Role of Toll-like receptor 4 for the pathogenesis of acute lung injury in Gram-negative sepsis.
OBJECTIVE
Proinflammatory cytokines as well as nitric oxide (NO) play a major role in mediating the response to lipopolysaccharide (LPS). The present study tested the hypothesis that LPS induces proinflammatory cytokines in the lung via the Toll-like receptor 4 (TLR4)/CD14 signalling cascade.


METHODS
Control mice and TLR4-deficient (TLR4-D) mice were used to test TLR4-mediated effects of LPS. Both strains received either Escherichia coli LPS (20 mg kg-1 intraperitoneal) or saline and their lungs were collected at different time points. Pulmonary nuclear factor kappaB (NFkappaB) activation was investigated with electromobility shift assay. mRNA expression of inflammatory mediators and their corresponding receptors were detected with Ribonuclease Protection Assay. Protein expression was detected by ELISA and western blotting. Inducible NO synthase (iNOS) expression was monitored by RT-PCR and iNOS activity by conversion of l-arginine to citrulline. Immune cells were sampled by bronchoalveolar lavage (BAL) and classified.


RESULTS
LPS application induced CD14-, but not TLR4 protein expression in control mice. Activation of pulmonary NFkappaB was observed within 60 min in control, but not in TLR4-D mice. Six hours of LPS administration induced a significant increase in pulmonary tumour necrosis factor alpha-, interleukin-1beta- and interleukin-6 mRNA and protein expression in control mice compared to TLR4-D mice. Furthermore, LPS induced a significantly higher increase of the iNOS expression and catalytic activity in control mice than in TLR4-D mice. BAL revealed an increase in total cell count in all LPS treated mice.


CONCLUSIONS
Our findings suggest that TLR4 plays a key role for regulating the expression of relevant cytokines within the lung during endotoxic shock.
Baumgarten
G
G
Universitätsklinikum Bonn, Department of Anesthesiology and Intensive Care Medicine, Bonn, Germany.
Knuefermann
P
P
Wrigge
H
H
Putensen
C
C
Stapel
H
H
Fink
K
K
Meyer
R
R
Hoeft
A
A
Grohé
C
C
D000818:Animals; D001992:Bronchoalveolar Lavage Fluid; D006090:Gram-Negative Bacteria / Q000378:metabolism*; D053583:Interleukin-1beta / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000096:biosynthesis; D008168:Lung / Q000382:microbiology*; D008171:Lung Diseases / Q000382:microbiology*; D055370:Lung Injury; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D009569:Nitric Oxide / Q000378:metabolism; D012128:Respiratory Distress Syndrome / Q000378:metabolism / Q000382:microbiology*; D012772:Shock, Septic / Q000378:metabolism*; D051197:Toll-Like Receptor 4 / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D053583:Interleukin-1beta; D015850:Interleukin-6; D018950:Lipopolysaccharide Receptors; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide