Activation of human neutrophils by mycobacterial phenolic glycolipids

Abstract

The interaction between mycobacterial phenolic glycolipids (PGLs) and phagocytes was studied. Human neutrophils were allowed to interact with each of four purified mycobacterial PGLs and the neutrophil production of reactive oxygen metabolites was followed kinetically by luminol-/isoluminol-amplified chemiluminescence. The PGLs from Mycobacterium tuberculosis and Mycobacterium kansasii, respectively, were shown to stimulate the production of oxygen metabolites, while PGLs from Mycobacterium marinum and Mycobacterium bovis BCG, respectively, were unable to induce an oxidative response. Periodate treatment of the M. tuberculosis PGL decreased the production of oxygen radicals, showing the importance of the PGL carbohydrate moiety for the interaction. The activation, however, could not be inhibited by rhamnose or fucose, indicating a complex interaction which probably involves more than one saccharide unit. This is in line with the fact that the activating PGLs from M. tuberculosis and M. kansasii contain tri- and tetrasaccharides, respectively, while the nonactivating PGLs from M. marinum and M. bovis BCG each contain a monosaccharide. The complement receptor 3 (CR3) has earlier been shown to be of importance for the phagocyte binding of mycobacteria, but did not appear to be involved in the activation of neutrophils by PGLs. The subcellular localization of the reactive oxygen metabolites formed was related to the way in which the glycolipids were presented to the cells.

Fig. 1

Neutrophil NADPH-oxidase activity triggered by mycobacterial PGL. (a) The time-courses of the neutrophil responses induced by M. tuberculosis PGL (□), M. kansasii PGL (▪), M. bovis BCG PGL (○), and M. marinum PGL (•), are given. The responses were measured with luminol and HRP in microtitre wells coated with PGL (coating concentration of 10 μg/ml). Abscissa, time (min); ordinata, chemiluminescence (arbitrary units). (b) The mean peak value (+ SD) of five (M. bovis BCG; and M. marinum; PGL) and 10 (M. tuberculosis; □ and M. kansasii; ▪ PGL) experiments. Ordinata, CL (arbitrary units).

Fig. 2

Neutrophil NADPH-oxidase activity triggered by PGL from M. tuberculosis and M. kansasii, respectively. The time-courses of the neutrophil responses induced by (a) M. tuberculosis PGL or (b) M. kansasii PGL are given. The responses were measured with luminol and HRP in microtitre wells coated with PGL at coating concentrations of 0.1 μg/ml (•), 10 μg/ml (□), or 30 μg/ml (▪). Abscissa, time (min); ordinata, chemiluminescence (arbitrary units).

Fig. 3

Effect of sodium meta-periodate treatment on the neutrophil NADPH-oxidase activity triggered by M. tuberculosis PGL. The figure shows the time-course of the CL response induced by periodate-treated (▪) or untreated (□) M. tuberculosis PGL (coating concentration 10 μg/ml). The responses were measured in the presence of luminol and HRP. Abscissa, time (min); ordinata, chemiluminescence (arbitrary units). The inset shows the mean peak value (+ SD) of three experiments.

Fig. 4

Effect of monosaccharides on the superoxide anion production in human neutrophils induced by M. tuberculosis PGL. The figure shows the mean peak value (+ SD) of the CL responses induced by M. tuberculosis PGL (coating concentration 10 μg/ml) in the absence (□) or presence of 3 mm rhamnose (▪) or fucose (). The responses were measured in the presence of luminol and HRP. Ordinata, chemiluminescence (arbitrary units).

Fig. 5

Extra- and intracellular NADPH-oxidase activity triggered by PGL from M. tuberculosis. The figure shows the time-course of the CL responses in neutrophils induced by M. tuberculosis PGL coated on a surface as measured by a Luminoskan (a) or coated on latex beads as measured by a six channel Biolumat LB 9505 (b). Coating concentration (a) 10 μg/ml and (b) 50 μg/ml. The intracellular responses to PGL (▪) were measured in the presence of SOD, catalase, and luminol, while the extracellular responses to PGL (□) were measured in the presence of HRP and isoluminol. In (b) the intracellular (♦) and extracellular response (⋄) to control latex beads is included. Abscissa, time (min); ordinata, chemiluminescence. (a) Arbitrary units, (b) Mcpm). The insets show the mean peak value (+ SD) of three experiments.

Fig. 6

Effect of cytochalasin B on the superoxide anion production in human neutrophils induced by M. tuberculosis PGL. The time-course of the CL response induced by M. tuberculosis PGL (coating concentration 10 μg/ml) in the absence (▪) or presence (□) of cytochalasin B (1 μg/ml final concentration) is shown. The responses were measured in the presence of luminol and HRP. Abscissa, time (min); ordinata, chemiluminescence (arbitrary units). The inset shows the oxidative response in neutrophils preincubated with (broken line) or without (solid line) 1 μg/ml cytochalasin B and subsequently stimulated with fMLP (10⁻⁷m final concentration). Abscissa, time (min); ordinata, CL (Mcpm).