Production of macrophage inflammatory protein 3alpha (MIP-3alpha) (CCL20) and MIP-3beta (CCL19) by human peripheral blood neutrophils in response to microbial pathogens

Abstract

Effects of bacterial pathogens on the production of macrophage inflammatory protein 3alpha (MIP-3alpha) and MIP-3beta from human peripheral blood neutrophils were investigated. Neutrophils produced both chemokines by coincubation with either gram-positive or gram-negative bacteria. Neutrophils may initiate antigen-specific immune responses through the release of these chemokines that are capable of promoting selective recruitment of dendritic cells and T-cell subsets.

FIG. 1.

Induction of MIP-3α and MIP-3β in neutrophils by microbial pathogens. (A) Neutrophils were incubated with killed E. coli at a ratio of 1:10 for the indicated times using RPMI 1640 medium supplemented with 5% heat-inactivated fetal calf serum. (B) Neutrophils were incubated with gram-positive or gram-negative bacteria at a ratio of 1:10 for 6 h. Gene expression of MIP-3α and MIP-3β was determined by reverse transcription-PCR followed by Southern blot hybridization. These results were representative of three separate experiments using neutrophils isolated from different donors.

FIG. 2.

Immunostaining of MIP-3α and MIP-3β in neutrophils. Neutrophils were incubated with E. coli at a ratio of 1:10 and fixed with 4% paraformaldehyde. Immunostaining of MIP-3α (A) and MIP-3β (B) was performed by using mouse antibodies against human MIP-3α and MIP-3β.

FIG. 3.

Production of chemokines by neutrophils stimulated with microbial pathogens. (A) Neutrophils were incubated with killed E. coli at a ratio of 1:10 for the indicated times. (B) Neutrophils were incubated with various microbial pathogens (1:10) or LPS (10 ng/ml) for 48 h. Chemokines in the culture supernatant were determined by enzyme-linked immunosorbent assay. Data represent means ± standard deviations. These results were representative of three separate experiments using neutrophils isolated from independent donors.