There is much variability between individuals in the response to inhaled toxins, but it is not known why certain people develop disease when challenged with environmental agents and others remain healthy. To address this, we investigated whether TLR4 (encoding the toll-like receptor-4), which has been shown to affect lipopolysaccharide (LPS) responsiveness in mice, underlies the variability in airway responsiveness to inhaled LPS in humans. Here we show that common, co-segregating missense mutations (Asp299Gly and Thr399Ile) affecting the extracellular domain of the TLR4 receptor are associated with a blunted response to inhaled LPS in humans. Transfection of THP-1 cells demonstrates that the Asp299Gly mutation (but not the Thr399Ile mutation) interrupts TLR4-mediated LPS signalling. Moreover, the wild-type allele of TLR4 rescues the LPS hyporesponsive phenotype in either primary airway epithelial cells or alveolar macrophages obtained from individuals with the TLR4 mutations. Our findings provide the first genetic evidence that common mutations in TLR4 are associated with differences in LPS responsiveness in humans, and demonstrate that gene-sequence changes can alter the ability of the host to respond to environmental stress.
Individuals exposed to inhaled endotoxin (lipopolysaccharide [LPS]) can develop airway symptomatology and exacerbations of asthma. Moreover, among those occupationally exposed to organic dusts, the progression of airflow obstruction is related to the endotoxin concentration in the bioaerosol. Not everyone exposed to high concentrations of LPS develops these problems. To determine whether individuals express a differential response to inhaled LPS, we challenged 72 healthy volunteers with increasing doses of LPS. Airflow was assessed after each dose and the protocol was terminated for decline in FEV1 >/= 20%. Marked differences in the response to inhaled LPS were observed: eight "sensitive" subjects had at least 20% decline in their FEV1 after inhaling 6.5 micrograms or less of LPS, whereas 11 "hyporesponsive" subjects maintained an FEV1 >/= 90% of their baseline even after inhaling 41.5 micrograms of LPS. Serial testing demonstrated that the response to inhaled LPS is reproducible. Sensitive subjects were more commonly female and hyporesponsive subjects were more often male (p = 0.016). Peripheral blood monocytes from hyporesponsive subjects, compared with sensitive subjects, released less interleukin (IL)-6 and IL-8. These findings demonstrate that an LPS phenotype can be reproducibly elicited in humans, which creates an opportunity to identify genes involved in this response to inhaled LPS.
To identify the role of endotoxin in grain dust-induced lung disease, we conducted a population-based, cross-sectional investigation among grain handlers and postal workers. The study subjects were selected by randomly sampling all grain facilities and post offices within 100 miles of Iowa City. Our study population consisted of 410 grain workers and 201 postal workers. Grain workers were found to be exposed to higher concentrations of airborne dust (p = 0.0001) and endotoxin (p = 0.0001) when compared with postal workers. Grain workers had a significantly higher prevalence of work-related (cough, phlegm, wheezing, chest tightness, and dyspnea) and chronic (usual cough or phlegm production) respiratory symptoms than postal workers. Moreover, after controlling for age, gender, and cigarette smoking status, work-related respiratory symptoms were strongly associated with the concentration of endotoxin in the bioaerosol in the work setting. The concentration of total dust in the bioaerosol was marginally related to these respiratory problems. After controlling for age, gender, and cigarette smoking status, grain workers were found to have reduced spirometric measures of airflow (FEV1, FEV1/FVC, and FEF25-75) and enhanced airway reactivity to inhaled histamine when compared with postal workers. Although the total dust concentration in the work environment appeared to have little effect on these measures of airflow obstruction, higher concentrations of endotoxin in the bioaerosol were associated with diminished measures of airflow and enhanced bronchial reactivity. Our results indicate that the concentration of endotoxin in the bioaerosol may be particularly important in the development of grain dust-induced lung disease.
To investigate the relationship between the physiologic and biologic effects of grain dust inhalation, we exposed 15 nonsmoking, nonasthmatic, nonatopic male grain handlers to buffered saline and aqueous corn dust extract by inhalation challenge in a crossover study. The inhalation challenges to buffered saline and corn dust extract were separated by at least 14 d. Compared with buffered saline, inhalation of corn dust extract resulted in significant airflow obstruction, which was observed within 30 min of exposure and persisted for 5 h. Inhalation of corn dust extract resulted in an acute inflammatory response characterized by higher concentrations of neutrophils (p = 0.001), IL-1 beta (p = 0.001), IL-1RA (p = 0.001), IL-6 (p = 0.001), IL-8 (p = 0.001), and TNF-alpha (p = 0.04) in bronchoalveolar lavage (BAL) fluid. mRNA levels specific for IL-1 beta, IL-1RA, IL-6, and IL-8 from cells present in the BAL fluid were significantly greater after challenge with corn dust extract than after challenge with buffered saline. Importantly, no significant differences were observed in the concentration of lymphocytes or eosinophils in the BAL fluid following inhalation of corn dust extract, and the concentrations of histamine and 15-HETE were similar in BAL fluid after the two challenges. The maximal percentage decrease in FEV1 was significantly associated with the absolute neutrophil concentration in the BAL fluid (p = 0.001), as well as the concentration of TNF-alpha (p = 0.03), IL-1 beta (p = 0.005), IL-1RA (p = 0.001), IL-6 (p = 0.001), and IL-8 (p = 0.001) in the BAL fluid.(ABSTRACT TRUNCATED AT 250 WORDS)
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