Bronchoalveolar Cell Counts Research Articles

Correlation of BAL Cell Count and Pulmonary Function Tests in the Era of Antifibrotics: Data From the Belgium-Luxembourg Idiopathic Pulmonary Fibrosis Registry

Bronchoalveolar cell count at diagnosis is not linked to lung function decline and exacerbations in a large prospective cohort of idiopathic pulmonary fibrosis patients.

Skin TLR7 Triggering Promotes Accumulation of Respiratory Dendritic Cells and Natural Killer Cells

The TLR7 agonist imiquimod has been used successfully as adjuvant for skin treatment of virus-associated warts and basal cell carcinoma. The effects of skin TLR7 triggering on respiratory leukocyte populations are unknown. In a placebo-controlled experimental animal study we have used multicolour flow cytometry to systematically analyze the modulation of respiratory leukocyte subsets after skin administration of imiquimod. Compared to placebo, skin administration of imiquimod significantly increased respiratory dendritic cells (DC) and natural killer cells, whereas total respiratory leukocyte, alveolar macrophages, classical CD4+ T helper and CD8+ T killer cell numbers were not or only moderately affected. DC subpopulation analyses revealed that elevation of respiratory DC was caused by an increase of respiratory monocytic DC and CD11bhi DC subsets. Lymphocyte subpopulation analyses indicated a marked elevation of respiratory natural killer cells and a significant reduction of B lymphocytes. Analysis of cytokine responses of respiratory leukocytes after stimulation with Klebsiella pneumonia indicated reduced IFN-γ and TNF-α expression and increased IL-10 and IL-12p70 production after 7 day low dose skin TLR7 triggering. Additionally, respiratory NK cytotoxic activity was increased after 7d skin TLR7 triggering. In contrast, lung histology and bronchoalveolar cell counts were not affected suggesting that skin TLR7 stimulation modulated respiratory leukocyte composition without inducing overt pulmonary inflammation. These data suggest the possibility to modulate respiratory leukocyte composition and respiratory cytokine responses against pathogens like Klebsiella pneumonia through skin administration of a clinically approved TLR7 ligand. Skin administration of synthetic TLR7 ligands may represent a novel, noninvasive means to modulate respiratory immunity.

Skin triggering of TLR7 modulates respiratory dendritic cells and natural killer cells - identification of a novel mode for manipulation of respiratory leukocytes

Objective: Dendritic cell (DC) maturation and activation in situ is a critical element of successful DC vaccination protocols whereas suppression of functional DC activation represents an important step in the development of immunoregulatory cellular therapy protocols. The TLR7 agonist imiquimod has been used successfully as novel topical immune adjuvant but the systemic use has been limited due to significant toxicity. We hypothesized that skin application of a synthetic TLR7 ligand may represent a novel means to modulate respiratory leukocytes. Methods: In a placebo-controlled experimental animal study we have used 7-colour flow cytometry to systematically analyze the modulation of murine respiratory leukocyte subsets after skin administration of the synthetic TLR7 ligand imiquimod. Results: Compared to placebo, skin administration of imiquimod markedly increased respiratory dendritic cells and innate lymphocytes within 24h, whereas total respiratory leukocytes, alveolar macrophages, T helper and killer lymphocyte numbers were not affected or reduced. DC subpopulation analyses revealed that elevation of respiratory DC was related to an increase of monocytic DC (CD11c+CD11b+MHC-classIIdimSiglecFneg,CD45+). Lymphocyte subpopulation analyses indicated a marked elevation of respiratory natural killer cells and a reduction of B cells. In contrast, bronchoalveolar cell counts and histopathology were not affected suggesting that skin TLR7 stimulation markedly modulated respiratory leukocyte composition without inducing a respiratory inflammatory response. Conclusion: These data suggest for the first time the possibility to modulate respiratory leukocyte subsets after skin administration of a clinically approved TLR7 ligand. Skin administration of synthetic TLR7 ligands may represent a novel and safe means to modulate respiratory immunity. DC vaccination trials targeting the respiratory system may be modulated by skin TLR7 triggering.

The extra domain A of fibronectin is essential for allergen-induced airway fibrosis and hyperresponsiveness in mice

Asthma is characterized by airway inflammation, airway remodeling, and airway hyperresponsiveness (AHR). Myofibroblast differentiation and subepithelial fibrosis are key features of airway remodeling. Extra domain A (EDA)-containing fibronectin (EDA-FN), an alternatively spliced form of the extracellular matrix protein fibronectin, has been implicated in fibroblast differentiation during wound healing and tissue fibrosis. We sought to investigate the role of EDA-FN in airway remodeling using a murine model of chronic allergen-induced experimental asthma. EDA(-/-) and wild-type (WT) mice were sensitized and exposed to inhaled ovalbumin (OVA) or saline for 5 weeks. EDA-FN expression was evaluated by means of PCR and immunostaining. Peribronchial fibrosis, smooth muscle area, mucus-producing cell numbers, bronchoalveolar cell counts, and lung function were assessed in WT and EDA(-/-) mice. Fibroblast activation and differentiation were evaluated ex vivo by using OVA-treated WT and EDA(-/-) lung fibroblasts. Exposure to OVA increased EDA-FN expression in lung tissue and primary lung fibroblasts. OVA-treated EDA(-/-) mice showed reduced airway fibrosis and AHR and impaired expression of TGF-β1 and IL-13 without changes in airway inflammation or other aspects of remodeling. Lung fibroblasts from OVA-treated EDA(-/-) mice exhibited reduced proliferation, migration, α-smooth muscle actin expression, and collagen deposition and impaired TGF-β1 and IL-13 release compared with that seen in WT mice. EDA-FN is essential for the development of OVA-induced airway fibrosis and AHR. The effect of the EDA domain on airway fibrosis after OVA challenge is through activation and differentiation of fibroblasts. Fibroblast activation and airway fibrosis are necessary for the development of AHR.

Osteopontin Induces Airway Remodeling and Lung Fibroblast Activation in a Murine Model of Asthma

Airway remodeling is a central feature of asthma; however, the mechanisms underlying its development have not been fully elucidated. We have demonstrated that osteopontin, an inflammatory cytokine and an extracellular matrix glycoprotein with profibrotic properties, is up-regulated in a murine model of allergen-induced airway remodeling. In the present study, we determined whether osteopontin plays a functional role in airway remodeling. Osteopontin (OPN)-deficient (OPN(-/-)) and wild-type mice were sensitized and exposed to inhaled ovalbumin (OVA) or saline for 5 weeks. Collagen production, peribronchial smooth muscle area, mucus-producing cell number, and bronchoalveolar cell counts were assessed. The functional behavior and phenotype of lung fibroblasts from OVA-treated OPN(-/-) and from wild-type mice were studied using ex vivo cultures. OVA-treated OPN(-/-) mice exhibited reduced lung collagen content, smooth muscle area, mucus-producing cells, and inflammatory cell accumulation as compared with wild-type mice. Reduced matrix metalloproteinase-2 activity and expression of transforming growth factor-beta1 and vascular endothelial growth factor were observed in OVA-treated OPN(-/-) mice. Lung fibroblasts from OVA-treated OPN(-/-) mice showed reduced proliferation, migration, collagen deposition, and alpha-smooth muscle actin expression in comparison with OVA-treated wild-type lung fibroblasts. Thus, OPN is key for the development of allergen-induced airway remodeling in mice. In response to allergen, OPN induces the switching of lung fibroblasts to a pro-fibrogenic myofibroblast phenotype.

Stress-induced increment on total bronchoalveolar cell count in OVA-sensitized rats

The influence of stress on total leukocyte count from bronchoalveolar lavage (BAL) was investigated in rats sensitized and challenged with ovalbumin (OVA). The animals were injected intraperitoneally with a suspension of OVA plus aluminum hydroxide in 0.9% NaCl (Day 0) and boosted at Day 7 with an identical OVA solution, administered subcutaneously. From the first to the 13th day after sensitization, rats were placed individually in a shuttle box where they received 50 escapable footshocks per day, always preceded by a sound signal (S); the responses that occurred during both S and shocks canceled the stressful stimulation. On Day 14, animals were submitted to a single session of 50 inescapable footshocks, preceded by the same S; immediately after, the animals were submitted to a 1% OVA-inhalation challenge. Results showed high levels of stress in the shocked animals as detected through both ultrasonic vocalizations (UVs) and social interaction test in an open field. Total leukocyte count in BAL from stressed animals (24 h post-OVA challenge) revealed a significant increase in the number of inflammatory cells in comparison to that measured in sensitized, nonstressed challenged rats. These data demonstrate that stress plays a relevant and important role on total bronchoalveolar cell count in OVA-sensitized rats.

Abstract 20-39

Summary19. Neuroinflammatory circuits in Alzheimer's disease: Interleukin-1 induces cyclooxygenase 2 in human neuroblastoma cells20. Neuroinflammatory circuits in Alzheimer's disease: Prostaglandin E-2 induces astroglial interleukin-6 synthesis independent of cAMP formation21. Autonomic dysfunction in patients with multiple sclerosis and fatigue22. Behaviorally conditioned immunosuppression23. Influence of stress on immune-competent cells in patients suffering from ulcerative colitis24. Fatigue and regulation of the hypothalamo-pituitary-adrenal axis (HPA axis) in multiple sclerosis (MS)25. Magnocellular hypothalamic neurohypophysial system under acute inflammation26. Diurnal variations of interleukin 6 plasma levels during sleep and wakefulness: Effects of particularities of blood drawing27. Orgasm-induced circulation of lymphocyte subsets28. Bronchodilatation by antidepressants in allergen sensitized guinea pigs29. Adrenergic modulation of cytokine production during MOG-induced EAE30. The suppressive effect of acetylcholine on monocyte activation is mediated by nicotinergic receptors31. G protein coupled receptors and neuroendocrine signalling in autoimmunity32. Local immune response in the spleen: Regulation by the nervous system and effects on behavior33. The critical first year in the life of European wild rabbits (Oryctolagus cuniculus)34. SLE and RA patients differ from healthy controls in their cytokine pattern after stress exposure35. Stress-induced increment on bronchoalveolar cell count in OVA-sensitized mice is mediated by tachykinin NK1 receptor36. Quantitative analysis of cytokine mRNA expression patterns in the disease course of adolescent patients with anorexia nervosa37. CD26 mutant rats: A model for psycho-neuro-immune interactions38. Failure of α-melanocyte stimulating hormone to attenuate cerebral complications in experimental pneumococcal meningitis39. Neuronal plasticity is mediated by neurotrophin receptor p75NTR in a murine model of allergic bronchial asthma

Biochemical and cytologic alterations in the respiratory tract of rats exposed for 4 hours to hydrogen sulfide

Fischer-344 rats were killed by exsanguination 1, 20, and 44 hr after a single 4-hr exposure to an atmosphere of 0, 10, 200, and 400 ppm of hydrogen sulfide (H 2S). Alterations in the activities of lactate dehydrogenase and alkaline phosphatase, and cytomorphology of epithelial cells in fluids obtained by nasal and bronchoalveolar lavage were used as indicators of cell injury. Changes in the number of leukocytes were used as indicators of inflammatory response, and changes in the concentration of protein were used as indicators of altered vascular permeability. Inhalation of H 2S resulted in 139, 483, and 817% increased cellularity in the nasal cavity of rats exposed to 10, 200, and 400 ppm, respectively. This was due to marked exfoliation of degenerated epithelial cells and exudation of neutrophils. The high dose of H 2S resulted in a moderate increase in lactate dehydrogenase and protein in nasal passages; values returned to baseline levels 20 hr later. Bronchoalveolar cell counts were decreased in rats exposed to 400 ppm and unchanged in those exposed to 10 and 200 ppm. Enzymatic activities in lung lavage fluid were moderately elevated (up to 90%), yet protein concentrations were increased by more than 3000% and remained significantly elevated up to 44 hr after exposure to 400 ppm. It was concluded that inhalation of H 2S has a severe cytotoxic effect on the nasal epithelium and a severe edematogenic effect on lung parenchyma. These results are in agreement with autopsy findings of individuals killed by accidental exposure to H 2S-containing sour gas.

Bronchoalveolar cell count during compensatory hypertrophy of the lung

Bronchoalveolar cell count during compensatory hypertrophy of the lung

Biochemical and Cytologic Alterations in the Respiratory Tract of Rats Exposed for 4 Hours to Hydrogen Sulfide

Fischer-344 rats were killed by exsanguination 1, 20, and 44 hr after a single 4-hr exposure to an atmosphere of 0, 10, 200, and 400 ppm of hydrogen sulfide (H2S) Alterations in the activities of lactate dehydrogenase and alkaline phosphatase, and cytomorphology of epithelial cells in fluids obtained by nasal and bronchoalveolar lavage were used as indicators of cell injury. Changes in the number of leukocytes were used as indicators of inflammatory response, and changes in the concentration of protein were used as indicators of altered vascular permeability. Inhalation of H2S resulted in 139, 483, and 817% increased cellularity in the nasal cavity of rats exposed to 10, 200, and 400 ppm, respectively. This was due to marked exfoliation of degenerated epithelial cells and exudation of neutrophils. The high dose of H2S resulted in a moderate increase in lactate dehydrogenase and protein in nasal passages; values returned to baseline levels 20 hr later. Bronchoalveolar cell counts were decreased in rats exposed to 400 ppm and unchanged in those exposed to 10 and 200 ppm. Enzymatic activities in lung lavage fluid were moderately elevated (up to 90%), yet protein concentrations were increased by more than 3000% and remained significantly elevated up to 44 hr after exposure to 400 ppm. It was concluded that inhalation of H2S has a severe cytotoxic effect on the nasal epithelium and a severe edematogenic effect on lung parenchyma. These results are in agreement with autopsy findings of individuals killed by accidental exposure to H2S-containing sour gas.

Processing of lung lavage fluid causes variability in bronchoalveolar cell count.

To study the effects on cell counts of different ways of processing bronchoalveolar lavage (BAL) fluid, we performed 77 BAL in 19 healthy control subjects and in 58 patients with sarcoidosis. We investigated the role of readers, hemocytometers, mode of identification of macrophages, cell washing, and speed of cytocentrifugation. No significant effect of reader or hemocytometer was observed. The percentage of macrophages, determined as large cells stained with neutral red in a Malassez hemocytometer (63.9 +/- 24.6%, mean +/- SD) was lower than the percentage of macrophages determined by May Grunwald Giemsa staining (76.3 +/- 19.2%, p less than 10(-9)). Cell counts decreased 34% after 2 washings (p less than 0.001), and more lymphocytes were counted after cytocentrifugation at 90 g (33.2 +/- 25.3%) than at 23 g (27.7 +/- 22.1%). We conclude that bronchoalveolar cell counts vary with changes in processing lung lavage fluid and that this variability should be considered when using BAL cell counts.