Low endotoxic potential of Legionella pneumophila lipopolysaccharide due to failure of interaction with the monocyte lipopolysaccharide receptor CD14

Abstract

Legionella pneumophila, a gram-negative bacterium causing Legionnaires' disease and Pontiac fever, was shown to be highly reactive in in vitro gelation of Limulus lysate but not able to induce fever and the local Shwartzman reaction in rabbits and mice. We analyzed the capacity of purified L. pneumophila lipopolysaccharide (LPS-Lp) to induce activation of the human monocytic cell line Mono Mac 6, as revealed by secretion of proinflammatory cytokines and desensitization to subsequent LPS stimulation. We showed that despite normal reactivity of LPS-Lp in the Limulus amoebocyte lysate assay, induction of cytokine secretion in Mono Mac 6 cells and desensitization to an endotoxin challenge required LPS-Lp concentrations 1,000 times higher than for LPS of Salmonella enterica serovar Minnesota. Therefore, we examined the interaction of LPS-Lp with the LPS receptor CD14. We demonstrated that LPS-Lp did not bind to membrane-bound CD14 expressed on transfected CHO cells, nor did it react with soluble CD14. Our results suggest that the low endotoxic potential of LPS-Lp is due to a failure of interaction with the LPS receptor CD14.

FIG. 1

Reactivities of LPS-Lp and LPS-sm in the Limulus amoebocyte lysate test. Data are means ± SD of three experiments.

FIG. 2

Secretion of proinflammatory cytokines by Mono Mac 6 cells after stimulation with various concentrations of LPS-Sm and LPS-Lp, with (▨) and without (▩) addition of human AB serum. Data are means ± SD of five experiments.

FIG. 3

Inactivation of LPS-Sm and LPS-Lp by polymxin B (PmB). Data are means ± SD of five experiments.

FIG. 4

Desensitization of Mono Mac 6 cells for proinflammatory cytokine production in response to a subsequent challenge with LPS-Ec after preincubation with LPS-Sm (•) in comparison to preincubation with LPS-Lp (■). Data are means ± SD of four experiments.

FIG. 5

Competition of LPS-Lp with [³H]LPS-Ec for binding to CD14⁺ CHO cells. Cells were incubated with [³H]LPS-Ec in the presence (bars 1 and 3 to 6) or absence (bar 2) of LBP as described in Materials and Methods, and cell-bound activity was counted by liquid scintillation counting. Bars 1 and 2, no competitor; bars 3 to 6, 1,000-fold excess of unlabeled LPS-Ec LCD 25, LPS-Lp, Lp-lipid A, and Lp-O chain, respectively. Data are means ± SD of three experiments.

FIG. 6

Native PAGE assay. sCD14 (50 μg/ml) (lane 1) was incubated with LPS-Ec LCD 25 (lane 2), LPS-Lp (lane 3), Lp-O chain (lane 4), Lp-lipid A (lane 5), lipid A of LPS-Ec (lane 6), LPS-Ec O55:B5 (lane 7), and LPS-Sm (lane 8) (each at 80 μg/ml) in a volume of 10 μl. Reactions were run on a 4 to 15% native polyacrylamide gel. sCD14 was detected by Western blotting using a rabbit anti-CD14 antiserum. Lane 9, mixture of all LPSs without CD14. Incubation was performed in the absence (A) or presence (B) of mouse recombinant LBP.