interleukin-8 and Corneal-Diseases

Dying cells release endogenous molecules known as alarmins that signal danger to surrounding tissue. We investigated the effects of necrotic cell-derived alarmins on cytokine expression and barrier function in human corneal epithelial cells.. The release of interleukin (IL)-6 and IL-8 from immortalized human corneal epithelial (HCE) cells in culture was measured with enzyme-linked immunosorbent assays. The abundance of IL-6 and 8 mRNAs was quantitated by reverse transcription and real-time polymerase chain reaction analysis. Barrier function of HCE cells was evaluated by measurement of transepithelial electrical resistance (TER). The subcellular localization of the p65 subunit of the transcription factor NF-κB was determined by immunofluorescence analysis, and phosphorylation of the endogenous NF-κB inhibitor IκBα was examined by immunoblot analysis.. A necrotic cell supernatant prepared from HCE cells induced the up-regulation of IL-6 and 8 expression at both mRNA and protein levels as well as reduced TER in intact HCE cells. Among alarmins tested, only IL-1α (not IL-33 or HMGB1) mimicked these effects of the necrotic cell supernatant. Furthermore, IL-1 receptor antagonist (IL-1RA) and neutralizing antibodies to IL-1α (but not those to IL-1β) each attenuated the effects of the necrotic cell supernatant. Exposure of HCE cells to the necrotic cell supernatant also induced the phosphorylation and degradation of IκBα as well as translocation of the p65 subunit of NF-κB to the nucleus.. IL-1α released from necrotic corneal epithelial cells may trigger inflammatory responses at the ocular surface, including cytokine production and barrier disruption.

Topics: Alarmins; Cells, Cultured; Corneal Diseases; Cytokines; Electric Impedance; Enzyme-Linked Immunosorbent Assay; Epithelium, Corneal; Gene Expression Regulation; Humans; Immunoblotting; Interleukin-1alpha; Interleukin-6; Interleukin-8; Necrosis; Reverse Transcriptase Polymerase Chain Reaction; RNA

Corneal limbal stem cell deficiency (LSCD) may be treated using ex vivo limbal epithelial stem cells (LESCs) derived from cadaveric donor tissue. However, continuing challenges exist around tissue availability, inflammation, and transplant rejection. Lipopolysaccharide (LPS) or recombinant human IL-1β stimulated primary human keratocyte and LESC models were used to investigate the anti-inflammatory properties of a short chain, IL-1 receptor antagonist peptide for use in LESC sheet growth to control inflammation. The peptide was characterized using mass spectroscopy and high performance liquid chromatography. Peptide cytotoxicity, patterns of cell cytokine expression in response to LPS or IL-1β stimulation, and peptide suppression of this response were investigated by MTS/LDH assays, ELISA, and q-PCR. Cell differences in LPS stimulated toll-like receptor 4 expression were investigated using immunocytochemistry. A significant reduction in rIL-1β stimulated inflammatory cytokine production occurred following LESC and keratocyte incubation with anti-inflammatory peptide and in LPS stimulated IL-6 and IL-8 production following keratocyte incubation with peptide (1 mg/mL) (P < 0.05). LESCs produced no cytokine response to LPS stimulation and showed no TLR4 expression. The peptide supported LESC growth when adhered to a silicone hydrogel contact lens indicating potential use in improved LESC grafting through suppression of inflammation.

Topics: Cadaver; Cell Proliferation; Cells, Cultured; Cornea; Corneal Diseases; Corneal Keratocytes; Epithelium, Corneal; Humans; Inflammation; Interleukin-1beta; Interleukin-8; Lipopolysaccharides; Peptides; Receptors, Interleukin-1; Recombinant Proteins; Stem Cells; Toll-Like Receptor 4

To compare the resolution of inflammation and long-term results of cultivated and conventional limbal stem cell transplantation (LSCT) in a patient with Stevens-Johnson syndrome (SJS).. Interventional case report.. A 32-year-old man with SJS and bilateral total limbal stem cell deficiency underwent cultivated LSCT in the right eye, followed by conventional LSCT in the left eye three weeks later. The postoperative medication included dexamethasone 0.1% and ofloxacin 0.3% eyedrops and a tapering dose of systemic corticosteroid, cyclosporine, and cyclophosphamide. Tear samples were collected and analyzed for interleukin (IL) 8 levels.. Complete corneal epithelialization was achieved 48 hours after cultivated LSCT, compared with three weeks after conventional LSCT. Ocular inflammation and IL-8 levels decreased more rapidly in the eye with cultivated LSCT. Four years after surgery, more severe corneal scarring and opacification were noted in the conventional LSCT eye.. Cultivated LSCT resulted in a better clinical result and vision, with less stromal scarring compared with conventional LSCT.

Topics: Adult; Adult Stem Cells; Cells, Cultured; Corneal Diseases; Epithelial Cells; Epithelium, Corneal; Humans; Interleukin-8; Limbus Corneae; Male; Stem Cell Transplantation; Stevens-Johnson Syndrome; Tears

To determine the levels of interleukin 6 (IL-6) and interleukin 8 (IL-8/CXCL-8) in tears collected from the eyes of normal individuals and of patients with different irritative eye diseases, in order to acquire information on the immunological changes occurring during the early postoperative period following various forms of eye surgery, including penetrating keratoplasty (PKP).. IL-6 and IL-8 levels were measured with the aid of human ultrasensitive ELISA kits in the non-stimulated tears of patients in the early postoperative period following PKP or cataract operation, and of patients with acute bacterial conjunctivitis or with a corneal foreign body. The IL-6 and IL-8 concentrations, the total amounts released in a given time and the rates of their release were calculated.. A significant increase in IL-6 release was observed in all patient groups compared with the normal controls (p < or = 0.003). The IL-8 release levels were significantly higher in the tears of all patient groups (p < or = 0.03), except for the cataract operation group, where the IL-8 release was not significantly higher (p = 0.053) than in the control samples. No significant differences in IL-6 or IL-8 release were observed when the various patient groups were compared with each other.. The release of IL-6 and IL-8 into the tears is enhanced in various anterior segment eye diseases, and this may be used as an indicator of various inflammatory reactions in the early postoperative period.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Anterior Eye Segment; Biomarkers; Cataract; Cataract Extraction; Conjunctivitis, Bacterial; Corneal Diseases; Enzyme-Linked Immunosorbent Assay; Follow-Up Studies; Humans; Interleukin-6; Interleukin-8; Middle Aged; Prognosis; Severity of Illness Index; Tears

The purpose of this study was to identify the growth factors and cytokines present in normal and diseased corneas. Total RNA was isolated from normal and diseased corneas. cDNA was synthesized from individual corneas and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed with primers to IL-1alpha, 1IL-8, PDGF-B, BMP-2, BMP-4, IGF-I, TGF-beta2, FGF-2, and VEGF. After normalization to beta2-microglobulin, several factors were identified that were significantly different from normal. Antibodies to IGF-I, BMP-2, VEGF and TGF-beta2 were used for immunohistochemistry. A total of 93 corneas were used for this study including 31 normal, 20 keratoconus, 19 bullous keratopathy (pseudophakic and aphakic, PBK/ABK), and 23 diabetic corneas. The VEGF RNA levels were significantly decreased in the keratoconus and PBK/ABK corneas but increased in the diabetic corneas. BMP-2 gene expression was lower than normal in the PBK/ABK and diabetic corneas. IGF-I and BMP-4 RNA levels were increased in PBK/ABK. In the immunohistochemical studies, the protein patterns paralleled those found at the mRNA level. The only exception was IGF-I in diabetic corneas that showed increased staining in the epithelium and its basement membrane without a significant increase in mRNA levels. TGF-beta2 mRNA and protein levels were similar to normal in all diseased corneas. Thus, no alterations in the tested growth factors/cytokines were unique to keratoconus corneas. In contrast, PBK/ABK corneas had specific significant elevations of BMP-4 and IGF-I. Diabetic corneas were unique in their increased VEGF mRNA levels. These data suggest that while some growth factor/cytokine alterations are non-specific and can be found in multiple corneal diseases, there are others that are unique to that disease.

Topics: Adult; Aged; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Case-Control Studies; Corneal Diseases; Cytokines; Diabetes Complications; Diabetes Mellitus; DNA, Complementary; Endothelial Growth Factors; Fibroblast Growth Factors; Gene Expression; Growth Substances; Humans; Insulin-Like Growth Factor I; Interleukin-1; Interleukin-8; Keratoconus; Platelet-Derived Growth Factor; Reverse Transcriptase Polymerase Chain Reaction; RNA; Transforming Growth Factor beta

To correlate tear fluorescein clearance with interleukin-1alpha (IL-1alpha) concentration and gelatinase B (matrix metalloproteinase [MMP]-9) activity in the tear fluid of patients with ocular rosacea and normal control subjects.. Gelatinase activity was evaluated by gelatin zymography in tear fluid obtained from 13 patients with ocular rosacea (including 1 patient with recurrent epithelial erosion, 2 with recurrent peripheral corneal infiltrates and vascularization, and 2 patients with epithelial basement membrane dystrophy) and 13 normal subjects with normal aqueous tear production and no irritation symptoms. Tear fluorescein clearance was evaluated by measuring fluorescence in tear fluid collected from the inferior meniscus 15 minutes after instillation of 5 microl of 2% Na-fluorescein with a CytoFluor II fluorometer. Pro-MMP-9 and IL-1alpha concentrations in the tear fluid were measured by enzyme-linked immunosorbent assay (ELISA).. Compared with normal control subjects, patients with ocular rosacea had a greater delay of tear fluorescein clearance (P < 0.001), a higher tear IL-1alpha concentration (P < 0.001), and a greater pro-gelatinase B (92 kDa) activity (P < 0.001) in their tear fluid. The 84-kDa active form of gelatinase B was observed in 46% of the rosacea tear samples and none of the controls. The zymographic results were confirmed by ELISA that showed a significantly greater concentration of pro-MMP-9 (92 kDa) in the tear fluid of rosacea patients than controls. Delayed tear clearance was correlated with elevated tear IL-1alpha concentration (p=0.67, P < 0.001) and increased tear gelatinase B activity (p=0.84, P < 0.001). Tear IL-1alpha concentration was correlated with tear gelatinase B activity (p=0.58, P < 0.002).. Gelatinase B (MMP-9) activity is greater in patients with ocular rosacea than in normal eyes. The majority of this activity is due to 92-kDa proform of this enzyme. This activity is correlated with delayed tear clearance and tear fluid concentration of interleukin-1alpha, a proinflammatory cytokine that has been reported to stimulate gelatinase B production. Elevated gelatinase B activity in ocular rosacea may be involved in the pathogenesis of the irritation symptoms, recurrent epithelial erosions, vascularization, and epithelial basement membrane dystrophy that develops in the corneas of patients with this condition.

Topics: Adult; Aged; Aged, 80 and over; Collagenases; Conjunctival Diseases; Corneal Diseases; Enzyme-Linked Immunosorbent Assay; Female; Fluorescein; Fluorophotometry; Humans; Interleukin-8; Male; Matrix Metalloproteinase 9; Middle Aged; Rosacea; Tears