Selective early production of CCL20, or macrophage inflammatory protein 3alpha, by human mast cells in response to Pseudomonas aeruginosa

Abstract

Mast cells are important as sentinel cells in host defense against bacterial infection. Much of their effectiveness depends upon recruiting other immune cells; however, little is known about the mechanisms of this response. CCL20, also known as macrophage inflammatory protein-3alpha (MIP-3alpha), Exodus, and LARC, is a chemokine known to be a potent chemoattractant for immature dendritic cells and T cells. In this study, we examined the human mast cell production of both CCL20 and granulocyte-macrophage colony-stimulating factor (GM-CSF), a critical cytokine for innate immune responses in the lung, in response to Pseudomonas aeruginosa. Reverse transcription-PCR and Western blot analysis demonstrated that the human mast cells (HMC-1) express CCL20 mRNA and are able to produce a significant amount (32.4 ng/ml) of CCL20 protein following stimulation by calcium ionophore and phorbol myristate acetate. Importantly, P. aeruginosa potently stimulated CCL20 production in human cord blood-derived mast cells (CBMC), with production peaking at 6 h after stimulation. This time course of expression was distinct from that of GM-CSF, which peaked after 24 to 48 h. Significant CCL20 production did not occur following immunoglobulin E-mediated activation of CBMC under conditions which induced a substantial GM-CSF response. Interestingly, the CCL20 response of mast cells to P. aeruginosa was relatively resistant to inhibition by the corticosteroid dexamethasone, interleukin-10, or cyclosporine, while GM-CSF production was potently inhibited. However, P. aeruginosa-induced CCL20 production was blocked by the protein kinase C (PKC) inhibitor Ro 31-8220 and a PKC pseudosubstrate. These results support a role for human mast cells in the initiation of immune responses to P. aeruginosa infection.

FIG. 1.

mRNA expression and protein secretion of CCL20 by mast cells. (a) RNA samples from HMC-1 cells were analyzed by RT-PCR for CCL20, yielding PCR products of 216 and 269 bp for CCL20 and β2M, respectively. Note the prominent single products at the appropriate sizes. Lanes 1 and 2, CCL20; lanes 3 and 4, β2M. (b) Supernatants from HMC-1 cells stimulated with A23187 (10⁻⁶ M) and PMA (10⁻⁷ M) for 24 h were analyzed for CCL20 by Western blotting. Lane 1, human recombinant CCL20; lanes 2 and 3, supernatants from A23187- and PMA-treated HMC-1 cells from two independent experiments. (c) CCL20 production by human mast cells. HMC-1 cells were stimulated with A23187 (10⁻⁶ M) and PMA (10⁻⁷ M) for 1 to 24 h. Cell-free supernatants were used to test CCL20 levels by ELISA. Results are the means ± standard errors of the means for three experiments.

FIG. 2.

P. aeruginosa but not L. pneumophila stimulated CCL20 production by human CBMC (a) and the human mast cell line HMC-1 (b). Human mast cells (10⁶ cells/ml) were stimulated with P. aeruginosa (CF-associated mucoid strain 8821 and nonmucoid strain PAO.1), L. pneumophila (cell-to-bacteria ratio, 1/50), or LPS (from P. aeruginosa serotype 10; Sigma) (10 μg/ml) for 24 h. CCL20 levels in cell-free supernatants were determined by ELISA. Results are means ± standard errors of the means from eight individual donors (a) and from four independent experiments (b).

FIG. 3.

Distinct time course of CCL20 (a and b) and GM-CSF (c and d) production by human CBMC as a result of CF-associated P. aeruginosa (8821) stimulation. CBMC (10⁶ cells/ml) were stimulated with P. aeruginosa (8821) (cell-to-bacteria ratio, 1/50) for various times. CCL20 and GM-CSF levels in cell-free supernatants were determined by ELISA. Mean values ± standard errors of the means of the results from triplicate assays are shown for each donor.

FIG. 4.

P. aeruginosa-induced production of CCL20 (a and b) by human mast cells is not inhibited by IL-10, but that of GM-CSF (c and d) is inhibited. Human CBMC (5 × 10⁵ cells/ml) were stimulated with P. aeruginosa strain 8821 (mast cell-to-bacteria ratio, 50:1) in the presence or absence of various concentrations (5, 50, and 500 ng/ml) of IL-10 for 24 h. Cell-free supernatants were used to determine cytokine levels by ELISA. Mean results from triplicate assays are shown for each of two representative cell donors.

FIG. 5.

Differential effects of dexamethasone (Dex) on P. aeruginosa-induced CCL20 (a and b) and GM-CSF (c and d) production by human mast cells. Human CBMC (5 × 10⁵ cells/ml) were treated with P. aeruginosa strain 8821 (mast cell-to-bacteria ratio, 50:1) in the absence (0 μM) or presence of various concentrations (0.1, 1, and 10 μM) of Dex for 24 h. Cell-free supernatants were used to determine cytokine levels by ELISA. Mean results from triplicate assays are shown for each of two representative cell donors.

FIG. 6.

Cyclosporine does not block P. aeruginosa-induced production of CCL20 (a and b) by human mast cells but blocks that of GM-CSF (c and d). Human CBMC (5 × 10⁵ cells/ml) were treated with P. aeruginosa strain 8821 (mast cell-to-bacteria ratio, 50:1) in the absence (0 ng/ml) or presence of various concentrations (10, 100, and 1,000 ng/ml) of cyclosporine for 24 h. Cell-free supernatants were used to determine cytokine levels by ELISA. Mean ELISA results from triplicate well assays are shown for each of two representative donors.

FIG. 7.

PKC inhibitors block P. aeruginosa-induced CCL20 production by human mast cells. (a and b) CBMC (5 × 10⁵ cells/ml) were treated with P. aeruginosa strain 8821 (Ps.a) for 24 h in the presence (10 μM) or absence of Ro 31-8220 (Ro). Results are the mean values from triplicate wells from representative donor responses. (c) HMC-1 5C6 cells were treated with a cell-permeable PKC pseudosubstrate, a sequence derived from PKCα and PKCβ (PKC peptide; 20 μM). Cell-free supernatants were used to determine CCL20 proteins by ELISA. Values are the means ± standard errors of the means for four similar experiments.