Abstract
Intestinal inflammation is exacerbated by defects in the epithelial barrier and subsequent infiltration of microbes and toxins into the underlying mucosa. Production of chemokines and antimicrobial peptides by an intact epithelium provide the first line of defense against invading organisms. In addition to its antimicrobial actions, human beta defensin-2 (HBD2) may also stimulate the migration of dendritic cells through binding the chemokine receptor CCR6. As human colonic epithelium expresses CCR6, we investigated the potential of HBD2 to stimulate intestinal epithelial migration. Using polarized human intestinal Caco2 and T84 cells and non-transformed IEC6 cells, HBD2 was equipotent to CCL20 in stimulating migration. Neutralizing antibodies confirmed HBD2 and CCL20 engagement to CCR6 were sufficient to induce epithelial cell migration. Consistent with restitution, motogenic concentrations of HBD2 and CCL20 did not induce proliferation. Stimulation with those CCR6 ligands leads to calcium mobilization and elevated active RhoA, phosphorylated myosin light chain, and F-actin accumulation. HBD2 and CCL20 were unable to stimulate migration in the presence of either Rho-kinase or phosphoinositide 3-kinase inhibitors or an intracellular calcium chelator. Together, these data indicate that the canonical wound healing regulatory pathway, along with calcium mobilization, regulates CCR6-directed epithelial cell migration. These findings expand the mechanistic role for chemokines and HBD2 in mucosal inflammation to include immunocyte trafficking and killing of microbes with the concomitant activation of restitutive migration and barrier repair.
Highlights
Defensins, like chemokines, are highly conserved key host defense molecules that participate in host defense through the direct killing of microbes [7]
Using epithelial cell model systems we demonstrate for the first time that human beta defensin-2 (HBD2) and CCL20 stimulate restitutive intestinal cell migration through mobilization of intracellular calcium, activation of phosphoinositide 3-kinase (PI3K), monomeric RhoGTPase, and myosin light chain (MLC) signaling pathways
Stimulation of migration was specific for the inducible defensin HBD2, because IEC-6 monolayers treated with 20 ng/ml of the constitutively expressed defensin HBD1 did not significantly increase migration (Fig. 1C)
Summary
Materials—Recombinant HBD2 and human CCL20 was purchased from Peprotech (Rocky Hill, NJ), and were 96 and 99% pure as defined by the manufacturer. To assess cell migration signaling mechanisms, monolayers were pre-treated for 30 min with Y27632 (10 M), LY294002 (2–50 M), or BAPTA-AM (10 M) and stimulated in the presence or absence of HBD2 and CCL20. Immunoblot Analysis—IEC-6 cells were grown to 80% confluence and serum-starved 24 h before stimulation with titrated doses of HBD2 or CCL20. Fluorescence Microscopy—IEC-6 cell were grown to 30% confluence, washed in PBS, and fixed in 4% (w/v) paraformaldehyde for 15 min. After a wash step in PBS, the cells were incubated in 1% (w/v) bovine serum albumin/PBS for 30 min, followed by an overnight incubation with a 1:50 dilution of rabbit polyclonal anti-CKR6 antibody (Santa Cruz Biotechnology) that binds CCR6. The cells were washed in buffer and incubated 1 h with 1 g/ml mouse-anti-GST (Cell Signaling) or mouse IgG (Molecular Probes) in 1% (w/v) bovine serum albumin/PBS at room temperature. Statistical Analysis—Differences between unstimulated controls, and experimental samples were analyzed using an unpaired Student’s t test using SigmaStat (Jandel Scientific Software, San Rafael, CA)
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