Beta-chemokines are induced by Mycobacterium tuberculosis and inhibit its growth

Abstract

Chemokines (CK) are potent leukocyte activators and chemoattractants and aid in granuloma formation, functions critical for the immune response to Mycobacterium tuberculosis. We hypothesized that infection of alveolar macrophages (AM) with different strains of M. tuberculosis elicits distinct profiles of CK, which could be altered by human immunodeficiency virus (HIV) infection. RANTES, macrophage inflammatory protein-1 alpha (MIP-1 alpha), and MIP-1 beta were the major beta-CK produced in response to M. tuberculosis infection. Virulent M. tuberculosis (H37Rv) induced significantly less MIP-1 alpha than did the avirulent strain (H37Ra), while MIP-1 beta and RANTES production was comparable for both strains. MIP-1 alpha and MIP-1 beta were induced by the membrane, but not cytosolic, fraction of M. tuberculosis. M. tuberculosis-induced CK secretion was partly dependent on tumor necrosis factor alpha (TNF-alpha). AM from HIV-infected individuals produced less TNF-alpha and MIP-1 beta than did normal AM in response to either M. tuberculosis strain. We tested the functional significance of decreased beta-CK secretion by examining the ability of beta-CK to suppress intracellular growth of M. tuberculosis. MIP-1 beta and RANTES suppressed intracellular growth of M. tuberculosis two- to threefold, a novel finding. Thus, beta-CK contribute to the innate immune response to M. tuberculosis infection, and their diminution may promote the intracellular survival of M. tuberculosis.

FIG. 1.

Less induction of MIP-1α by H37Rv than by H37Ra. AM from normal (n = 8) and HIV-infected (n = 8) individuals were infected with M. tuberculosis for 72 h at 37°C, and the resulting supernatants were assayed by ELISA for MIP-1α. Both H37Ra and H37Rv induced significant quantities of MIP-1α compared to uninfected control AM. H37Rv induced significantly less MIP-1α production than did H37Ra in normal AM. Single asterisks indicate a significant difference compared to uninfected AM; double asterisks indicate a significant difference compared to H37Ra-infected AM from HIV-positive individuals.

FIG. 2.

AM from HIV-positive individuals produce less MIP-1β than control AM in response to M. tuberculosis. Supernatants of AM from normal (n = 8) and HIV-infected (n = 8) individuals were infected with M. tuberculosis as described for Fig. 1 and assayed by ELISA for MIP-1β. Induction of MIP-1β in normal AM was comparable for both H37Ra and H37Rv. AM from the HIV group produced significantly less MIP-1β than did normal AM in response to both H37Ra and H37Rv. Asterisks indicate a significant difference compared to uninfected (Un) AM. Number signs indicate a significant difference compared to the corresponding condition for AM from HIV-infected individuals.

FIG. 3.

Infection of AM with M. tuberculosis induces production of RANTES. RANTES in supernatants from AM infected as described for Fig. 1 was assayed by ELISA. Although both H37Ra and H37Rv induced significant quantities of RANTES in AM from normal subjects (n = 6) and from HIV-infected individuals (n = 8), AM from the latter group produced significantly less RANTES than did normal AM. Asterisks indicate a significant difference compared to uninfected (Un) AM, and number signs indicate a P value of 0.055 (not statistically significant).

FIG. 4.

Infection of AM with M. tuberculosis induces production of MCP-1. MCP-1 in supernatants from AM infected as described for Fig. 1 was assayed by ELISA. Statistically significant increases in MCP-1 were seen with H37Ra and H37Rv in control AM. Only H37Ra induced statistically significant increases in MCP-1 in AM from HIV-infected individuals. Asterisks indicate a significant difference compared to uninfected (Un) AM.

FIG. 5.

Infection of control AM with M. tuberculosis induces production of IP-10. IP-10 in supernatants from AM infected as described for Fig. 1 was assayed by ELISA. Infection with either H37Ra or H37Rv induced significant quantities of IP-10 in control AM compared to uninfected AM but not compared to AM from HIV-infected individuals. Asterisks indicate a significant difference compared to uninfected (Un) AM, and number signs indicate a significant difference between control AM and AM from HIV-infected individuals.

FIG. 6.

The mycobacterial membrane fraction induces AM production of MIP-1α and MIP-1β. AM from normal individuals were treated with either soluble membrane (black bars) or cytosolic (stippled bars) fractions of H37Rv for 72 h at 37°C. Supernatants were assayed by ELISA for MIP-1α (a) and MIP-1β (b) (n = 5). Both of these β-CK were induced by the membrane fraction but not by the cytosolic fraction, especially at higher doses. Error bars indicate standard errors of the means. Ag, antigen.

FIG. 7.

MIP-1β and RANTES suppress intracellular growth of M. tuberculosis. AM from control individuals (n = 11) were infected for 4 h with H37Rv and washed, and either isotype control or neutralizing antibodies to MIP-1α, MIP-1β, or RANTES were added. After 6 days of culture, cells were lysed and cultured on Middlebrook 7H11 agar for 21 days, and the number of CFU was counted at 21 days. Neutralization of MIP-1β or RANTES induced significant growth enhancement of M. tuberculosis within AM compared to control antibody-treated AM Asterisks indicate a significant difference compared to the isotype control. Error bars indicate standard errors of the means.