Farmers and smokers are repeatedly exposed to airborne organic material. We hypothesised that farmers and smokers show altered airway responses to inhaled organic, proinflammatory agents.A total of 11 farmers, 12 smokers and 12 controls underwent lipopolysaccharide (LPS) bronchial challenge and spent 3 h in a pig barn. Lung function, exhaled nitric oxide and bronchial responsiveness were assessed and nasal lavage fluid and induced sputum were also collected. Symptoms and body temperature were recorded before and after exposures.Following exposure to the pig barn, bronchial responsiveness, exhaled nitric oxide, sputum interleukin (IL)-6, nasal lavage cell count and IL-8 were increased to a greater extent in controls compared to farmers. The sputum IL-6 response was also attenuated in farmers after LPS challenge. The response shown by smokers following exposure to the pig barn was similar to that of controls regarding measurements of exhaled nitric oxide, IL-8 in nasal lavage and IL-6 in sputum, but more similar to farmers concerning bronchial responsiveness and the cell numbers present in nasal lavage. Sputum IL-8 showed a greater increase in smokers than in the other groups following LPS challenge.We conclude that individuals who are repeatedly exposed to organic material develop an adaptation to the effects of acute exposure to inhaled organic material.

Pattern-recognition receptors (PRRs), that are expected to make a first line of defense against invading micro-organisms, are differently regulated in smokers with COPD compared with smokers without airflow limitation and non-smokers. This is likely of importance in COPD patho-physiology, in particular for exacerbations, which often are caused by micro-organisms.

BackgroundChronic obstructive pulmonary disease (COPD) is associated with local and systemic inflammation. The knowledge of interaction and co-variation of the inflammatory responses in different compartments is meagre.MethodHealthy controls (n = 23), smokers with (n = 28) and without (n = 29) COPD performed spirometry and dental examinations. Saliva, induced sputum, bronchoalveolar lavage (BAL) fluid and serum were collected. Inflammatory markers were assessed in all compartments using ELISA, flow cytometry and RT-PCR.ResultsNegative correlations between lung function and saliva IL-8 and matrix metalloproteinase-9 (MMP-9) were found in smokers with COPD. IL-8 and MMP-9 in saliva correlated positively with periodontal disease as assessed by gingival bleeding in non-smokers.Tumor necrosis factor-α (TNF-α) in saliva, serum and TNF-α mRNA expression on macrophages in BAL-fluid were lower in smokers than in non-smokers. There were positive correlations between soluble TNF-α receptor 1 (sTNFR1) and soluble TNF-α receptor 2 (sTNFR2) in sputum, BAL-fluid and serum in all groups. Sputum interleukin-8 (IL-8) or interleukin-6 (IL-6) was positively correlated with sTNFR1 or sTNFR2 in non-smokers and with sTNFR2 in COPD.ConclusionSaliva which is convenient to collect and analyse, may be suitable for biomarker assessment of disease activity in COPD. An attenuated TNF-α expression was demonstrated by both protein and mRNA analyses in different compartments suggesting that TNF-α response is altered in moderate and severe COPD. Shedding of TNFR1 or TNFR2 is similarly regulated irrespective of airflow limitation.

Endotoxin, tumor necrosis factor (TNF), and organic dust constitute proinflammatory stimuli involved in the initiation of inflammation. The major receptor for endotoxin (lipopolysaccharide [LPS]) is Toll-like receptor 4 (TLR4), whereas TLR2 binds to agents from gram-positive bacteria. The aim of the study was to elucidate whether TLR2 and TLR4, expressed on primary bronchial epithelial cells (PBECs), are influenced by exogenous (organic dust and LPS) and endogenous (TNF) stimuli and whether this interaction is influenced by a glucocorticosteroid. The cells were exposed to LPS (10 microg/ml), TNF (10 ng/ml), or dust (100 microg/ml) 1.5 and 6 h, in the presence or absence of budesonide (10(-6) M) in vitro. The mRNA expression of interleukin (IL)-6, IL-8, TLR2, and TLR4 were measured with real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and IL-6 and IL-8 release was assessed with enzyme-linked immunosorbent assay (ELISA). To elucidate the importance of TLR-signaling for IL-6 and IL-8 secretion, the effect of TLR-blockers was studied. Endotoxin, TNF, and dust stimulated the release of IL-6 and IL-8 in a time-dependent manner. Budesonide significantly attenuated the release and expression of IL-6 and IL-8 after exposure. Budesonide did not influence TLR expression, but costimulation with LPS, TNF, or dust together with budesonide increased TLR2 expression synergistically. Blocking of TLR2 and TLR4 reduced cytokine secretion in stimulated cells. Budesonide reduced IL-6 and IL-8 production and enhanced expression of TLR2 in PBECs only in the presence of a proinflammatory stimulus. These findings contribute to our understanding of the beneficial effects of glucocorticosteroids during chronic obstructive pulmonary disease (COPD) exacerbations and asthma, which are frequently caused by microorganisms.

IntroductionAlveolar macrophages (AMs) are equipped with innate immune receptors such as toll-like receptor 2 (TLR2) and toll-like receptor 4 (TLR4). In primary bronchial epithelial cells, exposure of toll-like receptor (TLR) ligands or tumor necrosis factor-alpha (TNF-α) increased TLR2 mRNA expression and reduced interleukin-8 (IL-8) release when coincubated with glucocorticosteroids. The aim of this study was to compare TLR2 and TLR4 expression levels and the effect of a glucocorticosteroid after stimulation with TLR ligands on AMs from smokers with and without COPD compared with the healthy controls.Subjects and methodsBronchoalveolar lavage was performed, and AMs were isolated from smokers with (n=10) and without COPD (n=11) and healthy controls (n=10) and stimulated ex vivo with peptidoglycan (PGN), lipopolysaccharide (LPS), or TNF-α ± budesonide (Bud). Blocking antibodies to TLR2 or TLR4 were added before stimulation with LPS or PGN ± Bud, respectively. The release of proinflammatory cytokine (TNF-α), chemoattractant (CXCL8), and TLR expression was analyzed by enzyme-linked immunosorbent assay and reverse transcription polymerase chain reaction.ResultsLPS, PGN, and TNF-α induced an increased release of IL-8 and TNF-α in the AMs in all the groups independent of smoking or disease. These responses were inhibited by a glucocorticosteroid (Bud) in all the three groups, except PGN-induced IL-8 secretion in smokers without COPD. Bud increased TLR2 expression in the healthy controls and smokers without COPD. Costimulation of TLR ligands and Bud significantly enhanced TLR2 mRNA expression in both groups of smokers compared with TLR ligands alone. In smokers, costimulation with PGN and Bud significantly increased TLR2 expression when compared with Bud alone. On stimulation with the TLR4 agonist, LPS downregulated TLR4 mRNA expression in all the three groups.ConclusionThe combination of glucocorticosteroids with TLR ligands can increase TLR2 expression, thereby improving host defense in smokers. Also this combination can decrease the secretion of proinflammatory cytokines and chemokines as an anti-inflammatory response. Our findings indicate that glucocorticosteroid therapy strengthens immune defense pathways, which may have implication during exacerbation caused by microorganisms.