A rapid flow cytometric method for determining the cellular composition of bronchoalveolar lavage fluid cells in mouse models of asthma

Rijt, Leonie S. van; Kuipers, Harmjan; Vos, Nanda; Hijdra, Daniëlle; Hoogsteden, Henk C.; Lambrecht, Bart N.

doi:10.1016/j.jim.2004.03.004

Cited by 158 publications

(136 citation statements)

References 29 publications

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“…Our use of AF to distinguish macrophages (AF ϩ ) from lung DCs (AF Ϫ ) represents a critical step in our identification process because both cell types express CD11c. This distinction, well described in two prior studies 41,45 and in other infectious and noninfectious models of pulmonary inflammation, 42,[51][52][53]68 permits more specific identification and enumeration of macrophage and DC populations in the lungs of mice. In our model, AF ϩ macrophages, purified by cell sorting, are large cells (with high forward and side scatter) displaying numerous intracytoplasmic vacuoles, some containing cryptococci, consistent with macrophage morphological features (Figure 1).…”

Section: Discussionmentioning

confidence: 85%

“…Next, we set consecutive gates which identified CD11c ϩ cells ( Figure 1A, left dot plot) and distinguished autofluorescent (AF ϩ ) macrophages from non-AF (AF Ϫ ) dendritic cells (DCs) ( Figure 1A, middle dot plot), as previously described. 41,45,[51][52][53] Last, we used relative expression of CD11b to distinguish CD11b-negative AMs (CD11c ϩ AF ϩ CD11b Ϫ ) 42,54,55 and CD11b-positive ExMs (CD11c ϩ AF ϩ CD11b ϩ ) 33,35 ( Figure 1A, right dot plot; gates labeled AM and ExM). Proper gate placement for AF and expression of CD11c and CD11b were determined using AMs obtained from bronchoalveolar lavage fluid of uninfected mice (which consists of Ͼ90% CD11c ϩ AF ϩ CD11b Ϫ AMs; data not shown).…”

Section: Cd11c ϩ Cd11b ϩ Autofluorescent Exms Are the Most Prevalent mentioning

confidence: 99%

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Chemokine Receptor 2-Mediated Accumulation of Fungicidal Exudate Macrophages in Mice That Clear Cryptococcal Lung Infection

Osterholzer

Chen

Olszewski

et al. 2011

The American Journal of Pathology

112

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Clearance of pulmonary infection with the fungal pathogen Cryptococcus neoformans is associated with the accumulation and activation of lung macrophages. However, the phenotype of these macrophages and the mechanisms contributing to their accumulation are not well-defined. In this study, we used an established murine model of cryptococcal lung infection and flow cytometric analysis to identify alveolar macrophages (AMs) and the recently described exudate macrophages (ExMs). Exudate macrophages are distinguished from AMs by their strong expression of CD11b and major histocompatibility complex class II and modest expression of costimulatory molecules. Exudate macrophages substantially outnumber AMs during the effector phase of the immune response; and accumulation of ExMs, but not AMs, was chemokine receptor 2 (CCR2) dependent and attributable to the recruitment and subsequent differentiation of Ly-6C high monocytes originating from the bone marrow and possibly the spleen. Peak ExM accumulation in wild-type (CCR2 ؉/؉ ) mice coincided with maximal lung expression of mRNA for inducible nitric oxide synthase and correlated with the known onset of cryptococcal clearance in this strain of mice. Exudate macrophages purified from infected lungs displayed a classically activated effector phenotype characterized by cryptococcal-enhanced production of inducible nitric oxide synthase and tumor necrosis factor ␣. Cryptococcal killing by bone marrow-derived ExMs was CCR2 independent and superior to that of AMs. We conclude that clearance of cryptococcal lung infection requires the CCR2-mediated massive accumulation of fungicidal ExMs derived from circulating Ly-6C high monocytes.

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Section: Discussionmentioning

confidence: 85%

Section: Cd11c ϩ Cd11b ϩ Autofluorescent Exms Are the Most Prevalent mentioning

confidence: 99%

Chemokine Receptor 2-Mediated Accumulation of Fungicidal Exudate Macrophages in Mice That Clear Cryptococcal Lung Infection

Osterholzer

Chen

Olszewski

et al. 2011

The American Journal of Pathology

112

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“…Expression of protein levels of iNOS, IL-12b, Arg-1, Fizz1, MGL-1/2, and IL-10 was determined by flow cytometry cell surface and intracellular staining, as previously described (41,42). Cells isolated from BALF were incubated with Golgi inhibitor (monensin; BD Biosciences), and cell surface staining was carried out by incubation with PerCP-Cy5.5 antimouse CD11c (BioLegend), fixation and permeabilization (BD Fixation and Permeabilization Solution Kit; BD Biosciences), and staining with allophycocyanin-conjugated mAb against murine IL-10, FITC-conjugated mAb against murine iNOS, or PE-conjugated mAb against murine IL-12b (all from BD Biosciences).…”

Section: Flow Cytometrymentioning

confidence: 99%

Akt2 Deficiency Protects from Acute Lung Injury via Alternative Macrophage Activation and miR-146a Induction in Mice

Vergadi

Vaporidi

Theodorakis

et al. 2014

The Journal of Immunology

142

106

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Acute respiratory distress syndrome (ARDS) is a major cause of respiratory failure, with limited effective treatments available. Alveolar macrophages participate in the pathogenesis of ARDS. To investigate the role of macrophage activation in aseptic lung injury and identify molecular mediators with therapeutic potential, lung injury was induced in wild-type (WT) and Akt2−/− mice by hydrochloric acid aspiration. Acid-induced lung injury in WT mice was characterized by decreased lung compliance and increased protein and cytokine concentration in bronchoalveolar lavage fluid. Alveolar macrophages acquired a classical activation (M1) phenotype. Acid-induced lung injury was less severe in Akt2−/− mice compared with WT mice. Alveolar macrophages from acid-injured Akt2−/− mice demonstrated the alternative activation phenotype (M2). Although M2 polarization suppressed aseptic lung injury, it resulted in increased lung bacterial load when Akt2−/− mice were infected with Pseudomonas aeruginosa. miR-146a, an anti-inflammatory microRNA targeting TLR4 signaling, was induced during the late phase of lung injury in WT mice, whereas it was increased early in Akt2−/− mice. Indeed, miR-146a overexpression in WT macrophages suppressed LPS-induced inducible NO synthase (iNOS) and promoted M2 polarization, whereas miR-146a inhibition in Akt2−/− macrophages restored iNOS expression. Furthermore, miR-146a delivery or Akt2 silencing in WT mice exposed to acid resulted in suppression of iNOS in alveolar macrophages. In conclusion, Akt2 suppression and miR-146a induction promote the M2 macrophage phenotype, resulting in amelioration of acid-induced lung injury. In vivo modulation of macrophage phenotype through Akt2 or miR-146a could provide a potential therapeutic approach for aseptic ARDS; however, it may be deleterious in septic ARDS because of impaired bacterial clearance.

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“…Differential cells counts were analysed by flow cytometry as previously described [42]. For the determination of DC activation in lung or LNs, cell suspensions were stained for 20 min with APClabelled anti-CD80, PE-labelled anti-OX40L, PE-labelled anti-CD40, APC-labelled anti-CD80, APC-labelled anti-CCR7, FITC-labelled anti-MHC class II antibodies.…”

Section: Flow Cytometry Stainingmentioning

confidence: 99%

IL‐33‐activated dendritic cells are critical for allergic airway inflammation

et al. 2011

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IL-33, a new member of the IL-1 family cytokine, is involved in Th2-type responses in a wide range of diseases and signals through the ST2 receptor expressed on many immune cells. Since the effects of IL-33 on DCs remain controversial, we investigated the ability of IL-33 to modulate DC functions in vitro and in vivo. Here, we report that IL-33 activates myeloid DCs to produce IL-6, IL-1b, TNF, CCL17 and to express high levels of CD40, CD80 OX40L and CCR7. Importantly, IL-33-activated DCs prime naive lymphocytes to produce the Th2 cytokines IL-5 and IL-13, but not IL-4. In vivo, IL-33 exposure induces DC recruitment and activation in the lung. Using an OVA-induced allergic lung inflammation model, we demonstrate that the reduced airway inflammation in ST2-deficient mice correlates with the failure in DC activation and migration to the draining LN. Finally, we show that adoptive transfer of IL-33-activated DCs exacerbates lung inflammation in a DC-driven model of allergic airway inflammation. These data demonstrate for the first time that IL-33 activates DCs during antigen presentation and thereby drives a Th2-type response in allergic lung inflammation. IntroductionAllergic asthma is a chronic disorder characterized by eosinophilic airway inflammation, mucus hypersecretion, antigenspecific-IgE antibodies, airway remodeling and increased airway hyperreactivity [1,2]. The process of airway inflammation involves various cells types, such as eosinophils, mast cells, epithelial cells, lymphocytes and DCs. Th2 cells have been shown to play a predominant role in allergic asthma and Th2 cytokines, such as IL-4, IL-5 and IL-13, exacerbate disease severity [3,4]. IL-33, the recently discovered Th2 cytokine, is found at high levels in the plasma of asthmatic patients [5,6] and in the lungs of mice during experimental allergic asthma [7,8].IL-33 is a member of IL-1 family [9][10][11]. Like IL-1b or IL-18, IL-33 is synthesized as a precursor and can be cleaved by caspase-1 and 3 but the cleavage products are biologically less active than the precursor [12,13]. In contrast to the other IL-1 family members, IL-33 is mainly expressed in non-hematopoietic cells such as fibroblasts, epithelial cells and endothelial cells [10,14,15]. Because of its nuclear localization sequence, IL-33 is usually present in the nucleus, where it acts as a potential transcriptional repressor [16]. Recently, IL-33 has been shown to be released from necrotic cells and may act as an alarmin in a similar manner to IL-1a [17] or high mobility group box1 protein HMGB1 [18,19]. [14]. In accordance with its Th2 functions, administration of IL-33 into naive mice induces severe inflammation in the lung and digestive tract with elevated levels of IL-4, IL-5 and IL-13, splenomegaly and increased serum Ig [10]. In vitro, IL-33 has also been reported to polarize naive CD4 1 T cells to produce IL-5 and IL-13, but not . Polarization of this atypical Th2 population is independent of IL-4, STAT6 and GATA3. On macrophages, IL-33 amplifies IL-13-mediated polarization...

show abstract

A rapid flow cytometric method for determining the cellular composition of bronchoalveolar lavage fluid cells in mouse models of asthma

Cited by 158 publications

References 29 publications

Chemokine Receptor 2-Mediated Accumulation of Fungicidal Exudate Macrophages in Mice That Clear Cryptococcal Lung Infection

Chemokine Receptor 2-Mediated Accumulation of Fungicidal Exudate Macrophages in Mice That Clear Cryptococcal Lung Infection

Akt2 Deficiency Protects from Acute Lung Injury via Alternative Macrophage Activation and miR-146a Induction in Mice

IL‐33‐activated dendritic cells are critical for allergic airway inflammation

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