Integrin adhesion in regulation of lacrimal gland acinar cell secretion

The extracellular microenvironment regulates lacrimal gland acinar cell secretion. Culturing isolated rabbit lacrimal gland acinar cells on different extracellular matrix proteins revealed that laminin enhances carbachol-stimulated secretion to a greater extent than other extracellular matrix proteins investigated. Furthermore, immunofluorescence indicated that integrin subunits, potentially functioning as laminin receptors are present in acinar cells. Among these, the integrin alpha6 and beta1 subunit mRNA expression was also confirmed by RT-PCR and sequence analysis. Secretion assays, which measured beta-hexosaminidase activity released in the culture media, demonstrated that function-blocking integrin alpha6 and beta1 monoclonal antibodies (mAbs) induce a rapid, transient and dose-dependent secretory response in cultured cells. To determine the intracellular pathways by which integrin alpha6 and beta1 mAbs could induce secretion, selected second messenger molecules were inhibited. Although inhibitors of protein kinase C and IP(3)-induced Ca(2+) mobilization attenuated carbachol-stimulated secretion, no effect on integrin mAb-induced release was observed. In addition, protein tyrosine kinases do not appear to have a role in transducing signals arising from mAb interactions. Our data clearly demonstrate, though, that cell adhesion through integrins regulates secretion from lacrimal gland acinar cells. The fact that the integrin mAbs affect the cholinergic response differently and that the integrin beta1 mAb secretion, but not the alpha6, was attenuated by the phosphatase inhibitor, sodium orthovanadate, suggests that each subunit utilizes separate intracellular signaling pathways to induce exocytosis. The results also indicate that the secretory response triggered by the beta1 integrin mAb is generated through dephosphorylation events.     

Laminin synthesis and the adhesion characteristics of immortalized human corneal epithelial cells to laminin isoforms

We have studied the synthesis of laminins (Ln) and determined the specific integrins mediating the adhesion of immortalized human corneal epithelial cells to mouse Ln-1, and human Lns-5 and -10. Immunofluorescence microscopy of the cells demonstrated integrin alpha(2), alpha(3), alpha(6), beta(1)and beta(4)subunits, integrins alpha(6)and beta(4)being found in a typical 'leopard-skin' like manner. Immunoprecipitation studies showed that the cells produced alpha 3, beta 3 and gamma 2 chains of Ln-5, but not Lns-1 or -10. In culture Ln-5 was found as small plaques beneath the adhering cells within 1 hr, while in 4 hr widely spread Ln-5 plaques were observed in colocalization with beta(4)integrin subunit. By using a quantitative cell adhesion assay and function-blocking monoclonal antibodies we showed that integrin beta(1)subunit plays a role in mediating corneal epithelial cell adhesion to mouse Ln-1. However, none of the available function-blocking antibodies to integrin alpha-subunits inhibited the adhesion. Integrin alpha(3)beta(1)complex mediated the adhesion of corneal epithelial cells to human Lns-5 and -10. Integrin complex alpha(3)beta(1), as well as laminin alpha(3)chain, was also shown to mediate cell adhesion to newly produced endogenous Ln-5. The present results show that integrin alpha(3)beta(1)complex mediates the adhesion of corneal epithelial cells to Lns-5 and -10, while a yet unknown integrin alpha subunit appears to play a role in the adhesion to Ln-1. The results also show that among corneal basement membrane laminins, Ln-5 is synthetized by epithelial cells while Ln-10 may be a product of keratocytes.     

Down-regulation of androgen receptor expression and inhibition of lacrimal gland cell proliferation by retinoic acid

Androgens and retinoids are known to be involved in control of lacrimal gland function. Because retinoids generally antagonize androgen function it was the purpose of this study to investigate interactions of retinoic acid and androgens in rabbit lacrimal acinar cells in culture by determining effects of retinoic acid on androgen receptor (AR) mRNA expression, AR protein levels and androgen-stimulated cell proliferation. Experiments were conducted using primary rabbit lacrimal acinar cells and a transformed rabbit lacrimal acinar cell line. Exposure of primary lacrimal acinar cells in culture to 10(-10)-10(-6)M all-trans retinoic acid for 4-24hr causes an approximately 50% decrease in AR mRNA expression. Expression of AR protein in primary and transformed rabbit lacrimal acinar cells was confirmed by immunohistochemistry. Exposure of the primary cells to 10(-6)M retinoic acid for 24hr caused a 40% decrease in AR protein levels as determined by measurement of binding of(3) [H]-dihydrotestosterone (DHT) to cells in culture and Scatchard analysis. Exposure to 10(-9)-10(-6)M DHT stimulates proliferation of transformed rabbit lacrimal acinar cells. This effect is receptor mediated since it is blocked by the AR antagonist, flutamide. Proliferation of the lacrimal acinar cells is inhibited by retinoic acid, as compared to control, and retinoic acid also completely inhibits androgen stimulation of cell proliferation. This study supports the hypothesis that androgens play a supportive role in lacrimal gland function. The antagonistic influences of androgens and retinoic acid suggests that, under physiologic conditions there is a balance between the effects of androgens and retinoids in the lacrimal gland. A decrease in androgen levels in a dry eye patient may alter the balance between the effects of these important controllers of gene expression. The antagonistic effect of retinoids on androgens in the lacrimal gland must also be considered when devising pharmaceutical treatments for dye eye.     

Insulin-like growth factor-1 induces migration and expression of laminin-5 in cultured human corneal epithelial cells

PURPOSE: The effects of insulin-like growth factor (IGF)-1 on laminin (Ln)-5 and the associated integrins during in vitro HCEC migration were examined. Also investigated were the effects of IGF-1 on the migration of human corneal epithelial cells (HCECs). METHODS: HCEC migration was examined by wound-healing and chemoattraction assays. For migration inhibition assays, HCECs were pretreated with inhibitors of the IGF-1 receptor (alphaIR3), the PI3-K/AKT pathway (LY294002), and the MEK-ERK pathway (PD98059). The expression levels of Ln-5 and fibronectin (Fn) were determined by Western blot analysis, and the expression levels of the beta1 and alpha3 integrins were determined by confocal microscopy and Western blot analysis. The migration inhibition with anti-integrin alpha3 and beta1 antibodies was also determined. RESULTS: HCEC migration was significantly increased in the presence of IGF-1 and Ln-5. IGF-1 enhanced the production of Ln-5 in both a dose- and time-dependent manner, and this upregulation was blocked by pretreatment with alphaIR3 or LY294002. IGF-1 treatment upregulated expression of beta1 integrin, but not alpha3 integrin. The HCEC migration facilitated by IGF-1 was inhibited with the anti-integrin antibody for beta1. However, there was no cross-talk between Ln-5 and integrin beta1 production. CONCLUSIONS: The results reveal that IGF-1 induces HCEC migration through the independent production of Ln-5 and beta1 integrin, which are directed at least in part by activation of the PI3-K/AKT pathway, but are not affected by the MEK-ERK pathway.     

Induction of nitric oxide synthase and over-production of nitric oxide by interleukin-1beta in cultured lacrimal gland acinar cells

PURPOSE: Inflammation of the lacrimal gland is one of the major causative factors in aqueous tear-deficient dry eye syndrome. Pro-inflammatory cytokine production is upregulated in lacrimal gland autoimmune disease (i.e. Sj?gren's syndrome) and is associated with cell death. The expression of inducible nitric oxide synthase (iNOS/NOS-2) is known to be induced in the presence of pro-inflammatory cytokines in several secretory epithelial cell types. We hypothesize that pro-inflammatory cytokines, such as interleukin-1beta (IL-1beta), cause a marked increase in nitric oxide (NO) production via induction of iNOS in lacrimal gland epithelial cells and that this may be a significant pathophysiological pathway of dry eye syndrome. METHODS: Cultured immortalized rabbit lacrimal gland acinar cells were incubated with IL-1beta, iNOS inhibitor, or IL-1 receptor antagonist (IL-1ra). Colorimetric detection of NO(2)(-) and NO(3)(-) in the media, measured by the Griess reaction, was used as an index of NO production. Expression of iNOS was determined by SDS-PAGE and Western blot. RESULTS: IL-1beta stimulated a concentration-dependent and time-dependent increase in NO production. IL-1beta-induced NO production was significantly antagonized by co-incubation with IL-1ra or the iNOS-specific inhibitor, 1400W. Expression of iNOS protein was greatest at 4hr after addition of IL-1beta, and was nearly undetectable at 12hr. IL-1ra greatly reduced IL-1beta-induced iNOS expression. CONCLUSIONS: Lacrimal gland acinar cells are able to produce iNOS in response to the pro-inflammatory cytokine IL-1beta. The amount of iNOS expressed and the subsequent levels of NO that are produced by lacrimal cells are far lower than those seen in macrophages, but are consistent with those reported for other cell types in the literature. This pathway of iNOS induction and overproduction of NO may be a factor in lacrimal gland cell death in dry eye syndrome. Inhibitors of iNOS or IL-1 receptor may be beneficial for controlling lacrimal gland inflammation.     

Role of proinflammatory cytokines in the impaired lacrimation associated with autoimmune xerophthalmia

PURPOSE: To determine the effects of the proinflammatory cytokines interleukin (IL)-1alpha, IL-1beta, and tumor necrosis factor (TNF)-alpha on neurally mediated lacrimal gland protein secretion and to determine whether the amount of IL-1beta protein is upregulated in inflamed lacrimal glands of the MRL/lpr mouse, a murine model of human Sj?gren syndrome. METHODS: Lacrimal gland lobules of BALB/c mice were prepared and incubated for 2 hours in the presence or absence of recombinant human (rh)IL-1alpha, rhIL-1beta (10 ng/mL each), or rhTNFalpha (50 ng/mL). Peroxidase secretion in response to depolarizing KCl (75 mM) solution was measured by spectrofluorometric assay. In another set of experiments, saline, rhIL-1beta (1 microg), or an antibody against IL-1 receptor type I (IL-1RI), with or without rhIL-1beta, was injected (2 microL) into the lacrimal glands of anesthetized BALB/c mice. Twenty-four hours later, lacrimal gland lobules were prepared and peroxidase secretion was measured. The amount of IL-1beta protein in lacrimal gland acinar cell lysates prepared from 3-, 9-, and 13-week-old BALB/c, MRL/(+), and MRL/lpr mice was determined by ELISA. RESULTS: KCl-induced peroxidase secretion was inhibited in vitro 62%, 66%, and 53% by rhIL-1alpha, rhIL-1beta, and rhTNFalpha, respectively. In vivo, rhIL-1beta inhibited KCl-induced peroxidase secretion by 72%. This inhibitory effect of IL-1beta was completely reversed by an antibody against IL-1RI. Compared with 3-week-old mice, the amount of IL-1beta protein was upregulated 15- and 21-fold in lacrimal gland acinar cells isolated from 9- and 13-week-old MRL/lpr mice, respectively. CONCLUSIONS: Proinflammatory cytokines inhibit neurally mediated lacrimal gland secretion. The amount of IL-1beta protein is upregulated in acinar cells prepared from lacrimal glands infiltrated with lymphocytes. These results suggest that elevated levels of IL-1beta, as they occur in Sj?gren syndrome exocrine glands, may impair the secretory function of these tissues.     

Interactions of testosterone and all-trans retinoic acid in regulation of androgen receptor expression in rat lacrimal gland

All-trans retinoic acid down-regulates androgen receptor (AR) expression in lacrimal gland acinar cells in culture. The goal of this study was to determine if retinoic acid inhibits androgen-stimulated up-regulation of AR protein and AR mRNA expression in lacrimal glands of orchiectomized rats in vivo. Delivery of androgens to orchiectomized rats was accomplished by subcutaneous implantation of a 25 or 50 mg 21-day slow-release testosterone pellet. Rats were treated with retinoic acid by gastric gavage at 20 mg kg(-1) day(-1). After 7 days of treatment lacrimal glands were removed, AR protein expression in frozen sections was determined by immunohistochemistry and total RNA was probed for AR mRNA expression. Serum testosterone was measured by ELISA and serum retinoic acid was detected by HPLC. Orchiectomy decreases serum testosterone to 17 +/- 8 ng dl(-1), compared to 143 +/- 27 ng dl(-1) in normal rats, and reduces the number of lacrimal acinar cell nuclei expressing ARs to less than 30% of normal. Implantation of testosterone pellets restored lacrimal AR expression, but increased serum testosterone to more than 10 times the normal levels. Retinoic acid failed to inhibit AR expression in rats with high serum testosterone. Therefore a dose-response study was conducted in which testosterone was delivered by injection of a single dose of Depotestosterone at 2.5-200 mg kg(-1). Treatment of orchiectomized rats with a dose of testosterone as low as 2.5 mg kg(-1) resulted in serum testosterone levels of 62 +/- 17 ng dl(-1) and significantly increased lacrimal gland AR expression. Delivery of retinoic acid at 20 or 50 mg kg(-1) day(-1) simultaneously with a 2.5 mg kg(-1) testosterone injection prevented restoration of lacrimal gland AR expression and significantly reduced AR mRNA expression. A pharmacologic dose of retinoic acid inhibits AR expression in lacrimal gland acinar cells in vivo, as well as in vitro. This indicates that effects of retinoic acid and testosterone are antagonistic and suggests that retinoic acid may modulate effects of testosterone on the lacrimal gland.     

A comparison of ocular melanocyte and uveal melanoma cell invasion and the implication of alpha1beta1, alpha4beta1 and alpha6beta1 integrins

BACKGROUND/AIMS: Posterior uveal melanoma is the most common intraocular tumour in adults, responsible for the death of approximately 35% of patients. Hepatic metastases are most frequent, and once diagnosed survival is usually less than 1 year. The beta1 family of integrins, alphavbeta3 and MMP-2 and MMP-9 have been implicated in the metastasis of several types of tumour. To study their involvement in uveal melanoma we analysed the expression of the beta1 integrins, alphavbeta3, MMP-2, and MMP-9 in 10 primary posterior uveal melanomas, and correlated expression with invasive potential in vitro. Comparable studies were undertaken on cultures of melanocytes. METHODS: Expression of integrins was studied by immunohistochemistry, secretion of MMP-2 and MMP-9 by zymography, and the invasive potential was assessed using a transwell model. RESULTS: MMP-2 was secreted by all uveal melanomas and seven of 10 secreted MMP-9. Among uveal melanoma, invasion levels of 4-25% were observed and the major integrins expressed were alpha1beta1, alpha2beta1, alpha3beta1, alpha5beta1, and avbeta3. Melanocytes did not express alpha1beta1, alpha4beta1, and alpha6beta1. CONCLUSION: The laminin binding alpha6beta1 integrin was not expressed by either melanocytes or tumours with spindle morphology, which are considered to have a better prognosis. It is possible that expression of the alpha6beta1 integrin may prove useful as a prognostic indicator.     

The functions of exogenous and endogenous laminin-5 on corneal epithelial cells

Recent evidence suggests that the basement membrane not only separates basal cells from Bowman's layer, but also has a crucial role in the proliferation, differentiation and migration of corneal epithelial cells. The basement membrane is composed of a mixture of matrix components including collagens, laminins and heparan sulfate proteoglycans. In these extracellular matrixes, laminin is a major component of the basement membrane. Of 11 laminin isoformes, laminin-5 is a variant, composed of three nonidentical subunits alpha3, beta3, gamma2 and is a major component of the corneal basement membrane. However, little is known about the interactions of laminin-5 with corneal epithelial cells. In this study, we investigated the functions of laminin-5 on SV-40 transfected human corneal epithelial cells (HCE cells). We also revealed different functions between exogenous and endogenous laminin-5 on HCE cells. Laminin-5 is synthesized initially as a 490 kDa molecule that undergoes specific processing to cleavaged isoforms after being secreted. The alpha3 subunit is processed from 200-190 kDa to 160 kDa/145 kDa. The gamma2 subunit is processed from 150 kDa to 105 kDa/80 kDa. The beta3 subunit (140 kDa) is not processed. Exogenously added laminin-5 (soluble form) in this study was purified from a serum-free, conditioned medium of a human gastric carcinoma cell line STKM-I. This soluble laminin is a processed isoform containing alpha3 (160 kDa), beta3 (140 kDa) and gamma2 (105 kDa) chains. On the other hand, immunocytochemical analysis showed that HCE cells themselves secreted laminin-5 endogenously. Western blotting analysis revealed that HCE cells initially produced unprocessed isoform containing 190 kDa alpha3, 140 kDa beta3 and 150 kDa gamma2 chains and that after being secreted, the alpha3 chain was processed to 160 kDa/145 kDa and the gamma2 chain was processed to 105 kDa.Initially we investigated the functions of exogenous (processed) laminin-5 on HCE cells. Exogenously added laminin-5 strongly promoted cell adhesion via alpha3beta1 integrin, cell spreading, assembly of hemidesmosomes and mildly inhibited cell migration. Next we estimated the effect of endogenous (unprocessed) laminin-5 on HCE cells. Using an anti laminin-5 monoclonal antibody (mAb) or anti integrin alpha3beta1 mAbs, the blocking of the interaction between endogenously secreted laminin-5 and HCE cells caused strong inhibition of cell migration. Integrin alpha3beta1 and alpha6beta4 were expressed in HCE cells. These integrins are receptors of laminin-5. But, anti integrin alpha6beta4 mAbs did not have any blocking ability against cell migration. These results indicated that endogenous (unprocessed) laminin-5 has a crucial role in cell migration on HCE cells via alpha3beta1 integrin.In conclusion, structural differences between exogenous (processed) and endogenous (unprocessed) laminin-5 regulated their functions on HCE cells. Exogenously added laminin-5 strongly promoted cell adhesion, cell spreading and assembly of hemidesmosomes. Endogenously secreted laminin-5 had a crucial role in cell migration. In the future, processed soluble laminin-5 could be a useful drug for the prevention of recurrent corneal erosion, and unprocessed soluble laminin-5 could be applied for the treatment of prolonged corneal epithelial defects.     

All-trans retinoic acid remodels extracellular matrix and suppresses laminin-enhanced contractility of cultured human retinal pigment epithelial cells

All-trans retinoic acid (atRA) has been reported to inhibit the proliferation of retinal pigment epithelial (RPE) cells and used in treatment of proliferative vitreoretinopathy (PVR) in animal model. This study aimed at examining the effectiveness of atRA in inhibiting the extracellular matrix (ECM) biosynthesis by RPE cells and the RPE cell-mediated collagen gel contraction. Cultured RPE cells were treated with atRA and the expression of four ECM proteins (collagen types I, III, IV and laminin β1) was profiled. The results indicated that atRA treatment up-regulated de novo synthesis of collagen type I, but decreased that of laminin β1 in a dose-dependent manner. Moreover, the effect of atRA on RPE cell contraction was evaluated by measuring the area of collagen gel where RPE cells populated. Treatment with atRA significantly inhibited RPE cell-mediated collagen gel contraction. Addition of exogenous laminin nonapeptide into gels promoted RPE cell contraction, while atRA reversed the laminin-enhanced contractility. atRA treatment significantly suppressed the gene expression of integrin β3 but not αV subunit, and effectively inhibited the tyrosine phosphorylation of integrin β3 at residue 747 in RPE cells grown on laminin-coated dish, suggesting that atRA may suppress the RPE contractility through either inhibiting integrin β3 expression or abrogating the integrin β3-mediated signaling. In conclusion, atRA pharmacologically possesses a tissue-remodeling capacity and inhibits contractility of RPE cells. Therefore, atRA might be potentially a therapeutic agent for certain ocular disorders such as PVR.     

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

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 Sj?gren's syndrome and six non-Sj?gren'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 Sj?gren'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.     

Laminin-binding integrins in rat lens morphogenesis and their regulation during fibre differentiation

Mammalian lens development involves cell-cell and cell-ECM interactions. As integrins are a major family of cell adhesion molecules, we examined the expression patterns of several integrin subunits (alpha3A, alpha3B, alpha6A, alpha6B, beta1 and beta4) during rat lens development. RT-PCR, in situ hybridisation, immunofluorescence and immunoblotting were used to investigate expression of integrin subunits during lens development and differentiation. RT-PCR showed expression of alpha3A, alpha6A, alpha6B and beta1A but not alpha3B or beta4 subunits in postnatal rat lenses. Each subunit displayed distinct spatio-temporal expression patterns. beta1 integrin was expressed in both epithelium and fibres. alpha3A subunit expression was restricted to the epithelium; expression ceased abruptly at the lens equator. Expression of the alpha6A subunit increased during fibre differentiation, whereas alpha6B expression was predominantly associated with epithelial cells during lens development. In lens epithelial explants, FGF induced some of the changes in integrin expression that are characteristic of fibre differentiation in vivo. One notable exception was the inability of FGF to reproduce the distinctive down-regulation of the alpha3 isoform that is associated with initiation of elongation in vivo. Interestingly, vitreous treatment was able to reproduce this shift in alpha3 expression indicating that another factor(s), in addition to FGF, may be required for full and complete transition from an epithelial cell to a fibre cell. Integrin subunit expression therefore appears to be highly regulated during lens development and fibre differentiation with evidence of major changes in alpha3 and alpha6 isoform expression. These results indicate that integrins may play important roles in development and growth of the lens. How specific integrin subunits influence the behaviour of cells in different developmental compartments of the lens remains to be determined.     

Expression of integrins and MMPs during alkaline-burn-induced corneal angiogenesis

PURPOSE: To determine in a corneal alkaline burn model of angiogenesis whether the expression of integrins and MMPs is consistent with a VEGF-induced angiogenic response. METHODS: Neovascularization in female Sprague-Dawley rats was induced by alkaline cauterization of the central cornea. RT-PCR for integrins alpha(1), alpha(2), beta(3), and beta(5); the endothelial marker CD31; and metalloproteinases MMP-2 and MT1-MMP was performed on naive corneas and on cauterized corneas 72 and 288 hours after cautery. Analyses of protein and MMP expression were conducted on naive corneas and on cauterized corneas 24, 72, 120, and 168 hours after cautery by immunofluorescence microscopy and gelatin zymography. RESULTS: RT-PCR indicated a correlation between the induced angiogenic response and the expression of alpha(1) and beta(3) integrin subunits and MT1-MMP. Immunohistochemical analysis indicated that alpha(1), alpha(2), alpha(5), and beta(5) integrins and MMP-2 and MT1-MMP were expressed on the newly developing vasculature. The beta(3) integrin was preferentially expressed on platelets. CONCLUSIONS: Integrin expression during neovascularization of rat corneas in response to alkaline injury correlates with an angiogenic response that uses the VEGF/alpha(v)beta(5) pathway. MMP-2 and MT1-MMP, but not MMP-9, are expressed in a pattern consistent with their involvement in the angiogenic response.     

Fibronectin fragments promote human retinal endothelial cell adhesion and proliferation and ERK activation through alpha5beta1 integrin and PI 3-kinase

PURPOSE: Extracellular matrix degradation is associated with neovascularization in diabetic retinas. Fibronectin fragments (Fn-fs) are generated during vascular remodeling. The effects of cellular fibronectin (Fn) and selected Fn-fs on adhesion, proliferation, and signal transduction in human retinal endothelial cells (HRECs) were characterized. METHODS: Relative quantitative RT-PCR, flow cytometry, and immunocytochemistry determined integrin expression on HRECs. Adhesion was evaluated by coating plastic with Fn or Fn-fs of 45, 70, 110, or 120 kDa, and MTT conversion was used to measure proliferation and survival. Peptide inhibitors and blocking antibodies determined adhesive sites and integrins used for adhesion. Pharmacologic inhibitors and Western analyses were used to evaluate intracellular signaling. RESULTS: HRECs produced significant levels of alpha(2), alpha(3), alpha(5), alpha(v), beta(1), beta(3), and beta(5) integrin subunit mRNA. Flow cytometry of surface integrin expression revealed high levels of alpha(3), alpha(5), and beta(1) and lower levels of alpha(1), alpha(v), beta(3), and beta(5). These results were confirmed by immunocytochemistry. For adhesion to Fn and Fn-fs. the alpha(5)beta(1) integrin was essential. Pharmacologic inhibitors of PI 3-kinase blocked adhesion to Fn and Fn-fs, whereas the mitogen-activated protein (MAP) kinase kinase (MEK) inhibitor PD98059 blocked phosphorylation. The 110- and 120-kDa Fn-fs showed a concentration-dependent increase in proliferation, whereas 500 ng of the 70 kDa Fn-f-induced proliferation. Addition of III1-C, a matrix assembly domain, increased the proliferative effect of these Fn-fs. CONCLUSIONS: Fn and its Fn-fs modulate HREC adhesion and proliferation through signal-transduction pathways involving coupling of the alpha(5)beta(1) integrin through PI 3-kinase. Mitogenic signals for endothelial cells from degraded extracellular matrix may contribute to the development of diabetic retinopathy.     

Peroxisome proliferator-activated receptor agonists inhibit interleukin-1beta-mediated nitric oxide production in cultured lacrimal gland acinar cells.

Development of dry eye disease often occurs in individuals with autoimmune disorders such as Sj?gren's syndrome. The cause of dry eye in these patients is thought to be due, at least in part, to lymphocytic infiltration of the lacrimal glands, with subsequent loss of secretion of the aqueous component of tear film. How this lymphocytic infiltration leads to loss of secretion is not fully understood. We have previously shown that the proinflammatory cytokine, interleukin-1beta (IL-1beta), can stimulate the production of nitric oxide (NO) in cultured lacrimal gland acinar cells. It is possible that IL-1beta, produced by the infiltrating macrophages, stimulates production of inducible nitric oxide synthase (iNOS), and subsequently excessive production of NO. Peroxynitrate and other radical byproducts associated with excessive synthesis of NO may be detrimental to normal function of the lacrimal gland. Here we show that the peroxisome proliferator-activated receptor (PPAR)alpha and gamma agonists can inhibit NO production in cultured lacrimal gland acinar cells. Further, this is accomplished without loss of iNOS expression or tetrahydrobiopterin. These data suggest that the use of ointments or eye drops containing these PPAR agonists may provide an effective therapeutic intervention for the prevention of dry eye in Sj?gren's syndrome patients.     

Distribution of very late activation integrins in the human cornea. An immunohistochemical study using monoclonal antibodies

There is growing evidence that cellular adhesion mechanisms characterized by cell-cell and cell-matrix interactions are a fundamental process in the immunobiology of the cornea. Interactions with various extracellular matrix components are mediated by the very late activation (VLA) subgroup of the integrin superfamily of adhesion molecules. The six different VLA dimers known thus far consist of a common beta 1 subunit and a variable alpha (1 to 6) subunit. They serve as receptors for laminin (alpha 3 and alpha 6), collagen (alpha 2 and alpha 3), and fibronectin (alpha 4 and alpha 5). Using in situ immunohistochemistry and monoclonal antibodies, the distribution of the common beta 1 and the variable alpha-chains of VLA molecules was studied in normal human cornea and in cases with scarring or subepithelial/retrocorneal fibrous tissue. Epithelial cells were VLA-beta 1 and VLA-alpha 2, -alpha 3, -alpha 4, -alpha 5, and -alpha 6 positive. This is consistent with their intercellular adhesion and may aid in their attachment to the basement membrane which is composed of collagen, laminin, and fibronectin. Keratocytes in normal stroma expressed only the common beta 1-chain and no detectable alpha-chains. In regions of scar or fibrous tissue, however, an upregulated expression of the alpha-chains was detected. The VLA- alpha 1, -alpha 3, -alpha 4, and -alpha 5 were expressed in young fibrous tissue; in older lesions, VLA- alpha 1, -alpha 2, -alpha 3, -alpha 4, and -alpha 5 could be detected. The corneal endothelium showed a strikingly strong positivity for all VLA integrins.(ABSTRACT TRUNCATED AT 250 WORDS)     

The expression of laminin-5 and ultrastructure of the interface between basal cells and underlying stroma in the keratoconus cornea

PURPOSE: We investigated the expression of laminin-5 and integrins, and the ultrastructure of the interface between basal cells and the basement membrane in the keratoconus cornea. These findings were compared to those in normal central cornea and limbus. METHODS: Frozen sections of the normal cornea (center and limbus) and the keratoconus cornea were immunostained with monoclonal antibodies against three chains of laminin-5 and integrins. To investigate the ultrastructure of the interface between basal cells and the underlying stroma, we used transmission electron microscopy. RESULTS: As compared to those in the normal central cornea, immunostaining patterns of the three chains of laminin-5 were thick and irregular in the keratoconus cornea and the normal limbus. Using electron microscopy analysis, the same characteristic structure of the interface between basal cells and the underlying stroma was recognized in the keratoconus cornea and the normal limbus. The expression of integrin alpha(6)beta(4) was restricted to the basal aspect of basal cells in the normal cornea. In the keratoconus cornea, however, integrin alpha(6)beta(4) was expressed in all aspects in basal and suprabasal cells. CONCLUSION The expression patterns of laminin-5 and the ultrastructure of the interface between basal cells and the basement membrane in the keratoconus cornea were similar to those in the normal limbus.     

Detection of integrins in human cataract lens epithelial cells and two mammalian lens epithelial cell lines

AIM: To compare the incidence of various integrin subunits in human cataract anterior lens epithelial cells (A-LEC) and in two mammalian LEC lines. METHODS: Circular sections of anterior capsules with attached LEC were obtained during cataract surgery. Integrin subunits were immunolocalised in these anterior LEC and in a human and rabbit LEC line, using four monoclonal antibodies specific for subunits alpha2, alpha3, and alpha5, and beta subunit 2. RESULTS: All of these subunits were found in at least a proportion A-LEC samples as follows: alpha2 71%, alpha3 92%, alpha5 62%, and beta2 24%. The human LEC line was immunoreactive for alpha2 and alpha3 only. The rabbit lens epithelial cell line was immunoreactive for alpha5 but there was no staining for alpha2, alpha3, or beta2. CONCLUSION: The A-LEC and mammalian LEC lines showed a similarity in their pattern of integrin expression. As these integrins are receptors for extracellular matrix (ECM) components, they are likely to be associated with the attachment and migration of LECs that precedes capsular opacification. Therefore these cell lines may be useful in the elucidation of mechanisms involved the pathogenesis of capsule opacification.