Characterization of the lung epithelium of wild-type and TLR9(-/-) mice after single and repeated exposures to chicken barn air.

Exposure to chicken barn air causes lung injury resulting in lower and upper respiratory symptoms in the poultry workers, and mechanisms of which are not fully understood. The lung injury can initiate modifications such as proliferation of the airway epithelial cells such as Clara cells, type II alveolar (T2) cells and mucus producing goblet cells as part of the innate immune response. Toll-like receptors (TLR) have been suggested to play a role in cell division and proliferation. To understand the effect of TLR9 on Clara cells, T2 and mucus-producing goblet cells, we quantified the numbers of these cells in the lungs of wild-type (WT) and TLR9(-/-) mice exposed to chicken barn air. The mice were exposed for either one day or five or 20 days for 8 h/day. Clara cells and T2 cells were labelled with antibodies, and the mucus cells were identified with Periodic-acid Schiff stain, and quantified in per unit tissue section area. The data show decrease in the number of Clara cells and increase in mucus-producing goblet cells after exposure to chicken barn air in both WT and TLR9(-/-) mice. Numbers of T2 cells increased and decreased in WT and TLR9(-/-) mice, respectively, after exposure to poultry barn air. These data show that exposure to chicken barn air can affect major lung epithelial cells, and allude to the role of TLR9 in regulation of some of these responses.

Elevated levels of beta-D-glucan in bronchoalveolar lavage fluid in patients with farmer's lung in Miyazaki, Japan.

BACKGROUND: Farmers may be often exposed to beta-D-glucan in moldy hay, since straw for feed can be stored throughout the year. OBJECTIVES: The aim of the present study was to clarify whether levels of beta-D-glucan, which modifies immune responses, are high in the respiratory tract in farmer's lung and whether beta-D-glucan participates in the pathogenesis of this condition. METHODS: We measured beta-D-glucan levels in the bronchoalveolar lavage fluid (BALF) of 10 patients with farmer's lung, 4 with summer-type hypersensitivity pneumonitis (HP) and 10 healthy volunteers. Interleukin (IL)-8 and tumor necrosis factor (TNF)-alpha levels in BALF were measured by enzyme-linked immunosorbent assay (ELISA). We investigated the effects of beta-D-glucan on nuclear factor (NF)-kappaB activation and on the release of IL-8 and TNF-alpha from small airway epithelial cells (SAECs) in vitro. RESULTS: beta-D-Glucan levels in the BALF of farmer's lung patients were increased compared to those in patients with summer-type HP and in healthy volunteers. Additionally, IL-8 levels in BALF were higher in farmer's lung than in summer-type HP, and TNF-alpha levels were equal in the two patient groups but raised compared to those in healthy volunteers. High, but not low, concentrations of beta-D-glucan were found to induce NF-kappaB activation in SAECs. IL-8 levels in the supernatant obtained from SAEC cultures were increased following the addition of beta-D-glucan in vitro. CONCLUSION: BALF from farmer's lung patients showed high beta-D-glucan levels, which may enhance the expression and release of cytokines through NF-kappaB activation in respiratory epithelial cells.

Experimental hypersensitivity pneumonitis: role of MCP-1.

Inhalation of Saccharopolyspora rectivirgula causes "farmer's lung" disease, a classic example of hypersensitivity pneumonitis (HP). Monocyte chemoattractant protein-1 (MCP-1) is increased in the bronchoalveolar lavage fluid of mice challenged with S rectivirgula, and S rectivirgula induces MCP-1 secretion by alveolar macrophages. We tested the hypothesis that MCP-1 and its receptor CC chemokine receptor-2 (CCR2) are essential to the development of experimental HP by treating mice with MCP-1 antibody and using CCR2(-/-) mice. Administration of anti-MCP-1 did not change the response to intratracheally administered S rectivirgula. CCR2(-/-) animals responded in a fashion similar to that of wild-type animals to intratracheally administered.S rectivirgula. To determine the influence of the MCP-1-CCR2 interaction in vitro, we transferred S rectivirgula-cultured spleen cells from S rectivirgula-sensitized mice, to naïve recipients. Later, challenge of the recipients with intratracheal S rectivirgula and examination of both lung histology and bronchoalveolar lavage fluid characteristics were used to determine whether adoptive transfer had occurred. We found that cultured cells from CCR2(-/-) animals were fully capable of adoptive transfer. We conclude that interaction of MCP-1 with CCR2 is not necessary for the development of pulmonary inflammation in response to intratracheally administered S rectivirgula or cells able to adoptively transfer experimental HP.

Role of interleukin-2 in the development and persistence of lymphocytic alveolitis in farmer's lung.

Farmer's lung (FL) is characterized by an intense lymphocytic alveolitis which persists after an acute episode with continuous exposure to the offending antigens. This study aimed to examine the role of interleukin-2 (IL-2) in the development and persistence of this lymphocytic alveolitis. Three groups of dairy farmers were studied: acute FL, ex-FL (past history of FL but no clinical evidence of active disease) and asymptomatic farmers (no lung disease). IL-2 was measured by enzyme immunosorbent assay and T-cell proliferation was evaluated by 3H-thymidine incorporation. Acute and ex-FL patients had more lymphocytes (p<0.01) and higher levels of IL-2 (p<0.05) in their bronchoalveolar lavage (BAL) than asymptomatic farmers. BAL T-lymphocytes from acute and ex-FL patients released considerable amounts of IL-2 after stimulation with concanavalin A and showed dose-dependent proliferative responses to IL-2. IL-2 production was decreased after treatment with prednisone. Acute FL patients, but not ex-FL, had higher levels of soluble CD25 in their serum than asymptomatics (p=0.009). These results suggest that interleukin-2 may play a role in farmer's lung by providing a stimulus not only for the accumulation of lymphocytes but also for their persistence at the site of hypersensitivity reaction, and that the lung is a likely source of this cytokine in vivo.

Elafin/elastase-specific inhibitor in bronchoalveolar lavage of normal subjects and farmer's lung.

Secretory leukocyte proteinase inhibitor (SLPI) and alpha1-proteinase inhibitor (alpha1(PI)) cannot fully explain the total neutrophil elastase (NE) inhibitory capacity detected in bronchoalveolar lavage (BAL) fluid, suggesting the existence of other NE inhibitor(s). In the present study, we measured the concentrations of elafin, a newly described, low-molecular-weight serine proteinase inhibitor, SLPI, and alpha1(PI) in BAL fluids from eight healthy subjects, 13 asymptomatic farmers, seven farmers with active farmer's lung (FL), and seven farmers with previous (Ex) FL. In addition to SLPI and alpha1(PI), elafin was present in BAL fluids from control subjects and asymptomatic farmers, 13 (7-31) and 12 (7-67) mmol/mol of albumin (median and range) respectively. Elafin concentration increased significantly to 105 (38-207) mmol/mol of albumin in farmers with active FL and was also elevated in farmers with Ex FL. Elafin levels were highly correlated with lung inflammatory cell numbers, especially lymphocytes, and the decrease in single-breath diffusion capacity (DLCO). Elafin and SLPI were linked to yet uncharacterized proteins in BAL fluids. In conclusion, elafin is a constituent of BAL fluid from normal subjects and is found in enhanced concentrations in FL and in farmers with lymphocytic alveolitis. This suggests that elafin may play a role in lung homeostasis and inflammation.

Proinflammatory cytokines in hypersensitivity pneumonitis.

We examined the synthesis and release of the proinflammatory cytokines macrophage inflammatory protein-1 alpha (MIP-1 alpha) and interleukin-8 (IL-8) in the context of subjects with hypersensitivity pneumonitis (HP). Subjects with acute HP were found to have alveolar macrophages that released high levels of MIP-1 alpha and IL-8, whereas control subjects had cells that released low levels of these factors. Bronchoalveolar lavage fluid from HP subjects contained low but significant levels of MIP-1 alpha and IL-8. Immunohistochemistry revealed that macrophages from acute HP subjects expressed MIP-1 alpha and IL-8 at high levels. Treatment of acute HP subjects by contact avoidance or by corticosteroids reduced the synthesis of both cytokines. Supernatants of alveolar macrophages from subjects with HP were shown to attract activated CD8+ T lymphocytes, and this activity was significantly inhibited by anti-MIP-1 alpha. MIP-1 alpha and IL-8 may be important inflammatory cytokines in the development of HP via their capacity to attract inflammatory cells (activated CD8+ T lymphocytes and neutrophils, respectively) into the airways of subjects with HP.

Mast cell and histamine involvement in farmer's lung disease.

The aim of this study was to evaluate the cellular and biochemical characteristics of the bronchoalveolar lavage (BAL) fluid in patients with farmer's lung disease (FLD). Total cell numbers in BAL fluids from patients with FLD (n = 30) were significantly higher than in normal subjects (n = 7; p < 0.01), and differential cell counts were significantly different. Lymphocytes were the most numerous cell type in BAL fluids from patients with FLD (65.4 +/- 2.5 percent vs 6.8 +/- 0.5 percent), and analysis of lymphocyte subsets revealed increased percentages of CD3+ and CD8+ cells (91.8 +/- 0.9 percent vs 68.8 +/- 3 percent, p < 0.01, and 54.3 +/- 3.1 percent vs 30.1 +/- 3.2 percent, p < 0.01, respectively). A marked increase in mast cell numbers, as revealed by the specific alcian blue/safranin staining, was observed in patients with FLD (4.2 +/- 0.57 percent, n = 12, vs 0.18 +/- 0.04 percent, n = 7, p < 0.001). Histamine levels in BAL supernatants were increased in patients with FLD (mean = SEM, 4.4 +/- 0.8 ng/ml vs 0.9 +/- 0.1 ng/ml; median, 2.4 ng/ml vs 0.9 ng/ml, p < 0.01), and correlated positively with mast cell numbers and percentages (r = +0.63, p < 0.03, and r = +0.69, p < 0.02, respectively); conversely, a negative correlation was found between histamine levels and CD8+ lymphocyte percentages (r = -0.48, p < 0.01). Raised neutrophil percentages (5.1 +/- 0.8 vs 0.5 +/- 0.18, p < 0.05) and albumin concentrations (29.2 +/- 3.9 mg/dl vs 3.4 +/- 1.3 mg/dl, p < 0.01) were also found in patients with FLD. These findings show that increased numbers of mast cells, lymphocytes, and neutrophils can be found in BAL fluids of patients with FLD. The increased histamine levels in the supernatants of BAL fluids indicate that mast cells are activated. These data allow us to postulate a role for mast cell accumulation and histamine release in the inflammatory process of FLD.

Elevated serum galactosylhydroxylysyl glucosyltransferase, a collagen synthesis marker, in fibrosing lung diseases.

The activity of galactosylhydroxylysyl glucosyltransferase, an enzyme catalyzing collagen biosynthesis, was measured in the sera of 101 patients with various pulmonary diseases to study whether detectable enzyme amounts are liberated into the serum from the lung tissue, and whether this is associated with the development of lung fibrosis. Increased serum galactosylhydroxylysyl glucosyltransferase activity was found in all the patients with progressive pulmonary fibrosis and in about half of the patients with acute stages of farmer's lung and infectious pneumonia. In one third of the patients with stage I sarcoidosis the serum enzyme activity was slightly increased, whereas in bronchial asthma and chronic bronchitis the values were mostly within the normal range. In conclusion, elevated serum enzyme activity was demonstrated in connection with those respiratory diseases in which pulmonary fibrosis was already verifiable or relatively often develops later. Measurements of serum galactosylhydroxylysyl glucosyltransferase may, thus, be useful in evaluating actual lung collagen synthesis in human pulmonary diseases.