ABSTRACT
Inhaled endotoxin (lipopolysaccharide, LPS) initiates an inflammatory response and leads to the expression of CR3 (CD11b/CD18) receptors on polymorphonuclear leukocytes (PMNs). We determined if PMN activation in nasal lavage fluid (NLF) is a possible biomarker of occupational endotoxin exposure. Seven subjects exposed to endotoxin provided NLF samples that were split into three aliquots (negative control--1 M nicotinamide; sham; positive control--11 etag of exogenous LPS) and PMN activation was measured using a chemiluminometer. Differences in mean PMN activation were apparent, negative control: 548 +/- 15.65 RLU 100 microl(-1); sham: 11469 +/- 2582 RLU 100 microl(-1); positive control: 42026 +/- 16659 RLU 100 microl (n = 7; p <0.05). This technique shows promise as a diagnostic method for measuring upper airway LPS exposure.
Subject(s)
Macrophage-1 Antigen/metabolism , Nasal Lavage Fluid/immunology , Neutrophil Activation/immunology , Neutrophils/immunology , Analysis of Variance , Animals , Animals, Laboratory/immunology , Bronchial Hyperreactivity/diagnosis , Bronchial Hyperreactivity/etiology , Bronchial Hyperreactivity/immunology , Humans , Lipopolysaccharides/immunology , Luminescent Measurements/instrumentation , Luminescent Measurements/methods , Nasal Lavage Fluid/chemistry , Nasal Lavage Fluid/cytology , Neutrophils/metabolism , Occupational Diseases/diagnosis , Occupational Diseases/etiology , Occupational Diseases/immunology , Occupational Exposure/adverse effectsABSTRACT
Tumor necrosis factor-alpha (TNF-alpha), an inflammatory cytokine, has been shown to activate the small GTPase Rho, but the underlying signaling mechanisms remained undefined. This general problem is particularly important in the kidney, because TNF-alpha, a major mediator of kidney injury, is known to increase paracellular permeability in tubular epithelia. Here we aimed to determine the effect of TNF-alpha on the Rho pathway in tubular cells (LLC-PK(1) and Madin-Darby canine kidney), define the upstream signaling, and investigate the role of the Rho pathway in the TNF-alpha-induced alterations of paracellular permeability. We show that TNF-alpha induced a rapid and sustained RhoA activation that led to stress fiber formation and Rho kinase-dependent myosin light chain (MLC) phosphorylation. To identify new regulators connecting the TNF receptor to Rho signaling, we applied an affinity precipitation assay with a Rho mutant (RhoG17A), which captures activated GDP-GTP exchange factors (GEFs). Mass spectrometry analysis of the RhoG17A-precipitated proteins identified GEF-H1 as a TNF-alpha-activated Rho GEF. Consistent with a central role of GEF-H1, its down-regulation by small interfering RNA prevented the activation of the Rho pathway. Moreover GEF-H1 and Rho activation are downstream of ERK signaling as the MEK1/2 inhibitor PD98059 mitigated TNF-alpha-induced activation of these proteins. Importantly TNF-alpha enhanced the ERK pathway-dependent phosphorylation of Thr-678 of GEF-H1 that was key for activation. Finally the TNF-alpha-induced paracellular permeability increase was absent in LLC-PK(1) cells stably expressing a non-phosphorylatable, dominant negative MLC. In summary, we have identified the ERK/GEF-H1/Rho/Rho kinase/phospho-MLC pathway as the mechanism mediating TNF-alpha-induced elevation of tubular epithelial permeability, which in turn might contribute to kidney injury.
