Chemokine production by a human alveolar epithelial cell line in response to Mycobacterium tuberculosis

Abstract

To investigate the role of chemokines during the initial local response to Mycobacterium tuberculosis in the human lung, we studied chemokine production by the human alveolar epithelial cell line A549 after infection with M. tuberculosis. M. tuberculosis-infected A549 cells produced mRNAs and protein for monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8) but not mRNAs for macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES. Chemokine production in response to M. tuberculosis was not dependent on production of tumor necrosis factor alpha, IL-1beta, or IL-6. Two virulent clinical M. tuberculosis isolates, the virulent laboratory strain H37Rv, and the avirulent strain H37Ra elicited production of comparable concentrations of MCP-1 and IL-8, whereas killed M. tuberculosis and three Mycobacterium avium strains did not. The three virulent M. tuberculosis strains grew more rapidly than the avirulent M. tuberculosis strain in the alveolar epithelial cell line, and the three M. avium strains did not grow intracellularly. These findings suggest that intracellular growth is necessary for mycobacteria to elicit production of MCP-1 and IL-8 by alveolar epithelial cells but that virulence and the rate of intracellular growth do not correlate with chemokine production. Alveolar epithelial cells may contribute to the local inflammatory response in human tuberculosis by producing chemokines which attract monocytes, lymphocytes, and polymorphonuclear cells.

FIG. 1

Chemokine mRNA expression of A549 cells infected with M. tuberculosis H37Ra. A549 cells were infected with H37Ra, and mRNA expression for five chemokines at various time points was determined by RT-PCR. The far-left lane shows molecular weight markers, the positive control was cDNA prepared from phytohemagglutinin-stimulated cells, and the negative control contained no cDNA.

FIG. 2

Competitive RT-PCR to quantitate mRNAs for MCP-1 and IL-8 in A549 cells infected with M. tuberculosis H37Ra. A549 cells were infected with H37Ra, and mRNA expression for MCP-1 and IL-8 at various time points was determined by competitive RT-PCR. The far-left lane shows molecular weight markers; the negative control contains no cDNA.

FIG. 3

Concentrations of MCP-1 (A) and IL-8 (B) in supernatants of A549 cells infected with M. tuberculosis (M. tb.) H37Ra or uninfected. A quantity of 10⁶ organisms represents approximately one bacillus per A549 cell.

FIG. 4

Mean concentrations of MCP-1 and IL-8 in supernatants of A549 cells infected with M. tuberculosis (M. tb.) H37Ra or uninfected. A quantity of 10⁶ organisms represents approximately one bacillus per A549 cell.

FIG. 5

Effect of anti-IL-6 (α-IL-6) on MCP-1 (A) and IL-8 (B) concentrations in supernatants of A549 cells infected with M. tuberculosis H37Ra. Exp., experiment.

FIG. 6

Mean concentrations of MCP-1 (A) and IL-8 (B) in supernatants of A549 cells cultured for 6 days with M. tuberculosis strains, M. avium, or lipopolysaccharide (LPS). The M. tuberculosis strains were H37Ra, H37Rv, and the clinical isolates S29 and S51.

FIG. 7

Growth of M. tuberculosis strains in A549 cells. A549 cells were infected with H37Ra, H37Rv, or clinical isolate S29 or S51 at a bacterium/cell ratio of 1:100, and CFU were measured at different time points. The values shown are the means and SEs for four separate experiments.