Flow Cytometry as an Alternative to Microscopy for the Differentiation of BAL Fluid Leukocytes.

BACKGROUND: Microscopy is currently the gold standard to differentiate BAL fluid (BALF) leukocytes. However, local expertise for microscopic BALF leukocyte differentiation is often unavailable in clinical practice. RESEARCH QUESTION: Can automated flow cytometry be used instead of microscopy to differentiate BALF leukocytes? STUDY DESIGN AND METHODS: A new automated flow cytometric method for BALF leukocyte differentiation, using four antibodies (anti-CD45, anti-CD66b, anti-HLA-DR, anti-CD52) given to human BALF in one tube, was developed and prospectively validated in 745 unselected, subsequent BALF samples from patients with interstitial lung diseases (455 patients), infectious diseases (196 patients), and other diseases (94 patients). Flow cytometry and traditional microscopy were performed by separate investigators in a double-blind fashion. Results were compared using Spearman`s correlation, Deming regression, and Bland-Altman analysis. RESULTS: There was a strong correlation between flow cytometric and microscopic results regarding macrophage/monocyte, lymphocyte, eosinophil, and neutrophil percentages in BALF (P < .001 for all leukocyte subpopulations). Bland-Altman analyses showed that the mean differences between the methods were ≤2% for all four cell types. Flow cytometric results differed less than 20% from microscopic results in more than 95% of all samples. Subgroup analyses confirmed that these results were independent from total leukocyte counts in BALF. INTERPRETATION: We report the first validated flow cytometric method for BALF leukocyte differentiation, which can be used in clinical settings where local expertise for microscopic analysis is unavailable and which can be combined easily with lymphocyte surface marker analysis.

Bronchoalveolar lavage for the diagnosis of Pulmonary Langerhans cell histiocytosis.

BACKGROUND: The histologic diagnosis of Pulmonary Langerhans cell histiocytosis (PLCH) is invasive and can cause complications. To confirm the diagnosis of PLCH, guidelines therefore recommend measuring CD1a-positive bronchoalveolar lavage fluid (BALF) cells despite its poor sensitivity and specificity. Thus, an improved diagnostic accuracy of BALF cell analysis would be desirable. METHODS: Using four-colour flow cytometry, plasmacytoid and myeloid dendritic cells (DCs) were analysed in BALF of 10 newly diagnosed, untreated, smoking patients with PLCH, and compared with BALF DCs from 40 asymptomatic smokers and 21 never-smokers. RESULTS: Compared with controls, myeloid DCs (median: 0.79% of BALF leukocytes) and their subpopulation of Langerhans cells (median: 0.44% of BALF leukocytes) were not increased in PLCH. Patients with PLCH displayed a normal expression of the maturity marker CD83 on BALF myeloid DCs. However, the expression of the co-signaling molecule CD80 on BALF myeloid DCs was significantly lower than in both control groups, with the lowest expression found in more severe disease (presence of cysts > 2 cm in diameter). Based on receiver operating characteristic (ROC) curve analysis, a cut-off of 53% CD80-positive BALF myeloid DCs was optimal for the diagnosis of PLCH, yielding a sensitivity of 0.90 and a specificity of 0.90. CONCLUSIONS: BALF Langerhans cells are not increased in PLCH. However, PLCH is characterised by a low expression of CD80 on BALF myeloid DCs. Due to its considerably higher sensitivity and specificity, this marker appears to be more appropriate to diagnose PLCH than the currently recommended marker CD1a.

Imbalance of dendritic cell co-stimulation in COPD.

BACKGROUND: Dendritic cells (DCs) control immunity and play a role in the pathogenesis of chronic obstructive pulmonary disease (COPD). However, the expression of function-associated surface molecules on circulating DCs in COPD is unknown. METHODS: Four-colour flow cytometry was used to compare blood DC surface molecules of 54 patients with COPD (median age: 59 years; median FEV1: 38% predicted, median CAT score: 24) with two age-matched control groups with normal lung function: 21 current smokers and 21 never-smokers. RESULTS: Concentrations of plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) and the mDC/pDC ratio did not differ between the groups. The increased expression of BDCA-1, BDCA-3, CD86 and CCR5 on mDCs in patients with COPD did not significantly differ from smokers with normal lung function. In contrast, COPD was specifically characterised by a decreased expression of the anti-inflammatory co-stimulatory molecule PD-L1 on pDCs and an increased expression of the pro-inflammatory co-stimulatory molecule OX40 ligand (OX40L) on mDCs. These changes were not confined to patients with elevated systemic inflammation markers (leukocytes, c-reactive protein, interleukin-6, fibrinogen). The ratio of OX40L to PD-L1 expression (OX40L/PD-L1 ratio), a quantitative measure of imbalanced DC co-stimulation, correlated with the severity of pulmonary emphysema in patients with COPD. CONCLUSION: An imbalance of DC co-stimulation might contribute to the pathogenesis of COPD.

Impact of smoking on dendritic cell phenotypes in the airway lumen of patients with COPD.

BACKGROUND: Myeloid dendritic cells (DCs) are increased in the airway wall of patients with chronic obstructive pulmonary disease (COPD), and postulated to play a crucial role in COPD. However, DC phenotypes in COPD are poorly understood. METHODS: Function-associated surface molecules on bronchoalveolar lavage fluid (BALF) DCs were analyzed using flow cytometry in current smokers with COPD, in former smokers with COPD and in never-smoking controls. RESULTS: Myeloid DCs of current smokers with COPD displayed a significantly increased expression of receptors for antigen recognition such as BDCA-1 or Langerin, as compared with never-smoking controls. In contrast, former smokers with COPD displayed a significantly decreased expression of these receptors, as compared with never-smoking controls. A significantly reduced expression of the maturation marker CD83 on myeloid DCs was found in current smokers with COPD, but not in former smokers with COPD. The chemokine receptor CCR5 on myeloid DCs, which is also important for the uptake and procession of microbial antigens, was strongly reduced in all patients with COPD, independently of the smoking status. CONCLUSION: COPD is characterized by a strongly reduced CCR5 expression on myeloid DCs in the airway lumen, which might hamper DC interactions with microbial antigens. Further studies are needed to better understand the role of CCR5 in the pathophysiology and microbiology of COPD.

Fluticasone impact on airway dendritic cells in smokers: a randomized controlled trial.

BACKGROUND: Myeloid Dendritic cells are key drivers of inflammation in smoke-related lung diseases, whereas plasmacytoid DCs play a crucial role in the defense against infections. Effects of inhaled corticosteroids (ICS) on airway DCs in smokers are unknown. METHODS: In this randomized, double-blind, placebo-controlled clinical trial, 45 active cigarette smokers inhaled placebo, fluticasone or fluticasone plus salmeterol twice daily for 4 weeks. Bronchoalveolar lavage fluid DCs were analyzed using four-color flow cytometry before and after the inhalation period. In addition, fluticasone effects were tested on T-cell proliferation in co-cultures with blood myeloid DCs from smokers. RESULTS: Inhalation of fluticasone plus salmeterol, but not fluticasone alone or placebo, reduced endobronchial concentrations of myeloid DCs (median decrease: 24%), macrophages (median decrease: 26%) and neutrophils (median decrease: 76%). In contrast, fluticasone reduced plasmacytoid DC concentrations independently of salmeterol. There were no changes in the expression of function-associated surface molecules on myeloid DC (such as CD1a, Langerin, BDCA-1, CD83 or CCR5) in all groups after treatment. Fluticasone (either alone or in combination with salmeterol) suppressed T-cell proliferation in co-cultures with blood myeloid DCs from smokers. CONCLUSIONS: Resistance to ICS monotherapy in smokers might in part be due to lacking effects on airway myeloid DCs, whereas the increased risk for infections during ICS therapy could be attributable to a reduction in plasmacytoid DCs. Combination therapy of fluticasone with salmeterol is associated with a reduction in airway myeloid DCs, but also airway macrophages and neutrophils. TRIAL REGISTRATION: Registered at ClinicalTrials.gov (identifier: NCT00908362) and the European Clinical Trial Database, EudraCT (identifier: 2009-009459-40).

Stage-dependent association of BDNF and TGF-ß1 with lung function in stable COPD.

BACKGROUND: Chronic Obstructive Pulmonary Disease (COPD) is characterised by complex inflammatory, neuronal and fibrotic changes. Brain-derived Neurotrophic Factor (BDNF) is a key regulator of neuronal plasticity, whereas Transforming Growth Factor-ß1 (TGF-ß1) plays a crucial role in tissue repair and emphysema pathogenesis. Both mediators are stored in platelets and released from platelets in inflammatory conditions and during serum preparation. In patients with asthma, it was previously shown that elevated serum BDNF concentrations correlate with disease severity, whereas TGF-ß1 concentrations were normal. METHODS: In the present study, 63 patients with stable COPD (spirometric GOLD stages 2-4) and 17 age- and comorbidity-matched controls were studied. Lung function, smoking history, medication, platelet concentrations in peripheral blood and serum concentrations of BDNF, TGF-ß1 and Serotonin (5-HT) were assessed in all participants. RESULTS: Serum levels of both BDNF and TGF-ß1 (but not concentrations of platelets in peripheral blood) were significantly elevated in all stages of COPD as compared to controls. Highest BDNF concentrations were found in spirometric GOLD stage 3, whereas highest TGF-ß1 serum levels were found in spirometric GOLD stage 4. There were specific, stage-dependent correlations of these mediators with lung function parameters of the patients. CONCLUSIONS: Taken together, we show that, in contrast to asthma, COPD is characterised by elevated concentrations of both BDNF and TGF-ß1 in serum. The stage-dependent association with lung function supports the hypothesis that these platelet mediators may play a role in the pathogenesis of COPD.

Plasmacytoid dendritic cells and their Toll-like receptor 9 expression selectively decrease with age.

Dendritic cells (DCs) play a crucial role in the initiation of immune responses against infectious particles and tumor cells; however, the impact of age, anthropometric parameters, and gender on the number and the expression of function-associated molecules of human DCs is poorly understood. In this study, blood DCs of 50 volunteers (19-84 years old) with no acute or chronic inflammatory diseases were examined using 4-color flow cytometry. Increasing age was associated with a decrease in blood plasmacytoid, but not myeloid DCs and a selective decrease in Toll-like receptor 9 (TLR9) expression by plasmacytoid DCs. In contrast, gender and body mass index did not impact the number of DC subsets or the expression of function-associated DC molecules. Thus, we demonstrate that age has a selective impact on plasmacytoid DCs and their TLR9 expression. This may contribute to an increased susceptibility to infections and tumors with increasing age.

Altered phenotype of blood dendritic cells in patients with acute pneumonia.

BACKGROUND: Dendritic cells (DCs) play a key role in the host defence against inhaled pathogens. However, the phenotype of blood DCs in patients with acute respiratory infections is unknown. OBJECTIVE: To investigate the number and the expression of function-associated molecules of blood DCs in patients with acute infectious pneumonia. METHODS: Sixteen patients with acute pneumonia and 19 controls without pneumonia were included in the study. The number as well as the expression of function-associated molecules of myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) were analysed in peripheral blood using four-colour flow cytometry. RESULTS: Elevated concentrations of procalcitonin (median: 0.55 ng/ml) and the rapid response to antibiotic treatment suggested a bacterial origin of the pneumonia in the patients. Total mDC (median: 27% of the controls) and pDC counts (median: 53% of the controls) were markedly reduced in patients with pneumonia, as compared to the controls. Percentages of blood mDCs, but not pDCs, were negatively correlated with serum concentrations of C-reactive protein. Patients with pneumonia were characterised by a significantly increased expression of Fc gamma receptors (CD32 and CD64) on mDCs and the Toll-like receptor 9 (TLR9) on pDCs. CONCLUSIONS: Circulating DCs are markedly reduced in patients with pneumonia, and characterised by an up-regulation of molecules recognising pathogen-associated molecular patterns and opsonised antigens.

Differential effect of clopidogrel and aspirin on the release of BDNF from platelets.

Anti-platelet treatment is a key therapeutic intervention in patients with cerebrovascular diseases. However, there is no information on its impact on the release of Brain-derived neurotrophic factor (BDNF), which is stored in large amounts in human platelets and essential for neuronal protection and repair. Here, we show that a single oral dose of clopidogrel, but not aspirin, significantly reduced the release of BDNF from platelets in healthy volunteers. These data point, for the first time, to possible differential effects of anti-platelet regimens on neuronal function in patients with cerebrovascular disorders.

Impact of immunotherapy on blood dendritic cells in patients with Hymenoptera venom allergy.

BACKGROUND: Modulation of T-cell differentiation, which is controlled by dendritic cells (DCs), plays a crucial role in specific immunotherapy (SIT). However, the number and the characteristics of blood DCs before and during immunotherapy are unknown. OBJECTIVE: To analyze the number and the characteristics of blood DC subsets in patients with Hymenoptera venom allergy before and after initiation of SIT. METHODS: In this clinical trial (NCT00947908), blood myeloid and plasmacytoid DCs were analyzed in 20 patients with Hymenoptera venom allergy (bee or wasp venom) by using 4-color flow cytometry at 3 time points: directly before SIT, and 52 hours and 12 months after initiation of SIT. In addition, 20 age-matched and sex-matched controls were examined. RESULTS: In patients with Hymenoptera venom allergy, the number of plasmacytoid DCs before SIT was comparable to that of controls. Plasmacytoid DCs decreased markedly 52 hours after initiation of SIT and returned to control levels after 12 months of treatment. Myeloid DCs were elevated in patients with Hymenoptera venom allergy before, during, and after the first 12 months of SIT. In addition, there were changes in the expression of function-associated surface molecules on myeloid DCs (such as Fc γ receptor 2 and Toll-like receptor 2) during SIT. CONCLUSION: Numbers of blood myeloid DCs are elevated in patients with Hymenoptera venom allergy, and there are specific changes in the expression of function-associated surface molecules on these cells during SIT. Numbers of plasmacytoid DCs in blood are profoundly but are only transiently decreased after initiation of SIT.

Extracellular adenosine triphosphate and chronic obstructive pulmonary disease.

RATIONALE: Extracellular ATP promotes inflammation, but its role in chronic obstructive pulmonary disease (COPD) is unknown. OBJECTIVES: To analyze the expression of ATP and its functional consequences in never-smokers, asymptomatic smokers, and patients with COPD. METHODS: ATP was quantified in bronchoalveolar lavage fluid (BALF) of never-smokers, asymptomatic smokers, and patients with COPD of different severity. The expression of specific ATP (purinergic) receptors was measured in airway macrophages and blood neutrophils from control subjects and patients with COPD. The release of mediators by macrophages and neutrophils and neutrophil chemotaxis was assessed after ATP stimulation. MEASUREMENTS AND MAIN RESULTS: Chronic smokers had elevated ATP concentrations in BALF compared with never-smokers. Acute smoke exposure led to a further increase in endobronchial ATP concentrations. Highest ATP concentrations in BALF were present in smokers and ex-smokers with COPD. In patients with COPD, BALF ATP concentrations correlated negatively with lung function and positively with BALF neutrophil counts. ATP induced a stronger chemotaxis and a stronger elastase release in blood neutrophils from patients with COPD, as compared with control subjects. In addition, airway macrophages from patients with COPD responded with an increased secretion of proinflammatory and tissue-degrading mediators after ATP stimulation. These findings were accompanied by an up-regulation of specific purinergic receptors in blood neutrophils and airway macrophages of patients with COPD. CONCLUSIONS: COPD is characterized by a strong and persistent up-regulation of extracellular ATP in the airways. Extracellular ATP appears to contribute to the pathogenesis of COPD by promoting inflammation and tissue degradation.

Interleukin-4 suppresses the cytotoxic potential of in vitro generated, adaptive regulatory CD4 T cells by down-regulation of granzyme B.

Regulatory CD4+ T cells (Tregs) control immune responses using secretion of anti-inflammatory cytokines and/or cytotoxic mechanisms and play a central role in the outcomes of several immune pathologies. Previous studies suggest an impaired function of Tregs in allergy, especially during allergen seasons, but the underlying mechanism is not known. Therefore, we analysed the impact of the T helper type 2 cytokine interleukin (IL)-4 on in vitro generated adaptive Tregs (aTregs), which have been reported to use the granzyme B (GrB)/perforin pathway to kill autologous immune cells. aTregs were generated by co-ligation of CD3 and CD46 on CD4+ T lymphocytes and granzyme expression was analysed using flow cytometry. To quantify GrB and perforin expression as well as IL-10 secretion in response to IL-4, specific enzyme-linked immunosorbent assays were performed in cell lysates and/or culture supernatants. Using a flow cytometry-based cytotoxicity assay the impact of IL-4 on the cytotoxic potential of aTregs was investigated. While IL-4 did not affect IL-10 secretion and perforin expression in aTregs, a significant suppression of GrB synthesis was detected in the presence of IL-4. In addition, IL-4-mediated suppression of GrB led to impaired cytotoxicity of aTregs against K562 target cells. In conclusion, our data suggest that IL-4 might play a role in impaired aTreg function in allergy.

Function-associated surface molecules on airway dendritic cells in cigarette smokers.

Airway dendritic cells (DCs) control pulmonary immune responses to inhaled particles. However, the profile of function-associated surface molecules on airway DCs in smokers is unknown. In this study, function-associated surface molecules were analyzed using four-color flow cytometry on myeloid DCs (mDCs) in bronchoalveolar lavage fluid (BALF) of cigarette smokers and never-smokers. Furthermore, the lung function was assessed directly before bronchoscopy in all participants. There was a 7-fold increase in total cell numbers in BALF of smokers, as compared with never-smokers. The percentage of mDCs among BALF cells and the expression of the maturation marker CD83 on mDCs did not differ between smokers and never-smokers. However, there was a strong increase in the expression of Langerin and CD1a (markers of Langerhans cells) on mDCs of smokers. Furthermore, mDCs of smokers were characterized by an increased expression of antigen presentation markers such as CD80 and CD86. By contrast, mDCs of smokers displayed a decreased expression of the lymph node homing receptor CCR7, as compared with mDCs of never-smokers. Decreased expression of CCR7 on mDCs, but not any of the other surface molecules studied, was specifically associated with airway obstruction and pulmonary hyperinflation in smokers. In conclusion, our data suggest that smoking affects the expression profile of function-associated surface molecules on airway mDCs. We provide the first evidence that a reduced CCR7 expression on airway mDCs is associated with airflow limitation in smokers.

Safety of segmental allergen challenge in human allergic asthma.

BACKGROUND: Segmental allergen challenge is widely used to study mechanisms of human allergic asthma. Despite the relatively large dissemination, limited information is available about the safety of this method. OBJECTIVE: Observational, retrospective study to report the adverse events of segmental allergen challenge in a large group of volunteers with asthma. METHODS: In total, 78 cases from several studies performed between 1994 and 2007 were pooled for this analysis. Volunteers underwent allergen challenge using either a fixed dose of allergen (7 cases) or an individually standardized allergen dose defined by an inhaled allergen test before the challenge (71 cases). A subgroup of 13 volunteers underwent repeated challenges, with more than 6 months between the challenges. RESULTS: With a fixed dose instilled during bronchoscopy, 43% of the participants developed wheezing and coughing, requiring 2-6 puffs of a ss(2)-agonist after segmental allergen challenge. In volunteers with individually standardized doses, a ss(2)-agonist was required in only 19% of the cases. No severe adverse events occurred in all cases studied. Volunteers who underwent repeated challenges did not develop more adverse events than those who underwent 1 challenge. CONCLUSIONS: Segmental allergen challenge is a safe tool to study the mechanisms of human allergic asthma, even when repeated challenges are performed in the same patient. It is associated with only a few, tolerable adverse events, especially when the dose of allergen is standardized individually.

A role for the neurotrophin receptor TrkB on maturing dendritic cells.

BACKGROUND: Neurotrophins are involved in neuroimmune interactions. However, the expression and role of neurotrophin receptors on dendritic cells have not been systematically studied. METHODS: The neurotrophin receptors p75NTR, TrkA, TrkB and TrkC were analyzed on immature and mature Human Monocyte-derived Dendritic Cells (HMDCs) using flow cytometry. In addition, we compared the impact of five different maturing protocols on the expression of neurotrophin receptors on HMDCs. Finally, the effect of neurotrophins on surface molecule expression and the survival of HMDCs was investigated. RESULTS: There was a low expression of p75NTR, TrkA and TrkB on immature HMDCs. HMDCs at different maturation stages did not show a significantly different expression of TrkA, as compared to immature HMDCs. By contrast, there was an upregulation of TrkB on suboptimally, but not optimally matured HMDC. In addition, there was a non-significant trend to a parallel increase of p75NTR expression on these cells. Functional experiments with maturing HMDCs revealed that both ligands of the TrkB receptor, Brain-derived neurotrophic factor (BDNF) and Neurotrophin 4 (NT-4), significantly increased the expression of markers of antigen presentation (HLA-DR and CD80) and the survival of maturing HMDCs. CONCLUSION: These data suggest that the TrkB ligands BDNF and NT-4 can directly influence human dendritic cells during their maturation.