Pseudomonas aeruginosa slime glycolipoprotein is a potent stimulant of tumor necrosis factor alpha gene expression and activation of transcription activators nuclear factor kappa B and activator protein 1 in human monocytes

Abstract

Pseudomonas aeruginosa, an opportunistic pathogen, causes infections associated with a high incidence of morbidity and mortality in immunocompromised hosts. Production of tumor necrosis factor alpha (TNF-alpha), primarily by cells of monocytic lineage, is a crucial event in the course of these infections. During in vivo infections with P. aeruginosa, both lipopolysaccharide (LPS) and extracellular slime glycolipoprotein (GLP) produced by mucoid and nonmucoid strains are released. In the present study, we sought to explore the relative contributions of these two bacterial products to TNF-alpha production by human monocytes. To this end, fresh human monocytes and THP-1 human monocytic cells were stimulated with P. aeruginosa LPS or GLP. GLP was found to be a more potent stimulus for TNF-alpha production (threefold higher) by human monocytes than LPS. Moreover, its effect was comparable to that of viable bacteria. Quantitative mRNA analysis revealed predominantly transcriptional regulation. Electrophoretic mobility shift assays and transfection assays demonstrated activation of NF-kappa B and activator protein 1 (AP-1). NF-kappa B activation by GLP was rapid and followed the same time course as that by viable bacteria, suggesting that bacteria could directly activate NF-kappa B through GLP. Moreover P. aeruginosa GLP induced the formation of AP-1 complex with delayed kinetics compared with NF-kappa B but much more efficiently than the homologous LPS. These results identify GLP as the most important stimulant for TNF-alpha production by human monocytes. Activation of NF-kappa B and AP-1 by P. aeruginosa GLP may be involved not only in TNF-alpha induction but also in many of the inflammatory responses triggered in the course of infection with P. aeruginosa.

FIG. 1.

(A) TNF-α production by human peripheral monocyte cultures stimulated with increasing doses of P. aeruginosa LPS (▪), slime GLP (▨), or E. coli LPS (□) as a control. Human monocytes were stimulated for 6 h with 10 ng to 100 μg of P. aeruginosa LPS/ml, 10 ng to 100 μg of slime GLP/ml, or 5 μg of E. coli LPS/ml as a control. TNF-α concentrations were determined as described in Materials and Methods. Results are means for triplicate cultures obtained in three different experiments. * and **, P < 0.001. (B) Time course of TNF-α levels in supernatants of human peripheral monocyte cultures stimulated with different components of P. aeruginosa. Cells (1 × 10⁶/ml) were challenged with 5 μg of LPS/ml (▪), 50 μg of slime GLP/ml (▴), or 10⁷ (10 bacteria/monocyte) whole viable (○) P. aeruginosa cells, as described in Material and Methods. E. coli LPS (5 μg/ml) (⧫) was used as a positive control. Results are means for triplicate cultures obtained in three separate experiments. (C) Kinetics of TNF-α production by monocytes stimulated with 10⁷ formalin-fixed heat-killed P. aeruginosa bacteria/ml or 10⁷ whole viable P. aeruginosa bacteria. Culture supernatants for cytokine measurements were removed at the times indicated on the graph. Results are means from two independent experiments.

FIG. 2.

(A) Time course of mRNA TNF-α isolated from human peripheral monocytes. Cells were stimulated with different components of P. aeruginosa. Monocytes (∼10⁷) were challenged with either 5 μg of E. coli LPS/ml (▴) as a control or with 5 μg of P. aeruginosa LPS/ml (○), 50 μg of P. aeruginosa slime GLP/ml (⧫), or 10⁸ viable P. aeruginosa cells (×), as described in Materials and Methods. Results are representative of four independent experiments. (B) Differential induction of TNF-α mRNA after treatment of human monocytes with (left to right) medium, 5 μg of E. coli LPS/ml, 5 μg of P. aeruginosa LPS/ml, 50 μg of P. aeruginosa slime GLP/ml, or 10⁸ P. aeruginosa viable cells. After 2 h of stimulation, monocytes were lysed and tested for levels of TNF-α mRNA as described in Materials and Methods. Results represent means ± standard deviations for three different experiments. * and **, P < 0.002.

FIG. 3.

Activation of NF-κB in response to different components of P. aeruginosa. NF-κB activation was measured by EMSA using radiolabeled oligonucleotide encompassing the NF-κB consensus motif. Human monocytes were stimulated with either 5 μg of LPS/ml, 50 μg of slime GLP/ml, or 10⁸ whole viable P. aeruginosa bacteria (10 bacteria/monocyte) for 2 and 6 h. Untreated cells were used as negative control, while 5 μg of E. coli LPS/ml was used as a positive control. Nuclear extracts and EMSAs were performed as indicated in Materials and Methods. The specificity of the DNA binding was assessed by preincubating extracts (5 μg) with unlabeled specific NF-κB or unspecific SP-1 competitor oligonucleotide at a 50-fold molar excess.

FIG. 4.

(A) NF-κB binding induction in the human monocytic THP-1 cell line after stimulation with the different components of P. aeruginosa. NF-κB binding was induced by stimulation with either 5 μg of E. coli LPS/ml, 50 μg of slime GLP/ml, or 10 viable P. aeruginosa bacteria/monocyte for 2 and 6 h. Nuclear extracts were prepared as described in Materials and Methods. Unstimulated monocytes were used as a negative control. The specificity of the DNA binding was assessed by preincubating extracts (5 μg) with unlabeled specific NF-κB or unspecific SP-1 competitor oligonucleotide at a 50-fold molar excess. (B) NF-κB-dependent transactivation in THP-1 cells stimulated with different components of P. aeruginosa. THP-1 cells were transfected with the pNF-κB-Luc reporter construct. Forty-eight hours after transfection, cells were treated for 6 h with either medium alone (▥), 5 μg of E. coli LPS/ml (▪), 5 μg of P. aeruginosa LPS/ml (□), or 50 μg of P. aeruginosa slime GLP/ml (▨). Transfection efficiency was monitored by cotransfection of pGEFP-C1 plasmid. Cell lysates were prepared, and aliquots were assayed for luciferase activity. Luciferase activity is expressed as relative light units/mg of total protein. Results are representative of three independent experiments. * and **, P < 0.001.

FIG. 5.

Identification of the NF-κB subunits induced by LPS or slime GLP of P. aeruginosa in monocytes. Nuclear extracts from cells stimulated with either 5 μg of P. aeruginosa LPS/ml or 50 μg of P. aeruginosa slime GLP/ml were preincubated with the indicated antiserum for 20 min at room temperature before the binding reaction with the NF-κB specific probe was performed. The arrow indicates supershift complexes by antibody binding.

FIG. 6.

(A) Activation of AP-1 in response to different components of P. aeruginosa. AP-1 activation was determined by EMSA using a radiolabeled oligonucleotide encompassing the AP-1 consensus motif. Human monocytes were stimulated with either 5 μg of LPS/ml, 50 μg of slime GLP/ml, or 10⁸ viable P. aeruginosa cells for 6 h as described in Materials and Methods. Untreated cells were used as a negative control. The specificity of the DNA binding was assessed by preincubating extracts with an unlabeled specific (AP-1) or unspecific (SP-1) competitor oligonucleotide at a 50-fold molar excess. (B) AP-1-dependent transactivation by different components of P. aeruginosa. THP-1 cells were transfected with the pAP-1 luciferase construct as described in Materials and Methods. Forty-eight hours after transfection, cells were stimulated with either (left to right) medium alone, 5 μg of E. coli LPS/ml, 5 μg of P. aeruginosa LPS/ml, or 50 μg of P. aeruginosa slime GLP/ml for 6 h. Cell lysates were prepared, and aliquots were assayed for luciferase activity. Luciferase activity is expressed as relative light units/mg of total protein. Results are representative of three independent experiments. * and **, P < 0.03.