Karen A. Kernacki

Prolonged Elevation of IL-1 in Pseudomonas aeruginosa Ocular Infection Regulates Macrophage-Inflammatory Protein-2 Production, Polymorphonuclear Neutrophil Persistence, and Corneal Perforation

The kinetics of IL-1 (α and β) production after Pseudomonas aeruginosa corneal infection was examined in susceptible (cornea perforates) C57BL/6J (B6) and resistant (cornea heals) BALB/cByJ (BALB/c) mice. IL-1α and -1β (mRNA and protein) were elevated in both mouse strains, and levels peaked at 1 day postinfection (p.i.). Significantly greater amounts of IL-1 protein were detected in B6 vs BALB/c mice at 1 and 3 days p.i. At 5 days p.i., IL-1α and -1β (mRNA and protein) remained elevated in B6, but began to decline in BALB/c mice. To test the significance of elevated IL-1 in B6 mice, a polyclonal neutralizing Ab against IL-1β was used to treat infected B6 mice. A combination of subconjunctival and i.p. administration of IL-1β polyclonal Ab significantly reduced corneal disease. The reduction in disease severity in infected B6 mice was accompanied by a reduction in corneal polymorphonuclear neutrophil number, bacterial load, and macrophage inflammatory protein-2 mRNA and protein levels. These data provide evidence that IL-1 is an important contributor to P. aeruginosa corneal infection. At least one mechanism by which prolonged and/or elevated IL-1 expression contributes to irreversible corneal tissue destruction appears to be by increasing macrophage inflammatory protein-2 production, resulting in a prolonged stimulation of polymorphonuclear neutrophil influx into cornea. In contrast, a timely down-regulation of IL-1 appears consistent with an inflammatory response that is sufficient to clear the bacterial infection with less corneal damage.

Macrophage Inflammatory Protein-2 Is a Mediator of Polymorphonuclear Neutrophil Influx in Ocular Bacterial Infection

Polymorphonuclear neutrophils (PMN) in Pseudomonas aeruginosa-infected cornea are required to clear bacteria from affected tissue, yet their persistence may contribute to irreversible tissue destruction. This study examined the role of C-X-C chemokines in PMN infiltration into P. aeruginosa-infected cornea and the contribution of these mediators to disease pathology. After P. aeruginosa challenge, corneal PMN number and macrophage inflammatory protein-2 (MIP-2) and KC levels were compared in mice that are susceptible (cornea perforates) or resistant (cornea heals) to P. aeruginosa infection. While corneal PMN myeloperoxidase activity (indicator of PMN number) was similar in both groups of mice at 1 and 3 days postinfection, by 5–7 days postinfection corneas of susceptible mice contained a significantly greater number of inflammatory cells. Corneal MIP-2, but not KC, levels correlated with persistence of PMN in the cornea of susceptible mice. To test the biological relevance of these data, resistant mice were treated systemically with rMIP-2. This treatment resulted in increased corneal PMN number and significantly exacerbated corneal disease. Conversely, administration of neutralizing MIP-2 pAb to susceptible mice reduced both PMN infiltration and corneal destruction. Collectively, these findings support an important role for MIP-2 in recruitment of PMN to P. aeruginosa-infected cornea. These data also strongly suggest that a timely down-regulation of the host inflammatory response is critical for resolution of infection.

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.

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.

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

The role of macrophage inflammatory protein‐1α (MIP‐1α) 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‐1α exacerbated disease and was associated with an increased number of neutrophils (PMNs) in the cornea. Treatment of BALB/c mice with rMIP‐1α 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‐1α polyclonal antibody also significantly reduced PMN numbers and pathology. Collectively, evidence is provided that MIP‐1α directly contributed to CD4+ T cell recruitment and indirectly to PMN persistence in the infected cornea.

Corneal cell proteins and ocular surface pathology.

The cornea is a transparent and avascular tissue that functions as the major refractive structure for the eye. A wide variety of growth factors, chemokines, cytokines and their receptors are synthesized by corneal epithelial and stromal cells, and are found in tears. These molecules function in corneal wound healing and in inflammatory responses. Proteoglycans and glycoproteins are essential for normal corneal function, both at the air-epithelial interface and within the extracellular matrix. The ocular MUC mucins may play roles in forming the mucus layer of the tear film, in regulating tear film spread, and in inhibiting the adhesion of pathogens to the ocular surface. Lumican, keratocan and mimecan are the major keratan sulfate proteoglycans of the corneal stroma. They are essential, along with other proteoglycans and interfibrillar proteins, including collagens type VI and XII, for the maintenance of corneal transparency. Corneal epithelial cells interact with a specialized extracellular matrix structure, the basement membrane, composed of a specific subset of collagen type IV and laminin isoforms in addition to ubiquitous extracellular matrix molecules. Matrix metalloprotein-ases have been identified in normal corneal tissue and cells and may play a role in the development of ulcerative corneal diseases. Changes in extracellular matrix molecule localization and synthesis have been noted in other types of corneal diseases as well, including bullous keratopathy and keratoconus.

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≤= 0.005) by 30 days post infection. 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≤= 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.

Murine Model of Bacterial Keratitis

Publisher Summary This chapter describes a murine model of bacterial keratitis. Both inbred strains of mice and outbred mice are used in this model. The choice of whether to use an outbred mouse or an inbred mouse with a resistant or susceptible phenotype depend on the nature of the study. The anesthetic is administered by inhalation by placing a mouse within a jar containing the anesthetic. The degree of anesthesia can be monitored by the animals loss of equilibrium and rapid but steady respiration. The length of time required to anesthetize a mouse varies with the construction of the anesthetizing jar and with the size and strain of mouse. Following anesthesia, the mice are placed beneath a stereoscopic microscope and the central cornea of each mouse is scarified with three 1-mm incisions using a 26G needle. The wounds are randomly examined histologically to ensure that they penetrate the epithelial basal lamina, but extend no deeper than the superficial corneal stroma. After wounding, the bacterial suspension is topically applied to the scarified cornea by using a calibrated micropipette with a sterile disposable tip. After infection, the mice are placed into a holding cage to recover from the anesthesia. After recovery, they are transferred to a cage containing fresh bedding. At daily intervals after infection, animals are grossly observed to ensure that animals are infected and to grade the progression of the disease. To facilitate grading of sealed eyes, the eyelid may be gently swabbed with a sterile cotton swab moistened with sterile saline and then gently opened sufficiently widely to grade the ocular disease.