PURPOSE: Respiratory failure due to alveolar inflammation during Pneumocystis pneumonia is a major cause of death in immunosuppressed patients. Recent data indicate that airway epithelial cells (AEC) release IL-8 in response to pulmonary pathogens through NF-δB. We have shown in rat epithelial cells that Pneumocystis carinii cell wall β-Glucan components (PCBG) elicit NF-δB-mediated release of MIP-2, the rat homolog of IL-8. However, the mechanisms that regulate IL-8 in PCBG activated human AEC are poorly understood. We therefore studied the roles of PI3K/AKT and MAPKs as effectors of IL-8 secretion in the PCBG activated human airway epithelial cell line; 1HAEo-.
METHODS: IL-8 was measured in the supernatant of PCBG activated AEC by ELISA. To elucidate the role of MAPKs and PI3K in IL-8 secretion, cells were pre-incubated with different pharmacological inhibitors prior stimulation: supernatants were colected and IL-8 was measure by ELISA. Phosphorylated proteins were detected by Immunoblot using specific antibodies. The relevance of the NF-δB site in the IL-8 promoter was evaluated by reporter assay in PCBG stimulated cells.
RESULTS: Our data demonstrates that PCBG induces IL-8 in AEC. This IL-8 activation was shown to be mediated by NF-δB through mutation of the NF-kB binding site in the IL-8 promoter, which fails to induce gene transcription. PCBG was also found to induce phosphorylation of ERK kinase and AKT. Additionally, pharmacological inhibitors of ERK and PI3K were demonstrated to decrease IL-8 release by AEC. Interestingly PI3K inhibitors blocks AKT phosphorylation but did not inhibit phosphorylation of ERK.
CONCLUSION: All together, these data provides evidence that PCBG-challenged AEC induces IL-8 secretion in an NF-δB-dependent manner. We have also demonstrated that IL-8 secretion involves activation of more than one pathway, since the ERK and PI3K/AKT pathways both get activated. Moreover, our data shows that inhibition of PI3K does not affect activation of ERK, indicating that AKT and ERK pathways are activated in different ways.
CLINICAL IMPLICATIONS: Our data provides new insights into the role of AEC in the host inflammatory response induced by PCBG.
DISCLOSURE: Eva Carmona, None.