interleukin-8 and Keratitis

Notable among the many communicable agents known to infect the human cornea is the human adenovirus, with less than ten adenoviruses having corneal tropism out of more than 100 known types. The syndrome of epidemic keratoconjunctivitis (EKC), caused principally by human adenovirus, presents acutely with epithelial keratitis, and later with stromal keratitis that can be chronic and recurrent. In this review, we discuss the current state of knowledge regarding the molecular biology of adenovirus infection of corneal stromal cells, among which the fibroblast-like keratocyte is the most predominant, in order to elucidate basic pathophysiologic mechanisms of stromal keratitis in the human patient with EKC.

Topics: Adenoviruses, Human; Animals; Cornea; Host Microbial Interactions; Humans; Interleukin-8; Keratitis; Keratoconjunctivitis; Organogenesis; Stromal Cells

Therapeutic management of inflammation in infectious keratitis (IK) requires new strategy and targets for selective immunomodulation. Targeting host cell-type specific inflammatory responses might be a viable strategy to curtail unnecessary inflammation and reduce tissue damage without affecting pathogen clearance. This study explores the possibility of pathogen and host cell-type dependent differences in the inflammatory pathways relevant in the pathogenesis of IK. Human corneal epithelial cell line (HCEC) and phorbol 12-myristate-13 acetate (PMA) differentiated THP-1 macrophage line were infected with either Aspergillus flavus conidia or Acanthamoeba castellanii trophozoites and the elicited inflammatory responses were studied in terms of gene expression and secretion of proinflammatory factors interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α) and an upstream inflammatory regulator and mediator protein-the Macrophage Migration Inhibitory Factor (MIF). Given the pleotropic mode of MIF function in diverse cell types relevant in many human diseases, we tested if MIF driven responses to infection is different in HCECs and THP-1 macrophages by studying its expression, secretion and involvement in inflammation by siRNA mediated knockdown. We also examined IK patient tear samples for MIF levels. Infection with A. flavus or A. castellanii induced IL-8 and TNF-α responses in HCECs and THP-1 macrophages but to different levels. Our preliminary human data showed that the level of secreted MIF protein was elevated in IK patient tear, however, MIF secretion by the two cell types were strikingly different in-vitro, under both normal and infected conditions. We found that HCECs released MIF constitutively, which was significantly inhibited with infection, whereas THP-1 macrophages were stimulated to release MIF during infection. MIF gene expression remained largely unaffected by infection in both the cell lines. Although MIF in HCECs appeared to be intracellularly captured during infection, MIF knockdown in HCECs associated with a partial reduction of the IL-8 and TNF-α expression produced by either of the pathogens, suggesting a pro-inflammatory role for MIF in HCECs, independent of its canonical cytokine like function. In contrast, MIF knockdown in THP-1 macrophages accompanied a dramatic increase in IL-8 and TNF-α expression during A. castellanii infection, while the responses to A. flavus infection remained unchanged. These data imply a host cell-type a

Topics: Humans; Immunomodulation; Inflammation; Interleukin-8; Intramolecular Oxidoreductases; Keratitis; Macrophage Migration-Inhibitory Factors; Tumor Necrosis Factor-alpha

Dimethyl itaconate (DI) is a membrane-permeable itaconate derivative with anti-inflammatory functions. However, the anti-inflammatory effect of DI has never been studied in fungal keratitis. In this study, we tested the protective effect of DI against fungal keratitis and assessed the role of NF-E2-related factor-2 (Nrf2)/heme oxygenase-1 (HO-1) signaling in this process. Eyes of C57BL/6 (B6) mice were treated with 2 mm DI after infection with Aspergillus fumigatus. Human corneal epithelial cells (HCECs) were pretreated with 0.25 mm DI and then incubated with A. fumigatus. Clinical scoring, slit-lamp photography, myeloperoxidase determination, flow cytometry and immunostaining were used to assess the disease response and treatment efficacy. PCR, Western blot and ELISA were used to assess the expression of interleukin-1β (IL-1β), chemokine (C-X-C motif) ligand 1, IL-6, IL-8, Nrf2 and HO-1. In addition, quantification of viable fungi, absorbance assays and fluorimetry were used to measure DI fungistatic activity. We observed that DI-treated eyes showed decreased clinical scores, fungal loads, polymorphonuclear neutrophil (PMN) infiltration and cytokine expression, compared with phosphate-buffered saline-treated infected eyes. DI treatment decreased the cytokine levels in infected corneas and in HCECs stimulated with A. fumigatus. Moreover, DI treatment increased Nrf2 and HO-1 expression in corneas and nuclear Nrf2 accumulation in HCECs. DI-induced cytokine downregulation was inhibited by pretreatment with an Nrf2 or HO-1 inhibitor. Finally, DI treatment reduced the A. fumigatus absorbance and fungal mass. These data indicate that DI protects against fungal keratitis by limiting inflammation via the Nrf2/HO-1 signaling pathway and that DI inhibits the growth of A. fumigatus.

Topics: Animals; Aspergillosis; Aspergillus fumigatus; Chemokine CXCL1; Cornea; Epithelial Cells; Epithelium, Corneal; Heme Oxygenase-1; Humans; Inflammation; Interleukin-6; Interleukin-8; Keratitis; Mice; NF-E2-Related Factor 2; Signal Transduction; Succinates

Lewisite (LEW), a potent arsenical vesicating chemical warfare agent, poses a continuous risk of accidental exposure in addition to its feared use as a terrorist weapon. Ocular tissue is exquisitely sensitive to LEW and exposure can cause devastating corneal lesions. However, detailed pathogenesis of corneal injury and related mechanisms from LEW exposure that could help identify targeted therapies are not available. Using an established consistent and efficient exposure system, we evaluated the pathophysiology of the corneal injury in New Zealand white rabbits following LEW vapor exposure (at 0.2 mg/L dose) for 2.5 and 7.5 min, for up to 28 day post-exposure. LEW led to an increase in total corneal thickness starting at day 1 post-exposure and epithelial degradation starting at day 3 post-exposure, with maximal effect at day 7 postexposure followed by recovery at later time points. LEW also led to an increase in the number of blood vessels and inflammatory cells but a decrease in keratocytes with optimal effects at day 7 postexposure. A significant increase in epithelial-stromal separation was observed at days 7 and 14 post 7.5 min LEW exposure. LEW also caused an increase in the expression levels of cyclooxygenase-2, IL-8, vascular endothelial growth factor, and matrix metalloproteinase-9 at all the study time points indicating their involvement in LEW-induced inflammation, vesication, and neovascularization. The outcomes here provide valuable LEW-induced corneal injury endpoints at both lower and higher exposure durations in a relevant model system, which will be helpful to identify and screen therapies against LEW-induced corneal injury.

Topics: Animals; Arsenicals; Blister; Blood Vessels; Chemical Warfare Agents; Cornea; Corneal Keratocytes; Corneal Neovascularization; Corneal Pachymetry; Corneal Stroma; Cyclooxygenase 2; Epithelium, Corneal; Interleukin-8; Keratitis; Matrix Metalloproteinase 9; Rabbits; Risk Assessment; Time Factors; Vascular Endothelial Growth Factor A

Our previous studies show that human corneal epithelial cells (HCEC) have a functional vitamin D receptor (VDR) and respond to vitamin D by dampening TLR-induced inflammation. Here, we further examined the timing of the cytokine response to combined vitamin D-TLR treatment and used genome-wide microarray analysis to examine the effect of vitamin D on corneal gene expression.. Telomerase-immortalized HCEC (hTCEpi) were stimulated with polyinosinic-polycytidylic acid (poly[I:C]) and 1,25-dihydroxyvitamin D3 (1,25D3) for 2 to 24 hours and interleukin (IL)-8 expression was examined by quantitative (q)PCR and ELISA. Telomerase-immortalized HCEC and SV40-HCEC were treated with 1,25D3 and used in genome-wide microarray analysis. Expression of target genes was validated using qPCR in both cell lines and primary HCEC. For confirmation of IκBα protein, hTCEpi were treated with 1,25D3 for 24 hours and cell lysates used in an ELISA.. Treatment with 1,25D3 increased poly(I:C)-induced IL-8 mRNA and protein expression after 2 to 6 hours. However, when cells were pretreated with 1,25D3 for 24 hours, 1,25D3 decreased cytokine expression. For microarray analysis, 308 genes were differentially expressed by 1,25D3 treatment in hTCEpi, and 69 genes in SV40s. Quantitative (q)PCR confirmed the vitamin D-mediated upregulation of target genes, including nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α (IκBα). In addition to increased transcript levels, IκBα protein was increased by 28% following 24 hours of vitamin D treatment.. Microarray analysis demonstrates that vitamin D regulates numerous genes in HCEC and influences TLR signaling through upregulation of IκBα. These findings are important in dissecting the role of vitamin D at the ocular surface and highlight the need for further research into the functions of vitamin D and its influence on corneal gene expression.

Topics: Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Epithelium, Corneal; Gene Expression Regulation; Humans; Interleukin-8; Keratitis; Real-Time Polymerase Chain Reaction; Receptors, Calcitriol; RNA, Messenger; Signal Transduction; Tissue Array Analysis; Transcription, Genetic; Vitamin D; Vitamins

The Toll-Like Receptor 2 (TLR2) plays an active and important role in Staphylococcus aureus-induced chronic ocular inflammation. The aim of this study was to investigate the expression and function of TLR2 of corneal stromal cells in ex vivo rabbit model of S. aureus keratitis. Corneal buttons with sclera rims placed in an ex vivo air-interface organ culture were assigned to two groups: corneas with epithelial and stromal abrasions. Each group was then divided into two sub-groups exposed to UV-killed S. aureus ATCC 6538P and S. aureus ATCC 29213, respectively. TLR2 and IL-8 mRNA expressions were analyzed by quantitative real-time RT-PCR. TLR2 localization was visualized by immunofluorescence analysis. The results demonstrated that TLR2 and IL-8 mRNA were significantly expressed in the stromal cells of the groups exposed to S. aureus strains. Moreover, it has been demonstrated that, after corneal injury, keratocytes differentiated into myofibroblasts became able to express TLR2 only when exposed to S. aureus. Identification of mechanisms regulation of corneal TLRs may lead to development of therapeutic interventions aimed at controlling corneal inflammation. This ex vivo model can be used to clarify the molecular events of bacterial-corneal tissue interactions and their inflammatory consequences.

Topics: Animals; Cell Differentiation; Corneal Keratocytes; Corneal Stroma; Enzyme Activation; Inflammation; Interleukin-8; Keratitis; Myofibroblasts; Organ Culture Techniques; Rabbits; RNA, Messenger; Staphylococcal Infections; Staphylococcus aureus; Stromal Cells; Toll-Like Receptor 2

Corneal infections are frequent and potentially vision-threatening diseases, and despite the significance of the immunological response in animal models of microbial keratitis (MK), it remains unclear in humans. The aim of this study was to describe the cytokine profile of tears in patients with MK. Characteristics of ocular lesions such as size of the epithelial defect, stromal infiltration, and hypopyon were analyzed. Immunological evaluation included determination of interleukine (IL)-1β, IL-6, IL-8, IL-10, IL-12 and tumor necrosis factor (TNF)-α in tear samples obtained from infected eyes of 28 patients with MK and compared with their contralateral non-infected eyes. Additionally, frequency of CD4+, CD8+, CD19+ and CD3-CD56+ cells was also determined in peripheral blood mononuclear cells in patients with MK, and compared with 48 healthy controls. Non-significant differences were observed in the size of the epithelial defect, stromal infiltration, and hypopyon. Nevertheless, we found an immunological profile apparently related to MK etiology. IL-8 > IL-6 in patients with bacterial keratitis; IL-8 > IL-6 > IL-1β and increased frequency of circulating CD3-CD56+ NK cells in patients with gram-negative keratitis; and IL-8 = IL-6 > IL-1β in patients with fungal keratitis. Characterization of tear cytokines from patients with MK could aid our understanding of the immune pathophysiological mechanisms underlying corneal damage in humans.

Topics: Adult; Aged; Aged, 80 and over; Antigens, CD; Female; Fungi; Gene Expression Regulation; Gram-Negative Bacteria; Humans; Interleukin-1beta; Interleukin-6; Interleukin-8; Keratitis; Killer Cells, Natural; Leukocytes, Mononuclear; Male; Middle Aged; Tears; Tumor Necrosis Factor-alpha

Pseudomonas aeruginosa (P. aeruginosa) microbial keratitis (MK) is a sight-threatening disease. Previous animal studies have identified an important contribution of the complement system to the clearance of P. aeruginosa infection of the cornea. Mannose-binding lectin (MBL), a pattern recognition receptor of the lectin pathway of complement, has been implicated in the host defense against P. aeruginosa. However, studies addressing the role of the lectin pathway in P. aeruginosa MK are lacking. Hence, we sought to determine the activity of the lectin pathway in human MK caused by P. aeruginosa.. Primary human corneal epithelial cells (HCECs) from cadaveric donors were exposed to two different P. aeruginosa strains. Gene expression of interleukin (IL)-6, IL-8, MBL, and other complement proteins was determined by reverse transcription-polymerase chain reaction (RT-PCR) and MBL synthesis by enzyme-linked immunosorbent assay and intracellular flow cytometry.. MBL gene expression was not detected in unchallenged HCECs. Exposure of HCECs to P. aeruginosa resulted in rapid induction of the transcriptional expression of MBL, IL-6, and IL-8. In addition, expression of several complement proteins of the classical and lectin pathways, but not the alternative pathway, were upregulated after 5 h of challenge, including MBL-associated serine protease 1. However, MBL protein secretion was not detectable 18 h after challenge with P. aeruginosa.. MK due to P. aeruginosa triggers activation of MBL and the lectin pathway of complement. However, the physiologic relevance of this finding is unclear, as corresponding MBL oligomer production was not observed.

Topics: Complement Activation; Complement System Proteins; Epithelial Cells; Epithelium, Corneal; Gene Expression Regulation; Hep G2 Cells; Humans; Interleukin-6; Interleukin-8; Keratitis; Mannose-Binding Lectin; Pseudomonas aeruginosa; Pseudomonas Infections

Free-living amoebae of the Acanthamoeba species are the causative agent of Acanthamoeba keratitis (AK), a sight-threatening corneal infection that causes severe pain and a characteristic ring-shaped corneal infiltrate. Innate immune responses play an important role in resistance against AK. The aim of this study is to determine if Toll-like receptors (TLRs) on corneal epithelial cells are activated by Acanthamoeba, leading to initiation of inflammatory responses in the cornea. Human corneal epithelial (HCE) cells constitutively expressed TLR1, TLR2, TLR3, TLR4, and TLR9 mRNA, and A. castellanii upregulated TLR4 transcription. Expression of TLR1, TLR2, TLR3, and TLR9 was unchanged when HCE cells were exposed to A. castellanii. IL-8 mRNA expression was upregulated in HCE cells exposed to A. castellanii. A. castellanii and lipopolysaccharide (LPS) induced significant IL-8 production by HCE cells as measured by ELISA. The percentage of total cells positive for TLR4 was higher in A. castellanii stimulated HCE cells compared to unstimulated HCE cells. A. castellanii induced upregulation of IL-8 in TLR4 expressing human embryonic kidney (HEK)-293 cells, but not TLR3 expressing HEK-293 cells. TLR4 neutralizing antibody inhibited A. castellanii-induced IL-8 by HCE and HEK-293 cells. Clinical strains but not soil strains of Acanthamoeba activated TLR4 expression in Chinese hamster corneas in vivo and in vitro. Clinical isolates but not soil isolates of Acanthamoeba induced significant (P< 0.05) CXCL2 production in Chinese hamster corneas 3 and 7 days after infection, which coincided with increased inflammatory cells in the corneas. Results suggest that pathogenic species of Acanthamoeba activate TLR4 and induce production of CXCL2 in the Chinese hamster model of AK. TLR4 may be a potential target in the development of novel treatment strategies in Acanthamoeba and other microbial infections that activate TLR4 in corneal cells.

Topics: Acanthamoeba; Animals; Cornea; Cricetinae; Cricetulus; Epithelial Cells; HEK293 Cells; Humans; Inflammation; Interleukin-8; Keratitis; Protein Binding; Protein Transport; Species Specificity; Toll-Like Receptor 4; Trophozoites; Up-Regulation

The aim of this study was to evaluate the role of trans-resveratrol on Staphylococcus aureus-induced keratitis. Rabbit corneas (intact corneas, abraded corneas and abraded corneas exposed to inactivated S. aureus strains) were placed in an ex vivo culture model. The abraded corneas exposed to S. aureus were divided into two 1-h-treatment sub-groups: corneas treated with trans-resveratrol and corneas treated with vehicle. The tissues were examined by immunohistochemical analyses and quantitative real-time RT-PCR to determine whether resveratrol could reduce TLR2-mediated recognition of S. aureus on epithelial cells and, if so, whether this reduction repressed the expression of inflammatory cytokines. The results demonstrated that resveratrol treatment effectively downregulated cell surface TLR2 on cells stimulated by S. aureus and reduced the expression of interleukin-8 gene. In addition, the corneal culture model tested, which is simple and reproducible, could be an alternative to in vivo animal testing for the development of novel specific therapies.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Cornea; Disease Models, Animal; Gene Expression Profiling; Immunohistochemistry; Interleukin-8; Keratitis; Rabbits; Real-Time Polymerase Chain Reaction; Resveratrol; Staphylococcal Infections; Stilbenes; Toll-Like Receptor 2; Treatment Outcome

Keratitis caused by Pseudomonas aeruginosa is a potentially vision-threatening condition that requires prompt treatment to prevent vision loss. The recognition of infectious agents by the Toll-like receptor (TLR) system initiates primary innate and later adaptive immune responses. In this study, in late cases of corneal P. aeruginosa infection, the expression of TLR2, 4, 5 and 9 mRNA were all upregulated. In early infection cases, only TLR9 mRNA expression was upregulated. In late cases, the protein expression of TLR2, 4, 5, 9 and pIkappaB-alpha were elevated. In early cases, only TLR9 and pIkappaB-alpha expression were upregulated. Concentrations of IL-6 and IL-8 increased in infected corneas, especially in late cases. Myeloperoxidase (MPO) activity suggested that polymorphonuclear leukocyte (PMN) numbers were higher in late than in early stages of infection. The delayed response of TLRs may explain why P. aeruginosa infection exacerbates rapidly at the early infection stage. This finding may have important implications for the treatment of innate immunologic responses to corneal infections.

Topics: Adult; Cornea; Female; Gene Expression Profiling; Humans; Interleukin-6; Interleukin-8; Keratitis; Male; Middle Aged; Neutrophils; Peroxidase; Pseudomonas aeruginosa; Pseudomonas Infections; Time Factors; Toll-Like Receptors; Up-Regulation; Young Adult

The purpose of this study was to consider the use of a hydroxyapatite (HA) coated porous carbon matrix as a synthetic dental laminate substitute in osteo-odonto-keratoprosthetic (OOKP) design. 3 types of carbon meshes were coated with HA by sonoelectrochemical deposition. The materials were characterised by scanning electron microscopy (SEM) and HA deposition was characterised by elemental analysis and X-ray diffractometry (XRD). In vitro assays were carried out to quantify the effects of HA coating on human keratocyte adhesion. Cellular cytokine production was used to assess inflammatory potential. HA coating significantly increased keratocyte adhesion to the carbon matrix (p<0.01). The materials did not induce excessive cytokine production by the adherent keratocytes. In addition, the matrices themselves adsorbed significant levels of the cytokine IL-8 (p<0.05). The results indicate that HA coated carbon matrices provide a suitable environment to enhance in-growth of corneal cells without inducing further inflammation. The materials may also suppress excessive inflammation by adsorption of the cytokine IL-8 into the porous, internal carbon structure.

Topics: Adsorption; Carbon; Cells, Cultured; Coated Materials, Biocompatible; Cornea; Corneal Opacity; Durapatite; Electron Probe Microanalysis; Humans; Implants, Experimental; Interleukin-6; Interleukin-8; Keratitis; Materials Testing; Microscopy, Electron, Scanning; Surgical Mesh; X-Ray Diffraction

Corneal inflammation associated with ocular adenoviral infection is caused by leukocytic infiltration of the subepithelial stroma in response to expression of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) by infected corneal cells. We have shown that these two chemokines are activated by the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase (ERK) and p38 for IL-8, and Jun-terminal kinase (JNK) for MCP-1. It is also well established that transcription of each of these chemokines is tightly controlled by the nuclear factor kappa B (NFkappaB) transcription factor family. Therefore, we sought to better understand the differential regulation of chemokine expression by NFkappaB in adenoviral infection of the cornea.. Primary keratocytes derived from human donor corneas were treated with signaling inhibitors and small interfering RNA specific to MAPKs, and infected with adenovirus for different time periods before analysis. Activation of specific NFkappaB subunits was analyzed by western blot, confocal microscopy, electromobility shift assay, and chromatin immunoprecipitation, and chemokine expression was quantified by enzyme-linked immunosorbent assay.. Upon adenoviral infection, NFkappaB p65, p50, and cREL subunits translocate to the nucleus. This translocation is blocked by inhibitors of specific MAPK signaling pathways. Confocal microscopy showed that inhibitors of the p38, JNK, and ERK pathways differentially inhibited NFkappaB nuclear translocation, while PP2, an inhibitor of Src family kinases, completely inhibited NFkappaB nuclear translocation. Western blot analysis revealed that activation of specific NFkappaB subunits was time dependent following infection. Chromatin immunoprecipitation experiments indicated that binding of NFkappaB p65 and p50 subunits to the IL-8 promoter upon viral infection was differentially reduced by chemical inhibitors of MAPKs. Electromobility shift assay and luciferase assay analysis revealed that transactivation of IL-8 occurred with binding by the NFkappaB p65 homodimer or NFkappaB p65/p50 heterodimer as early as 1 h post infection, whereas MCP-1 expression was dependent upon the NFkappaB cREL but not the p65 subunit, and occurred 4 h after IL-8 induction. Finally, knockdown of NFkappaB p65 by short interfering RNA abrogated IL-8 but not MCP-1 expression after adenoviral infection.. The kinetics of NFkappaB subunit activation are partly responsible for the observed pattern of acute inflammation in the adenoviral-infected cornea. MAPKs differentially regulate chemokine expression in adenoviral keratitis by differential and time-dependent activation of specific NFkappaB subunits.

Topics: Adenoviridae; Adenoviridae Infections; Cell Nucleus; Chemokine CCL2; Chromatin Immunoprecipitation; Enzyme Activation; Humans; I-kappa B Kinase; Interleukin-8; Keratitis; Kinetics; NF-kappa B; Promoter Regions, Genetic; Protein Binding; Protein Subunits; Protein Transport; Time Factors; Transcription Factor RelA

In the present study, we examined the role of Staphylococcus aureus protein A (SpA) in inducing inflammatory response in human corneal epithelial cells (HCECs). Exposure of HCECs to SpA induces rapid NF-kappaB activation and secretion of proinflammatory cytokine/chemokines (TNF-alpha and IL-8) in both concentration and time-dependent manner. Challenge of HCECs with live SpA(-/-) mutant S. aureus strains resulted in significantly reduced production of the cytokines when compared to the wild-type S. aureus strain. SpA also elicited the activation of MAP Kinases P38, ERK, but not JNK, in HCECs. SpA-induced production of proinflammatory cytokine were completely blocked by the NF-kappaB and p38 inhibitors and partially inhibited by the Jnk inhibitor. Pretreatment with anti-TLR2 neutralizing antibody had no effect on SpA-induced inflammatory response in HCECs, suggesting that this response is independent of TLR2 signaling. Moreover, unlike TLR2 ligands, SpA failed to induce the expression of antimicrobial peptides (hBD2 and LL-37) in HCECs. These studies indicate that SpA is a S. aureus virulence factor that stimulates HCEC inflammatory response through a pathway distinct from TLR2 in HCECs.

Topics: Enzyme Activation; Epithelium, Corneal; Interleukin-8; Keratitis; Mitogen-Activated Protein Kinases; NF-kappa B; Staphylococcal Protein A; Staphylococcus aureus; Toll-Like Receptor 2; Tumor Necrosis Factor-alpha

To investigate in vivo expression of chemokines in normal and inflamed human corneas, to determine whether chemokines are responsible for the recruitment of inflammatory cells.. In situ hybridization of the CXC chemokines growth-related oncogene-alpha (Gro-alpha) (CXCL-1), interleukin 8 (CXCL-8), macrophage interferon-gamma inducible gene (CXCL-9), and interferon-gamma inducible protein 10 (CXCL-10) and of the CC chemokines macrophage chemoattractant protein 1 (MCP-1) (CCL-2), macrophage inflammatory protein 1alpha (CCL-3), and regulated on activation, normal T-cell expressed and secreted (CCL-5) was performed to localize chemokine messenger RNA. Immunohistochemistry was used to identify the cellular infiltrate within the cornea. Three normal human eyes were compared with eyes enucleated because of chronic inflammation (n = 10), secondary to perforating injuries.. In normal corneas, no chemokine expression was detected. In inflamed lesions, a high intensity of signals from Gro-alpha (CXCL-1) and MCP-1 (CCL-2) messenger RNA was observed in limbal epithelium and from Gro-alpha (CXCL-1), interleukin 8 (CXCL-8), and MCP-1 (CCL-2) in corneal stroma. The Gro-alpha (CXCL-1) was the only chemokine expressed by central corneal epithelium. All other examined chemokines were only moderately expressed in limbus and corneal stroma, or barely detectable.. These cytokines are important agents in the cytokine network and contribute to the cell-specific and spatially restricted recruitment of neutrophils and mononuclear cells in acute inflammatory lesions of the human cornea. Clinical Relevance Understanding the role of chemokines in corneal inflammation may lead to the development of a selective receptor blockage of highly expressed chemokines to inhibit the recruitment of leukocyte subsets.

Topics: Antibodies, Monoclonal; Chemokine CCL2; Chemokine CCL4; Chemokine CXCL1; Chemokine CXCL10; Chemokine CXCL9; Chemokines; Chemokines, CXC; Chemotactic Factors; Cornea; Eye Injuries, Penetrating; Humans; Immunoenzyme Techniques; In Situ Hybridization; Inflammation; Intercellular Signaling Peptides and Proteins; Interleukin-8; Keratitis; Langerhans Cells; Macrophage Inflammatory Proteins; Macrophages; Neutrophils; RNA, Messenger; T-Lymphocytes

To investigate the cell populations in diffuse lamellar keratitis (DLK) infiltration after laser in situ keratomileusis and the possible mechanism underlying the infiltration.. Department of Ophthalmology, Tokyo Dental College, Chiba, Japan.. To develop DLK in rabbit eyes, 25 microL of lipopolysaccharide (LPS) solution at a concentration of 50 microg/mL was applied to the stromal bed beneath corneal flaps. For control rabbits, phosphate-buffered saline was applied. Postoperative examination by slitlamp microscopy was performed for 3 days after surgery. Rabbit eyes were excised and examined for histopathology with hematoxylin and eosin staining. Immunohistochemical analysis for interleukin (IL)-8 was performed.. Diffuse lamellar keratitis-like inflammation composed mainly of neutrophils was reproduced by LPS instillation in rabbit eyes. In eyes with severe inflammation, IL-8 immunoreactivity was found in the stromal keratocytes and infiltrating neutrophils.. The major cell type in the DLK infiltration induced by LPS instillation in rabbit eyes was the neutrophil. Interleukin-8, a prototype of CXC chemokine produced by keratocytes and neutrophils, may contribute to the development of DLK.

Topics: Animals; Cell Movement; Corneal Stroma; Disease Models, Animal; Immunoenzyme Techniques; Interleukin-8; Keratitis; Keratomileusis, Laser In Situ; Lipopolysaccharides; Neutrophils; Rabbits; Salmonella typhimurium

Adenovirus type 19 (Ad19) infection of the human cornea results in a chronic, multifocal, subepithelial keratitis. Existing evidence suggests that early subepithelial corneal infiltrates are composed of polymorphonuclear neutrophils. In this study, the capacity of Ad19-infected human corneal stromal fibroblasts (HCFs) to produce neutrophil chemotactants (chemokines) was tested.. HCFs grown from human donor corneas and passaged thrice were infected with a corneal isolate of Ad19 or mock-infected with virus-free media. Bioactivity of the cell supernatants was tested by a neutrophil chemotaxis assay. Supernatants were assayed by enzyme-linked immunosorbent assay for the neutrophil chemotactants interleukin-8 (IL-8) and GRO-alpha. Corneal facsimiles were generated with HCFs and collagen type I, infected with Ad19, and assayed by immunohistochemistry.. Ad19 infection of HCFs increased neutrophil chemotaxis from a baseline of 0.4+/-0.7 cells/high-powered field (hpf; mock-infected) to 21.8+/-2.3 cells/hpf (Ad19-infected). Chemotaxis was reduced by the addition of neutralizing antibodies against IL-8 and GRO-alpha. Infection of HCFs induced quantities of IL-8 protein 300- and 1000-fold over mock-infected controls at 4 and 24 hours, respectively (33 versus 11,813 pg/mL at 4 hours, and 57 versus 76,376 pg/mL at 24 hours, P< or = 0.001 for both). In contrast, GRO-alpha protein levels were only sevenfold higher at 24 hours postinfection (118 pg/mL in mock-infected controls versus 880 pg/mL in Ad19-infected cell supernatants). Neither chemokine was induced by infection of an immortalized human corneal epithelial cell line. Immunohistochemistry of infected corneal facsimiles demonstrated IL-8 in the extracellular matrix within 3 days after infection.. Production of chemokines in infected tissues facilitates an early innate immune response to infection, and in the infected corneal stroma represents an elementary defense mechanism. Interleukin-8 may play a role in the development of subepithelial infiltrates in adenovirus keratitis.

Topics: Adenoviridae; Adenoviridae Infections; Cells, Cultured; Chemokine CXCL1; Chemokines, CXC; Chemotactic Factors; Chemotaxis, Leukocyte; Corneal Stroma; Enzyme-Linked Immunosorbent Assay; Eye Infections, Viral; Fibroblasts; Growth Substances; Humans; Immunoenzyme Techniques; Intercellular Signaling Peptides and Proteins; Interleukin-8; Keratitis; Neutrophils

Contact lens induced acute red eye (CLARE) and contact lens induced peripheral ulcer (CLPU) are among the most common contact lens induced inflammatory reactions. Both CLARE and CLPU are characterized by corneal infiltration which indicates the presence of chemoattractants and other inflammatory mediators. The aim of this study was to characterize the cytokine and chemotactic lipid inflammatory mediator profile in the tears of people experiencing CLARE or CLPU. Cytokines IL-1 beta, IL-6, IL-8, GM-CSF and LTB4 in tears were measured by antibody sandwich and competition inhibition enzyme-linked immunosorbent assays (ELISA). Platelet activating factor-like activity was measured by a degranulation assay by measuring the release of labelled serotonin from platelets. The functional role GM-CSF and chemoattractants were determined by flow cytometry and chemotaxis. Increased levels of cytokines and chemoattractants were detected in both CLARE and CLPU tears. CLPU tears showed increased levels of LTB4 (P = 0.002) and PAF-like activity (P = 0.047) whereas CLARE tears showed increased levels of GM-CSF (P = 0.002). IL-8 (P < 0.05). LTB4 (P = 0.002) and PAF-like activity (P = 0.047) compared to control tears. Flow cytometric analysis revealed that incubation of PMN with CLARE tears increased the number of IgA receptors indicating that the GM-CSF in CLARE tears was active. Combinations of suboptimal concentrations (which were found in CLARE and CLPU tears) of IL-8 with either LTB4 or PAF significantly (P < 0.0001) enhanced the chemotactic activity for PMN compared to their individual effects. Our data highlight the possible pathophysiological roles of these inflammatory mediators in leukocyte recruitment and activation during ocular inflammatory responses. The results suggests that GM-CSF, IL-8 and LTB4 are active during corneal pathology and LTB4 or IL-8 may maintain the contact lens induced PMN response in vivo.

Topics: Acute Disease; Analysis of Variance; Cells, Cultured; Contact Lenses; Corneal Ulcer; Cytokines; Dose-Response Relationship, Drug; Flow Cytometry; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Inflammation Mediators; Interleukin-8; Keratitis; Leukotriene B4; Lipids; Neutrophil Activation; Neutrophils; Platelet Activating Factor; Receptors, Fc; Tears