The interaction between Lyn and FcεRIß is indispensable for FcεRI-mediated human mast cell activation.

BACKGROUND: FcεRIß reportedly functions as an amplifier of the FcεRIγ-mediated activation signal using a reconstitution system. However, the amplification mechanisms in human mast cells (MCs) are poorly understood. We previously reported the hyperexpression of FcεRIß of MCs in giant papillae from vernal keratoconjunctivitis patients, compared with that in conjunctivae from nonallergic conjunctivitis patients. Elucidation of the molecular mechanisms of the amplification induced by FcεRIß should provide new targets for novel therapeutic interventions. The aim is to understand in greater details the function of FcεRIß in human MC FcεRI expression and signaling. METHODS: FcεRIß and Lyn expression was reduced using a lentiviral shRNA silencing technique. Localization of Lyn and FcεRIß in cultured MCs was examined by confocal microscopic analysis. Mediators were measured by ELISAs. RESULTS: The diminution of FcεRIß significantly downregulated cell surface FcεRI expression and FcεRI-mediated mediator release/production. The downregulation of FcεRI-mediated degranulation was not only due to the decrease in FcεRI expression. The diminution of FcεRIß inhibited the redistribution of Lyn within the cell membrane following IgE sensitization. The diminution of Lyn in MCs significantly downregulated FcεRI-mediated degranulation. The recombinant cell-penetrating forms of phosphorylated FcεRIß immunoreceptor tyrosine-based activation motif (ITAM) for intracellular delivery disturbed the interaction between Lyn and phosphorylated endogenous FcεRIß ITAM, resulted in inhibiting IgE-dependent histamine release from MCs in vitro and from giant papillae specimens ex vivo. CONCLUSION: The interaction between Lyn and FcεRIß is indispensable for FcεRI-mediated human MC activation, and specific inhibition of the interaction may represent a new therapeutic strategy for the treatment of human allergic diseases.

Basophils in the giant papillae of chronic allergic keratoconjunctivitis.

AIMS: The essential role of basophils as an initiator of chronic allergic reaction has been elucidated in mouse models. The aim of this present study was to analyse the in situ immunolocalisation of basophils and other relevant inflammatory cells in chronic allergic keratoconjunctivitis. METHODS: Transmission electron microscopic (TEM) analysis was carried out to examine the existence of basophils in the giant papillae obtained from atopic keratoconjunctivitis (AKC) and vernal keratoconjunctivitis (VKC) patients. Cryostat sections of giant papillae were immunostained with basophil-specific antibody BB-1, and with anti-CD4, anti-CD8, anti-CD20, anti-major basic protein (MBP), anti-IgE and anti-FcepsilonRI-beta antibodies. RESULTS: TEM analysis confirmed the existence of basophils in the giant papillae. Small clusters of basophils were observed in the substantia propria of giant papillae, especially at the vicinity of vascular endothelium and subepithelial regions. BB-1-positive basophil clusters were surrounded by T cells, B cells, IgE-positive cells and MBP-positive eosinophils. No BB-1-positive basophils were observed in the control conjunctivae. CONCLUSION: Basophils may infiltrate from either vascular endothelium into the giant papillae. The existence of basophils at the centre of inflammatory cells suggests the role of basophils as an initiator of chronic allergic conjunctivitis.

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.

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.

Expression of stem cell factor in pterygium.

PURPOSE: To investigate the possible role of stem cell factor (SCF) in the pathogenesis of pterygium. METHODS: The localization of SCF was examined immunohistochemically in excised tissue from 4 primary pterygia and 5 normal conjunctival specimens. RESULTS: Three of the four pterygia showed strong immunoreactivity of SCF in the subepithelial connective tissue at the cap area. This immunoreactivity was completely blocked by using a primary antibody preincubated with recombinant SCF. The SCF-positive cells were identified as a population of fibroblasts by immunostaining for vimentin and prolyl 4-hydroxylase in adjacent sections. No apparent immunoreactivity of SCF was observed in the subepithelial connective tissues in the head and body of the pterygia and in the normal conjunctiva. CONCLUSION: Stem cell factor is overexpressed in fibroblasts at the cap area of most pterygia.

Anti VLA-4 monoclonal antibody inhibits eosinophil infiltration in allergic conjunctivitis model of guinea pig.

PURPOSE: To determine the contribution of very late activation antigen-4 (VLA-4) molecule to eosinophil infiltration into the conjunctiva in an actively sensitized allergic conjunctivitis model of guinea pig, effects of a monoclonal antibody against VLA-4 was examined in vivo. METHODS: A rat anti-mouse VLA-4 mAb (PS 2. 3), which cross-reacts with guinea pig VLA-4, inhibited the adhesion and transmigration of guinea pig eosinophils to /through human umbilical vein endothelial cells (HUVEC) in vitro. Hartley guinea pigs were actively sensitized by intraperitoneal injection of ovalbumin with aluminum hydroxide. Two weeks later, 2.5% ovalbumin was dropped into the eye for the antigen challenge. In the treatment group, 4 mg/kg (body weight) of the anti VLA-4 mAb (PS2.3), and in the control group, the same amount of a control Ab was intraperitoneally injected respectively at 4 hours before the antigen challenge. From both groups, the eyelids and eyeballs were excised at 2, 4, 10, and 24 hours after the antigen challenge, fixed, stained with Hansel solution and the number of the infiltrating eosinophils in the conjunctiva was counted. RESULTS: In the control group, infiltration of eosinophils to the conjunctiva increased with time, peaked at 12 hours, and then gradually decreased until 24 hours after the antigen challenge. In the anti VLA4-mAb treated group, eosinophil infiltration was almost completely inhibited at least until 24 hours after the antigen challenge. CONCLUSION: VLA-4 molecule was elucidated to play a critical roll in the eosinophil infiltration in experimentally-induced allergic conjunctivitis model of guinea pig.

Mast cells in pterygium: number and phenotype.

PURPOSE: To investigate the pathogenesis of pterygium. METHODS: The number and phenotype of mast cells were examined in excised tissue from 35 pterygia patients and compared with those in normal conjunctival specimens obtained during cataract or other intraocular surgery. RESULTS: Toluidine blue staining showed that the mean number of mast cells in the pterygia specimens was twice as high as that in the normal conjunctival tissues. Immunohistochemistry with a primary antibody to tryptase, specific for mast cells, also revealed a twofold increase in the mast cell number in the pterygia specimens compared with the normal conjunctival tissues. In the pterygia, more than 94% of the tryptase-positive mast cells were found to express chymase and c-kit. Almost all mast cells in the pterygia were tryptase-positive, chymase-positive mast cells (MC(TC)S). There was no phenotypic difference between the mast cells in the pterygia and those in the normal conjunctival tissues. CONCLUSIONS: The MC(TC)S appear not to be immune system-related and to have functions in angiogenesis and tissue remodeling. The increase in the number of mast cells caused by nonallergic stimulation may contribute to the pathogenesis of pterygium.

[Inhibitory effects of matrix metalloproteinase inhibitor on corneal neovascularization].

We evaluated the inhibitory effects of matrix metalloproteinase inhibitor (MMPI) on corneal neovascularization in vivo. MMPI drops in the right eye and MMPI basic drops in the left eye were administrated six times per day after wearing a large polymethylmethacrylate-hard contact lens (PMMA-HCL) and intralamellar transplantation of a basic fibroblast growth factor (basic-FGF) containing polymer disk. We observed the status of corneal neovascularization with a slit lamp biomicroscopically after two weeks. Corneal buttons were also investigated histologically with light and electron microscopes. The findings of both investigations showed MMPI eye drops tended to inhibit the corneal neovascularization.

[Pterygium and mast cells--mast cell number, phenotype, and localization of stem cell factor].

We examined the number and phenotype of mast cells, and the localization of stem cell factor (SCF) as a growth factor of mast cells in the excised tissue of 38 cases of pterygium. In histopathology with toluidine blue stain and immunohistochemistry with a monoclonal antibody to tryptase, the mean mast cell count in pterygium specimens was twice as high as in normal conjunctiva. In pterygium specimens more than 94% of tryptase-positive mast cells were found to express chymase and c-kit. There was no phenotypic difference between mast cells in pterygium and normal conjunctiva. In all immunohistochemical specimens in which we could examine the head of the pterygium, SCF was expressed in subepithelial fibroblasts at the central edge of pterygium. The results suggest that overexpression of SCF was accompanied with the augmentation of mast cells in the pterygium.