A melanin pigment purified from an epidemic strain of Burkholderia cepacia attenuates monocyte respiratory burst activity by scavenging superoxide anion

Abstract

The acquisition of Burkholderia cepacia in some cystic fibrosis patients is associated with symptoms of acute pulmonary inflammation that may be life threatening. The ability of lipopolysaccharide (LPS) from B. cepacia to prime a monocyte cell line for enhanced superoxide anion generation was investigated and compared with the priming activities of LPSs from Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Escherichia coli. The human monocyte cell line MonoMac-6 (MM6) was primed overnight with different LPSs (100 ng/ml), and the respiratory burst was triggered by exposure to opsonized zymosan (125 micrograms/ml). Superoxide generation was detected by enhanced chemiluminescence with Lucigenin. B. cepacia LPS was found to prime MM6 cells to produce more superoxide anion than P. aeruginosa or S. maltophilia LPS, and this priming response was CD14 dependent. In addition, the inhibition of respiratory burst responses in monocytes by a bacterial melanin-like pigment purified from an epidemic B. cepacia strain was investigated. The melanin-like pigment was isolated from tyrosine-enriched media on which B. cepacia had been grown and was purified by gel filtration, anion ion-exchange chromatography, and ethanol precipitation. The scavenging potential of the melanin-like pigment for superoxide anion radical (*O2-) generated during the respiratory burst was confirmed with superoxide produced from a cell-free system with xanthine-xanthine oxidase and detected by electron paramagnetic resonance spectroscopy with the spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-n-oxide. The addition of melanin during the LPS priming stage had no effect on the subsequent triggering of the respiratory burst, but melanin inhibited *O2- detection when added at the triggering stage of the respiratory burst. We conclude that melanin-producing B. cepacia may derive protection from the free-radical-scavenging properties of this pigment.

FIG. 1

Superoxide anion production by MM6 cells primed with LPS and triggered with opsonized zymosan, as measured by enhanced chemiluminescence. Error bars indicate the standard error of the mean for chemiluminescence in priming experiments performed in quadruplicate with each different LPS. n, number of strains of each organism.

FIG. 2

Superoxide anion production by MM6 cells primed with LPS, as measured by enhanced chemiluminescence. Bars represent the means for eight B. cepacia LPSs, four P. aeruginosa LPSs, and four S. maltophilia LPSs. Chemiluminescence in priming experiments with each LPS was measured in quadruplicate, and error bars indicate the standard error of the mean. P values were obtained against B. cepacia LPS activity (asterisk). The double asterisks indicate cells primed with B. cepacia LPS in the presence of anti-CD14 antibody.

FIG. 3

(A) Superoxide anion production in MM6 cells primed with LPS from the B. cepacia Cardiff epidemic strain (P1), as measured by chemiluminescence. Different doses of melanin added with opsonized zymosan at the triggering stage of the respiratory burst scavenged superoxide anion. For comparison, 400 U of SOD was added at the triggering stage. (B) Like panel A, except that melanin was added at the peak of superoxide anion production (55 min [arrow]).

FIG. 4

EPR spectrum of the purified melanin-like pigment powder.

FIG. 5

EPR spectra of superoxide anions detected by the spin trap DEPMPO. The melanin-like pigment scavenged superoxide radicals in a dose-dependent manner. (a) Xanthine-xanthine oxidase plus DEPMPO after 5 min of reaction. (b) As in panel a but with 50 μM melanin. (c) As in panel a but with 250 μM melanin. (d) As in panel a but with 2,500 μM melanin. (e) As in panel a but with 400 U of SOD.