Human {beta}-defensin 2 is expressed and associated with Mycobacterium tuberculosis during infection of human alveolar epithelial cells

Abstract

To determine the role of human beta-defensin 2 (HBD-2) in human tuberculosis, we studied the in vitro induction of HBD-2 gene expression by Mycobacterium tuberculosis H37Rv infection in the human lung epithelial cell line A549, in alveolar macrophages (AM), and in blood monocytes (MN) by reverse transcription-PCR. We also studied the induction of HBD-2 gene expression by mannose lipoarabinomannan (manLAM) from M. tuberculosis. Intracellular production of HBD-2 peptide was detected by immunocytochemistry and electron microscopy. Our results demonstrated that there was induction of HBD-2 mRNA in A549 cells after infection with M. tuberculosis at various multiplicities of infection (MOI) and that there was stimulation with manLAM. AM expressed the HBD-2 gene only at a high MOI with M. tuberculosis. MN did not express HBD-2 at any of the experimental M. tuberculosis MOI. Immunostaining revealed the presence of intracellular HBD-2 peptide in A549 cells following infection with M. tuberculosis, and the staining was more intense in areas where there were M. tuberculosis clusters. By using electron microscopy we also demonstrated production of HBD-2 after M. tuberculosis infection and adherence of HBD-2 to the membranes of M. tuberculosis. Alveolar epithelial cells are among the first cells to encounter M. tuberculosis following aerogenic infection. As HBD-2 has been shown to control growth of M. tuberculosis and has chemotactic activity, our results suggest that HBD-2 induction by M. tuberculosis may have a role in the pathogenesis of human tuberculosis.

Figures

FIG. 1.

Expression of the HBD-2 gene in A549 cells infected with M. tuberculosis at different MOI for 24 h. Lane 1, molecular weight marker; lane 2, positive control; lane 3, negative control; lane 4, MOI of 0.1:1; lane 5, MOI of 1:1; lane 6, MOI of 5:1; lane 7, MOI of 10:1.

FIG. 2.

HBD-2 gene expression in alveolar macrophages. Lane 1, molecular weight marker; lane 2, positive control; lane 3, negative control; lanes 4 and 7, nonstimulated alveolar macrophages; lanes 5 and 8, HBD-2 expression in alveolar macrophages infected with M. tuberculosis at an MOI of 70:1 at 18 and 24 h postinfection, respectively; lanes 6 and 9, HBD-2 expression in alveolar macrophages infected at an MOI of 350:1 at 18 and 24 h postinfection, respectively.

FIG. 3.

Expression of HBD-2 in A549 cells upon stimulation with manLAM. At 24 h after stimulation of A549 cells with manLAM at concentrations of 1 μg/ml (lane 8), 5 μg/ml (lane 9), 10 μg/ml (lane 10), and 20 μg/ml (lane 11) expression of the HBD-2 gene was detected by reverse transcription-PCR. Stimulation for 18 h with the same concentrations of manLAM (lanes 3 to 7) did not induce expression of HBD-2. A positive control band is shown in lane 2. Lane 12 contained a negative control.

FIG. 4.

(A) Noninfected cells (negative control). (B) Immunodetection of HBD-2 in A549 cells 24 h after infection with M. tuberculosis at an MOI of 5:1.

FIG. 5.

(A) Immunofluorescence of M. tuberculosis 24 h after infection of A549 cells at an MOI of 5:1. (B) Colocalization and association of HBD-2 with M. tuberculosis in the same microscopic field (arrows).

FIG. 6.

Immunoelectron microcopy of HBD-2 in A549 cells following a 24-h in vitro infection with M. tuberculosis. (A) Low-power micrograph showing phagocytosed bacilli (arrows) in the cytoplasm of an A549 cell. Magnification, ×8,000. (B) Immunoelectron micrograph of a mycobacterium on the surface of the A549 cell membrane. There is HBD-2 immunoreactivity on the bacterial cell wall. Magnification, ×50,000. (C) Intracellular mycobacterium showing HBD-2 immunolabeling in the cell wall and cytoplasm. Magnification, ×50,000. (D) Endocytosed and disrupted mycobacterium showing HBD-2 immunoreactivity in its cytoplasm and cell wall. Magnification, ×40,000.