Bronchoalveolar Lavage Differential Cell Counts Research Articles

The role of serum and bronchoalveolar lavage fluid chitotriosidase activity on diagnosis, disease characteristics and prognosis of sarcoidosis.

Sarcoidosis is a multisystem granulomatous disease with an unpredictable clinical course. Chitotriosidase is a chitinase mainly expressed by activated macrophages. Increased chitotriosidase activity has been reported in serum and bronchoalveolar lavage (BAL) of sarcoidosis patients compared to healthy controls. This study aims to evaluate the role of serum and BAL chitotriosidase activity on diagnosis, disease characteristics, and prognosis of sarcoidosis. Patients referred with suspected sarcoidosis or other interstitial lung disease were prospectively included in the study. All patients underwent bronchoscopy with BAL. Serum and BAL chitotriosidase activity, BAL differential cell counts, and lymphocyte phenotypes were determined. Sarcoidosis patients were followed up regularly. Forty-two sarcoidosis and 28 non-sarcoidosis patients were included in the study. Serum chitotriosidase activity was higher in sarcoidosis group 247.5 (2.78-461) vs 108 (2.78-272) nmol/h/mL (p< 0.001). BAL chitotriosidase activity tended to be higher in sarcoidosis group 11 (2-308) vs 6.95 (2.27-44) nmol/h/mg but was not found to be statistically significant (p= 0.11). Serum and BAL chitotriosidase activities were correlated with each other (p= 0.023, r= 0.355). No significant difference was found between the diagnostic performance of BAL CD4/CD8 ratio and serum chitotriosidase activity (p= 0.079). Serum chitotriosidase and ACE activities were correlated with each other (p= 0.004, r= 0.457). No significant difference was found between serum or BAL chitotriosidase activity and stage or extrapulmonary involvement. Serum chitotriosidase activity was higher in patients who needed systemic therapy at diagnosis (p= 0.046). However, no significant difference was found between serum or BAL chitotriosidase activities and disease progression (p= 0.395 and p= 0.723, respectively). Serum chitotriosidase activity can be helpful in the differential diagnosis of sarcoidosis with a similar diagnostic performance with BAL CD4/CD8 ratio. Although serum chitotriosidase activity at diagnosis does not predict progressive disease, it is associated with the need for systemic therapy at diagnosis. Serial chitotriosidase measurements may be useful in monitoring disease progression during follow-up.

Airway Eosinophilia on Bronchoalveolar Lavage and the Risk of Exacerbations in COPD.

The associations between airway eosinophilia, measured in sputum or peripheral blood, and acute exacerbations of chronic obstructive pulmonary disease (AECOPD) are inconsistent. We therefore aimed to determine the association between eosinophilia in bronchoalveolar lavage (BAL) fluid and AECOPD in a clinical cohort. We analyzed differential cell counts from baseline BAL fluid in participants in the DISARM clinical trial (Clinicaltrials.gov #NCT02833480) and classified participants by the presence or absence of BAL eosinophilia (>1% of total leukocytes). We determined the association between BAL eosinophilia and AECOPD over 1 year of follow-up using negative binomial regression and Cox proportional hazards test. N = 63 participants were randomized, and N = 57 had BAL differential cell counts available. Participants with BAL eosinophilia (N = 21) had a significantly increased rate of acute exacerbations (unadjusted incidence rate ratio (IRR) 2.0, p = 0.048; adjusted IRR 2.24, p = 0.04) and a trend toward greater probability of acute exacerbation (unadjusted hazard ratio (HR) 1.74, p = 0.13; adjusted HR 2.3, p = 0.1) in the year of follow-up compared to participants without BAL eosinophilia (N = 36). These associations were not observed for BAL neutrophilia (N = 41 participants), BAL lymphocytosis (N = 27 participants) or peripheral blood eosinophilia at various threshold definitions (2%, N = 37; 3%, N = 27; 4%, N = 16). BAL may therefore be a sensitive marker of eosinophilic inflammation in the distal lung and may be of benefit for risk stratification or biomarker-guided therapy in COPD.

Bronchoalveolar lavage differential cell count on prognostic assessment of patients with stable or acute interstitial lung disease: A retrospective real-life study

BAL cell differential counts of 133 therapy naive ILD patients and 43 patients during acute exacerbation of ILD (AE-ILD) were retrospectively evaluated.In the 20 patients who underwent BAL both at baseline and during an AE-ILD, there was an increase in neutrophils but a decrease in macrophages and eosinophils in the BAL obtained during AE-ILD. A detectable number of basophils at the baseline was a novel risk factor for earlier death and the occurrence of AE-ILD. Total BAL cell count >160 × 109/L during AE-ILD was correlated with a more favorable prognosis. BAL cell counts <20% of lymphocytes or > 20% of neutrophils during AE-ILD were associated with shorter survival.AE-ILD exerted significant changes in BAL cell profiles in individual patients. Several BAL-parameters correlated with survival of ILD patients; of these, baseline basophils and total cell count during AE-ILD were novel prognostic markers.

Bronchoalveolar lavage as a diagnostic procedure: a review of known cellular and molecular findings in various lung diseases.

Bronchoalveolar lavage (BAL) is a commonly used procedure in the evaluation of lung disease as it allows for sampling of the lower respiratory tract. In many circumstances, BAL differential cell counts have been reported to be typical of specific lung disorders. In addition, more specific diagnostic tests including molecular assays such as polymerase chain reaction (PCR) or enzyme-linked immunosorbent assay, special cytopathologic stains, or particular microscopic findings have been described as part of BAL fluid analysis. This review focuses on common cellular and molecular findings of BAL in a wide range of lung diseases. Since the performance of the first lung irrigation in 1927, BAL has become a common and important diagnostic tool. While some pulmonary disorders have a highly characteristic signature of BAL findings, BAL results alone often lack specificity and require interpretation along with other clinical and radiographic details. Development of new diagnostic assays is certain to reinforce the utility of BAL in the future. Our review of the BAL literature is intended to serve as a resource to assist clinicians in the care of patients with lung disorders.

Aspirin reduces lipopolysaccharide-induced pulmonary inflammation in human models of ARDS

RationalePlatelets play an active role in the pathogenesis of acute respiratory distress syndrome (ARDS). Animal and observational studies have shown aspirin's antiplatelet and immunomodulatory effects may be beneficial in ARDS.ObjectiveTo...

P121: Morphological and functional changes in the population of alveolar macrophages of mice with induced carcinogenesis in the lung

Background Alveolar macrophages (AM) are pulmonary residents of the bone marrow-derived mononuclear phagocyte system that play a critical role in several diverse lung functions and in lung host defense. The role of AM in lung cancer is multifaceted. AM secretion of proinflammatory cytokines has been found to enhance antitumor functions, cytostasis and cytotoxicity. AM have also demonstrated to have protumor functions resulting in tumor progression. Materials and methods 24-week-old male A/f mice were kept in plastic cages under standard laboratory conditions and received food and water ad libitum. Animals were randomly assigned to the following experimental groups: I – control, II – radiation only, III – sulphur dioxide only, IV – sulphur dioxide + radiation. Animals were subjected to total irradiation at dose 1.0 Gy (dose density of 16.6 cGy/min, using a 60Co gamma equipment). After irradiation mice were inhaled air with sulphur dioxide (20 mg/m 3 for 1 h) in separate exposure chamber. At the 20th week of experiment the 20 mice per each group were subjected to autopsy. Excised lungs were fixed in neutral buffered 10% formalin. To obtain an index of tumor incidence, the percentage of tumor – bearing mice per total number of mice in each group was calculated. Tumor multiplicity was defined as the average number of tumors per mice, obtained by dividing the total number of tumors by the total number of mice per group, including non-tumor bearing animals. Bronchoalveolar lavage (BAL) of lungs was performed on 6 mice (animals were anesthetized by an ip injection of sodium thiopental) from each study group at the 1, 7, 15 and 30 days after irradiation and SO2 inhalation. Total cells of BAL were counted using hemocytometer chamber and an optical microscope with a 400× zoom. BAL differential cell (macrophages, lymphocytes, eosinophiles and neutrophils) counts were performed on slides (Romanowsky stain). Phagocytic activity of AM was counted on slides. AM morphometric study was done by measuring the area of each cell (square of cell) and the area of nuclear of each cell (square of nuclear) with use of the original image analyzer computer system. Results Results of the combined effects of radiation and inhalation of sulfur dioxide showed the growth of tumor quantity in the lung: the frequency of adenomas/mouse was more than 42% higher than that of control group. Synergism coefficient was 1.1 in this case. Irradiation at a dose of 1.0 Gy modifies the reaction of free cells of lung on the action of sulfur dioxide, which is manifested in the change of the number of cells in the BAL fluid, the phagocytic activity of the AM, as well as of the changes the morphometric parameters of their cells. Conclusion Thus, if the amount of radiation exposure of BAL cells was significantly reduced by the 1st and 7th days, and after inhalation of sulfur dioxide – tended to increase, then the combined action on the 7th day it was almost half the theoretical (hypothetical) the sum of the separate effects of the studied factors. Exposure to sulfur dioxide significantly slowed the activation of cell count. It was found, that after combined action of the investigated factors the morphological characteristics of macrophages (reducing the square of cells and their nuclei) have changed (7th and 15th day). The absorption activity of phagocytes was significantly reduced (7 day).

Anti-malarial drug artesunate protects against cigarette smoke-induced lung injury in mice

Cigarette smoking is the primary cause of chronic obstructive pulmonary disease (COPD), which is mediated by lung infiltration with inflammatory cells, enhanced oxidative stress, and tissue destruction. Anti-malarial drug artesunate has been shown to possess anti-inflammatory and anti-oxidative actions in mouse asthma models. We hypothesized that artesunate can protect against cigarette smoke-induced acute lung injury via its anti-inflammatory and anti-oxidative properties. Artesunate was given by oral gavage to BALB/c mice daily 2h before 4% cigarette smoke exposure for 1h over five consecutive days. Bronchoalveolar lavage (BAL) fluid and lungs were collected for analyses of cytokines, oxidative damage and antioxidant activities. Bronchial epithelial cell BEAS-2B was exposed to cigarette smoke extract (CSE) and used to study the mechanisms of action of artesunate. Artesunate suppressed cigarette smoke-induced increases in BAL fluid total and differential cell counts; levels of IL-1β, MCP-1, IP-10 and KC; and levels of oxidative biomarkers 8-isoprostane, 8-OHdG and 3-nitrotyrosine in a dose-dependent manner. Artesunate promoted anti-oxidant catalase activity and reduced NADPH oxidase 2 (NOX2) protein level in the lungs from cigarette smoke-exposed mice. In BEAS-2B cells, artesunate suppressed pro-inflammatory PI3K/Akt and p44/42 MAPK signaling pathways, and increased nuclear Nrf2 accumulation in response to CSE. Artesunate possesses anti-inflammatory and anti-oxidative properties against cigarette smoke-induced lung injury, probably via inhibition of PI3K and p42/22 MAPK signaling pathways, augmentation of Nrf2 and catalase activities, and reduction of NOX2 level. Our data suggest that artesunate may have therapeutic potential for treating COPD.

Usefulness of cellular analysis of bronchoalveolar lavage fluid for predicting the etiology of pneumonia in critically ill patients.

BackgroundThe usefulness of bronchoalveolar lavage (BAL) fluid cellular analysis in pneumonia has not been adequately evaluated. This study investigated the ability of cellular analysis of BAL fluid to differentially diagnose bacterial pneumonia from viral pneumonia in adult patients who are admitted to intensive care unit.MethodsBAL fluid cellular analysis was evaluated in 47 adult patients who underwent bronchoscopic BAL following less than 24 hours of antimicrobial agent exposure. The abilities of BAL fluid total white blood cell (WBC) counts and differential cell counts to differentiate between bacterial and viral pneumonia were evaluated using receiver operating characteristic (ROC) curve analysis.ResultsBacterial pneumonia (n = 24) and viral pneumonia (n = 23) were frequently associated with neutrophilic pleocytosis in BAL fluid. BAL fluid median total WBC count (2,815/µL vs. 300/µL, P<0.001) and percentage of neutrophils (80.5% vs. 54.0%, P = 0.02) were significantly higher in the bacterial pneumonia group than in the viral pneumonia group. In ROC curve analysis, BAL fluid total WBC count showed the best discrimination, with an area under the curve of 0.855 (95% CI, 0.750–0.960). BAL fluid total WBC count ≥510/µL had a sensitivity of 83.3%, specificity of 78.3%, positive likelihood ratio (PLR) of 3.83, and negative likelihood ratio (NLR) of 0.21. When analyzed in combination with serum procalcitonin or C-reactive protein, sensitivity was 95.8%, specificity was 95.7%, PLR was 8.63, and NLR was 0.07. BAL fluid total WBC count ≥510/µL was an independent predictor of bacterial pneumonia with an adjusted odds ratio of 13.5 in multiple logistic regression analysis.ConclusionsCellular analysis of BAL fluid can aid early differential diagnosis of bacterial pneumonia from viral pneumonia in critically ill patients.

Pneumopathie infiltrante diffuse associée à une dermatomyosite amyopathique avec auto-anticorps anti-MDA5

Amyopathic dermatomyositis associated with anti-MDA5 autoantibodies is a rare and very recently described clinical entity. A 58-year-old woman was admitted with subacute onset of dyspnea (NYHA class IV) associated with cough, oligoarthritis of the wrists, myalgia and intermittent fever. Examination demonstrated skin lesions with heliotrope rash, Gottron's papules, "mechanics hands", and basal inspiratory crackles on lung auscultation. Pulmonary function tests showed a restrictive ventilatory defect, with decreased carbon monoxide diffusion capacity and marked hypoxemia (PaO2 61 mmHg). The chest high-resolution computed tomography appearances were consistent with organizing pneumonia. Bronchoalveolar lavage differential cell count demonstrated 22 % neutrophils. Serum creatine kinase and electromyography were normal ; the serum ferritin level was elevated. Antinuclear antibodies were present and anti-MDA5 autoantibodies were identified. Significant improvement was obtained with systemic corticosteroids, later converted to mycophenolate mofetil as a steroid-sparing agent. Amyopathic dermatomyositis associated with anti-MDA5 autoantibodies shares some characteristics with those associated with anti-synthetase antibodies. Muscular involvement may be mild or absent. Early diagnosis and treatment may improve outcome.

Primary Antiphospholipid Syndrome–Associated Diffuse Alveolar Hemorrhage

Diffuse alveolar hemorrhage (DAH) is an uncommon complication of primary antiphospholipid syndrome (APS). We aimed to describe the clinical characteristics, treatment, and outcomes of primary APS-associated DAH in a single center. We conducted a retrospective review of all adults with primary APS-associated DAH evaluated at Mayo Clinic over a 15-year period. DAH was defined as bilateral pulmonary infiltrates and bronchoalveolar lavage (BAL) fluid documenting progressively bloody returns and/or the presence of >20% hemosiderin-laden macrophages. Patients with other causes of DAH were excluded. Eighteen patients were identified (median age 43 years). Capillaritis was present in surgical lung biopsy samples of 3 patients. BAL differential cell counts revealed predominantly neutrophils. All patients were treated initially with glucocorticoids. Cyclophosphamide (CYC) was used in 8 patients; complete remission was achieved in 3 patients treated with CYC alone and in 1 patient receiving combination therapy with rituximab (RTX). RTX was used in 9 patients; 2 patients achieved remission with RTX alone, whereas 3 patients required combination therapy with CYC or mycophenolate mofetil (MMF). No patient achieved complete remission while receiving single therapy with MMF, azathioprine, or plasma exchange. Intravenous gamma globulin therapy was administered in 5 patients; no patient achieved control of the disease. Six patients died, all because of complications related to uncontrolled DAH or its therapy. We present the largest case series of primary APS-associated DAH reported in the literature. DAH carries a very poor prognosis and therapeutic options are limited. Immunosuppression with either CYC or RTX is associated with the highest likelihood of remission induction and should be considered early.

IL-4 and IL-5 Secretions Predominate in the Airways of Wistar Rats Exposed to Toluene Diisocyanate Vapor

ObjectivesWe established a Wistar rat model of asthma caused by toluene diisocyanate (TDI) exposure, and investigated the relationship between TDI exposure concentrations and respiratory hypersensitivity, airway inflammation, and cytokine secretions in animals, to better understand the mechanism of TDI induced occupational asthma.MethodsWistar rats were exposed to two different concentrations of TDI vapor four hours a day for five consecutive days. Bronchoalveolar lavage (BAL) was performed, and differential leucocytes from the BAL fluid were analyzed. Lung histopathological examination was carried out to investigate the inflammatory status in the airways. Production of cytokines interleukin (IL)-4 and IL-5 productions in the BAL fluid in vivo was determined with enzyme-linked immunosorbent assay kits.ResultsThe TDI-exposed rats exhibited greater airway hypersensitivity symptoms than the control rats. The BAL differential cell count and lung histopathological examination demonstrated that inflammation reactions were present in both the central and peripheral airways, characterized with marked infiltration of eosinophils in the TDI-exposed rats. The cytokine assay showed that IL-4 and IL-5 were predominantly produced in the BAL fluid in vivo.ConclusionsThese findings imply that TDI exposure concentrations may greatly affect the occurrence and extent of inflammatory events and that Th2 type cytokines may play an important role in the immunopathogenesis of TDI-induced occupational respiratory hypersensitivity.

Bronchoalveolar lavage in infants with recurrent lower respiratory symptoms.

BackgroundFew data are available about the inflammatory cytokine profile of bronchoalveolar lavage (BAL) from young children with frequent wheeze. The first aim was to investigate the BAL cellular and cytokine profiles in infants with recurrent lower respiratory symptoms in whom bronchoscopy was indicated for clinical symptom evaluation. The second aim was to relate the BAL results with the histological findings of the endobronchial carina biopsies.MethodsThirty-nine infants (median age 0.9 years) underwent lung function testing by whole-body plethysmography prior to the bronchoscopy. The BAL differential cell counts and cytokine levels were quantified. These findings were compared with the histological findings of the endobronchial carina biopsies.ResultsThe differential cytology reflected mainly that described for healthy infants with lymphocyte counts at the upper range level. A positive association between BAL CD8+ lymphocytes and neutrophils and endobronchial reticular basement membrane was found. Detectable levels of pro-inflammatory cytokine proteins IL-1β, IL-17A, IL-18, IL-23, and IL-33 were found, whereas levels of Th2-type cytokine proteins were low. Frequent wheeze was the only clinical characteristic significantly related to detectable combined pro-inflammatory cytokine profile. Lung function did not correlate with any cytokine.ConclusionsA positive association between BAL CD8+ lymphocytes and neutrophils and endobronchial reticular basement thickness was found. Detectable production of pro-inflammatory cytokines associated positively with frequent wheeze.

Combined pulmonary fibrosis and emphysema syndrome associated with ABCA3 mutations

To the Editor: Herein, we present the first report of combined pulmonary fibrosis and emphysema (CPFE) in an adult patient who was compound heterozygous for mutations of the ATP-binding cassette subfamily A member 3 gene ( ABCA3 , MIM 601615). A 41-year-old nonsmoking male presented with dyspnoea on mild exertion. The patient’s medical history indicated neonatal respiratory distress, gastro-oesophageal reflux and pneumonia 8 years previously that resolved with antibiotics. His physical examination revealed a mild pectus excavatum, finger clubbing and bilateral basal crackles. High-resolution computed tomography (HRCT) of the chest showed voluminous emphysema in the upper zones of the lungs associated with honeycomb fibrosis and ground-glass opacity in lower lobes, predominating in left lung (fig. 1). The bronchoalveolar lavage differential cell count was 67% macrophages, 22% neutrophils and 8% lymphocytes. Pulmonary function tests showed: total lung capacity of 75%, vital capacity (VC) of 50%, residual volume of 134%; forced expiratory volume in 1 s (FEV1) of 49%, diffusing capacity of the lung for carbon monoxide of 38% predicted, FEV1/VC of 74%, and arterial oxygen tension at room air was 96 mmHg. During a 6-min walk test the peripheral oxygen saturation decreased from 96% at rest to 90% after 630 m (80% of predicted value). A lung biopsy was not performed. …

No exacerbation but impaired anti-viral mechanisms in a rhinovirus-chronic allergic asthma mouse model

Severe asthma and viral-induced asthma exacerbations represent a high unmet medical need as no therapy is currently available for these patients. HRV (human rhinovirus) is prominently associated with asthma exacerbations in humans. The aim of the present study was to establish a mouse model of severe asthma with additional rhinovirus infection to investigate the interplay between chronic allergic airway inflammation and acute respiratory viral infection. Balb/c mice were sensitized with HDM (house dust mite) extract (25μg in 50μl of saline) by i.n. (intranasal) delivery to the lung over 7weeks. HRV1B (HRV serotype 1B) inoculation was performed i.n. on the last 3days. Therapeutic treatment with FP (fluticasone propionate) was performed to assess steroid efficacy. Lung resistance was measured invasively to assess AHR (airway hyper-responsiveness). BAL (bronchoalveolar lavage) differential cell count, cytokines, lung histology and the proliferative and cytokine response of MLN (mediastinal lymph node) cells upon invitro restimulation were analysed. Chronic HDM application induced a strong Th2-skewed eosinophilic airway inflammation and AHR, which was not exacerbated by superimposed HRV1B infection. Therapeutic steroid intervention in the chronic HDM model reduced BAL eosinophil cell counts, cytokine levels and AHR, while neutrophil numbers were unaffected. Steroid efficacy against inflammatory readouts was maintained during additional HRV1B infection. Animals with chronic allergic airway inflammation exhibited a diminished immune response towards superimposed HRV1B infection compared with HRV1B alone, as induction of the anti-viral and pro-inflammatory cytokines IFN (interferon)-α, IFN-γ and IL (interleukin)-12 were suppressed. Although superimposed HRV1B infection did not provoke asthma exacerbation in this severe model, a deficient anti-viral immune response to HRV1B was present under chronic allergic airway inflammatory conditions. Thus, this model is able to reflect some aspects of the complex interplay of respiratory virus infection in chronic allergic asthma.

Alveolar Foamy Macrophages and Mast Cells in Bronchoalveolar Lavage of Methotrexate-induced Pneumonitis

Methotrexate-induced pneumonitis (MTX-P) is an unusual adverse drug reaction that typically manifests as an interstitial lung disease (ILD). Early diagnosis of this complication is desirable because discontinuation of MTX with or without steroid administration often results in a fast recovery. Conversely, a delay or a misdiagnosis may lead to death. Diagnostic workup is often problematic because underlying diseases leading to methotrexate administration, notably rheumatoid arthritis (RA), and opportunistic infections because of drug-induced immune impairment can also produce an ILD. In this clinical setting, bronchoalveolar lavage (BAL) plays a key role either in excluding infectious agents, especially in febrile presentations, or in looking for malignant cells. Unfortunately, BAL does not have specific features to allow clinicians to differentiate MTX-P from RA-associated ILD. Here, we present 2 definite cases of MTX-P in RA patients. BAL differential cell counts and alveolar CD4/CD8 values were found to be in agreement with those reported in the literature, but an increase in mast cells and the presence of foamy macrophages have never been reported among previously published cases. The presence of both mast cells and foamy macrophages could be helpful in differentiating MTX-P from many drug-unrelated ILDs, but does not assist in distinguishing MTX-P from Pneumocystis jiroveci pneumonia and RA-associated bronchiolitis obliterans organizing pneumonia.

Immune Modulatory Effects of IL-22 On Allergen-Induced Pulmonary Inflammation

RationaleAllergen-induced pulmonary inflammation in asthma is mainly TH2 dominated. A recently discovered TH17/TH22 cell-derived cytokine, IL-22, has been found to be increased in asthma. However, several studies showed controversial findings in the immune modulatory effects of IL-22 in animal models of allergic asthma. Our objective was to determine the regulatory role of IL-22 in OVA-induced allergic inflammation using a novel lung-specific IL-22 transgenic overexpression system.MethodsInducible IL-22 transgenic (CC10-rtTA- and SPC-rtTA-TRE-tight-IL-22) mice on C57BL/6 genetic background were generated. IHC of lung tissue and ELISA of bronchoalveolar lavage (BAL) fluid for IL-22 were performed to confirm the location and quantity of IL-22 expression. OVA-induced pulmonary inflammatory responses were compared between IL-22 transgenic and wild type control mice. BAL total and differential cell counts, lung histology, mucous metaplasia, IHC of major basic protein for eosinophils, and serum OVA-specific IgE were determined.ResultsIL-22 transgene was successfully activated in the large (CC10 promoter) and small (SPC promoter) airway epithelium cells by doxycycline in the drinking water. OVA sensitization and challenge did not induce endogenous IL-22 expression. Compared to wild type mice, IL-22 transgenic mice showed decreased percentage of eosinophils in the BAL and slight reduction in eosinophilic inflammation and mucus metaplasia in the airways. Interestingly, inflammatory cell infiltration in lung parenchyma appeared slightly accentuated. In addition, there was no statistic difference in serum OVA-specific IgE.ConclusionsThese findings indicate that IL-22 may have immune modulatory effects on pulmonary inflammatory responses in the development of allergen-induced asthma. RationaleAllergen-induced pulmonary inflammation in asthma is mainly TH2 dominated. A recently discovered TH17/TH22 cell-derived cytokine, IL-22, has been found to be increased in asthma. However, several studies showed controversial findings in the immune modulatory effects of IL-22 in animal models of allergic asthma. Our objective was to determine the regulatory role of IL-22 in OVA-induced allergic inflammation using a novel lung-specific IL-22 transgenic overexpression system. Allergen-induced pulmonary inflammation in asthma is mainly TH2 dominated. A recently discovered TH17/TH22 cell-derived cytokine, IL-22, has been found to be increased in asthma. However, several studies showed controversial findings in the immune modulatory effects of IL-22 in animal models of allergic asthma. Our objective was to determine the regulatory role of IL-22 in OVA-induced allergic inflammation using a novel lung-specific IL-22 transgenic overexpression system. MethodsInducible IL-22 transgenic (CC10-rtTA- and SPC-rtTA-TRE-tight-IL-22) mice on C57BL/6 genetic background were generated. IHC of lung tissue and ELISA of bronchoalveolar lavage (BAL) fluid for IL-22 were performed to confirm the location and quantity of IL-22 expression. OVA-induced pulmonary inflammatory responses were compared between IL-22 transgenic and wild type control mice. BAL total and differential cell counts, lung histology, mucous metaplasia, IHC of major basic protein for eosinophils, and serum OVA-specific IgE were determined. Inducible IL-22 transgenic (CC10-rtTA- and SPC-rtTA-TRE-tight-IL-22) mice on C57BL/6 genetic background were generated. IHC of lung tissue and ELISA of bronchoalveolar lavage (BAL) fluid for IL-22 were performed to confirm the location and quantity of IL-22 expression. OVA-induced pulmonary inflammatory responses were compared between IL-22 transgenic and wild type control mice. BAL total and differential cell counts, lung histology, mucous metaplasia, IHC of major basic protein for eosinophils, and serum OVA-specific IgE were determined. ResultsIL-22 transgene was successfully activated in the large (CC10 promoter) and small (SPC promoter) airway epithelium cells by doxycycline in the drinking water. OVA sensitization and challenge did not induce endogenous IL-22 expression. Compared to wild type mice, IL-22 transgenic mice showed decreased percentage of eosinophils in the BAL and slight reduction in eosinophilic inflammation and mucus metaplasia in the airways. Interestingly, inflammatory cell infiltration in lung parenchyma appeared slightly accentuated. In addition, there was no statistic difference in serum OVA-specific IgE. IL-22 transgene was successfully activated in the large (CC10 promoter) and small (SPC promoter) airway epithelium cells by doxycycline in the drinking water. OVA sensitization and challenge did not induce endogenous IL-22 expression. Compared to wild type mice, IL-22 transgenic mice showed decreased percentage of eosinophils in the BAL and slight reduction in eosinophilic inflammation and mucus metaplasia in the airways. Interestingly, inflammatory cell infiltration in lung parenchyma appeared slightly accentuated. In addition, there was no statistic difference in serum OVA-specific IgE. ConclusionsThese findings indicate that IL-22 may have immune modulatory effects on pulmonary inflammatory responses in the development of allergen-induced asthma. These findings indicate that IL-22 may have immune modulatory effects on pulmonary inflammatory responses in the development of allergen-induced asthma.

Neutrophil and Eosinophil Granulocytes as Key Players in a Mouse Model of Chemical-Induced Asthma

Diisocyanates are an important cause of chemical-induced occupational asthma. This type of immunologically mediated asthma is often characterized by a predominant granulocytic inflammation in the airways, rather than an infiltration by lymphocytes. We sought to determine the contribution of granulocytes in the outcome of chemical-induced asthma using general and specific leukocyte depletion strategies in an established mouse model of isocyanate asthma. On days 1 and 8, BALB/c mice received dermal applications with toluene-2,4-diisocyanate (TDI) or vehicle (acetone olive oil), followed by two ip injections of cyclophosphamide (CP, days 11 and 13), or one iv injection of antigranulocyte receptor 1 (aGR1, day 13) monoclonal antibody (mAb), or two ip injections of Ly6G-specific mAb (1A8, days 13 and 14). On day 15, the mice were challenged (oropharyngeal administration) with TDI or vehicle. The next day, we assessed methacholine airway hyperreactivity (AHR); bronchoalveolar lavage differential cell count; histopathology and total serum IgE; and auricular lymphocyte subpopulations and release of interleukin (IL)-2, IL-4, IL-10, IL-13, and gamma interferon by these lymphocytes. CP depleted all leukocyte types and completely prevented AHR and airway inflammation. aGR1 depleted granulocytes and CD8(+) lymphocytes, which resulted in a partial prevention in AHR but no decrease in airway inflammation. Depletion of Ly6G-positive granulocytes, i.e., both neutrophils and eosinophils, prevented AHR and lung epithelial damage and significantly reduced airway inflammation. Injection of aGR1 or 1A8 led to significantly changed cytokine release patterns in TDI-treated mice. Granulocytes, both neutrophils and eosinophils, are key cellular players in this model of chemical-induced asthma.

Diagnostic Value of Antibodies Against Pseudomonas aeruginosa in Bronchoalveolar Lavage Fluid After Lung Transplantation

Background Pseudomonal airway colonization is a risk factor for chronic allograft dysfunction after lung transplantation (LTx). Pseudomonas aeruginosa exoproteases are involved in initiating colonization, and immune complexes directed against these proteases may activate innate immune responses. Objective To investigate whether specific antibodies against pseudomonal proteases could be measured in bronchoalveolar lavage (BAL) fluid, whether they are associated with innate immune responses, and whether they could identify patients with chronic P aeruginosa colonization after LTx. Materials and Methods BAL fluid from 40 noncolonized and 25 chronically colonized LTx recipients was retrospectively assayed for IgG antibodies against P aeruginosa alkaline protease (AP), elastase (Ela), and exotoxin (Exo), and for BAL total and differential cell counts and IL-8 protein concentration. Results BAL anti-Ela and anti-Exo antibody titers were significantly increased in colonized compared with noncolonized patients ( P = .009 and P = .02, respectively), whereas anti-AP titers were comparable ( P = .79). Antibody titers strongly correlated with each other, and anti-Ela and anti-Exo titers, but not anti-AP titers, also correlated with BAL total cellularity, neutrophilia, and IL-8 protein concentration. Anti-Ela antibodies demonstrated the greatest diagnostic value in receiver operating characteristic analysis to detect chronic airway colonization ( P = .009), followed by anti-Exo ( P = .02) and anti-AP ( P = .79). A combination of all 3 antibodies resulted in overall sensitivity of 45% (95% confidence interval [CI], 29.3–61.5), specificity of 88% (95% CI, 68.8–97.5), and positive predictive value of 55% (95% CI, 38.5–70.7). Conclusion P aeruginosa proteases in BAL may be associated with local innate immune responses, and could have the potential to enable detection of chronic colonization after LTx.

Thioredoxin as a biomarker for graft rejection in lung transplant recipients

Primary graft dysfunction and rejection are common complications in lung transplant recipients. Increased expression of thioredoxin-1 (Trx), a 12-kDa redox-regulatory protein, has been reported in multiple lung pathophysiological conditions involving oxidative and inflammatory mediated injury including graft rejection in canine and rat models of lung transplantation. Our objective was to determine whether increased Trx expression is associated with progression of rejection pathophysiology in human lung transplant recipients. Bronchoalveolar lavage (BAL) fluid and transbronchial biopsy samples were collected as a routine part of post-transplant clinical care from 18 lung transplant patients from our adult lung transplant programme. Lung transplant recipient profile included age/sex, ethnic background, days on ventilator, total ischaemic time, and cytomegalovirus (CMV) status. Based on histopathological grading criteria, patients were divided into two groups, rejecting (A1/A2 or B1) and non-rejecting (A0/B0). Rejecting and non-rejecting group total BAL cell counts and differential cell counts for neutrophils, macrophages, lymphocytes and eosinophils as well as total BAL cell Trx levels were analysed. Total BAL cell counts were significantly (p<0.05) elevated in graft rejecting versus non-rejecting patients. Differential BAL macrophage counts were comparable in rejection and non-rejection groups, whereas there were significant increases in neutrophils and lymphocytes but not eosinophils in patients with rejection versus non-rejection pathology (p<0.05). Total ischaemic time and days on ventilator in rejection and non-rejection groups were comparable. However, Trx levels were significantly elevated in BAL cells from graft-rejecting patients compared with non-rejecting patients (p<0.05). These data suggest that surveillance monitoring of BAL Trx levels after lung transplantation can serve as a biomarker to assess severity of graft rejection.

Protective effect of orally administered carnosine on bleomycin-induced lung injury

Carnosine is an endogenously synthesized dipeptide composed of beta-alanine and L-histidine. It acts as a free radical scavenger and possesses antioxidant properties. Carnosine reduces proinflammatory and profibrotic cytokines such as transforming growth factor-beta (TGF-beta), IL-1, and TNF-alpha in different experimental settings. In the present study, we investigated the efficacy of carnosine on the animal model of bleomycin-induced lung injury. Mice were subjected to intratracheal administration of bleomycin and were assigned to receive carnosine daily by an oral bolus of 150 mg/kg. One week after fibrosis induction, bronchoalveolar lavage (BAL) cell counts and TGF-beta levels, lung histology, and immunohistochemical analyses for myeloperoxidase, TGF-beta, inducible nitric oxide synthase, nitrotyrosine, and poly(ADP-ribose) polymerase were performed. Finally, apoptosis was quantified by terminal deoxynucleotidyltransferase-mediated UTP end-labeling assay. After bleomycin administration, carnosine-treated mice exhibited a reduced degree of lung damage and inflammation compared with wild-type mice, as shown by the reduction of 1) body weight, 2) mortality rate, 3) lung infiltration by neutrophils (myeloperoxidase activity and BAL total and differential cell counts), 4) lung edema, 5) histological evidence of lung injury and collagen deposition, 6) lung myeloperoxidase, TGF-beta, inducible nitric oxide synthase, nitrotyrosine, and poly(ADP-ribose) polymerase immunostaining, 7) BAL TGF-beta levels, and 8) apoptosis. Our results indicate that orally administered carnosine is able to prevent bleomycin-induced lung injury likely through its direct antioxidant properties. Carnosine is already available for human use. It might prove useful as an add-on therapy for the treatment of fibrotic disorders of the lung where oxidative stress plays a role, such as for idiopathic pulmonary fibrosis, a disease that still represents a major challenge to medical treatment.