Quantification of ocular inflammation: Evaluation of polymorphonuclear leucocyte infiltration by measuring myeloperoxidase activity

Hobden

et al. 2000

123

134

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.

Section: Quantitation Of Pmn In Corneal Tissuesmentioning

confidence: 99%

“…A myeloperoxidase (MPO) assay was used to quantitate the total number of PMN infiltrating the cornea following infection (23,24). At selected times after infection (between 1 and 7 days p.i.…”

Section: Quantitation Of Pmn In Corneal Tissuesmentioning

confidence: 99%

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

Kernacki

Hobden

et al. 2000

123

134

“…The change in absorbance at 460 nm was monitored for 5 min at 30-s intervals, and the results were expressed as units of MPO per cornea. One unit of MPO activity is equivalent to 2 ϫ 10 5 PMN (24).…”

Section: Myeloperoxidase (Mpo) Assaymentioning

confidence: 99%

β-Defensins 2 and 3 Together Promote Resistance toPseudomonas aeruginosaKeratitis

McClellan

et al. 2009

Defensins play an important role in both innate and adaptive immunity due to their antimicrobial, regulatory, and chemotactic effects. Nonetheless, the role of murine β-defensins (mBD) 3 and 4, the murine homologs of human β-defensins (hBD) 2 and 3, remains unknown in Pseudomonas aeruginosa keratitis. This study explored their role in corneal infection and potential synergy with mBD2, a defensin associated with better outcome in this disease. Immunostaining and real-time RT-PCR data demonstrated that mBD3 and mBD4 expression was inducible and differentially regulated in the infected cornea of resistant BALB/c vs susceptible C57BL/6 (B6) mice. Knockdown studies using small interfering RNA treatment indicated that mBD3, but not mBD4, is required in ocular defense. Moreover, in vivo studies demonstrated individual and combined effects of mBD2 and mBD3 that modulate bacterial load, polymorphonuclear neutrophil (PMN) infiltration, and production of IFN-γ, MIP-2, IL-1β, TNF-α, inducible NO synthase (iNOS), TLR2, TLR4, MyD88, and NF-κB. Most notably, bacterial load was increased at 5 days postinfection by silencing either mBD2 or mBD3, but it was elevated at both 1 and 5 days postinfection when silencing both defensins. PMN infiltration was increased at 1 day postinfection by silencing both defensins or mBD3, but not mBD2 alone. iNOS expression was elevated by silencing mBD2, but it was reduced after silencing mBD3 or both defensins. Additionally, cell sources of mBD2 (macrophages, PMN and fibroblasts) and mBD3 (PMN) in corneal stroma were identified by dual label immunostaining after infection. Collectively, the data provide evidence that mBD2 and mBD3 together promote resistance against corneal infection.

“…The slope of the line was determined for each individual sample and used to calculate the number of units of MPO in the tissue. One unit of MPO activity is defined as that degrading 1 mol of peroxide per minute (21). To ensure reproducibility of the data, the MPO assay was repeated once similarly.…”

Section: Quantitation Of Pmn In Corneamentioning

confidence: 99%

Prolonged Elevation of IL-1 inPseudomonas aeruginosaOcular Infection Regulates Macrophage-Inflammatory Protein-2 Production, Polymorphonuclear Neutrophil Persistence, and Corneal Perforation

Rudner

Kernacki

et al. 2000

147

124

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.