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CYSTIC FIBROSIS ASSOCIATED-PSEUDOMONAS AERUGINOSA STIMULATES CCL20/MIP-3a PRODUCTION BY HUMAN MAST CELLS THROUGH DISTINCT MECHANISMS

Lin TJ,^1,2, Maher LH¹, McCurdy J¹,GomiK¹, Issekutz TB^1,2, Garduno R¹, and Marshall JS^1,3.

¹Depts. Of Microbiology-Immunology, ²Pediatrics, and ³Pathology,

Dalhousie University, Halifax, Nova Scotia, Canada

Objective: To investigate if mast cells (MC) produce chemokine macrophage inflammatory protein 3a (MIP-3a), also known as CCL20, in response to Pseudomonas aeruginosa, and to investigate the regulation of MIP-3a production by anti-inflammatory agents.

Methods: Human cord blood-derived MC (CBMC), human mast cell line HMC-1, and cystic fibrosis-associated P.aeruginosa were used. MIP-3a production was determined by RT-PCR, Western blot and ELISA.

Results: We identified that human MC are able to produce MIP-3a, in response to P.aeruginosa, but not following IgE mediated activation. Significant MIP-3a production in response to P. Aeruginosa stimulation was observed in both HMC-1 and CBMC. MIP-3a production by CBMC peaked at 6 h after P.aeruginosa-stimulation, a time-course of expression distinct from other cytokines, such as GM-CSF which peaked after 24 to 48 h. Using protein kinase C (PKC) activators PMA or diacylglycerol, and PKC inhibitor Ro 31-8220, we demonstrated that PKC is involved in MIP-3a production by CBMC. Interestingly, P.aeruginosa-induced production of MIP-3a by CBMC was resistant to treatment of IL-10 and dexomethasone (Dex). In contrast, production of TNFα and GM-CSF by CBMC was completely inhibited by IL-10 and Dex.

Conclusion: MC produce MIP-3a through distinct mechanisms. This study supports the notion that MC function as sentinel cells in host defense against pathogens through production of chemokines such as MIP-3a.

Supported by Canadian Institutes of Health Research and Nova Scotia Health Research Foundation.