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Expression of macrophage migration inhibitory factor during Pseudomonas keratitis
1Cooperative Research Centre For Eye Research and Technology, The University of New South Wales, Sydney, Australia. a.thakur@cclru.unsw.edu.au
Abstract:
Macrophage migration Inhibitory factor (MIF) is a recently rediscovered pro-inflammatory cytokine, and has been shown to play a role in the regulation of neutrophil chemokines and angiogenesis. Corneal epithelial and endothelial cells have been shown to express MIF. This study evaluated the expression of MIF during Pseudomonas keratitis in mice and in vitro using a corneal epithelial cell line. Three strains of P. aeruginosa, 6294 (invasive strain), 6206 (cytotoxic strain) and Paer1 (non-infectious strain) were used. Both cytotoxic and invasive strains were isolated from human corneal ulcers and the Paer1 strain was isolated from a non-infectious condition. Following challenge in mouse corneas or a corneal epithelial cell line, corneal homogenates or lysed corneal epithelial cells were used to isolate RNA. Migration inhibitory factor mRNA expression in the mouse cornea or human corneal epithelial cells was examined by reverse transcription-polymerase chain reaction analysis, and was found to be expressed as early as 4 h after the injury (scratch controls) or infection in the mouse corneas. Migration inhibitory factor mRNA in scratch controls and Paer1-inoculated corneas showed peak levels at 4 h post-challenge and this dropped by 24 h post-challenge. Corneas challenged with invasive and cytotoxic strains showed peak expression 24 h post-challenge. Migration inhibitory factor mRNA levels were significantly higher in invasive and cytotoxic strain inoculated corneas compared to Paer1 inoculated corneas. Challenging the corneal epithelial cell line with Pseudomonas 6294 and 6206 strains induced peak expression at 8 h and levels were decreased by 12 h. Epithelial cells inoculated with recombinant human interleukin-1beta protein induced very high levels of MIF mRNA at all time points compared to infected and control corneal epithelial cells. High expression of MIF in the infected corneas suggests that it may have a role in the pathogenesis of corneal disease induced by invasive and cytotoxic strains of P. aeruginosa.
Insights
Macrophage migration inhibitory factor (MIF) expression increases in corneas infected with invasive or cytotoxic Pseudomonas aeruginosa strains. This suggests MIF plays a role in the pathogenesis of bacterial keratitis.
Area of Science:
- Ophthalmology
- Immunology
- Microbiology
Background:
- Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine involved in neutrophil chemokine regulation and angiogenesis.
- Corneal epithelial and endothelial cells express MIF.
- The role of MIF in Pseudomonas keratitis is not fully understood.
Purpose of the Study:
- To evaluate MIF expression during Pseudomonas keratitis in mice.
- To investigate MIF expression in vitro using a corneal epithelial cell line.
- To determine the correlation between MIF expression and P. aeruginosa strain virulence.
Main Methods:
- Mice and a human corneal epithelial cell line were challenged with three strains of P. aeruginosa (invasive, cytotoxic, non-infectious).
- MIF mRNA expression was analyzed using reverse transcription-polymerase chain reaction (RT-PCR) at various time points post-infection.
- Interleukin-1beta was used as a positive control for MIF induction.
Main Results:
- MIF mRNA was detected as early as 4 hours post-injury or infection in mouse corneas.
- Invasive and cytotoxic P. aeruginosa strains induced significantly higher MIF mRNA levels compared to the non-infectious strain.
- Peak MIF mRNA expression occurred at 24 hours post-challenge with invasive/cytotoxic strains in mice and 8 hours in cell lines.
- Interleukin-1beta strongly induced MIF mRNA expression in corneal epithelial cells.
Conclusions:
- High MIF expression in infected corneas suggests its involvement in the pathogenesis of Pseudomonas keratitis.
- Virulent strains of P. aeruginosa elicit a stronger MIF response.
- MIF may be a key mediator in the inflammatory response to bacterial keratitis.