Chemokines in allergic conjunctivitis.

PURPOSE OF REVIEW: Chemokines are a large group of low molecular weight cytokines that attract and activate leukocytes throughout the body and therefore have a key role in the framework of late-phase allergic responses. The purpose of this article is to provide an overview of the main chemokines involved in allergic conjunctivitis, their primary functions and their physiological roles, and therapies targeted at chemokines and their receptors for ocular allergic diseases. RECENT FINDINGS: In recent years, there have been considerable advances in the understanding of ocular pathophysiology of ocular surface inflammatory diseases including both allergic eye diseases and dry eye syndrome. Several therapies being developed for dry eye inflammation are recognized as possible therapies for ocular allergic diseases as there are often common chemokines involved in both disease spectra. SUMMARY: Chemokines represent an integral part of the late-phase cascade of ocular allergic inflammation. A deep understanding of specific chemokines and their interactions will help in targeting therapies to effectively manage ocular clinical findings and symptoms of allergic eye disease.

Laser Scanning In Vivo Confocal Microscopy of Clear Grafts after Penetrating Keratoplasty.

PURPOSE: To evaluate the changes of keratocytes and dendritic cells in the central clear graft by laser scanning in vivo confocal microscopy after penetrating keratoplasty (PK). METHODS: Thirty adult subjects receiving PK at Shandong Eye Institute and with clear grafts and no sign of immune rejection after surgery were recruited into this study, and 10 healthy adults were controls. The keratocytes and dendritic cells in the central graft were evaluated by laser scanning confocal microscopy, as well as epithelium cells, keratocytes, corneal endothelium cells, and corneal nerves (especially subepithelial plexus nerves). RESULTS: Median density of subepithelial plexus nerves, keratocyte density in each layer of the stroma, and density of corneal endothelium cells were all lower in clear grafts than in controls. The dendritic cells of five (16.7%) patients were active in Bowman's membrane and stromal membrane of the graft after PK. CONCLUSIONS: Activated dendritic cells and Langerhans cells could be detected in some of the clear grafts, which indicated that the subclinical stress of immune reaction took part in the chronic injury of the clear graft after PK, even when there was no clinical rejection episode.

Novel model of innate immunity in corneal infection.

The cornea functions as the major refractive interface for vision and protects the internal eye from insult. Current understanding of innate immune responses to corneal infection derives from a synthesis of in vitro and in vivo analyses. However, monolayer cell cultures and mouse models do not accurately duplicate all aspects of innate immunity in human patients. Here, we describe a three-dimensional culture system that incorporates human cells and extracellular matrix to more completely simulate the human cornea for studies of infection. Human corneal stromal fibroblasts were mixed with type I collagen in 3-µm pore size transwell inserts, and overlayed with Matrigel to simulate a human corneal stroma and epithelial basement membrane. These were then infected with a cornea-tropic adenovirus, and exposed on their inferior side to leukocytes derived from human peripheral blood. Subsequent analyses were performed with histology, confocal microscopy, ELISA, and fluorescence-activated cell sorting (FACS). CXCL8, a neutrophil chemokine shown previously as the first cytokine induced in infection of human corneal cells, increased upon adenovirus infection of facsimiles in a dose-responsive fashion. Myeloperoxidase-positive cells infiltrated infected corneal facsimiles in a sub-Matrigel location, possibly due to CXCL8 colocalization with heparan sulfate, a Matrigel constituent. Cellular infiltration was significantly inhibited by treatment with chemical inhibitors of p38 MAPK and Src kinase, both constituents of a signaling cascade previously suggested to regulate inflammation after adenovirus infection. FACS analysis determined that both virus and corneal fibroblasts were necessary for the induction of leukocyte migration into the facsimiles. The corneal facsimile, literally a cornea in a test tube, permits mechanistic studies on human tissue in a highly tractable system.

hCTLA4-Ig transgene expression in keratocytes modulates rejection of corneal xenografts in a pig to non-human primate anterior lamellar keratoplasty model.

BACKGROUND: Human corneal allografting is an established procedure to cure corneal blindness. However, a shortage of human donor corneas as well as compounding economic, cultural, and organizational reasons in many countries limit its widespread use. Artificial corneas as well as porcine corneal xenografts have been considered as possible alternatives. To date, all preclinical studies using de-cellularized pig corneas have shown encouraging graft survival results; however, relatively few studies have been conducted in pig to non-human primate (NHP) models, and particularly using genetically engineered donors. METHODS: In this study, we assessed the potential benefit of using either hCTLA4-Ig transgenic or α1,3-Galactosyl Transferase (GT) Knock-Out (KO) plus transgenic hCD39/hCD55/hCD59/fucosyl-transferase pig lines in an anterior lamellar keratoplasty pig to NHP model. RESULTS: Corneas from transgenic animals expressing hCTLA4-Ig under the transcriptional control of a neuron-specific enolase promoter showed transgene expression in corneal keratocytes of the stroma and expression was maintained after transplantation. Although a first acute rejection episode occurred in all animals during the second week post-keratoplasty, the median final rejection time was 70 days in the hCTLA4-Ig group vs. 21 days in the wild-type (WT) control group. In contrast, no benefit for corneal xenograft survival from the GTKO/transgenic pig line was found. At rejection, cell infiltration in hCTLA4Ig transgenic grafts was mainly composed of macrophages with fewer CD3+ CD4+ and CD79+ cells than in other types of grafts. Anti-donor xenoantibodies increased dramatically between days 9 and 14 post-surgery in all animals. CONCLUSIONS: Local expression of the hCTLA4-Ig transgene dampens rejection of xenogeneic corneal grafts in this pig-to-NHP lamellar keratoplasty model. The hCTLA4-Ig transgene seems to target T-cell responses without impacting humoral responses, the control of which would presumably require additional peripheral immunosuppression.

[CD34 and alpha-smooth muscle actin expression of keratocytes following photodynamic inactivation (PDI)]. / CD34 und Alpha-smooth-Muscle-Actin-Keratozyten-Expression nach photodynamischer Inaktivierung (PDI).

PURPOSE: Photodynamic inactivation (PDI) may be a potential treatment alternative in therapy-resistant infectious keratitis. PDI may eliminate the microorganisms from the infected cornea by damage caused through free oxygen radicals, or even by supporting different stages of activation of keratocytes and inflammatory cell response. The purpose of this study was to determine the impact of PDI on activation of human keratocytes in culture. METHODS: Primary human keratocytes were isolated by digestion in collagenase A (1 mg/mL) from human corneal buttons, and cultured in DMEM/Ham's culture medium supplemented with 10% foetal calf serum. Keratocytes underwent illumination (670 nm) for 13 minutes following exposure to 0, 50, 150 and 250 nMol/ml concentrations of the photosensitizer chlorin e6 (Ce6) in the culture medium. Twenty-four hours after treatment CD34 and α-smooth-muscle actin expression of the cells was analysed using flow-cytometry (FACS). RESULTS: Using Ce6 or illumination only, α-smooth-muscle actin expression of the cells did not change significantly. Twenty-four hours after PDI the percentage of CD34-positive keratocytes did not change significantly using 50-250 nM Ce6, however, the percentage of α-smooth-muscle actin-positive keratocytes decreased significantly at 250 nM Ce6 (p = 0.01). CONCLUSIONS: As a short-term effect, PDI seems to inhibit myofibroblastic transformation of keratocytes, but does not have an impact on activation of CD34-positive keratocytes.With this impact PDI possibly may reduce the antimicrobial defence of keratocytes.

Corneal cell response after flap creation using a mechanical microkeratome or a 200 kHz femtosecond laser.

PURPOSE: To compare the inflammatory cell response within the corneal flap interface created by a mechanical microkeratome and a femtosecond laser. SETTING: Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany. DESIGN: Experimental in vitro study. METHODS: Corneoscleral buttons of 12 enucleated human eyes not suitable for transplantation were put into organ culture. Corneal flaps were created using a 200 kHz femtosecond laser (Visumax) (femtosecond group) or a mechanical microkeratome (Amadeus) (microkeratome group). Flaps were not lifted after treatment. In 2 corneas, no treatment was performed (control group). Corneas were kept in organ culture for 12 hours thereafter. To evaluate cell-mediated immune reaction, immunofluorescent staining for leucocytes (cluster of differentiation 45) and specifically for dendritic cells (human leukocyte antigen-DR) was performed in every group. A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was used to determine apoptosis reaction. RESULTS: The ratio of dendritic cells in the femtosecond group compared with the microkeratome group was 1.2 (P=.02), the ratio of leucocytes was 1.4 (P=.06), and the ratio of apoptotic cells was 1.0 (P=.59). There was no marked significant difference in the distribution of inflammatory cell reaction. The control group showed neither specific inflammatory reaction nor apoptosis. CONCLUSION: This in vitro series of human corneas showed similar inflammatory tissue reaction after femtosecond laser-assisted and microkeratome-assisted flap creation (P<.05). FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Pretreatment with TLR2 and TLR4 ligand modulates innate immunity in corneal fibroblasts challenged with Aspergillus fumigatus.

PURPOSE: To study the innate immunity in telomerase-immortalized human stroma fibroblasts (THSFs) challenged with Aspergillus fumigatus hyphae after copretreatment with TLR2 and TLR4 ligand. METHODS: THSFs were pretreated with different concentrations of zymosan and/or lipopolysaccharide (LPS) at different time periods, and challenged with high-dose Aspergillus fumigatus hyphae. The gene expression and protein secretion of inflammatory cytokines (TNF-α, IL-6, and IL-8) were detected by RT-PCR and ELISA. The effects of stimulation or pretreatment of TLR ligands on proliferation of THSFs were measured by MTT analysis. RESULTS: In the certain concentration range, pretreatment of THSFs with zymosan suppressed gene expression of inflammatory cytokines (TNF-α and IL-6). Copretreatment with zymosan and LPS suppressed gene expression and protein secretion more strongly compared with pretreatment with zymosan or LPS alone. Zymosan and/or LPS pretreatment suppressed lethal effect of A. fumigatus to THSFs in a certain period. CONCLUSIONS: Pretreatment of THSFs with TLR2-specific ligand zymosan results in a state of A. fumigatus hyphae tolerance. Copretreatment with TLR2 and -4 ligands (zymosan and LPS) leads to a stronger state of A. fumigatus hyphae tolerance, and suppresses the lethal effect of A. fumigatus.

Expression of Stat3 and indoleamine 2, 3-dioxygenase in cornea keratocytes as factor of ocular immune privilege.

PURPOSE: Ocular immune privilege is a multifactorial phenomenon evolutionally selected to prevent immunogenic inflammation from disrupting the visual axis and causing blindness. Here, we investigated the role of signal transducers and activators of transcription (Stat3) and indoleamine 2,3-dioxygenase (IDO) in ocular immune privilege in corneal stromal cells. METHODS: Human keratocytes were isolated and cultured in vitro, and Stat3 and IDO expression on keratocytes was investigated by reverse transcription polymerase chain reaction (RT-PCR). The active form of Stat3 was detected by flow-cytometry, and IDO enzyme activity following IFN-γ stimulation of keratocytes was measured by tryptophan to kynurenine conversion with photometric determination of kynurenine concentration in the supernatant. RESULTS: Stat3 was constitutively expressed in cultured keratocytes and up-regulated following IFN-γ stimulation. The active form of Stat3 was also up-regulated following IFN-γ stimulation. IDO expression and enzyme activity was markedly induced following IFN-γ stimulation, but this induction was prevented by the IDO specific inhibitor, 1-methyl tryptophan (1-MT). CONCLUSIONS: On the basis of this study, Stat3 and IDO may act as a factor of ocular immune privilege in corneal keratocytes. Thus, focus on these inhibitory molecules should be considered in studies aimed at developing therapeutic agents for controlling ocular inflammatory or immune diseases.

Role of the IL-6 classic- and trans-signaling pathways in corneal sterile inflammation and wound healing.

PURPOSE: To investigate the role of the IL-6 classic- and trans-signaling pathways in corneal sterile inflammation and wound healing. METHODS: To assess the production of inflammatory molecules by corneal fibroblasts treated with supernatant derived from necrotic corneal epithelial cells, the authors used an antibody array. Expressions of membrane IL-6 receptor (mIL-6R) and soluble IL-6R (SIL-6R) by fibroblasts and epithelial cells were detected with flow cytometry and RT-PCR. Expressions of signal transducer and activator of transcription 3 (STAT3), vascular endothelial growth factor (VEGF), and monocyte chemotactic protein-1 (MCP-1) by fibroblasts stimulated with IL-6 alone or IL-6/SIL-6R were determined by ELISA. The effect of IL-6 or IL-6/SIL-6R on epithelial cell migration was investigated in vitro by the scratch assay, whereas expressions of IL-6R and S100 A4 in the corneas of mice were detected by immunohistochemistry after incision of the corneal stroma. RESULTS: IL-1 derived from necrotic corneal epithelial cells induced the production of IL-6 by corneal fibroblasts. mIL-6R and SIL-6R mRNAs were expressed by both types of cells, although IL-6R protein at the cell surface was expressed only by epithelial cells. Expression of gp130 was detected in both types of cells. Activation of the IL-6 trans-signaling pathway induced the phosphorylation of STAT3, resulting in an increase of VEGF and MCP-1 production by corneal fibroblasts. Activation of the IL-6 classic-signaling pathway promoted the migration of corneal epithelial cells. IL-6R expression was also detected in activated fibroblasts and basal cells of the epithelium during the processes of wound healing in vivo. CONCLUSIONS: The IL-6 classic- and trans-signaling pathways have an important role in corneal sterile inflammation and wound healing.