Mast Cells In Bronchoalveolar Lavage Fluid Research Articles

Cytokine mRNA expression in the bronchoalveolar lavage cells from horses affected by different equine asthma subtypes

Equine asthma (EA) is a respiratory syndrome associated with the increase of different leukocyte populations in the bronchoalveolar lavage fluid (BALF). Its pathogenetic mechanisms remain unclear. This study aimed to evaluate the associations between the mRNA expression of different cytokines in the BALF, different EA subtypes and lung function. Fifteen horses underwent physical examination, airway endoscopy, BALF cytology and lung function testing (8/15). One horse did not have evidence of EA and was used as healthy reference, while the others were classified as affected by neutrophilic or mixed granulocytic EA. Cells isolated from the residual BALF were used for IL-1β, IL-2, IFN-γ, IL-4, IL-17A genes expression by quantitative RT-PCR., Cytokine expression was compared between groups, and their correlations with BALF leukocyte and lung function were evaluated. IL-1β expression was positively correlated with BALF neutrophils count (p=0.038, r=0.56) and with increased expiratory resistance (p=0.047, r=0.76). IFN-γ was correlated with BALF mast cells (p=0.029, r=0.58). IL-4 was higher in horses with mixed granulocytic EA than neutrophilic (p=0.008), positively correlated with BALF mast cells (p=0.028, r=0.59) and inversely with whole-breath (p=0.046, r=-0.76) and expiratory reactance (p=0.003, r=-0.93). Finally, IL-17A was inversely correlated with expiratory reactance (p=0.009, r=-0.92). These results support that multiple immune responses are involved in EA pathogenesis; innate, Th2, and Th17 responses. Innate immunity appeared associated with neutrophilic inflammation, and Th2 response with increased mast cells. The role of Th1 response in EA remains questionable.

Quantitative Transcriptome Analysis of Purified Equine Mast Cells Identifies a Dominant Mucosal Mast Cell Population with Possible Inflammatory Functions in Airways of Asthmatic Horses.

Asthma is a chronic inflammatory airway disease and a serious health problem in horses as well as in humans. In humans and mice, mast cells (MCs) are known to be directly involved in asthma pathology and subtypes of MCs accumulate in different lung and airway compartments. The role and phenotype of MCs in equine asthma has not been well documented, although an accumulation of MCs in bronchoalveolar lavage fluid (BALF) is frequently seen. To characterize the phenotype of airway MCs in equine asthma we here developed a protocol, based on MACS Tyto sorting, resulting in the isolation of 92.9% pure MCs from horse BALF. We then used quantitative transcriptome analyses to determine the gene expression profile of the purified MCs compared with total BALF cells. We found that the MCs exhibited a protease profile typical for the classical mucosal MC subtype, as demonstrated by the expression of tryptase (TPSB2) alone, with no expression of chymase (CMA1) or carboxypeptidase A3 (CPA3). Moreover, the expression of genes involved in antigen presentation and complement activation strongly implicates an inflammatory role for these MCs. This study provides a first insight into the phenotype of equine MCs in BALF and their potential role in the airways of asthmatic horses.

Effects of human mesenchymal stem cells on airway inflammation in allergic asthma mice and the underlying mechanism

Objectives: To investigate the effects of human umbilical cord mesenchymal stem cells (hUC-MSCs) on airway inflammation in an ovalbumin (OVA) induced asthma mouse model and the underlying mechanism. Methods: Twenty-four BALB/c mice were randomly divided into four equal groups: normal control group, OVA-induced asthmatic model group, hUC-MSCs treated group (50 μl of hUC-MSCs was transplanted into the trachea of asthmatic mice ) and hUC-MSCs control group (50 μl of hUC-MSCs was transplanted into the trachea of control mice). Human umbilical cord mesenchymal stem cells from umbilical cord of healthy new born babies were used as the source of hUC-MSCs for this study. The asthmatic conditions of the airways and the lungs were assessed by examining: (1) histopathological changes of the airways and the lungs; (2) expression of cytokines IL-6 and TGF-β mRNA by real-time PCR; (3) total leukocytes and mast cell count in bronchoalveolar lavage fluid (BALF) and number of IL-17-expressing CD4(+) cells (Th17 cells) in the lung tissue using flow cytometry. Results: Typical histopathological changes of asthma were confirmed in the asthmatic model group. These changes included intensive inflammatory cell infiltration around the airways and patchy airway occlusion by hyperviscous mucus. The number of total leukocytes and mast cells in BALF were significantly increased in the asthmatic mice when compared with the control group (P<0.05). Mice in the asthmatic model group had significantly higher percentage of Th17 cells in lung tissues when compared with the control group (2.90% vs 0.76%, P<0.05). In contrast, in the asthmatic mice treated with hUC-MSCs, the inflammatory cell infiltration was significantly reduced compared with asthmatic mice, as observed by significantly lower leukocytes and mast cells in BALF (P<0.05) and significant reduction in the percentage of Th17 cells in the lung of OVA-challenged mice following hUC-MSCs treatment (percentage of Th17 cells: 0.24% vs 2.90%, P<0.05). The expression of mRNA for IL-6 and TGF-β was significantly suppressed in the hUC-MSCs treatment group (0.23 vs 2.30 and 0.56 vs 6.60, both P<0.01). No asthmatic pathological changes in both normal and hUC-MSCs control groups were observed. Conclusions: hUC-MSCs significantly inhibit the airway inflammation in OVA-induced asthmatic mice. This inhibition is associated with the suppression of Th17 cells and the down-regulation of inflammatory factors such as IL-6 and TGF-β in the lungs.

Neurogenic inflammation in allergen-challenged obese mice: a missing link in the obesity-asthma association?

ABSTRACTAim: A number of studies have shown an association between obesity and asthma. Controversy remains on the mechanisms supporting this association. In this study we aimed to assess neurogenic inflammation in a model of diet-induced obesity and allergen-challenged mice. Methods: High fat diet-induced (HFD) obese Balb/c mice were sensitized and challenged with ovalbumin (OVA). Glucose, insulin, OVA-specific IgE and substance P (SP), and the main tachykinin involved in neurogenic inflammation, were quantified in sera. Cell counts were performed in bronchoalveolar lavage fluid (BALF). The extent of peribronchial infiltrates was estimated on lung tissue sections and inflammation was score based on inflammatory cell counts surrounding the bronchi. Results: Obesity per se and allergen-sensitization per se increased serum SP (P = .027, P = .004, respectively). Further increased was observed in obese-sensitized mice (P = .007). Obese-sensitized mice also showed higher insulin (P = .0016), OVA-specific IgE (P = .016), peribronchial inflammatory score (P = .045), and tendency for higher glycemia. The interaction of obesity and asthma on SP levels was confirmed (P = .005, R2 = 0.710). SP was positively correlated with metabolic (glycemia, r = 0.539, P = .007) and allergic inflammation parameters (BALF eosinophils, r = 0.445, P = 0.033; BALF mast cells, r = 0.574, P = .004; peribronchial inflammation score, r = 0.661, P < .001; and OVA-specific IgE, r = 0.714, P < .001). Conclusions: Our findings provide support to the neurogenic inflammation link between obesity and asthma in mice. These two conditions independently increased SP and the presence of both pathologies further increased this level. Neurogenic inflammation may be a previously unrecognized mechanism beyond the obese-asthma phenotype. Further studies are need to confirm this role of SP in human obesity-asthma association.

Oral administration of Enterococcus faecalis FK-23 suppresses Th17 cell development and attenuates allergic airway responses in mice

Evidence is increasing that oral administration of probiotics can attenuate asthmatic responses both in murine models and clinical trials. T-helper 17 (Th17) cells, a subset of CD4+ T cells have been implicated as having an important role in the development of several allergic disorders, but the relationship between oral administration of probiotics and Th17 development has not been well studied. BALB/c mice were given lysed Enterococcus faecalis FK-23 (LFK) orally for 28 days. After sensitization by subcutaneous injection of ovalbumin (OVA) on Days 14 and 21 and 1% OVA inhalation on Days 25, 26 and 27, they were challenged with a 5% OVA aerosol on Day 28. Twenty-four hours later, airway resistance and accumulation of inflammatory cells in bronchoalveolar lavage fluid (BALF) and lung tissues were determined. Ιnterleukin (IL)-17-expressing CD4+ lymphocytes isolated from lung, spleen and lamina propria of the intestine were detected by flow cytometry. The expression of IL-6 and TGF-β mRNA was assessed by real-time PCR. Increases in airway hyperresponsiveness, and numbers of total leukocytes and mast cells in BALF induced by OVA challenge were significantly suppressed by oral administration of LFK. The increased percentage of IL-17-expressing CD4+ cells from lung, spleen and intestine in OVA-challenged mice was reduced following LFK treatment. We conclude that the oral administration of LFK suppresses the asthmatic response and that this is associated with attenuation of Th17 cell development.

Comparison of CytokinemRNAExpression in the Bronchoalveolar Lavage Fluid of Horses with Inflammatory Airway Disease and Bronchoalveolar Lavage Mastocytosis or Neutrophilia UsingRESTSoftware Analysis

The pathophysiology of inflammatory airway disease (IAD) is unknown, but in some cases involves the accumulation of mast cells, neutrophils, or both in the bronchoalveolar lavage fluid (BALF). The objective of this study was to characterize cytokine gene expression in the BALF cells of horses with IAD, including a comparison of cytokine gene expression between IAD horses with increased BALF mast cells (IAD-Mast) or neutrophils (IAD-Neutro). The mRNA expression of IL-4, IFN-γ, IL-17, IL-8, IL-1β, IL-5, IL-6, IL-10, IL-12p35, and eotaxin-2 was studied by quantitative polymerase chain reaction (QPCR) with efficiency correction in BALF samples of 17 horses with IAD (IAD-total), also subcategorized as 8 IAD-Mast and 9 IAD-Neutro, and 10 controls. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a reference gene. Relative expression software tool (REST) analysis provided ratios of expression, statistical analysis, and confidence intervals for the results. Compared with the control group, IL-5, IL-1β, IL-6, IL-8, and IL-10 mRNA expression was upregulated 3.5-, 3.4-, 2.8-, 2.2-, and 1.9-fold, respectively, in the IAD-total group. The IAD-Neutro group showed increased expression of IL-17, IL-8, and IL-5 (4.7-, 2.5-, and 2.9-fold, respectively) and a decreased expression of IL-4 (3.4-fold) compared with the IAD-Mast group. Cytokines from the Th2 family plays a key role in IAD and a different pathophysiology may be involved in mast cell versus neutrophil BALF accumulation in IAD horses.

Comparison of Four Staining Methods for Detection of Mast Cells in Equine Bronchoalveolar Lavage Fluid

Mast cells normally are present in equine bronchoalveolar lavage fluid (BALF), but usually represent <2% of all cells in healthy horses. An increased percentage of mast cells has been associated with airway hyperactivity and inflammatory airway diseases, but marked differences are reported between studies in normal and diseased horses. Because an abnormal mast cell count may be of clinical relevance, we compared the ability of a fast Romanowsky method to stain mast cell granules with that of 3 metachromatic stains: automated Romanowsky, May-Grünwald Giemsa, and toluidine blue stains. The BALF cells from 24 horses were studied. A differential cell count was performed blindly on 400 cells. The percentages of mast cells obtained were analyzed by means of repeated-measures analysis of variance and Fischer's PLSD test. The Bland and Altman method was used to assess agreement among stains. The mean percentage of mast cells in BALF was significantly lower with the fast Romanowsky than with the automated Romanowsky, May-Grünwald Giemsa, and toluidine blue stains. With the fast Romanowsky stain, the metachromatic granules of mast cells were not stained, and their identification was based on morphologic criteria. Toluidine blue staining allowed detection of the highest mean percentage of mast cells, but was inadequate for performing a differential cell count on other cell types. In conclusion, fast Romanosky stain may be inadequate for detection of mast cells in equine BALF, whereas automated Romanowsky, May-Grünwald Giemsa, and toluidine blue stains provide metachromatic staining of mast cell granules.

Phenotypic analysis of alveolar macrophages and lymphocytes following allergen inhalation by atopic subjects with mild asthma

The airway inflammation associated with allergic asthma is initiated through a complex interaction of antigen-presenting cells (APC) and T lymphocytes resulting in the release of a cascade of cytokines regulating the progress of the allergic inflammatory response. In the present study the state of alveolar macrophage (AM) and T cell activation was investigated following induction of allergic airway inflammation in individuals with atopic asthma. Eleven individuals with mild, atopic asthma received cumulated allergen inhalations. Before and one day after challenge, bronchoalveolar lavage (BAL) was performed, and peripheral blood samples obtained. Ten healthy individuals served as controls. The expression of cell surface markers by BAL fluid AMs and T cells, and by blood T cells, was investigated by flow cytometry. All patients developed early asthmatic reactions (EAR) with increased numbers of eosinophils and mast cells in BAL fluid following allergen challenge. After allergen challenge, patients had relatively fewer pulmonary CD4+ T cells expressing CD69 and HLA class II and also relatively fewer pulmonary CD8+ T cells expressing HLA class II, compared to before challenge. An increased quantitative expression of CD14 and CD86 was seen within the AM population following allergen challenge. The results indicate a recruitment of non-activated, immature macrophages and CD4+ T cells to the airways as well as an altered phenotype pattern within the AM population following induction of allergic airway inflammation by allergen inhalation challenge in asthma.

Extracellular matrix remodeling in children with airway foreign-body aspiration.

The purpose of this study was to observe extracellular matrix remodeling in children with airway foreign-body aspiration (FBA) by detecting the expression of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), and hydroxyproline (HYP) in bronchoalveolar lavage fluid (BALF). Forty-six children with FBA and 12 control subjects from 2002-2003 were enrolled in this study. The former were divided into three subgroups according to duration of foreign-body (FB) retention (subgroup 1, <7 days; subgroup 2, 7-30 days; subgroup 3, >30 days). Cell count, and levels of MMP-9, TIMP-1, and HYP in BALF were measured. The total number of cells and differential counts detected in BALF did not differ significantly between patients and controls (P > 0.05), while mast cells were found only in the BALF of patients. The positive rates of MMP-9 in controls, subgroup 1, subgroup 2, and subgroup 3 were 33.3%, 62.5%, 80.0%, and 93.3%, respectively, with a significant difference (P = 0.006). The positive rate of TIMP-1 in controls and subgroups 1, 2, and 3 were 33.3%, 62.5%, 80.0%, and 93.3%, respectively, with a significant difference (P < 0.001) as well. HYP levels in subgroups 2 and 3 were significantly more increased than in subgroup 1 and controls (all P < 0.001). Levels of MMP-9, TIMP-1, and HYP in patients were positively correlated with duration of FB retention (all P < 0.05), and levels of MMP-9 and TIMP-1 were also positively correlated with percentage of mast cells in BALF (all P < 0.05). The differences in MMP-9 and TIMP-1 before and after FB removed were not significant (P > 0.05), while HYP levels decreased significantly after FB were removed (P = 0.001). Our findings support the hypothesis that FBA may contribute not only to mechanical airway obstruction, but also possibly to eventual airway remodeling by generation of MMPs and TIMPs.

La suberosis: implicación de los mastocitos broncoalveolares en la génesis de la afectación intersticial

Suberosis--the lung disease suffered by cork industry workers--may present in the form of either hypersensitivity pneumonitis (HP) or obstructive pulmonary disease (OPD) with asthma-like symptoms or chronic bronchitis. Mast cells play an important role in pulmonary inflammation and are particularly implicated in the rapid release of mediators in bronchoconstriction and the production of cytokines and mediators of fibroblast activity. Increased numbers of mast cells are present in bronchoalveolar lavage (BAL) fluid in interstitial lung diseases, suggesting that these cells also participate in chronic inflammatory processes and in pulmonary fibrosis. To assess the participation of mas cells in interstitial pulmonary inflammation in cork industry workers by histochemically analyzing their presence in BAL fluid. Foreseeing the possible implication of bronchoalveolar mast cells in the pathogenesis of suberosis, we also studied their relation to various signs and symptoms of the disease, to respiratory function parameters and to degree of alveolitis. Thirty-one cork industry workers with respiratory symptoms related to occupational exposure were enrolled. Occupational and case histories were taken. Physical examinations were complemented by chest X-rays, plethysmography/spirometry, fiberoptic bronchoscopy with BAL, and determination of carbon monoxide diffusing capacity (DLCO) and arterial blood gases at rest. Patient classification (20 with HP and 11 with OPD) was based on clinical and functional criteria and analysis of BAL fluid. Mast cells in cytospinned samples treated with two different stains [May-Grunwald-Giemsa (MGG) and Toluidine Blue (Tol.Bl.)] were counted by two observers and the results were compared. Good correlation between the two staining methods was confirmed (rs = 0.86, p < 0.0001). Correlation between the two observers was also good (MGG rs = 0.86, Yol.Bl. rs = 0.87, p < 0.0001). The number of mast cells in BAL fluid was significantly higher in patients with HP [13.4 +/- 4.5 (x +/- SEM)] than in those with OPD (0.9 +/- 0.3; p < 0.002, Mann Whitney test). The subgroup of eight patients with poorer respiratory function (CV and/or DLCO < 80% of reference value) also had higher mast cell counts in BAL (19.9 +/- 7.7 versus 3.5 +/- 1.7; p = 0.002). We also saw a negative relation between mast cell counts in BAL fluid and lung function parameters: total lung capacity (rs = -0.68, p = 0.005) and DLCO (rs = -0.54, p = 0.008). Mast cell recovery from BAL fluid was positively related to severity of alveolitis in terms of total cell counts (rs = 0.62, p = 0.002), absolute lymphocyte counts (rs = 0.56, p = 0.006) and albumin levels (rs = 0.68, p = 0.003). Our findings suggest that mast cells participate in interstitial lung cell response to the inhalation of organic cork dust, particularly when HP is the form of presentation. Moreover, mas cell recruitment on the alveolar surface seems to be related to the intersity of lymphocytosis and interstitial pulmonary inflammation and to lung function deterioration in affected patients.

Bronchoalveolar mast cells in summer-type hypersensitivity pneumonitis: increase in numbers and ultrastructural evidence of degranulation.

To investigate the role of mast cells in the development of summer-type hypersensitivity pneumonitis (SHP), which is the most common type of hypersensitivity pneumonitis in Japan, bronchoalveolar lavage fluid (BALF) cells from 13 patients with this disease was studied by light and electron microscopy. The proportion and number of BALF mast cells recovered from SHP patients were significantly greater than those from patients with sarcoidosis and from control subjects. The BALF mast cells from the SHP patients showed various ultrastructural changes such as the transformation of granules, cytoplasmic vacuolation, and an increase of lipid droplets. These findings appeared to reflect activation and degranulation of bronchoalveolar mast cells, suggesting that mast cells play a role as inflammatory cells in the development of SHP.

Exposure to peat dust: acute effects on lung function and content of bronchoalveolar lavage fluid.

Mechanised production of peat for fuel consumption is associated with high concentrations of organic dust, which is inhaled by the peat workers. In the present study 17 workers at two peat bogs in northern Sweden were examined. Personal sampling of total dust and the respirable fraction was performed during several workshifts. Dynamic spirometry was carried out before and at the end of shifts. Bronchoscopy with bronchoalveolar lavage (BAL) was performed in six subjects at the end of the working season and the results were compared with unexposed reference subjects. Peat workers using modern machines with ventilated cabins containing air filters were found to be exposed to low concentrations of peat dust. The recorded dust concentrations were below the threshold limit value for organic dust (5 mg/m3 air) in all but one worker. The respirable fraction of peat dust recorded in the breathing zone of the workers correlated significantly with a decrease in forced expiratory volume in one second (FEV1). The effect on lung function in non-asthmatic peat workers was, however, small. The concentration of lysozyme positive alveolar macrophages in BAL fluid was significantly lower in the peat workers compared with reference subjects. An inverse correlation was found between the mentioned cells and exposure to the respirable fraction of the peat dust. Furthermore, one particularly dust exposed worker had pronounced increases in alveolar macrophages, fibronectin concentration, and mast cells in BAL fluid.

Histochemical Comparison of Mast Cells Obtained from the Airways of Mongrel Dogs and Basenji-Greyhound Dogs by Bronchoalveolar Lavage

An abnormally large number of mast cells in the airway lumen may be an important factor in the pathogenesis of bronchial hyperreactivity. However, it is unclear just how many mast cells are present in the lumen of normal or hyperreactive airways, in part because of differences in the histochemical techniques that have been used to identify mast cells and questions about the heterogeneity of mast cells. The present study was done (1) to compare the effectiveness of six techniques in the identification of mast cells obtained from dogs by bronchoalveolar lavage (BAL), (2) to compare the mast cells in the airways of normal mongrel dogs with those from a breed of dog (Basenji-Greyhound) known to have bronchial hyperreactivity, and (3) to determine whether the two-type histochemical classification used for rodent mast cells (formaldehyde-resistant or typical and formaldehyde-sensitive or atypical) applies meaningfully to the mast cells in BAL fluid from dogs. Cells obtained by BAL were fixed with Mota's basic lead acetate or formaldehyde. Mast cells were identified by metachromatic staining with toluidine blue or methylene blue, staining of highly sulfated proteoglycans with Alcian blue or berberine sulfate, and a histochemical reaction for chloroacetate esterase (mast cell chymase). After Mota's fixation, the methods were relatively similar in their effectiveness in determining the number of mast cells in lavage fluid from the mongrel dogs, in that all of the values fell within a narrow range: 0.53 to 0.96% of the total number of cells.(ABSTRACT TRUNCATED AT 250 WORDS)