M. Ni

Surfactant Protein D Is Present in Human Tear Fluid and the Cornea and Inhibits Epithelial Cell Invasion by Pseudomonas aeruginosa

ABSTRACT 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 ∼2 to 5 μg/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.

Human Tear Fluid Protects against Pseudomonas aeruginosa Keratitis in a Murine Experimental Model

ABSTRACT Pseudomonas aeruginosa keratitis is an acute sight-threatening infection. We previously reported that human tear fluid could protect individual human corneal epithelial cells in vitro against invasion by and cytotoxicity due to clinical and laboratory isolates of P. aeruginosa and that the protective mechanism was independent of bacteriostatic activity. In the present study, we examined the effects of human tear fluid in vivo. Tears were collected from healthy human volunteers and were studied in vivo in mice. The effects on the virulence of both invasive and cytotoxic clinical isolates of P. aeruginosa were examined. Tear fluid was found to reduce the severity of disease when corneas were challenged with cytotoxic bacteria immediately after scratch injury, and it completely protected against susceptibility to infection by a cytotoxic strain in a model in which corneas were infected during the healing process 6 h after scratching. Visible protection correlated with the inhibition of bacterial colonization 1, 4, and 48 h postinoculation. Tear fluid also significantly reduced the severity of infections caused by invasive P. aeruginosa in the 6-h-healing model. This result also coincided with significantly reduced bacterial colonization at 48 h. In vitro, human tear fluid significantly reduced the ability of invasive and cytotoxic bacteria to translocate across corneal epithelia and increased transepithelial resistance with or without bacterial inoculation. These data show that human tear fluid can protect against P. aeruginosa corneal infection in vivo and that the mechanism likely involves enhanced epithelial barrier function in addition to protection of individual epithelial cells against bacterial internalization and cytotoxicity.

Expression of surfactant protein D in human corneal epithelial cells is upregulated by Pseudomonas aeruginosa

We reported previously that surfactant protein D (SP-D) was present in human tears and corneal epithelial cells, and that it contributed to tear fluid protection of those cells against Pseudomonas aeruginosa invasion. This suggested a role in ocular innate immunity. Here, we explored the effects of bacterial challenge on SP-D expression by human corneal epithelial cells. Results showed that these cells produced and secreted SP-D constitutively in culture, and that production (mRNA, protein) and secretion of SP-D were upregulated after exposure to heat-killed P. aeruginosa or to purified flagellin or lipopolysaccharide. To begin exploring the mechanism for flagellin-mediated SP-D induction, cells were exposed to purified flagellin or flagellin mutated in the TLR-5-binding domain (L94A, L88A) which reduces IL-8 secretion by A549 respiratory cells. Mutated flagellin did not upregulate IL-8 expression in corneal epithelial cells, but did induce SP-D responses. Mitogen-activated protein kinase inhibitors, especially the JNK inhibitor SP600125, reduced secretion of SP-D, but not production, in the presence of P. aeruginosa. These data show that while SP-D and IL-8 corneal responses are each induced by P. aeruginosa or its antigens, they can involve different regions of the same ligand. The data suggest that separate mechanisms may regulate SP-D secretion and production by human corneal epithelia.

Protein Array Characterization of Bioactive Proteins Secreted By Immortalized Human Corneal Epithelium in Response to Pseudomonas Constituents

Purpose: To use protein arrays to delineate the spectrum of angiogenic bioactive protein modulators that might be secreted and up-regulated by the corneal epithelium in response to killed bacterial products. Methods: Immortalized human corneal epithelial cells were grown in culture, serum starved, and exposed to heat-killed Pseudomonas aeruginosa in a dose-dependent manner. The resultant culture medium was screened by antibody arrays for 43 proteins that can modulate angiogenesis and immune and inflammatory processes. Parallel analysis was carried out on tears recovered in the open and closed eye phases (OTF and CTF) of the diurnal cycle. Results: Array analysis reveals that the immortalized cells constitutively secrete several proteins and up-regulate the secretion of IL-6, IL-8, and GRO in response to killed bacteria. Also evident was the emergence of a strong signal for GM-CSF and moderate/weak signals for MCP-1, MMP-9, Leptin, and INFγ in a dose-dependent manner. Several of these proteins, including IL-6, IL-8, GRO, MMP-9, TIMP-1, and MCP-1, accumulate in the CTF. Other proteins are unique to tear fluid. Conclusions: Nine proteins were identified that are secreted by epithelium in response to killed bacteria that contribute to the innate and adaptive defense system through potentiating PMN and macrophage recruitment, activation, and opsonization in a cooperative manner. The vast majority of these proteins are angiogenic modulators, perhaps contributing to the imbalance between angiogenic and angiostatic processes and risk of corneal vascularization.