Expression of membrane-type matrix metalloproteinases 4, 5, and 6 in mouse corneas infected with P. aeruginosa.

PURPOSE: To investigate the expression and regulation of membrane-type matrix metalloproteinases (MT-MMPs) 4, 5, and 6 in the mouse corneas infected with Pseudomonas aeruginosa. METHODS: C57BL/6J mice were intracorneally infected with P. aeruginosa. The expression of MT4-, MT5-, and MT6-MMP was detected at both the mRNA and protein levels by RT-PCR and immunoblot analysis. Immunohistochemical staining was performed to localize the expression of MT4- and MT5-MMP in the mouse corneas. RESULTS: Expression of MT4- and MT5-MMP was detected in the normal (uninfected) cornea by RT-PCR and immunoblot analysis. When infected with P. aeruginosa, the corneas showed significant induction of each MT-MMP. Localization of MT4- and MT5-MMP revealed that the expression of MT5-MMP was restricted to the epithelial tissue in the normal cornea, whereas the induced expression of MT4- and MT5-MMP was predominantly in the substantia propria, which contained most of the infiltrating cells. MT6-MMP expression was not detected in the uninfected cornea but was upregulated in the infected corneas. CONCLUSIONS: Expression of MT4-, MT5-, and MT6-MMP was induced in corneas infected with P. aeruginosa. Immunohistochemistry showed predominant immunoreactivity of MT4- and MT5-MMP in the substantia propria. Previous histologic studies have revealed different patterns of inflammatory cell infiltration with an increased number of polymorphonuclear neutrophils (PMNs) during the early stage of inflammation and increased macrophages during the late stage. These results indicate a good correlation between the overexpression of the MT-MMPs in the infected corneas and the inflammatory response-that is, leukocyte infiltration-indicating that inflammatory cells such as macrophages and PMNs may play a role in the upregulation of MT-MMPs during corneal infection, which in turn can cause the destruction of corneal tissue.

Expression of cathepsins B, D and L in mouse corneas infected with Pseudomonas aeruginosa.

C57BL/6J naïve and immunized mice were intracorneally infected with Pseudomonas aeruginosa. Semi-quantitative RT-PCR was performed to detect cathepsin gene expression and the results were further confirmed by immunoblot analysis. The enzymatic activities of cathepsins B, D and L were measured by peptidase assays. Immunohistochemical staining was carried out to localize the expression of the cathepsins. Cathepsins B, D and L were detected in the normal cornea by RT-PCR. A peptidase assay revealed activities of all three cathepsins under normal physiological conditions. In naïve mice, enzymatic activities of cathepsins B, D and L were all significantly enhanced when the corneas were infected with P. aeruginosa and the peak of the induction appeared around day 6 postinfection. Immunoblot analysis showed increased expression of cathepsins B, D and L. The infected corneal samples from immunized mice exhibited much lower induction of enzymatic activities compared to those from naïve mice. Immunohistochemistry showed that the expression of cathepsins in the normal cornea was restricted to the epithelial tissue while the induced expression of cathepsins was predominantly in the substantia propria. Our data revealed up-regulated enzymatic activities of cathepsins B, D and L in the naïve corneas infected with P. aeruginosa, which correlated well with the inflammatory response. Immunization of mice against P. aeruginosa attenuated the inducing effect on cathepsin expression caused by infection. The time sequence for induction of cathepsin proteins and enzymatic activities suggests a mechanism of host proteolytic degradation of the extracellular matrix resulting in corneal destruction after P. aeruginosa infection.

Plasminogen Activators and Inhibitors in the Corneas of Mice Infected with Pseudomonas aeruginosa.

PURPOSE: To characterize the presence of plasminogen activators and their inhibitors in the corneas during the inflammatory response in naïve and immunized mice intracorneally infected with Pseudomonas aeruginosa. METHODS: RT-PCR was used to detect gene expression for plasminogen activators and their inhibitors in naïve and immunized mice. Immunoblot analysis, zymography, and ELISA were used to demonstrate the syntheses of these proteins. RESULTS: Naïve mice intracorneally infected with P. aeruginosa showed a temporally enhanced expression of tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), its receptor (uPAR), and plasminogen activator inhibitors 1 and 2 (PAI-1 and PAI-2), over a several-day holding period. Immunized mice demonstrated a lower and shorter expression of these factors over the same period. Expression of these factors at the mRNA and protein levels may have been due to enzymes and inhibitors present in inflammatory cells and in resident corneal cells. CONCLUSIONS: These results show a correlation between the overexpression of the PA system in infected naïve mice as part of the inflammatory response, with eventual ocular destruction. Immunized mice exhibit a more balanced and shorter expression of the PA system, which may contribute to the restoration of corneal clarity.

Membrane-type matrix metalloproteinases in mice intracorneally infected with Pseudomonas aeruginosa.

PURPOSE: To establish the presence of membrane-type matrix metalloproteinases (MT-MMPs) in the cornea and their expression in naive and immunized mice intracorneally infected with Pseudomonas aeruginosa. METHODS: Naive (unimmunized) and immunized C57BL/6J mice were infected with P. aeruginosa, and gene expression of MT-MMPs were detected by RT-PCR. Immunoblot analysis and immunostaining were also used to characterize the MT-MMP response in both sets of animals. RESULTS: Expression of MT1-MMP, MT2-MMP, and MT3-MMP (MMP 14, 15, and 16) was detected by RT-PCR and immunoblot analysis. Of the three MT-MMPs detected, MT1-MMP exhibited the greatest expression at protein levels. In general, a bell-shaped curve was obtained for each of the MT-MMPs in naive mice, but all of them showed much less expression in the immunized mice. MT1-MMP was localized in the epithelial tissue of the cornea, whereas MT2-MMP and MT3-MMP were mainly found in the interface between the epithelium and substantia propria. CONCLUSIONS: MT1-MMP was detected and expressed to a greater extent in naive mice than MT2-MMP and MT3-MMP. Peak expression of all three MT-MMPs showed a good correlation with the overall inflammatory response.

In vivo characterization of host and bacterial protease expression during Pseudomonas aeruginosa corneal infections in naive and immunized mice.

PURPOSE: The corneal destruction resulting from P. aeruginosa keratitis is thought to be due, in part, to the actions of host and bacterial proteases on corneal tissue. The intent of the current study was to compare host and bacterial protease expression in the ocular tissues of mice that have been shown to be susceptible to corneal infection with mice that have been protected from such infection as a result of prior immunization. METHODS: Supernatants of infected corneal homogenates from naive mice or from mice immunized as a result of prior corneal infection or that had been immunized against bacterial protease were analyzed by gelatin zymography for the presence of host gelatinases and bacterial proteases at various times after infection. In addition, studies were carried out to examine the effect of immunization with bacterial protease on numbers of ocular bacteria. RESULTS: Mice that were protected against the tissue destruction during infection were found to have reduced proteolytic activity in corneal tissues when compared with susceptible mice. More specifically, the resistant mice displayed lower levels of bacterial alkaline protease, latent MMP-2, and both the active and latent forms of MMP-9. In addition, immunization against either bacterial alkaline protease or elastase failed to reduce bacterial numbers in ocular tissues from 6 to 72 hours after infection. CONCLUSIONS: These results suggest that increased proteolytic activity in ocular tissues during P. aeruginosa infection may contribute to the irreversible corneal damage observed during the infection.

Anti-receptor antibodies inhibit Pseudomonas aeruginosa binding to the cornea and prevent corneal perforation.

A polyclonal antibody (pAb) against gangliotetraosylceramide (asialo GM1), a glycolipid to which bacterial pili and LPS bind, and a mAb against a 66 kDa pilus-binding protein purified from adult mouse corneal epithelium were used to determine if antibodies against host receptors for bacterial adhesins could inhibit bacterial binding to wounded corneal epithelium and protect ocularly challenged mice from corneal perforation when topically applied. Bacteria were mixed with anti-66 kDa mAb, a mixture of anti-asialo GM1 pAb and anti-66 kDa mAb, an irrelevant control mAb (anti-human histocompatibility Ag HLA-DR5) or PBS prior to application to scarified corneas in organ culture. The combination of the two antibodies or the anti-66 kDa mAb alone was effective in reducing bacterial adherence compared with either PBS or the antibody control. To determine if these antibodies were protective in vivo, corneas of C57BL/6J mice were scarified and inoculated with Pseudomonas aeruginosa. Eyes were treated topically with anti-asialo GM1 pAb, anti-66 kDa mAb, a mixture of the two or control mouse serum. More serum-treated corneas perforated compared to corneas from any other group (P < or = 0.005) by 30 days postinfection. Treatment with a combination of the two antibodies resulted in significantly less corneal pathology 30 days p.i. when compared to any other treatment (P < or = 0.005). These data provide evidence that antibodies against host corneal receptors significantly inhibit bacterial binding in vitro and when applied topically in vivo, lessen the severity of ocular disease characteristic of P. aeruginosa keratitis.

Bacterial proteases and adherence of Pseudomonas aeruginosa to mouse cornea.

The goal of this study was to test whether bacterial exoproducts, such as elastase or alkaline protease contribute to the initial binding of Pseudomonas aeruginosa to mouse corneal epithelium. Each protease, purified from P. aeruginosa, when applied exogenously at concentrations of either 25 or 50 ng ml-1, elevated binding of Pseudomonas to mouse cornea in organ culture. Polyclonal antibodies against bacterial alkaline protease, but not elastase, interfered with bacterial binding and reduced significantly the number of organisms bound to cornea in an organ culture binding inhibition assay. Zymographic analysis of conditioned media from additional organ culture experiments showed that the P. aeruginosa strain used, which is highly virulent in cornea in vivo, secretes detectable levels of alkaline protease, but not elastase in vitro and that secretion was enhanced if the corneal epithelium was wounded. Lastly, how alkaline protease enhanced bacterial binding to the corneal epithelium of the organ cultured eye was examined. Data from this study suggest that exposure of lipase-sensitivity epithelial receptors represents at least one mechanism.

In vivo bacterial protease production during Pseudomonas aeruginosa corneal infection.

PURPOSE: To establish if active pseudomonal proteases are present in vivo during corneal infection with Pseudomonas aeruginosa and to determine if the mouse strains used in these and previous studies have the ability to mount a nonocular antibody response to the purified proteases because antibodies to the bacterial proteases were not detected previously during in vivo ocular infection. METHODS: At certain times after corneal infection with P. aeruginosa, corneas were harvested and supernatants from the corneal homogenates were analyzed for proteolytic activity by zymography and immunoreactivity by immunoblotting. The efficiency of the extraction procedures used in these studies was determined by incubating uninfected corneal homogenates with the purified proteases. The resultant supernatants were analyzed for alkaline protease and elastase activity. Additionally, mice were immunized intraperitoneally with the purified proteases with and without adjuvant to determine if the animals could mount a nonocular antibody response. RESULTS: Corneas infected with P. aeruginosa demonstrated the presence of alkaline protease, but not elastase, by the two methods examined. The kinetics of the in vivo alkaline protease response closely parallels previously reported bacterial clearance studies in that peak alkaline protease activity was detected in corneal tissue when peak bacterial numbers also were observed in the eye, and it was absent when the eyes were sterile or nearly sterile. In addition, C57BL/6J mice were capable of mounting a nonocular antibody response to microgram quantities of both proteases only in the presence of adjuvant. CONCLUSIONS: In the model described, enzymatically active alkaline protease, but not elastase, was demonstrated in corneal tissues during in vivo infection. Concentrations of these proteases were much lower than those required to stimulate an antibody response.

Characterization of arachidonic acid metabolism and the polymorphonuclear leukocyte response in mice infected intracorneally with Pseudomonas aeruginosa.

PURPOSE: To examine the activity of myeloperoxidase (MPO) and the concentrations of the proinflammatory metabolites of arachidonic acid (AA) in ocular tissue of mice that are either capable or incapable of restoring corneal clarity during an intraocular Pseudomonas aeruginosa infection. METHODS: For a period of 11 days after infection, whole eyes were enucleated and homogenized in buffer from mice given only an initial infection as well as from mice given a subsequent infection in the previously uninfected eye either 4 or 8 weeks after the initial infection. Tissue-free supernatants from the ocular homogenates were used for the determination of MPO activity by quantitating the conversion of specific substrate by spectrophotometric methods and for the quantitation of AA metabolites by ELISA: RESULTS: Overall, animals reinfected at 4 and 8 weeks had a lower inflammatory response when compared to the mice given only the initial infection. The lowest levels of LTB4 and MPO activity, indicators of PMN involvement, were observed in the the 8-week reinfected mice, which restored corneal clarity in an enhanced manner. CONCLUSIONS: These results suggest that induced ocular PMN responses may play a role, in part, in the inflammatory response leading to the tissue destruction observed during ocular P. aeruginosa infection.

Pili and lipopolysaccharide of Pseudomonas aeruginosa bind to the glycolipid asialo GM1.

This study investigated which adhesins of Pseudomonas aeruginosa interact with the glycolipid asialo GM1, using solid-phase binding and thin-layer chromatography assays. Radioiodinated pili and flagella contaminated with lipopolysaccharide (LPS) bound to the glycolipid. When LPS was reduced to acceptable levels in pilus and flagellum samples, only pili specifically bound to the glycolipid. Commercial, radiolabeled LPS as well as whole bacteria of strain ATCC 19660 also bound to asialo GM1. Binding was specific, competitive, and saturable. Organ cultures of whole mouse eyes and scanning electron microscopy techniques were used also, and strain ATCC 19660 was inhibited from corneal binding by exogenous pili or commercial LPS and inhibition was concentration dependent for both. Binding of radiolabeled strain ATCC 19660 bacteria to neutral lipids extracted from bovine corneal epithelial tissue showed that the bacteria bound to a glycolipid which migrated at a position similar to that of an asialo GM1 standard and that the glycolipid stained positively with an antibody specific for asialo GM1. The data provide evidence that pili (reduced LPS) and LPS of P. aeruginosa bind to asialo GM1 glycolipid and that the glycolipid is not restricted to the mouse cornea.

Characterization of the inflammatory response induced by corneal infection with Pseudomonas aeruginosa.

In order to characterize the inflammatory response to corneal infection by Pseudomonas aeruginosa, ocular cytokine and arachidonic acid metabolite levels were determined in the C57BL/6J strain of mice. The effects of topical anti-inflammatory drugs on the ability of the mice to clear viable P. aeruginosa from the eyes during the 12 day infection period was also examined. Ocular IL-1 alpha, IL-6, and TNF-alpha were detected over an 11 day time period. Little or no bacteria, as determined by quantitative plate counts, was detected after this time period. The kinetics of the cytokine production varied from one another, with an immediate release of peak levels of IL-1 alpha within 24 hours after infection which did not begin to approach baseline until 9 to 11 days after infection. Five to ten-fold lower concentrations of IL-6 and TNF-alpha were detected. IL-6 levels were induced at 24 hours after infection but there was essentially no distinct peak time-point. Peak levels of TNF-alpha were detected at 6 days post-infection. The kinetics of arachidonic acid metabolite release from infected eyes were also examined. Peak levels of PGE2 and TxB2 were observed at 6 days post-infection whereas peak LTB4 levels were determined at 3 days post-infection. Topical treatment of infected eyes with the two anti-inflammatory drugs, prednisolone or quercetin, resulted in higher ocular bacterial levels throughout the infection.

Immunization with homologous Pseudomonas aeruginosa pili protects against ocular disease.

The prophylactic effect of pili in prevention or amelioration of Pseudomonas aeruginosa ocular disease was examined in mice, using both systemic and topical protection approaches. At 30 days postinfection, a significant number of animals actively or passively immunized with pili homologous to pseudomonal strain American Type Culture Collection 19660 were protected from ocular disease when compared with similarly infected phosphate-buffered saline controls. Although exogenously mixing strain 19660 with either homologous or heterologous (PAK/PR1) pili before topical application of the inoculum significantly inhibited bacterial adhesion in vitro, in similarly designed in vivo studies, animals were not protected from corneal disease. Neither was significant ocular protection conferred using pili (PAK/PR1) heterologous to the infecting strain (19660) for active or passive immunization of mice, nor in studies involving exogenous mixing of PAK/PR1 pili or its pili-specific monoclonal antibody with strain 19660 prior to topical application of the latter. These results provide evidence that significant ocular protection is achieved by either active or passive, but not topical, immunization with pili homologous to the infecting bacterial strain and that immunization with pili heterologous to the infecting bacterial strain fails to provide protection against ocular disease, despite the fact that heterologous pili are highly effective at decreasing bacterial binding to cornea in vitro.

Kinetics of serum and ocular antibody responses in susceptible mice that received a secondary corneal infection with Pseudomonas aeruginosa.

When susceptible C57BL/6J mice were infected with Pseudomonas aeruginosa in one eye and then reinfected in the previously uninfected contralateral control eye either 4 or 8 weeks after the primary infection, approximately 20 to 30% of the mice receiving a 4-week reinfection regimen restored corneal clarity within 4 weeks, while almost all of the 8-week-reinfected mice restored corneal clarity within 3 to 6 days postinfection. However, the rate of bacterial clearance was the same in both sets of mice despite the presence of opsonophagocytic antibodies only in the 8-week-reinfected mice. As determined by enzyme-linked immunosorbent assay, immunoglobulin G was the major immunoglobulin in both serum and ocular tissue of both mouse sets, and the immunoglobulin G level was two- to fourfold higher after the 8-week secondary infection than in the 4-week-reinfected mice.

Proteinase K decreases Pseudomonas aeruginosa adhesion to wounded cornea.

The present study was designed to obtain further information regarding the molecular nature of the corneal receptor(s) facilitating Pseudomonas aeruginosa adhesion to cornea. Scarified adult mouse corneas in organ culture were treated for 10 or 60 min with a panel of lipase-free proteases [each at 20 micrograms ml-1 or 0.22 Units (U) ml-1, activity] including trypsin, chymotrypsin, V8 protease, elastase, subtilisin A, pronase protease and proteinase K. All of these, except proteinase K treatment (20 micrograms ml-1 for 60 min), either significantly elevated or had no effect (proteinase K 20 micrograms ml-1 for 10 min) on subsequent bacterial adhesion at 60 min following topical application of the inoculum to the scarified corneal surface. Enzyme treatment times of 10, 30 or 60 min at a higher concentration (50 micrograms ml-1) of proteinase K, significantly decreased binding at 60 min after bacterial application for each enzyme treatment time. The combined effects of proteases and lipase on bacterial binding also was examined. Eyes treated with proteinase K (20 micrograms ml-1 for 1 hr) or protease-free lipase (50,000 U ml-1 for 1 hr) alone or in combination, all reduced bacterial binding, but the effect was not additive. Trypsin or lipase alone significantly enhanced or decreased binding, respectively. In contrast, trypsin (20 micrograms ml-1 for 1 hr) followed by lipase treatment (50,000 U ml-1 for 1 hr) resulted in binding which was not significantly different than phosphate-buffered saline (PBS) control binding, indicating that the trypsin exposed receptor was lipase sensitive.(ABSTRACT TRUNCATED AT 250 WORDS)

Systemic and topical protection studies using Pseudomonas aeruginosa flagella in an ocular model of infection.

Purified flagella from P. aeruginosa ATCC 19660 were used for active, passive, or topical immunization prior to corneal challenge with strain 19660. At 30 days post-infection, a significant number of mice actively or passively immunized with flagella and infected with the homologous bacterial strain were protected from ocular disease when compared to control animals. In topical immunization studies, premixing of 19660 flagella with the bacterial inoculum prior to ocular challenge with strain 19660, provided results similar to those of the active or passive immunization studies. A reduced lipopolysaccharide (LPS:1 E.U./mg) flagella preparation was also produced and used similarly. Again, significant protection was achieved in mice immunized by flagella regardless of the immunization route. An in vitro adherence assay also was performed to examine quantitatively the effect of exogenously applied flagella, or an antiflagella monoclonal antibody (MAb) on bacterial adhesion. Premixing of the bacterial inoculum with flagella or the MAb prior to applying it topically to corneas in organ culture all significantly inhibited bacterial binding. These results strongly suggest that significant ocular protection is achieved with either active or passive immunization, or premixing of the bacterial inoculum with flagella from strain 19660 prior to ocular challenge with the homologous bacterial strain. They also indicate that topical application of flagella or antiflagella MAb provide protection against ocular disease by decreasing bacterial adhesion to cornea.

Corneal epithelial glycoproteins exhibit Pseudomonas aeruginosa pilus binding activity.

Adherence of Pseudomonas aeruginosa to the cornea is a requisite step in the pathogenesis of bacteria-induced corneal disease. P. aeruginosa is capable of attaching to host epithelial cells by its pili, but there is little information regarding the epithelial receptors of this adhesin in the cornea. Using nitro-cellulose blotting of polyacrylamide gels of solubilized adult mouse corneal epithelium, four major proteins (molecular weights: 38, 42, 57, and 66 kD) and several minor proteins were identified that bound purified pili from strain PAK and its hyperpiliated mutant PAK/PR1. These proteins were identified by immunoblotting either with pilus-specific monoclonal antibodies, XLR-3 and PK 3B, or using peptide PAK 128-144 (OX). The glycosylated nature of the proteins was determined using similar gel electrophoresis of corneal epithelial proteins, blotting onto nitrocellulose, and staining the blots with lectins conjugated to either horseradish peroxidase or alkaline phosphatase. All four major pilus-binding proteins were stained with concanavalin A lectin (mannose and glucose) and either wheat germ agglutinin lectin (WGA, specific for sialic acid and N-acetylglucosamine) or succinylated WGA lectin (only N-acetylglucosamine). Staining for peanut agglutinin lectin (galactose beta(1-3) N-acetylgalactosamine) was seen for the 42-, 57-, and 66-kD proteins. The importance of the carbohydrate portions of these corneal proteins in pili binding was confirmed by preincubation of corneal epithelial blots with periodate or pili with sialic acid, both of which abolished the pili binding. These studies indicate that corneal epithelial pilus-binding proteins are glycoproteins in nature and that sialic acid may be a constituent of these pilus-specific receptors in the adult mouse corneal epithelium.

Kinetics of serum, tear, and corneal antibody responses in resistant and susceptible mice intracorneally infected with Pseudomonas aeruginosa.

The studies described here are aimed at determining the kinetics of antibody responses specific to Pseudomonas aeruginosa ATCC 19660 in sera, tears, and corneas of naturally resistant DBA/2 mice and susceptible C57BL/6 mice after intracorneal infection. Immunoglobulin (IgG) and IgM responses in sera were significantly greater in DBA/2 mice for the first 2 weeks postinfection. Little or no IgA was detected in the sera of mice from either strain. IgG was the predominant immunoglobulin class present in the corneas of the infected eyes from both mouse strains. However, differences in both the magnitude and the kinetics of the corneal IgG responses were noted between mouse strains. The kinetics of the corneal IgG responses were more similar to those of the serum IgG response than to those of the tear IgG response. Tear antibody responses in DBA/2 mice differed from those of C57BL/6 mice in two ways. First, there was a sharp increase in tear IgG levels 2 weeks after infection in DBA/2 mice that was not present in C57BL/6 mice. Second, IgA levels present in tears from the infected eyes of C57BL/6 mice dropped to nearly preinfection levels after the first week, whereas in DBA/2 mice, IgA levels remained elevated in the infected eyes after the first week. Determination of P. aeruginosa-specific antibody responses in the uninfected, contralateral control eyes revealed that IgA was detectable in the tears but not in the corneas of DBA/2 mice. Very little IgA was detected in the tears of the uninfected eyes of C57BL/6 mice. IgG was the only immunoglobulin class present in the uninfected corneas in both mouse strains tested. These results suggest that ocular IgA was made locally, whereas most ocular IgG may have originated from the serum, with some possible local synthesis. These immunological results indicate that DBA/2 and C57BL/6 mice respond differently to corneal challenge with P. aeruginosa.

In vivo and in vitro toxicity of phospholipase C from Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces phospholipase C (PLC), a heat-labile hemolysin. Histopathological analysis of PLC-treated mice revealed that the primary target organs involved in PLC-induced toxicity were the liver and kidney. Mice treated i.v. with PLC demonstrated significant tubular epithelial necrosis of the kidney with hematuria, while when given i.p. they exhibited hepatonecrosis with cellular infiltration. Splenomegaly was also a consistent finding. Results from in vitro studies indicate that PLC is toxic for mouse peritoneal cells and human leukocytes.

Distribution and kinetics of the inflammatory cell response to ocular challenge with Pseudomonas aeruginosa in susceptible versus resistant mice.

This study examined and characterized the distribution and kinetics of the inflammatory cell response to pseudomonas ocular challenge in susceptible C57BL/6J and resistant DBA/2J mice. Initially, in the cornea, the number of neutrophilic leukocytes (PMNs), macrophages/monocytes or lymphocytes was 2-2.5 times greater in resistant versus susceptible mice. The peak cellular response in the cornea was also greater in resistant than in susceptible animals. Further, resistant mice, when compared with susceptible mice, had a shorter duration of inflammatory cells as well as bacteria in the cornea. These data were similar for the cell infiltrate in the anterior chamber, with the exception that PMNs were initially greater in number in C57BL/6J than in DBA/2J mice. Collectively these data suggest that susceptible mice are, in general, cellularly hyporesponsive to pseudomonas ocular challenge when compared with resistant animals. This, together with persistence of inflammatory cells and bacteria in the cornea of susceptible animals, may contribute to their failure to restore corneal clarity following pseudomonas ocular challenge.

Characterization of pseudomonal adherence to unwounded cornea.

This study examined binding of Pseudomonas aeruginosa to unwounded postnatal day (P) 5 immature mouse cornea. To determine whether the receptor molecule was protein or lipid in nature, the eyes were incubated in vitro with trypsin or lipase before bacteria were applied. Trypsin significantly increased bacterial binding, whereas lipase had no effect. To determine if enhanced binding after trypsin indicated exposure of a lipid or a protein receptor, the eyes were treated sequentially with trypsin, then lipase. Subsequent binding did not differ significantly from trypsin alone, indicating that the exposed receptor was not a lipid. Incubation of the P 5 eye with neuraminidase to remove sialylated residues also significantly enhanced binding at all times, and premixing of the inoculum with the enzyme before adherence testing increased binding at 15 and 30 min. The effects of a monosialoganglioside (GM1) and gangliotetraosylceramide (asialo GM1) were also examined. Incubation of eyes with GM1 or asialo GM1 produced no significant inhibition of bacterial binding, but premixing of the bacterial inoculum with GM1 or asialo GM1 before corneal application transiently decreased adherence. Fibronectin (FN) treatment of the P 5 eye, premixing FN with the bacterial inoculum before its ocular application, or similar treatment with N-acetylneuraminic acid (NANA) transiently inhibited binding. These data demonstrate that pseudomonal binding to the unwounded eye is not lipase sensitive and is enhanced by trypsin treatment which exposes a lipase insensitive receptor and by neuraminidase which removes sialylated residues. It is not inhibited by pretreatment of the eye with either GM1 or asialo GM1 and is transiently inhibited by pretreatment of the eye or the inoculum with NANA or FN.