Total BAL Cells Research Articles

Euthanasia- and Lavage-mediated Effects on Bronchoalveolar Measures of Lung Injury and Inflammation.

Accurate and reproducible assessments of experimental lung injury and inflammation are critical for basic and translational research. In particular, investigators use various methods for BAL and euthanasia; however, the impact of these methods on assessments of injury and inflammation is unknown. To define potential effects, we compared methods of lavage and euthanasia in uninjured mice and after a mild lung injury model (ozone). C57BL/6J male mice (8-10 weeks old) underwent BAL after euthanasia with ketamine/xylazine, carbon dioxide (CO2), or isoflurane. BAL methods included 800 μl of isotonic solution instilled and withdrawn three times, and one or three passive fills and drainage to 20 cm H2O. Parallel experiments were performed 24 hours after 3 hours of ozone (O3) exposure at 2 ppm. BAL total cell counts/differentials and total protein/albumin were determined. Lung histology was evaluated for lung inflammation or injury. BAL cells were cultured and stimulated with PBS, PMA, or LPS for 4 hours and supernatants were evaluated for cytokine content. In uninjured mice, we observed differences due to the lavage and euthanasia methods used. The lavage method increased total cells and total protein/albumin in uninjured and O3-exposed mice, with the 800-μl instillation having the highest values. Isoflurane increased total BAL cells, whereas CO2 euthanasia increased the total protein/albumin levels in uninjured mice. These effects limited our ability to detect differences in BAL injury measures after O3 exposure. In conclusion, the method used for lavage and euthanasia affects measures of lung inflammation/injury and should be considered a variable in model assessments.

Anti-angiogenic Nanotherapy Inhibits Airway Remodeling and Hyper-responsiveness of Dust Mite Triggered Asthma in the Brown Norway Rat.

Although angiogenesis is a hallmark feature of asthmatic inflammatory responses, therapeutic anti-angiogenesis interventions have received little attention. Objective: Assess the effectiveness of anti-angiogenic Sn2 lipase-labile prodrugs delivered via αvβ3-micellar nanotherapy to suppress microvascular expansion, bronchial remodeling, and airway hyper-responsiveness in Brown Norway rats exposed to serial house dust mite (HDM) inhalation challenges. Results: Anti-neovascular effectiveness of αvβ3-mixed micelles incorporating docetaxel-prodrug (Dxtl-PD) or fumagillin-prodrug (Fum-PD) were shown to robustly suppress neovascular expansion (p<0.01) in the upper airways/bronchi of HDM rats using simultaneous 19F/1H MR neovascular imaging, which was corroborated by adjunctive fluorescent microscopy. Micelles without a drug payload (αvβ3-No-Drug) served as a carrier-only control. Morphometric measurements of HDM rat airway size (perimeter) and vessel number at 21d revealed classic vascular expansion in control rats but less vascularity (p<0.001) after the anti-angiogenic nanotherapies. CD31 RNA expression independently corroborated the decrease in airway microvasculature. Methacholine (MCh) induced respiratory system resistance (Rrs) was high in the HDM rats receiving αvβ3-No-Drug micelles while αvβ3-Dxtl-PD or αvβ3-Fum-PD micelles markedly and equivalently attenuated airway hyper-responsiveness and improved airway compliance. Total inflammatory BAL cells among HDM challenged rats did not differ with treatment, but αvβ3+ macrophages/monocytes were significantly reduced by both nanotherapies (p<0.001), most notably by the αvβ3-Dxtl-PD micelles. Additionally, αvβ3-Dxtl-PD decreased BAL eosinophil and αvβ3+ CD45+ leukocytes relative to αvβ3-No-Drug micelles, whereas αvβ3-Fum-PD micelles did not. Conclusion: These results demonstrate the potential of targeted anti-angiogenesis nanotherapy to ameliorate the inflammatory hallmarks of asthma in a clinically relevant rodent model.

Reduced expression of peroxisome proliferator-activated receptor alpha in BAL and blood T cells of non-löfgren’s sarcoidosis patients

BackgroundSarcoidosis is a granulomatous disease affecting in particular the lungs. The peroxisome proliferator-activated receptors (PPARs) play important regulatory roles in inflammation. The aim of this study was to gain more insight about the expression of all three PPARs (α, β/δ and γ) in sarcoidosis.MethodsBronchoalveolar lavage (BAL) cells and peripheral blood cells were obtained from healthy controls (HC) and sarcoidosis patients with Löfgren’s syndrome (LS) and without Löfgren’s syndrome (non-LS). PPARs mRNA expression was analyzed in total BAL cells and in FACS (Fluorescence-activated cell sorting) sorted alveolar macrophages (AM) and CD4+ T cells respectively by comparative RT-PCR. PPARs protein expression was analyzed in AM, and in BAL and blood CD4+ and CD8+ T cells by flow cytometry.ResultsIn BAL CD4+ T cells, we noticed a significantly lower PPARα mRNA expression in sarcoidosis patients compared with HC. In non-LS patients, a significantly lower PPARα protein expression in BAL CD4+ T cells was detected as compared with LS patients. In peripheral blood CD4+ T cells, non-LS patients had a significantly lower expression of PPARα and PPARγ compared with LS patients.ConclusionThe lower protein expression of PPARα and PPARγ could contribute to the persistent T-cell driven inflammation noted especially in non-resolving sarcoidosis, common in non-LS patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12950-015-0071-6) contains supplementary material, which is available to authorized users.

Pulmonary response of mice to a sequential exposure of side-stream cigarette smoke and multi-walled carbon nanotubes

Due to their unique properties, nano-sized carbon materials are predicted to have numerous applications in industry, but significant evidence exists to suggest their potential to cause toxicity. To determine if pre-exposure to side-stream cigarette smoke (SSCS) influences their toxicity, we examined the pulmonary response of smoke-exposed mice to multi-walled carbon nanotubes (MWCNT). Female A/J mice were exposed to SSCS in a whole body exposure chamber at approximately 40 mg/m3 for 4 weeks (6 h/d, 5 d/wk) and challenged with a single dose of MWCNT (40 µg) by the pharyngeal aspiration technique. A total of four groups were compared: air/phosphate buffered saline (PBS)-control, SSCS/PBS, air/MWCNT, and SSCS/MWCNT. At days 1 and 3 post-MWCNT treatment, lung tissues and bronchoalveolar lavage fluid (BALF) were collected and analyzed. In comparison with controls, significantly higher levels of total BAL cells were obtained from mice exposed to SSCS and MWCNT alone or combination. Influx of polymorphonuclear leukocytes (PMN) into BALF greatly increased in MWCNT alone and SSCS/MWCNT groups at both days 1 and 3 compared with controls. However, pre-exposure to SSCS significantly suppressed PMN response to MWCNT on day 1 but not day 3. Total BALF protein, lactate dehydrogenase, and mucin were significantly elevated in MWCNT and SSCS/MWCNT groups but not in the SSCS group, except mucin at day 3, when compared with controls. These results demonstrate that MWCNT markedly increases pulmonary toxicity in mice and SSCS pre-exposure plays a minor role in modulating MWCNT-induced lung toxicity at the concentrations and time points selected in the present study.

Characterization of MMP-9 and -12 KO mice in a model of acute smoke exposure

Short-term cigarette smoke exposure in mice has been shown to exhibit some similarities to cell and cell-mediated inflammation seen in COPD patients. We sought to identify biomarkers in the lungs and bronchoalveolar lavage fluid (BALF) that would characterize this inflammation. Mice were exposed; nose only, to 600 mg/m3 of cigarette smoke from 2R4F Kentucky Reference cigarettes twice daily for three consecutive days. Sham smoked animals were exposed to room air. Short-term cigarette smoke exposure induced a mild but statistically significant increase in total BAL cells in addition to a significant increase in neutrophils as compared with sham-exposed animals. Oral administration of dexamethasone did not attenuate total BAL cell or neutrophil counts. However, administration of an oral PDE4 inhibitor, significantly reduced percent neutrophil and macrophage counts. To assess expression of genes involved in this model, transcriptional profiling was performed on whole lungs harvested on day 4. Genes with the greatest fold increase included SAA3, CXCL1, 2 and 5 and NOXO1. In addition, multiplex analysis of BALF collected on day 4, revealed significant elevation in inflammatory mediators including cytokines, chemokines and metalloproteinases. Matrix metalloproteinase (MMP)–9 and –12 knockout (KO) mice were also tested in this model to evaluate whether or not these deficiencies influence acute smoke-induced inflammation. Total BAL inflammation and neutrophil counts were decreased in smoke exposed MMP-9 and -12 KO mice compared to wild type controls. These effects, however seem to be strain dependent as they were noted only in BALB/c KO animals. Deficiencies in MMP-9 and –12 appear to be beneficial in having an anti-inflammatory effect following acute exposure to smoke.

Electroacupuncture Attenuates Ovalbumin-Induced Allergic Asthma via Modulating CD4(+)CD25(+) Regulatory T Cells.

A mouse pulmonary hypersensitivity experimental model that mimics human asthma was developed, and electroacupuncture (EA) treatment was shown to reduce allergic inflammatory processes. In addition, we also assessed whether the beneficial effects of EA on allergic asthma could be correlated with CD4+CD25+Foxp3+ regulatory T cells (Treg). Cellular profiles and histopathologic analysis demonstrated that peribronchial and perivascular inflammatory cell infiltrates were significantly decreased in the EA-treated groups when compared to the OVA and anti-CD25 Ab-injected (Treg depletion) groups. Furthermore, total BAL cells were reduced in the EA groups when compared to other groups. Interestingly, the population of CD4+CD25+Foxp3+Tregs in pneumonocytes increased in EA-treated group when compared to OVA and Treg depletion groups. These results imply that EA stimulation at ST 36 may affect CD4+CD25+Foxp3+ Treg in an OVA-induced experimental model and may enhance Treg function by suppressing other T cells and limiting the immune response.

No evidence of altered alveolar macrophage polarization, but reduced expression of TLR2, in bronchoalveolar lavage cells in sarcoidosis

BackgroundSarcoidosis is a granulomatous inflammatory disease, possibly of infectious aetiology. We aimed to investigate whether the degree of functional polarization of alveolar macrophages (AMs), or Toll-like receptor (TLR) expression, is associated with sarcoidosis or with distinct clinical manifestations of this disease.MethodsTotal BAL cells (cultured four or 24 h in medium, or stimulated 24 h with LPS) from 14 patients and six healthy subjects, sorted AMs from 22 patients (Löfgren's syndrome n = 11) and 11 healthy subjects, and sorted CD4+ T cells from 26 patients (Löfgren's syndrome n = 13) and seven healthy subjects, were included. Using real-time PCR, the relative gene expression of IL-10, IL-12p35, IL-12p40, IL-23p19, CCR2, CCR7, iNOS, CXCL10, CXCL11, CXCL16, CCL18, CCL20, CD80, and CD86, and innate immune receptors TLR2, TLR4, and TLR9, was quantified in sorted AMs, and for selected genes in total BAL cells, while IL-17A was quantified in T cells.ResultsWe did not find evidence of a difference with regard to alveolar macrophage M1/M2 polarization between sarcoidosis patients and healthy controls. TLR2 gene expression was significantly lower in sorted AMs from patients, particular in Löfgren's patients. CCL18 gene expression in AMs was significantly higher in patients compared to controls. Additionally, the IL-17A expression was lower in Löfgren's patients' CD4+ T cells.ConclusionsOverall, there was no evidence for alveolar macrophage polarization in sarcoidosis. However, there was a reduced TLR2 mRNA expression in patients with Löfgren's syndrome, which may be of relevance for macrophage interactions with a postulated sarcoidosis pathogen, and for the characteristics of the ensuing T cell response.

Inflammatory mediators induced by intratracheal instillation of ultrafine amorphous silica particles

In order to evaluate the pulmonary effects and inflammatory mechanisms of ultrafine amorphous silica particles (UFASs), the UFASs suspension was prepared in PBS and intratracheally administered to A/J mice at doses of 0, 2, 10 and 50 mg/kg ( n = 5 per group). Animals were sacrificed at 24 h, and 1, 4 or 14 weeks following exposures. At each time point, a bronchoalveolar lavage fluid analysis, histopathological examination, quantitative real-time PCR and immunohistochemistry of the lung tissues were assessed. The intratracheal instillation of UFASs significantly increased the lung weights and total BAL cells following exposures. The histopathological examination revealed that UFASs-induced severe inflammation, with neutrophils, at an early stage and chronic granulomatous inflammation at the later stage. The mRNA and protein levels of IL-1β, IL-6, IL-8, TNF-α, MCP-1 and MIP-2 in lung tissues were significantly increased during the early stages, but there were no changes after weeks 1 (TNF-α) or 4 (IL-1β, IL-6, IL-8, MCP-1 and MIP-2). Instillation of UFASs-induced transient, but very severe lung inflammation. Therefore, the cytokines (IL-1β, IL-6, IL-8 and TNF-α) and chemokines (MCP-1 and MIP-2) play important roles in the inflammation induced by the intratracheal instillation of UFASs.

천식유발 마우스에서의 폐 내 세포조성 변화와 IL-4 및 IL-10의 발현 양상

Asthma is a chronic inflammatory disorder of the airways, which characterized by bronchial hyperresponsiveness, reversible airflow limitation and respiratory symptoms. Internationally, the prevalence of asthma has been increased over last 3 decades. Recently, several studies of asthma have been reported with gradually increasing importance. To tesify the hypothesis that interleukin (IL)-4 and IL-10 may be an important determinant of the severity of airway inflammation, their expression was studied in mouse model of asthma. BALB/c mouse, IL-4 Knockout (KO) mouse and IL-10 KO mouse were sensitized with intraperitoneal injection of ovalbumin adsorbed to aluminum potassium sulfate, followed by challenges with intranasal ovalbumin on 3 consecutive days. The severity of pulmonary inflammation was assessed by eosinophilia in BAL fluid, number of total BAL cells, histopathological changes in lung tissues, and immunohistochemical staining against IL-4 and IL-10. In BAL fluid, the number of total cells was significantly increased in asthma induced mouse compare to the control. In asthma induced mouse, eosinophil was increased to 56% and neutrophil was 0.2%. In H & E stains, eosinophilic infiltration and epithelium hyperplasia were clearly noticed in asthma induced mouse. In immunohistochemical staining for IL-4 and IL-10, there was no positive reaction in control group. However, very strong reactions were appeared in asthma induced group. In this research, IL-4 and IL-10, which seem to play a central role in allergic asthma, KO mouse was utilized to test the causative relationship between airway inflammation and role of specific cytokine. Asthma induced IL-4 and IL-10 KO mice showed much decreased inflammatory reactions in the number of total BAL cells, in eosinophilic infiltration, and in immunohistochemical stains against diverse inflammatory proteins. These results suggest that IL-4 and IL-10 increase the asthmatic reactions in vivo mice model.

Expression of Matrix Metalloproteinase-9 and Tissue Inhibitor of Metalloproteinase-1 after Administration of Endotoxin in Diabetic Rats

Results: The total BAL cells of the DM-LPS groups were significantly lower than the LPS groups (p < 0.01). The MMP-9 activities in the serum were higher in the DM+LPS groups than in the other groups. The MMP-9 activities in the BAL fluids were significantly higher in the DM+LPS group than in the normal and diabetic rats (p < 0.05). TIMP-1 expressions in the BAL fluids were significantly lower in the D M+LPS group than other groups (p < 0.05). The ratio between MMP-9 and TIMP-1 in the BAL fluids was significantly hi gher in the DM+LPS groups (p < 0.05). Conclusion: In ALI in diabetics the higher MMP-9 activity and lower TIMP-1 level are believed to prolonged and intensify the course of inflammation. (Tuberc Respir Dis 2006; 61: 256-264)

Effect of fungal antigen exposure on airway allergen sensitization

RATIONALE: Repeated exposure to innocuous antigens often induces immunologic tolerance unless if primed with an adjuvant. Fungi, such as Alternaria and Candida, are ubiquitous in the air and their potential pro-inflammatory properties could overcome immunologic tolerance. In this study, we examined the effect of Alternaria and Candida on the airway immune response to OVA using a mouse model.METHODS: BALB/c female mice were exposed intranasally on days 0 and 7 with fungal extracts (Alternaria alternata or Candida albicans), either alone or in combination with OVA. Plasma was obtained on day 14 for measurement of total and OVA-specific IgE and IgG1. The mice were challenged intranasally with OVA on days 21, 22 and 23. Bronchoalveolar lavage fluid was collected on day 24 and analyzed for airway inflammation.RESULTS: Mice exposed to both Alternaria and OVA developed more OVA-specific IgE (0.098±0.034 absorbance at 450 nm (O.D); p=0.053) and IgG1 (0.347±0.033 O.D; p<0.001), and a robust eosinophilic airway inflammatory response (19.8±6.7% of total BAL cells; p<0.05) compared to mice exposed to OVA alone (0.011±0.003, 0.046±0.027 and 2.5±1.3%, respectively). In contrast, mice exposed to both Candida and OVA produced no increase in OVA-specific IgE or IgG1. Furthermore, airway neutrophilia, and not eosinophilia, was observed when mice previously exposed to Candida plus OVA were subsequently exposed to OVA.CONCLUSIONS: Alternaria, but not Candida, is a potent catalyst for Th2-like acquired immunity to bystander antigens. The ability of ubiquitous fungi to enhance airway immune response to bystander antigens may play an important role in mounting an allergic response to aeroallergen. RATIONALE: Repeated exposure to innocuous antigens often induces immunologic tolerance unless if primed with an adjuvant. Fungi, such as Alternaria and Candida, are ubiquitous in the air and their potential pro-inflammatory properties could overcome immunologic tolerance. In this study, we examined the effect of Alternaria and Candida on the airway immune response to OVA using a mouse model. METHODS: BALB/c female mice were exposed intranasally on days 0 and 7 with fungal extracts (Alternaria alternata or Candida albicans), either alone or in combination with OVA. Plasma was obtained on day 14 for measurement of total and OVA-specific IgE and IgG1. The mice were challenged intranasally with OVA on days 21, 22 and 23. Bronchoalveolar lavage fluid was collected on day 24 and analyzed for airway inflammation. RESULTS: Mice exposed to both Alternaria and OVA developed more OVA-specific IgE (0.098±0.034 absorbance at 450 nm (O.D); p=0.053) and IgG1 (0.347±0.033 O.D; p<0.001), and a robust eosinophilic airway inflammatory response (19.8±6.7% of total BAL cells; p<0.05) compared to mice exposed to OVA alone (0.011±0.003, 0.046±0.027 and 2.5±1.3%, respectively). In contrast, mice exposed to both Candida and OVA produced no increase in OVA-specific IgE or IgG1. Furthermore, airway neutrophilia, and not eosinophilia, was observed when mice previously exposed to Candida plus OVA were subsequently exposed to OVA. CONCLUSIONS: Alternaria, but not Candida, is a potent catalyst for Th2-like acquired immunity to bystander antigens. The ability of ubiquitous fungi to enhance airway immune response to bystander antigens may play an important role in mounting an allergic response to aeroallergen.

Aluminum lactate treatment alters the lung biological activity of quartz.

Previous studies of surface modification of quartz particles have suggested that the biological activity of silica is at least in part related to its surface properties. In the present study, we exposed the tracheal lobe of 8 sheep to either 100 ml saline (saline group), 100 mg of quartz (Minusil-5) in 100 ml saline (SI group) or 100 mg of Al lactate treated quartz in 100 ml saline (SI-Al group). The 24 sheep were studied by bronchoalveolar lavage at days 0, 12, 24, 40, 60 and by autopsy at day 60. In the saline group, BAL analyses were as previously reported [1]. In the SI group, we found significant and sustained increases in total BAL cells (x 2, P less than 0.05), macrophages and lymphocytes (x 2, P less than 0.05), neutrophils (x 5-10, P less than 0.01), IgG (x 1.2-1.8, P less than 0.05), fibronectin (x 2-3, P less than 0.05), lactate dehydrogenase (x 3, P less than 0.01) and alkaline phosphatase (x 2, P less than 0.05). Histologically, a macrophagic and lymphocytic alveolitis was observed at day 60. In the SI-Al group, these changes were significantly attenuated and in the above parameters, SI-Al group did not differ from saline group after day 24. These data of BAL and histology of the sheep tracheal lobe model document clearly that aluminum lactate treatment alters the biological activity of quartz.