin-2 in allergic keratoconjunctivitis

OBJECTIVE: Eotaxin-1 and eotaxin-2 are potent eosinophil chemotactic and activating peptides that may be implicated in the pathogenesis of the chronic allergic eye diseases vernal keratoconjunctivitis (VKC) and atopic keratoconjunctivitis (AKC). The purpose of this study was to measure these chemokines in tear and mucus samples of active-disease patients and in vitro cultured conjunctival epithelial cells and fibroblasts. DESIGN: Comparative, observational case series and in vitro study. PARTICIPANTS: Sixteen patients with clinically active and untreated VKC or AKC, six age-matched control patients, and five nonactive seasonal allergic conjunctivitis patients. METHODS: Tears were collected from the active VKC and AKC patients, and from the normal patients. Mucus was collected from six of these VKC patients. Tears were also collected from an additional five allergic patients after obtaining a positive reaction to conjunctival allergen challenge. Conjunctival epithelial cell and conjunctival fibroblast cultures were exposed to interleukin (IL)-4, IL-13, and tumor necrosis factor-alpha (TNF-alpha), or to combinations of these cytokines. MAIN OUTCOME MEASURES: Levels of eotaxin-1 and eotaxin-2 in tears, mucus, and culture medium. RESULTS: High levels of eotaxin-1 and eotaxin-2 were found in mucus of VKC patients, whereas only eotaxin-2 was found to have increased significantly in tears of VKC and AKC patients compared with those of normal patients. Mucus contained higher levels of chemokines than did tears. Both tear eotaxin-1 and eotaxin-2 were correlated significantly with the percent of eosinophils in tear fluid. Eotaxin-1 also was correlated significantly with the sum clinical score and corneal involvement in VKC patients. Conjunctival epithelial cells in culture did not produce eotaxin-1 or eotaxin-2, either at baseline or after cytokine exposure. Conjunctival fibroblasts produced eotaxin-1 only after exposure to IL-4, TNF-alpha, and the combination of IL-4 plus TNF-alpha or IL-13 plus TNF-alpha. CONCLUSIONS: Eotaxin-1 and eotaxin-2 are implicated in eosinophil recruitment and in the pathogenesis of VKC and AKC. Cytokine-stimulated conjunctival fibroblasts may be one source of eotaxin-1 in severely allergic tissues.     

The implications of the upregulation of ICAM-1/VCAM-1 expression of corneal fibroblasts on the pathogenesis of allergic keratopathy

OBJECTIVE: The present study investigated the expression of ICAM-1 and VCAM-1 on fibroblasts with interleukin (IL)-4 and/or tumor necrosis factor (TNF)-alpha stimulation and assessed the effect of eosinophil adhesion on fibroblast viability. METHODS: Primary cultured human corneal fibroblasts were incubated with IL-4, TNF-alpha, or their combination for 24 hours. Expression of ICAM-1 and VCAM-1 was examined by real-time quantitative PCR and flow cytometric analysis. Purified eosinophils were cocultured with activated fibroblasts, and the number of eosinophils adhered to fibroblasts and the number of damaged fibroblasts were counted using microscopy. In a separate trial, conjunctival and corneal impression cytology was performed on patients with atopic keratoconjunctivitis and corneal ulcers (eight eyes) to assess the status of the ocular surface epithelium and the presence of inflammatory cell infiltrates. RESULTS: Real-time quantitative PCR and flow cytometric analysis revealed that both mRNA and protein of VCAM-1 and ICAM-1 were upregulated by IL-4 and TNF-alpha. IL-5-primed eosinophils adhered to the corneal fibroblasts treated with IL-4 and TNF-alpha, and the fibroblasts were damaged by eosinophil adherence. Anti-ICAM-1 antibody and anti-VCAM-1 antibody inhibited the eosinophil adherence to fibroblasts and the fibroblast damage. Impression cytology revealed extensive infiltration of neutrophil and eosinophils among isolated ocular surface epithelial cells with advanced squamous metaplasia. CONCLUSIONS: Corneal fibroblasts expressed ICAM-1 and VCAM-1 when activated with IL-4 and TNF-alpha. Eosinophils can adhere to the activated fibroblasts and can induce subsequent fibroblast damage through these adhesion molecules. Eosinophil adhesion to fibroblasts may possibly contribute to the pathogenesis of severe persistent allergic corneal ulcers.     

TGF-beta1, IL-1beta, and Th2 cytokines stimulate vascular endothelial growth factor production from conjunctival fibroblasts

Giant papillary formation containing newly formed vessels is a major characteristic of severe allergic conjunctivitis, such as atopic keratoconjunctivitis (AKC) or vernal keratoconjunctivitis (VKC). We examined production of vascular endothelial growth factor (VEGF) from cultured conjunctival fibroblasts from normal volunteers under stimulation with type 1-, type 2-helper T cell derived and proinflammatory cytokines to investigate the mechanism of giant papillae formation in AKC/VKC. Primary cultured conjunctival fibroblasts were incubated with interleukin (IL)-4, IL-13, IL-1beta, IL-2, tumor necrotizing factor (TNF)-alpha, interferon (IFN)-gamma, or transforming growth factor (TGF)-beta1. Effects of cytokines on VEGF protein secretion in supernatant were assessed by ELISA, and VEGF mRNA expression in cultured cells were assessed by quantitative PCR. TGF-beta1 most effectively increased VEGF concentration with dose- and time-dependent manner IL-1beta, IL-4, and IL-13 significantly increased VEGF concentration. Though IL-2 also showed slight increase of VEGF concentration, it was not statistically significant. TNF-alpha and INF-gamma did not increase VEGF concentration. Quantitative PCR showed significant increase of VEGF mRNA in TGF-beta1, IL-1beta, and IL-4 stimulated fibroblasts. TGF-beta1, IL-1beta, and Th2 cytokines from allergic inflammatory cells induced VEGF production in conjunctival fibroblasts, and may play a crucial role in neovascularization and formation of giant papillae in AKC/VKC.     

Inhibition by triptolide of chemokine, proinflammatory cytokine, and adhesion molecule expression induced by lipopolysaccharide in corneal fibroblasts

PURPOSE: The production of proinflammatory cytokines and chemokines as well as the surface expression of intercellular adhesion molecule (ICAM)-1 by corneal fibroblasts contribute to corneal inflammation. The effects of triptolide on the expression of these proteins induced by lipopolysaccharide (LPS) in human corneal fibroblasts were examined in comparison with those of dexamethasone. METHODS: The release of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, IL-6, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1beta, and IL-8 from cultured corneal fibroblasts was measured with assay kits. Surface expression of ICAM-1 on the cultured cells was measured with a whole-cell enzyme-linked immunosorbent assay. RESULTS: Lipopolysaccharide (LPS) induced the release of the proinflammatory cytokine IL-6 and that of the chemokines G-CSF, MCP-1, MIP-1beta, and IL-8 as well as surface expression of ICAM-1 by corneal fibroblasts, whereas IL-1beta, TNF-alpha, and GM-CSF were not detected in the culture supernatants of cells incubated with or without LPS. Triptolide and dexamethasone each inhibited in a concentration-dependent manner the LPS-induced release of IL-6, G-CSF, MCP-1, and IL-8 by corneal fibroblasts. Whereas the inhibitory effect of dexamethasone on LPS-induced IL-6 release was greater than that of triptolide, the inhibitory effect of triptolide on LPS-induced G-CSF release was more pronounced than was that of dexamethasone. Dexamethasone also inhibited LPS-induced MIP-1beta release, whereas triptolide did not. Both compounds inhibited the LPS-induced surface expression of ICAM-1. CONCLUSIONS: Triptolide inhibits the LPS-induced expression of IL-6, chemokines (G-CSF, MCP-1, IL-8), and ICAM-1 in cultured human corneal fibroblasts. This compound might thus be expected to limit the infiltration of immune cells into the cornea.     

Histamine-induced cytokine production and ICAM-1 expression in human conjunctival fibroblasts

PURPOSE: Conjunctival fibroblasts stimulated with histamine (H) may be directly involved in the inflammatory and remodeling processes of chronic allergic conjunctival diseases. METHODS: Proinflammatory cytokine and growth factor production, and the expression of intercellular adhesion molecule-1 (ICAM-1) were studied in conjunctival fibroblast cultures challenged with different concentrations of H (from 10(-9) M to 10(-) (4) M). Interleukin (IL)-1, IL-4, IL-6, IL-8, tumor necrosis factor-alfa (TNF-alpha), fibroblast growth factor (FGF), epidermal growth factor (EGF) and transforming growth factor-beta (TGFbeta-1) were measured in supernatants. ICAM-1 expression was evaluated by a fluorescence activated cell sorter (FACS). Inhibitory effects of the H-1 antagonists (antiH): emedastine, levocabastine, and azelastine, and of the antiH-2, cimetidine, on H-stimulated fibroblasts were evaluated by measuring both cytokines in supernatants and the cellular expression of ICAM-1. RESULTS: Histamine increased the production of IL-1, IL-6 and IL-8, and ICAM-1 expression. TNF-alpha, IL-4 and growth factor production were not modified by histamine. The antiH-1, emedastine, significantly reduced H-induced production of IL-1, IL-6 and IL-8, while azelastine reduced only IL-1. Levocabastine and cimetidine were less effective. The histamine-induced increase in ICAM-1 expression was inhibited by emedastine but not by azelastine and levocabastine. CONCLUSIONS: Histamine has pro-inflammatory effects on conjunctival fibroblasts, inducing the production of cytokines and the expression of ICAM-1. Emedastine significantly reduced cytokine and ICAM-1 expression from H-stimulated fibroblasts. Conjunctival fibroblasts may contribute to the maintenance of inflammation in chronic allergic diseases.     

Role of macrophage migration inhibitory factor in conjunctival pathology in ocular cicatricial pemphigoid

PURPOSE: Macrophage migration inhibitory factor (MIF) is a pleiotropic, proinflammatory cytokine that mediates various immunoinflammatory processes. Ocular cicatricial pemphigoid (OCP) is an autoimmune disease in which affected conjunctivae show features of an immunoinflammatory disease. In this study, the role of MIF in the pathogenesis of OCP was examined. METHODS: The expression of MIF in conjunctival tissues of patients with OCP (n = 10) and normal subjects (n = 5) was studied by quantitative real-time PCR and immunohistochemistry. The production of MIF by conjunctival fibroblasts of normal control subjects and patients with OCP was determined, by using quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA). In addition, the effects of interleukin (IL)-1, tumor necrosis factor (TNF)-alpha, and transforming growth factor (TGF)-beta1 on the induction of MIF by conjunctival fibroblasts were studied by quantitative real-time PCR. To determine the relationship between conjunctival expression of MIF and accumulation of macrophages, in patients with OCP, a correlation study was performed. RESULTS: An increased conjunctival expression of MIF was detected in patients with OCP, both at the mRNA (by real-time PCR) and protein level (by immunohistochemistry), compared with normal control patients. The expression of MIF was detected in the epithelial cells and occasionally in the stromal cells in control conjunctival tissues, by immunohistochemistry. In contrast, a statistically significant increase (P < 0.0001) in the expression of MIF was detected in the stromal cells of conjunctival tissues obtained from patients with OCP (control: 4.89 +/- 0.5; OCP: 19.82 +/- 1.34). By quantitative real-time PCR, compared with control conjunctiva, an increase in the expression of MIF was detected in the conjunctiva obtained from patients with OCP. A similar increase in the expression of MIF was also detected in conjunctival fibroblasts of patients with OCP, compared with control fibroblasts, by quantitative real-time PCR. A significantly increased (P < 0.001) level of MIF was also detected in supernatant collected from conjunctival fibroblasts of patients with OCP (186 +/- 5.4), compared with supernatant collected from control fibroblasts (9.3 +/- 7.6). Moreover, IL-1, TNF-alpha, and TGF-beta1, known factors involved in the pathogenesis of OCP, were found to induce the expression of MIF by conjunctival fibroblasts. A statistically significant correlation (P < 0.0001, r(2) = 0.4465) was observed between the expression of MIF and accumulation of CD68-positive macrophages in conjunctiva of patients with OCP. CONCLUSIONS: This study demonstrated an increased conjunctival expression of MIF in patients with OCP. MIF may be actively involved in the pathogenesis of OCP, possibly regulating the inflammatory events of the disease process.     

Role of enhanced expression of m-CSF in conjunctiva affected by cicatricial pemphigoid

PURPOSE: Local proliferation of macrophages has been reported to augment the inflammatory response in various human and experimental diseases. Macrophage accumulation in the submucosa is also an important feature in the pathogenesis of ocular cicatricial pemphigoid (OCP). In the present study, the role of local proliferation of macrophages in conjunctiva affected by OCP and the relationship between local proliferation of macrophages and expression of macrophage-colony-stimulating factor (m-CSF) in such conjunctiva were examined. METHODS: Biopsy specimens from the conjunctiva of 10 untreated patients with active OCP and from 5 normal subjects were studied for the expression of m-CSF, macrophages, and proliferating cell nuclear antigen (PCNA), a cell cycle protein, by immunohistochemistry. Dual staining for CD68 (a cell surface marker for macrophages) and PCNA was also performed to identify proliferating macrophages. In addition, fibroblasts isolated from conjunctiva of normal individuals and from patients with OCP were studied for the expression of m-CSF by immunostaining and real-time PCR. To identify the factors that induce m-CSF in conjunctival fibroblasts, the fibroblasts were incubated with different concentrations of interleukin (IL)-1alpha and tumor necrosis factor (TNF)-alpha, and the levels of m-CSF mRNA were determined by real-time PCR and the amount of m-CSF produced was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Normal conjunctiva showed weak expression of m-CSF in the conjunctival epithelial cells and stroma. Conjunctival expression of m-CSF protein was significantly (P < 0.0001) increased in conjunctival biopsy specimens from patients with OCP. m-CSF was detected in the infiltrating macrophages, stromal cells (presumably fibroblasts), and conjunctival epithelial cells. Compared with normal control conjunctival tissue, a 1.2-fold increase in the expression of mRNA for m-CSF was detected by real-time PCR in the conjunctival tissue obtained from patients with OCP. Increased expression of m-CSF correlated significantly (P < 0.0004) with an increased stromal accumulation of macrophages in conjunctival biopsy specimens of patients with OCP. A number of these accumulated macrophages (CD68-positive) were found to be proliferating (PCNA-positive). In addition, fibroblasts isolated and cultured from conjunctiva of patients with OCP showed significantly increased (1.7-fold) expression of m-CSF compared with normal conjunctival fibroblasts. When conjunctival fibroblasts were treated with IL-1alpha or TNF-alpha, real-time PCR and ELISA detected an increased level of m-CSF. CONCLUSIONS: An increased expression of m-CSF was observed in conjunctiva from patients with active OCP. There was a positive correlation between expression of m-CSF and accumulation of macrophages in conjunctival biopsy sections obtained from patients with OCP. Increased expression of m-CSF, mainly by conjunctival fibroblasts and infiltrating inflammatory cells, may play an important role in the regulation of local proliferation of macrophages in OCP. In the conjunctiva of patients with OCP, this process could augment or enhance the local inflammatory response and tissue injury consequent to it.     

Role of connective tissue growth factor in the pathogenesis of conjunctival scarring in ocular cicatricial pemphigoid

PURPOSE: Conjunctival fibrosis due to excessive accumulation of collagens is an important histologic feature in ocular cicatricial pemphigoid (OCP). Studies have suggested a role of transforming growth factor (TGF)-beta1 in conjunctival fibrosis in patients with OCP. Connective tissue growth factor (CTGF) is an important downstream mediator of TGF-beta1-induced collagen synthesis. CTGF usually acts synergistically with TGF-beta1 during the process of fibrosis in various organs. Hence, studying the mechanism by which CTGF influences TGF-beta1-induced synthesis of collagen in conjunctiva of patients with OCP would provide insight into the mechanism of conjunctival fibrosis in patients with OCP. METHODS: Biopsy specimens from conjunctiva of 10 patients with OCP and 5 normal subjects, were studied, with immunohistochemistry and real-time PCR, for the expression of CTGF and interstitial type I collagen. Using fibroblasts cultured from conjunctival biopsies we determined the effects of TGF-beta1 on the induction of CTGF and type I collagen by immunostaining, and quantitative real-time PCR. The effects of blocking the bioactivity of TGF-beta1 on the expression of CTGF and type I collagen were determined in TGF-beta1-stimulated fibroblasts, before and after treatment with type II receptor neutralizing antibody. RESULTS: An increased stromal accumulation of interstitial type I collagen with an increased expression of CTGF was observed in biopsy sections of patients with OCP, compared with the control. By quantitative real-time PCR, a 3.2-fold increase in the expression of CTGF was detected in conjunctival tissues obtained from patients with OCP, compared with control conjunctiva. Fibroblasts isolated from conjunctiva of patients with OCP expressed 4.4-fold more CTGF, compared with control conjunctival fibroblasts, by real-time PCR. When these cultured fibroblasts were immunostained, an increased expression of CTGF was detected in fibroblasts isolated from patients with OCP, compared with control. Furthermore, when conjunctival fibroblasts were treated with TGF-beta1, an approximately ninefold increase in the expression of CTGF and an approximately threefold increase in the expression of type I collagen were detected by real-time PCR, compared with unstimulated fibroblasts. Finally, when antibody to TGF-beta type II receptor was added before TGF-beta1 treatment of these fibroblasts, the expression of type I collagen and CTGF was significantly reduced. CONCLUSIONS: In the present study, an increased expression of CTGF was recorded in conjunctiva of patients with OCP. TGF-beta1 can induce production of CTGF and type I collagen by fibroblasts obtained from conjunctiva in OCP. This induction of CTGF by TGF-beta1 can be blocked by antibody to TGF-beta type II receptors. The findings lead to the conclusion that CTGF is one of the molecules involved in the pathogenesis of conjunctival fibrosis in patients with OCP.     

Effects of IL-4 on conjunctival fibroblasts: possible role in ocular cicatricial pemphigoid

PURPOSE: Increased stromal accumulation of macrophages and submucosal fibrosis due to excessive accumulation of collagens are central histologic features in ocular cicatricial pemphigoid (OCP). Interleukin (IL)-4 plays an important role in both the inflammatory and fibrotic events in several human and experimental diseases. In the present study, the possible role of IL-4 in the pathogenesis of OCP was investigated. METHODS: Biopsy specimens from the conjunctivae of 10 patients with OCP and 5 normal subjects were studied for the expression of IL-4 by immunohistochemistry. The expression level of IL-4 was also examined in conjunctival fibroblasts of normal control subjects and patients with OCP. The effects of IL-4 in the induction of inflammatory and fibrogenic molecules was studied in IL-4-treated conjunctival fibroblasts, and the expression levels of macrophage colony-stimulating factor (m-CSF), heat shock protein (HSP)-47 and type I collagen was determined by quantitative real-time PCR. The level of IL-4 was also measured by enzyme-linked immunosorbent assay (ELISA) in serum samples obtained from patients with OCP during active stage and remission and were compared with the levels in control sera. RESULTS: Compared with the weak expression of IL-4 in the normal conjunctival sections, an increased expression of IL-4 was noted in conjunctival sections of patients with OCP. A similar increase in the expression of IL-4 was also detected in fibroblasts isolated from conjunctiva of patients with OCP, compared with control fibroblasts. Real-time PCR and ELISA detected a significantly increased level of m-CSF, at both the mRNA and protein levels in IL-4-stimulated cells. Similarly, IL-4 treatment resulted in the induction of type I collagen and collagen-binding HSP47 by conjunctival fibroblasts, as detected by real-time PCR. However, no apparent changes in the levels of IL-4 were detected by ELISA in serum samples of patients with OCP and control subjects. CONCLUSIONS: Increased conjunctival expression of IL-4 may play an important role in the regulation of local accumulation of macrophages (by inducing m-CSF), and matrix accumulation (by inducing HSP47 and collagen) during conjunctival scarring in patients with OCP. IL-4, therefore, may augment or enhance both conjunctival inflammatory and subsequent fibrotic responses in patients with OCP.     

多西环素对培养的人结膜上皮细胞炎性反应及凋亡的影响

目的探讨多西环素对体外培养的人结膜上皮细胞细胞黏附分子1（ICAM-1,CD54）、人类白细胞抗原DR（HLA—DR）、白细胞介素1β（IL-1β）表达及凋亡的影响。方法混合消化液培养法培养人结膜上皮细胞,免疫组织化学方法进行细胞鉴定。培养至第3代或第4代,在细胞培养液中分别加入γ干扰素（IFN-γ）0U／ml、IFN-γ300U／ml、IFN-γ300U／ml＋多西环素10μg／ml、IFN-γ300U／ml＋多西环素20μg／ml、IFN-γ300U／ml＋多西环素40μg／ml、IFN-γ300U／ml＋地塞米松100μg／ml,24h后收集细胞,流式细胞学检测CD54、HLA—DR及IL-1β的表达,Western blot检测CD54的表达。72h后收集细胞,碘化丙啶（PI）染色,流式细胞学检测凋亡。结果培养的正常人结膜上皮细胞可以表达少许CD54和IL-1β,加入IFN-γ后表达明显增加,加入多西环素和地塞米松后CD54和IL-1β的表达下降（P〈0．01）。随着所加多西环素浓度的增加,CD54和IL-1β的表达逐渐降低（P〈0．01）。培养的正常人结膜上皮细胞未发现明显的HLA—DR的表达及凋亡的产生,加入IFN-γ及多西环素后HLA—DR的表达及凋亡水平无明显变化（P〉0．05）。结论多西环素可抑制培养的正常人结膜上皮细胞CD54和IL-1β等炎性相关因子的产生,提示多西环素对干眼等非感染性眼表炎性疾病的治疗可能具有一定的应用前景。（中华眼科杂志,2005,41：842-846）     

Role of TGF-β in tissue eosinophilia associated with vernal keratoconjunctivitis

To determine the role of TGF-β₁ in the tissue eosinophilia associated with vernal keratoconjunctivitis (VKC), we investigated the immunohistochemical expression of TGF-β₁ and TGF-β₁-related proteins in giant papillae obtained from VKC patients. We also investigated the effect of TGF-β₁ on production of eotaxin by cultured conjunctival and corneal fibroblasts using ELISA. Finally, the effects of glucocorticoids, cyclosporine, and tacrolimus on eotaxin production by corneal fibroblasts were assessed. Our investigations revealed that eosinophils expressing TGF-β₁ and TGF-β₁-related proteins (such as phosphorylated Smad2, integrin αvβ₆, α-smooth muscle actin, type I procollagen, and tenascin-C) were expressed in the giant papillae. TGF-β₁ and IL-4/IL-13 caused a synergistic increase of eotaxin production in cultured conjunctival and corneal fibroblasts. This effect of TGF-β₁ and IL-4/IL-13 was inhibited by glucocorticoids, but neither by cyclosporine nor by tacrolimus. In conclusion, TGF-β₁ has an important role in the tissue eosinophilia associated with VKC.