AsialoGM1-mediated IL-8 release by human corneal epithelial cells requires coexpression of TLR5.

PURPOSE: In this study, it was determined that human corneal epithelial cells (HCECs) express asialoganglioside ganliotetraosylceramide (asialoGM1) and toll-like receptor (TLR)-5, and their interaction induces interleukin (IL)-8 release through Ca(2+) transient activation and mitogen-activated protein kinase (MAPK) stimulation. METHODS: Expression of asialoGM1 and TLR5 was detected in SV40 HCECs by Western blot and flow cytometry analyses and their association by coimmunoprecipitation. Single-cell fluorescence imaging was used to measure intracellular free Ca(2+) transients in fura-2-loaded cells. The enzyme-linked immunosorbent assay (ELISA) was used to quantify IL-8 production in both cultured and primary HCECs. RESULTS: The HCECs expressed both asialoGM1 and TLR5 receptors. Ligation of asialoGM1 resulted in protein-protein interaction with TLR5, followed by transient increases in Ca(2+) influx through L-type voltage-dependent Ca(2+) channels. This led to P2Y receptor stimulation along with membrane depolarization, resulting from increases in ATP release into the medium. Intracellular Ca(2+) transients led to time-dependent extracellular signal-regulated kinase (ERK) MAPK pathway stimulation, followed by a 9.5-fold increase in IL-8 release. Similarly, in primary HCECs, asialoGM1 receptor stimulation resulted in an 8.1-fold increase. With a TLR5 neutralizing antibody, no asialoGM1-induced increases in IL-8 release occurred, and this response was not suppressed in the presence of a TLR2 neutralizing antibody. CONCLUSIONS: IL-8 release by HCECs is mediated through ligand-induced asialoGM1 protein-protein interactions with TLR5. This response is dependent on ATP efflux into the medium, followed by P2Y receptor stimulation. Such activation, in turn, results in increases in Ca(2+) influx through L-type voltage-dependent Ca(2+) channels, as well as stimulation of the ERK pathway.

Tear glucose dynamics in diabetes mellitus.

This study compares tear glucose dynamic differences between 121 diabetic and nondiabetic subjects after the administration of a carbohydrate load. A quantitative chromatographic analysis of tear glucose was used and the values correlated to blood glucose values. Diabetic and nondiabetic tear glucose mean values were 0.35 +/- 0.04 mmol/L and 0.16 +/- 0.03 mmol/L, respectively. Significant differences were observed among the subject groups in both the tear and capillary blood glucose values. A correlation between tear glucose and capillary blood glucose was observed. The concentration of glucose in the tear fluid changes proportionately with respect to capillary blood glucose after a carbohydrate challenge. Although it is possible to determine the diabetic status of a subject using tear glucose values alone, in the clinical setting this may not prove to be practical due to technical limitations.

Membrane array characterization of 80 chemokines, cytokines, and growth factors in open- and closed-eye tears: angiogenin and other defense system constituents.

PURPOSE: To adapt membrane-bound antibody array (MA) technology to characterize the distribution of a wide range of bioactive trace proteins in reflex (RTF) and open-eye (OTF) and closed-eye (CTF) tear samples. METHODS: Tears were collected by capillary tube and centrifuged. A commercially available standard MA and a custom array were modified to maximize the sensitivity of detection and the signal-to-noise ratio, to assay RTF and individually pooled CTF and OTF samples for 80 chemokines, growth factors, cytokines, and angiogenic modulators. The reliability of data was assessed by Western blot and other methods. RESULTS: Coupling an ultrasensitive chemiluminescence substrate system to an MA and optimizing conditions enhanced the sensitivity several hundredfold, allowing the detection of approximately 40 of the 79 probed proteins on the standard array, most of which were shown to be elevated in CTF. Identified entities include the known constituents epidermal growth factor (EGF), monocyte chemoattractant protein (MCP)-1, IL-8, tissue inhibitor of metalloproteinase (TIMP)-1 and -2, and numerous previously undetected tear components, such as angiogenin (ANG), growth factors, and the CXC and CC chemokines IFN-gamma inducible protein (IP)-10, growth-related oncogene (GRO), epithelial neutrophil-activating protein (ENA)-78, and macrophage inflammatory protein (MIP)-3alpha. Identification of other proteins was hindered by high background on the negative control array. Using a less complex custom array dramatically reduced background and allowed the visualization in CTF of proteins, such as VEGF, that were not detected with the standard array. CONCLUSIONS: MAs are powerful tools for differential screening of tears for large numbers of trace proteins. Analysis allowed the identification of previously undetected proteins that may participate in the host defense system as well as demonstrated the profound change in tear composition associated with prolonged eye closure in a manner reflective of physiological function.

Interaction of Pseudomonas aeruginosa with human tear fluid components.

PURPOSE: Reflex human tears bind Pseudomonas aeruginosa bacteria and prevent them from invading corneal epithelial cells. In this study, we assessed the effect of eye closure and the role of sialoglycoprotein (SG) in tears on bacterial binding and invasion. METHODS: Human tears (reflex and closed-eye) were collected using a microcapillary tube. Reflex tears were separated into 13 fractions by high-performance liquid chromatography while high-molecular-weight components from closed-eye tears were separated into an SG/mucin fraction and a nonmucin fraction. Bacterial binding was quantified by viable counts and bacterial invasion was tested using the gentamicin survival technique. RESULTS: Closed-eye tears bound significantly more bacteria than open-eye tears. Fractionation of reflex tears showed that 11 out of the 13 fractions bound bacteria, while all 13 fractions significantly reduced bacterial invasion of corneal epithelial cells. Surprisingly, the SG/mucin component of closed-eye tears resisted bacterial binding and had no significant effect on bacterial invasion. CONCLUSIONS: P. aeruginosa bacteria bind more efficiently to closed-eye tears than to open-eye tears. The mechanism by which tears bind bacteria and protect against invasion does not require SG/mucin, as this fraction of closed-eye tears does not contain either activity.

Surfactant protein D is present in human tear fluid and the cornea and inhibits epithelial cell invasion by Pseudomonas aeruginosa.

We have previously shown that human tear fluid protects corneal epithelial cells against Pseudomonas aeruginosa in vitro and in vivo and that protection does not depend upon tear bacteriostatic activity. We sought to identify the responsible tear component(s). The hypothesis tested was that collectins (collagenous calcium-dependent lectins) were involved. Reflex tear fluid was collected from healthy human subjects and examined for collectin content by enzyme-linked immunosorbent assay (ELISA) and Western blot with antibody against surfactant protein D (SP-D), SP-A, or mannose-binding lectin (MBL). SP-D, but not SP-A or MBL, was detected by ELISA of human reflex tear fluid. Western blot analysis of whole tears and of high-performance liquid chromatography tear fractions confirmed the presence of SP-D, most of which eluted in the same fraction as immunoglobulin A. SP-D tear concentrations were calculated at approximately 2 to 5 microg/ml. Depletion of SP-D with mannan-conjugated Sepharose or anti-SP-D antibody reduced the protective effect of tears against P. aeruginosa invasion. Recombinant human or mouse SP-D used alone reduced P. aeruginosa invasion of epithelial cells without detectable bacteriostatic activity or bacterial aggregation. Immunofluorescence microscopy revealed SP-D antibody labeling throughout the corneal epithelium of normal, but not gene-targeted SP-D knockout mice. SP-D was also detected in vitro in cultured human and mouse corneal epithelial cells. In conclusion, SP-D is present in human tear fluid and in human and mouse corneal epithelia. SP-D is involved in human tear fluid protection against P. aeruginosa invasion. Whether SP-D plays other roles in the regulation of other innate or adaptive immune responses at the ocular surface, as it does in the airways, remains to be explored.

Is the cystatin-like domain of TSL functionally active in external ocular infections and during the normal diurnal cycle?

PURPOSE: To test whether the cystatin-like functional domain in tear specific lipocalin (TSL) is functionally active in tears during the normal diurnal cycle and during external ocular infections. METHODS: Capillary tube collected reflex (RTF), open (OTF) and closed (CTF) eye tear samples were recovered from six normals and semi-quantitatively western blot assayed for cystatin C and TSL. CTF samples were immunoprecipitated with antibodies raised against TSL, cystatin C and other antiproteases and screened for the co-precipitation of proteases by casein and gelatin zymography. OTF samples recovered from individuals with viral, fungal and bacterial keratitis were similarly screened for TSL-bound proteases. Human tissue was subjected to immunohistochemical study. RESULTS: Western blot analysis reveals a progressive increase in cystatin C in going from RTF to OTF to CTF samples (approximately 3, 7 and 30 ng microl(-1), respectively). In contrast, the concentration of TSL remains constant (approximately 1500 ng microl(-1)). Immunocytochemistry data show staining of the apical surface of the human conjunctiva and some intra-cellular staining for cystatin C, but not for cystatin A. Zymography confirms earlier data that CTF contains exceptionally high levels of proteases bound to a wide range of specific inhibitors. However, only trace amounts of proteases are complexed with cystatin C and no protease can be detected bound to TSL in either the pathological or CTF samples. CONCLUSION: Although TSL contains a functional cystatin-like domain, it is not physiologically active during the normal diurnal cycle or during external ocular infections. Reactive proteases in CTF are most likely controlled by the presence of excess levels of more reactive cystatins, especially cystatin C, which accumulates during prolonged eye closure. Immunohistochemical data suggest that the apical conjunctiva may be a contributing source for the accumulating cystatin C.