MIP-1alpha regulates CD4+ T cell chemotaxis and indirectly enhances PMN persistence in Pseudomonas aeruginosa corneal infection.

The role of macrophage inflammatory protein-1alpha (MIP-1alpha) in cell infiltration into Pseudomonas aeruginosa-infected cornea and subsequent disease was examined. Greater amounts of the chemokine (protein and mRNA) were found in the infected cornea of susceptible B6 ("cornea perforates") versus resistant BALB/c ("cornea heals") mice from 1 to 5 days postinfection. Treatment of BALB/c mice with recombinant (r) MIP-1alpha exacerbated disease and was associated with an increased number of neutrophils (PMNs) in the cornea. Treatment of BALB/c mice with rMIP-1alpha also induced recruitment of activated CD4+ T cells into the affected cornea, converting resistant to susceptible mice. Depleting CD4+ T cells in r-treated BALB/c mice significantly decreased PMNs in cornea tissue, suggesting that T cells regulate persistence of PMNs at this site. In B6 mice, administration of neutralizing MIP-1alpha polyclonal antibody also significantly reduced PMN numbers and pathology. Collectively, evidence is provided that MIP-1alpha directly contributed to CD4+ T cell recruitment and indirectly to PMN persistence in the infected cornea.

Corneal and ocular surface histochemistry.

In summary, this review has provided information concerning the application of histochemical and cytochemical procedures used to detail the normal versus pathological cornea and ocular surface. Specifically, histochemical analysis has been used to study protein and peptide degradation in cornea, to analyze stromal non-collagenous and collagenous fibers and associated extracellular matrix. Cytochemistry of the ocular surface has been used to detail the morphology of corneal and conjunctival mucin. Use of small cationic probes as well as lectin-gold binding was advantageous to quantitatively demonstrate that ocular mucin contains sialylated residues and that the number of these residues significantly changes (increases) with age. These data are important in that the degree of sialylation has been shown to correlate with the ability of bacterial organisms to adhere to and infect the immature in contrast to the mature corneal surface. The use of lectin analysis of diseased ocular tissue also has shown that there are specific alterations in glycoconjugates which occur in the diseased versus normal human cornea. Wound healing in cornea is an important problem which has been studied at length using combined histochemical and biochemical approaches. Results support the hypothesis that apical cell surfaces of the leading edge of a migrating sheet differ from those of the normal epithelium. During wound healing, alpha 6 integrin expression by corneal epithelial cells has been demonstrated, but another protein, syndecan was only seen in non-migrating epithelium which had restratified. The association of immunoglobulins with the ocular surface epithelium of the cornea, their change with age and kinetics of appearance also has been demonstrated using a cytochemical approach. Histochemical procedures have been used to localize Class I and Class II molecules in cornea and conjunctiva. Class II antigen expression has been shown to be absent on corneal endothelium, but it can be induced by treatment with IFN-gamma. These data are of importance in corneal pathology such as that resulting in rejection of corneal transplants. Langerhans cells (Class II, Ia positive) also are not found in normal central cornea. They are localized in the peripheral cornea and are stained histochemically by ADPase, ATPase and by specific anti-Ia and other antisera. Increased numbers of LC have been demonstrated in cornea following various stimuli and in diseases of the cornea including both bacterial and viral induced keratitis.

Maintaining corneal integrity how the "window" stays clear.

The anterior surface of the eye is composed of the cornea, conjunctiva, and the zone between the two called the limbus. The cornea must maintain optical clarity to retain good vision. However, the ocular surface is vulnerable to trauma, microbial infection, and exposure to environmental toxins. This places the cornea, especially, at risk for disruptions of the epithelial barrier and subsequent immunopathological events. Cell-cell and cell-matrix attachment junctions incorporating adhesion molecules ensure that the epithelial barrier remains intact. Protein components of the basement membrane, including laminins, are vital to the adhesion of corneal epithelial cells to the underlying stroma and function to enhance the strength of the bond between epithelium and connective tissue. Epithelial cells also play an early and crucial role in the initiation of ocular surface responses should a potentially antigenic molecule enter into deeper corneal tissues. For example, epithelial cells may produce and release cytokines such as interleukin-1 (IL-1). The delicate balance between the matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are central to mechanisms regulating dissolution of the extracellular matrix that may be a consequence of infection or wound healing. Adhesion molecules, cytokines and chemokines, and MMPs and TIMPs thus participate in the corneal response to immunologic challenge or wounding. They may also be involved in corneal pathologies associated with genetic diseases, diabetes, and vitamin A deficiency. In addition these molecules are components of cellular pathways underlying the clinical complications often observed with contact lens wear and refractive surgeries used to improve visual acuity.

Glycoconjugates on corneal epithelial surface: effect of neuraminidase treatment.

The purpose of this study was to develop a procedure to quantitatively examine corneal epithelial apical cell membrane-associated glycoconjugates. Saccharide moieties on young, mature, and aged corneal epithelial cells were detected and localized in corneas of immature and adult mice by using colloidal gold-labeled lectins and transmission electron microscopy (TEM). In general, dense binding to the corneal epithelial apical surface cell membranes with wheat germ agglutinin (WGA) was seen in the adult, whereas the immature cornea bound less WGA-gold. Neuraminidase digestion decreased binding of the conjugate on epithelial plasma membranes of young and mature cells in adult cornea. Lectin-gold binding was decreased in the immature cornea on mature and aged cells. WGA-gold binding after neuraminidase was elevated on young cells of immature and on aged cells of adult animals. No binding of peanut agglutinin (PNA) or horse gram agglutinin (DBA) to the corneal epithelial surface was seen in animals of either age. After neuraminidase digestion, PNA binding sites were exposed only on the adult corneal surface. These data suggest that a terminal trisaccharide sequence, sialic acid-galactose beta(1----3)-N-acetylgalactosamine, is present at the adult corneal surface but is absent or at undetectable levels at the corneal surface of the immature animal. These data may be of significance in light of the dissimilar pattern of P. aeruginosa recognition and binding to the immature vs adult corneal epithelium.

Evidence for TIMP-1 protection against P. aeruginosa-induced corneal ulceration and perforation.

PURPOSE: To determine the biological significance of individual endogenous tissue inhibitors of metalloproteinases (TIMPs) in protection against tissue destruction using a Pseudomonas aeruginosa-induced model of corneal ulceration. METHODS: Corneal TIMP-1, -2, and -3 mRNA levels were compared between young adult (resistant) and aged (susceptible) mice challenged with P. aeruginosa. Resistant mice that demonstrated greater amounts of an individual TIMP were treated with polyclonal antibody (pAb) to that TIMP. To determine whether TIMP neutralization exacerbated P. aeruginosa-induced corneal disease, TIMP pAb- and normal rabbit serum (NRS)- (control) treated mice were examined macroscopically and histopathologically after infection. Corneal neutrophil (PMN) myeloperoxidase (MPO) levels also were examined in these mice. RESULTS: Greater amounts of TIMP-1 mRNA only were found in corneas of resistant versus suscep tible mice after P. aeruginosa challenge. Systemic treatment of resistant mice with TIMP-1 pAb resulted in corneal perforation by 5 to 7 days after infection (PI). Histopathologic evaluation of corneal tissues from TIMP-1 pAb- versus NRS-treated mice confirmed that TIMP-1 pAb treatment resulted in extensive stromal dissolution. This treatment also was associated with loss of epithelium within the central cornea. Both the histopathology and PMN MPO enzyme assays also showed an increase in corneal PMN number following TIMP-1 pAb treatment. CONCLUSIONS: These studies provide evidence that, after P. aeruginosa infection, adequate endogenous expression of TIMP-1 in cornea protects against extensive corneal tissue destruction. The protective effects of TIMP-1 may be multifactorial. In addition to directly protecting extracellular matrix components from active matrix metalloproteinases, TIMP-1 may either directly or indirectly influence recruitment of PMNs into infected cornea. Finally, TIMP-1 also may affect wound healing and resurfacing of the corneal epithelium.

C57BL/6 mice lacking Muc1 show no ocular surface phenotype.

PURPOSE: To test the hypothesis that a membrane-spanning mucin, Muc1, facilitates the spread of tear film and protects against bacterial adherence. METHODS: Age-matched, Muc1 null mice and wild-type mice of C57BL/6 genetic background were used for comparison. Eyes were examined by slit lamp biomicroscopy with fluorescein solution to assess epithelial damage and tear film stability. Structure of the ocular surface epithelia was examined by light microscopy, scanning and transmission electron microscopy, and wholemount confocal microscopy. Bacterial adherence assay was performed on in vivo corneas with Pseudomonas aeruginosa containing a plasmid encoding green fluorescent protein, followed by wholemount confocal microscopy. Real-time reverse transcription-polymerase chain reaction was performed using Muc4-specific primers to quantitate Muc4 mRNA expression in ocular surface tissues. RESULTS: No differences were found between Muc1 null and control mice in any parameter tested. Ocular surface epithelia of Muc1 null mice of the C57BL/6 strain had a normal appearance of surface microplicae, a well-developed glycocalyx on the apical cell membrane, and a normal appearance of goblet cell mucin packets. There was no convincing evidence that bacterial adherence on the cornea was increased in Muc1 null mice. Muc4 mRNA expression was not upregulated in Muc1 null mice compared with control. No ocular surface infections were observed in Muc1 null mice of the C57BL/6 strain (n = 204), which were housed in the animal facility over a period of 26 months. CONCLUSIONS: Muc1 null mice of C57BL/6 background appeared normal in all respects tested. These data differ from the reported phenotype in the mice of the C57BL/6 x SVJ129 background, which show development of blepharitis and conjunctivitis.

Acanthamoebae bind to rabbit corneal epithelium in vitro.

PURPOSE: Selection of an appropriate animal model is crucial for the investigation of the pathogenesis of Acanthamoeba keratitis. To this end, it has been reported that fluorescein isothiocyanate (FITC)-labeled Acanthamoeba castellanii bind to human, pig, and hamster corneas, but not to rabbit corneas in organ culture. However, 35S-labeled A. polyphaga and A. culbertsoni have been found to bind to rabbit corneal epithelium grown in tissue culture. The purpose of the current study was to establish whether A. castellanii bind to rabbit corneal epithelium in organ culture. METHODS: Two different adhesion assays were used to determine whether 35S-labeled and FITC-labeled A. castellanii bind to epithelium of corneal buttons in vitro and, if so, whether the binding is temperature-dependent, enhanced by injury, and inhibited by specific saccharides. Ameba binding to rabbit corneal epithelium was also evaluated by scanning electron microscopy. The binding of A. castellanii to corneal epithelium of three different species (human, pig, and rabbit) was compared. RESULTS: Both 35S-labeled as well as FITC-labeled parasites were found to bind to epithelium of rabbit corneal buttons in vitro. Although the parasites bound avidly to the corneas at 25 degrees C and 35 degrees C, little binding was observed at 4 degrees C. Injury enhanced the binding. Methyl alpha-D-mannopyranoside, but not other saccharides (alpha-L-fucose and beta-galactose), inhibited binding of the parasites to the epithelium of rabbit corneas. By scanning EM, A. castellanii were found to adhere, invade, and penetrate the epithelium of rabbit corneas. Compared with rabbit corneas, ameba binding to pig, and human corneas was only 1.2 and 1.4 times higher, respectively. CONCLUSIONS: Rabbit animal model may prove useful for investigation of the molecular mechanisms that mediate adhesion of Acanthamoeba to corneal epithelium.

Evidence for N-acetylmannosamine as an ocular receptor for P. aeruginosa adherence to scarified cornea.

This study provides evidence for an N-acetylmannosamine (manNac) receptor mediating in vivo bacterial adherence of P. aeruginosa to the scarified corneal epithelial surface. Bacteria, whether exposed to the inhibiting sugar or not, did not adhere immediately after inoculation, but required time in contact with the scarified corneal surface to adhere and adherence increased with time. Organisms were observed primarily adherent to the surface of epithelial cells lining the wound and were less frequently seen on areas of denuded stroma. Saturation of binding sites on the bacterial surface by manNac inhibited attachment of the organisms to newly exposed surface membrane receptors. In vivo protection studies showed excellent correlation with quantitative analysis of scanning electron micrographs, in that the number of adherent organisms at the corneal surface at 60 min following scarification and bacterial inoculation was decreased significantly by prior treatment of the bacteria with manNac when compared with PBS or the other monosaccharides. These data were confirmed by quantitative bacterial culture of excised corneas which had been similarly infected in vivo with PBS or manNac treated organisms.

Penetration of the unwounded immature mouse cornea and conjunctiva by Pseudomonas: SEM-TEM analysis.

Scanning and transmission electron microscopy were used to study the early ocular response to infection by Pseudomonas aeruginosa inoculated under the fused eyelids of immature mice. In the absence of experimental corneal wounding, the organisms produced lysis of the epithelium of the peripheral cornea and conjunctiva and penetrated and lysed the respective stromas as early as 15 min after inoculation. Bacteria were infrequently detected on the ocular surface at 3 hr after infection. At this time, corneal epithelial cells in the vicinity of the inoculation were markedly shrunken.

Pseudomonas eye infections in cyclophosphamide-treated mice.

Swiss-Webster mice challenged intracorneally with varying doses of P. aeruginosa exhibit eye infection within 24 hr. as detected by corneal opacity. The infection remains localized and spontaneously heals within 4 to 6 weeks. However, when mice are pretreated with a single i.p. injection of cyclophosphamide 4 days prior to either intracorneal or anterior-chamber challenge with various bacterial cell suspensions (10(1) to 10(8)), 83% of the anterior chamber and 54% of the intracorneally challenged mice (most at higher dilutions of the bacteria) died of Pseudomonas septicemia within 48 hr. Corneal damage was histochemically and ultrastructurally observed in surviving animals by 24 to 48 hr. following challenge by either route.

Increased severity of Pseudomonas aeruginosa corneal infection in strains of mice designated as Th1 versus Th2 responsive.

PURPOSE: Mice favoring Th1 (C57BL/6, C57BL/10, and B10.D2/nSn) versus Th2 (BALB/c, BALB/cBy, BALB.B, and BALB.K) response development were evaluated for their response to infection with Pseudomonas aeruginosa. This study addresses the question of whether Th1 versus Th2 response propensity affects the pathogenesis of bacterial keratitis in mice. METHODS: Ocular disease was determined by mean clinical score, slit lamp, plate counts, and histopathology, and antigen-specific cellular responses were assessed by immunostaining and measurement of delayed type hypersensitivity (DTH). RESULTS: Strains of mice favoring Th1 (B6, BL10, and B10.D2) versus Th2 (BALB/c, BALB/cBy, BALB.B, and BALB.K) responsiveness were infected with P. aeruginosa. Mice favoring Th1 response development exhibited a similar course of disease and the infected eyes of all mice perforated by 7 days postinfection (p.i.). Strains (BALB/c, BALB/cBy, BALB.B, and BALB.K) favoring Th2 response development exhibited a milder course of disease, and none of the infected corneas perforated at 7 days p.i. In a Th1-responsive strain (B10.D2), positive immunostaining for CD4+ and CD8+ T cells was observed in the cornea by 3 days p.i. and by 5 days p.i., respectively, some cells stained positively for IL2-R, indicating that the cells were activated. In contrast, in a Th2 responder strain (BALB/c), there was no detectable positive immunostaining in cornea for any of the T-cell markers tested and DTH was significantly elevated in B10.D2 versus BALB/c mice. CONCLUSIONS: These studies are the first to provide evidence that in P. aeruginosa ocular infection, mouse strains favoring development of a Th1-type response are susceptible (cornea perforates), whereas strains favoring Th2 response development are resistant (no corneal perforation).

Monoclonal antibodies provide protection against ocular Pseudomonas aeruginosa infection.

A panel of well characterized monoclonal antibodies (MAbs) directed against outer membrane proteins H2, or F (porin) of Pseudomonas aeruginosa were examined to determine whether they exhibited any protective effect against subsequent ocular challenge with the bacteria topically applied to the scarified corneal surface. Mice were observed macroscopically following bacterial challenge and the degree of ocular disease graded on a scale of 0 to 4 (0, normal, fully protected cornea; 4, corneal perforation or phthisis, not protected). Mice treated intravenously with either MAb MA1-6 (anti-H2) or MA2-10 (anti-F), or a combination of these two MAbs and MAb MA4-4 (anti-F), two hours before corneal challenge with the viable bacteria, exhibited significantly less corneal disease than mice either not treated with the MAbs, treated with MA4-4 alone or treated with MAb MA1-3 (anti-I). The latter MAb is directed against an outer membrane epitope that is not surface exposed. Light and transmission electron microscopic histopathology also was employed and provided confirmatory evidence to support the macroscopic analyses.

Aging and PMN response to P. aeruginosa infection.

PURPOSE: Alterations in immune system function associated with aging may contribute to increased morbidity in this population of individuals. The current studies were performed to determine aging-related changes in polymorphonuclear neutrophil (PMN) function after corneal infection with Pseudomonas aeruginosa. METHODS: Total PMN number, macrophage inflammatory protein (MIP)-2 mRNA and protein expression, and ocular bacterial load were determined in 8-week- and 12-month-old inbred BALB/c mice at various times after infection with P. aeruginosa. In addition, 12-month-old mice were treated systemically with the MIP-2 polyclonal antibody (pAb) to determine the effects of MIP-2 neutralization on ocular disease and PMN recruitment. RESULTS: Histologically, PMN infiltration into the cornea of 12-month-old mice was delayed initially and was associated with an inability to reduce bacterial load at later postinfection (PI) times. In addition, a significantly greater number of PMNs were found in the cornea of 12-month-old mice at later PI times. The increase in PMN number in 12-month-old mice correlated with a persistence of MIP-2 expression in cornea at these later times. Systemic treatment of 12-month-old mice with neutralizing MIP-2 pAb versus normal rabbit serum (NRS) resulted in reduced corneal PMN number and ocular disease. CONCLUSIONS: These data provide evidence that persistence of PMN in the cornea of 12-month-old mice contributes to corneal tissue destruction after P. aeruginosa challenge. Further evidence also is provided that the chemoattractant MIP-2 contributes to the altered PMN response in these animals.

Early TIMP gene expression after corneal infection with Pseudomonas aeruginosa.

PURPOSE: To evaluate the expression of tissue inhibitor of metalloproteinases (TIMP) mRNA in corneal tissues before and at early time points (6 and 12 hours and 1, 3, and 5 days) after corneal infection with Pseudomonas aeruginosa. METHODS: Ribonuclease protection assays were used to detect and quantitate TIMP mRNA expression in uninfected (wounded and unwounded) and in wounded corneas inoculated with P. aeruginosa. RESULTS: Expression of TIMP-1 mRNA was undetectable in either wounded or unwounded, uninfected corneal tissues, but it increased in a time-dependent manner with peak expression occurring at 3 days after corneal wounding and bacterial inoculation. Constitutive low-level mRNA expression of TIMP-2 was detected in both groups of uninfected corneal tissues; these groups remained essentially unchanged after corneal abrasion and bacterial inoculation. TIMP-3 mRNA was detected in uninfected (wounded and unwounded) corneal tissues and appeared to decrease in a time-dependent manner after corneal abrasion and bacterial inoculation. CONCLUSIONS: The evidence indicated that TIMPs were expressed in infected and in uninfected (wounded and unwounded) corneal tissues. In addition, these data suggested that TIMP expression appeared to be independently regulated.

Secretory IgA inhibits Pseudomonas aeruginosa binding to cornea and protects against keratitis.

PURPOSE: To determine whether secretory IgA (SIgA) antibody inhibits Pseudomonas aeruginosa binding to cornea in vitro and if boosting SIgA antibody in tears using heat-killed P. aeruginosa as an immunizing antigen is protective in vivo in experimentally induced bacterial keratitis in the mouse. METHODS: SIgA, immunoglobulin-G, immunoglobulin-M, and an undiluted crude human milk preparation were tested in vitro for their ability to inhibit P. aeruginosa binding to the scarified corneas of adult (6 weeks to 6 months of age) mice by topical application of each before similar delivery of the bacterial inoculum. Scanning electron microscopy (scanning EM) was used to quantitate bacterial adherence. In vivo mice were immunized topically with heat-killed P. aeruginosa or sham immunized by application of a similar volume of phosphate-buffered saline (PBS). Tears were collected from both groups of mice and levels of immunoglobulins (Igs) measured by enzyme-linked immunosorbent assay (ELISA). After the second immunization, the same two groups were challenged ocularly with 5.0 x 10(7) colony forming units P. aeruginosa and the response to infection graded. RESULTS: In vitro, after a 30-minute preincubation with Igs, SIgA (250 micrograms/ml) significantly decreased P. aeruginosa binding to cornea in vitro when compared to the number of bacteria bound in PBS control specimens, and binding reduction was concentration dependent. In vivo, 15 days after a second ocular topical immunization, tear SIgA was elevated significantly and was specific for P. aeruginosa when measured by ELISA. In vivo, corneal disease response grades in the heat-killed antigen immunized mice also were significantly less severe when compared to sham-immunized mice. CONCLUSIONS: SIgA significantly inhibits binding of P. aeruginosa to the wounded mouse cornea in vitro, and inhibition is concentration dependent. In vivo, specific antipseudomonal SIgA in mouse tears can be elicited by topical ocular immunization with heat-killed P. aeruginosa, and a significant number of immunized animals with elevated levels of SIgA in their tears exhibited less severe ocular disease after bacterial challenge.

Role of complement in murine corneal infection caused by Pseudomonas aeruginosa.

The role of complement components C3 and C5 was examined in murine corneal infection induced by Pseudomonas aeruginosa. DBA/2 and Swiss-Webster mice are naturally resistant to experimental P. aeruginosa corneal infection. Mice of these two strains are able to restore a clear cornea within 4-6 weeks following Pseudomonas corneal challenge and are genetically deficient in the fifth component of complement. In contrast, ocular infection of congenic C5-deficient B10.D2o/Sn or normocomplementemic B10.D2n/Sn mice results in an identical pattern of susceptibility in which corneal perforation and phthisis bulbi occur. Taken together, these results indicate that C5 plays little or no role in susceptibility or resistance to Pseudomonas eye infection. In contrast, depletion of C3 in normally resistant DBA/2 mice using cobra venom factor (CoVF) diminishes the ability of these mice to restore a clear cornea following Pseudomonas infection. A single inoculation of CoVF, 24 hours prior to ocular challenge, is as effective in altering the response of DBA/2 mice as is continuous depletion of C3 during the course of infection using multiple inoculations of CoVF. Mice that are unable to clear Pseudomonas ocular infection following CoVF treatment regain this ability when the contralateral eye is infected after recovery of normal levels of C3. Depletion of C3 by CoVF treatment of DBA/2 mice, which had previously restored a clear cornea following Pseudomonas eye infection, again renders these mice susceptible.

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.

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.

Aged mice fail to upregulate ICAM-1 after Pseudomonas aeruginosa corneal infection.

PURPOSE: In young Swiss (HSD:ICR) outbred mice, corneal clarity is restored after Pseudomonas aeruginosa ocular infection, whereas disease in aged outbred mice progresses to corneal perforation. This study was conducted to elucidate further the mechanism responsible for this age-related disparity in disease response. METHODS: Corneas of young (6 to 8 weeks of age) and aged (1.5 to 2 years) female mice were scarified and inoculated with 1.0 x 10(8) colony-forming units of P. aeruginosa ATCC 19660. Eyes were scored for corneal pathology (0 to +4) at 6, 12, 24, 48, 72, 96, and 120 hours after infection. At each time point, six mice were killed from each age group, and both eyes were enucleated. Eyes (three infected, three uninfected) were embedded in OCT compound, frozen in liquid nitrogen, sectioned on a cryostat, and stained for ICAM-1 and LFA-1 immunoreactivity. The remaining six eyes (three infected, three uninfected) were embedded in eponaraldite resin, thick sectioned, and stained for light microscopic histopathologic examination. RESULTS: Immunostaining of slight to moderate intensity for ICAM-1 was seen on conjunctival fibroblasts, stromal keratocytes, corneal epithelium, and endothelium and conjunctival blood vessel endothelium of uninfected contralateral eyes in both age groups. In response to P. aeruginosa infection, only young animals were capable of upregulating ICAM-1 (as evidenced by an increase in the intensity of immunostaining) on these cells when compared to aged mice. Conversely, the intensity of immunostaining for LFA-1, a ligand for ICAM-1 on infiltrating leukocytes, was similar despite animal age. On gross observation, corneal pathology was more severe in young mice 24 to 96 hours after infection. Histopathologically, in contrast to young mice, eyes of aged animals 24 to 48 hours after infection had significantly fewer inflammatory cells, such as polymorphonuclear leukocytes (PMNs), infiltrating the corneal stroma and adhering to the endothelium near wound sites. CONCLUSION: These data suggest that the disparate response to ocular P. aeruginosa infection in young versus aged mice is due, at least in part, to the inability of aged animals to upregulate ICAM-1 above constitutively expressed levels. Consequently, the migration of inflammatory cells (PMNs) into infected corneas of aged mice is delayed, perhaps facilitating bacterial growth and contributing to a poor prognosis.

Immunization against experimental Pseudomonas aeruginosa and Serratia marcescens keratitis. Vaccination with lipopolysaccharide endotoxins and proteases.

Rabbits vaccinated with lipopolysaccharide endotoxins or with purified protease preparations from Pseudomonas aeruginosa and Serratia marcescens before corneal challenge with the viable bacteria exhibited significantly less corneal damage than rabbits not vaccinated with the bacterial products. However, the rabbits vaccinated with the lipopolysaccharide endotoxin preparations were significantly better protected than rabbits vaccinated with the bacterial proteases. Rabbits vaccinated with antisera raised against the proteases showed significantly less corneal damage than rabbits vaccinated with normal rabbit serum, and the passive protection was not significantly different than that elicited by active immunization against the bacterial proteases. The ability of the antiserum raised against the pseudomonas elastolytic protease to passively protect against severe corneal damage produced by experimentally induced pseudomonas keratitis was confirmed in mice. These findings support the idea that the bacterial endotoxins and proteases are virulence factors during the development of pseudomonas and serratia keratitis.