Technical Advance: Autofluorescence as a tool for myeloid cell analysis

Mitchell, Andrew J.; Pradel, Lydie; Chasson, Lionel; Rooijen, Nico van; Grau, Georges E.; Hunt, Nicholas H.; Chimini, Giovanna

doi:10.1189/jlb.0310184

Cited by 61 publications

(57 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cells were isolated from enzymatically digested mouse lungs, and after the exclusion of doublets and debris, immune cells were identified by CD45 staining. (A) A sequential gating strategy was first used to identify populations expressing specific markers: alveolar macrophages (MF) (Siglec F antigen/fluorochrome (21)(22)(23). Moreover, the autofluorescence of myeloid cells in the lung may further increase after exposure to environmental particulate matter.…”

Section: Discussionmentioning

confidence: 99%

Flow Cytometric Analysis of Macrophages and Dendritic Cell Subsets in the Mouse Lung

Misharin

Morales‐Nebreda

Mutlu

et al. 2013

Am J Respir Cell Mol Biol

754

712

View full text Add to dashboard Cite

The lung hosts multiple populations of macrophages and dendritic cells, which play a crucial role in lung pathology. The accurate identification and enumeration of these subsets are essential for understanding their role in lung pathology. Flow cytometry is a mainstream tool for studying the immune system. However, a systematic flow cytometric approach to identify subsets of macrophages and dendritic cells (DCs) accurately and consistently in the normal mouse lung has not been described. Here we developed a panel of surface markers and an analysis strategy that accurately identify all known populations of macrophages and DCs, and their precursors in the lung during steady-state conditions and bleomycin-induced injury. Using this panel, we assessed the polarization of lung macrophages during the course of bleomycin-induced lung injury. Alveolar macrophages expressed markers of alternatively activated macrophages during both acute and fibrotic phases of bleomycin-induced lung injury, whereas markers of classically activated macrophages were expressed only during the acute phase. Taken together, these data suggest that this flow cytometric panel is very helpful in identifying macrophage and DC populations and their state of activation in normal, injured, and fibrotic lungs.Keywords: pulmonary macrophages; alveolar macrophages; interstitial macrophages; macrophage polarization; lung fibrosis Cells of the innate immune system, and especially myeloid cells such as neutrophils, eosinophils, monocytes, macrophages (alveolar and interstitial), and dendritic cells (DCs, i.e., plasmacytoid DCs, CD1031 DCs, and CD11b 1 DCs), play an important role in lung development and physiology, and contribute to important lung diseases, including pulmonary infection, cancer, asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis (1-5). Alveolar and interstitial lung macrophages exhibit different origins and life spans in lungs, and have been identified as key regulators of pathological and reparative processes. Alveolar macrophages, which are considered tissue-resident macrophages, populate lung tissue during early embryogenesis and remain viable for prolonged periods, with minimal replenishment from bone marrow-derived monocytes (6). In contrast, interstitial macrophages originate from bone marrow-derived monocytes and have a shorter half-life (7,8). In recent studies, several groups of investigators suggested that these two populations of lung macrophages play opposing roles in lung injury. Alveolar macrophages appear to limit neutrophil influx into the lung during acute lung injury (9) or chronic exposure to organic dust (10), whereas interstitial macrophages promote neutrophil extravasation (11,12). An additional layer of complexity is added by the phenotypic plasticity of macrophages. Classically activated macrophages (sometimes referred to as M1-polarized) have been suggested to promote the development of acute lung injury, whereas alternatively activated macrophages (M2) may play a role in limiting or resolving lu...

show abstract

Section: Discussionmentioning

confidence: 99%

Flow Cytometric Analysis of Macrophages and Dendritic Cell Subsets in the Mouse Lung

Misharin

Morales‐Nebreda

Mutlu

et al. 2013

Am J Respir Cell Mol Biol

754

712

View full text Add to dashboard Cite

show abstract

“…18 In brief, cells in the erythroblastic islands were suspended in PBS containing 0.1% BSA and 1 mM EDTA (PBS/BSA/EDTA), stained with 200 nM SytoxBlue, and SytoxBlue low population was regarded as macrophages. In some cases, cells in the erythroblastic islands were incubated on ice for 30 minutes with 1 mg/mL biotinylated anti-F4/80 or anti-MerTK, or 1 mg/mL PE-anti-Vcam1, followed by incubation with 1 mg/mL APC-streptavidin and 200 nM SytoxBlue.…”

Section: Binding Of Erythroblasts To Central Macrophagesmentioning

confidence: 99%

MerTK-mediated engulfment of pyrenocytes by central macrophages in erythroblastic islands

Toda

Segawa

Nagata

2014

Blood

View full text Add to dashboard Cite

Key Points• An in vitro system for the engulfment of pyrenocytes was established using erythroblastic islands.• MerTK, a receptor kinase, was essential for the engulfment of pyrenocytes by the central macrophages at erythroblastic islands.Definitive erythropoiesis takes place at erythroblastic islands, where erythroblasts proliferate and differentiate in association with central macrophages. At the final stage of erythropoiesis, pyrenocytes (nuclei surrounded by plasma membranes) are excluded from erythroblasts, expose phosphatidylserine (PtdSer), and are engulfed by the macrophages in a PtdSer-dependent manner. However, the molecular mechanism(s) involved in the engulfment of pyrenocytes are incompletely understood. Here, we constructed an in vitro assay system for the enucleation and engulfment of pyrenocytes using a methylcellulose-based culture. As reported previously, erythroblasts were bound to macrophages via interactions between integrin-a 4 b 1 on erythroblasts and Vcam1 on macrophages. After enucleation, the resulting pyrenocytes exhibited a reduced affinity for Vcam1 that correlated with the presence of inactive integrin-a 4 b 1 complexes. The pyrenocytes were then engulfed by the macrophages via a MerTK-protein S-dependent mechanism. Protein S appeared to function as a bridge between the pyrenocytes and macrophages by binding to PtdSer on the pyrenocytes and MerTK on the macrophages. Normally, NIH3T3 cells do not engulf pyrenocytes, but when they were transformed with MerTK, they efficiently engulfed pyrenocytes in the presence of protein S. These results suggest that macrophages use similar mechanisms to engulf both pyrenocytes and apoptotic cells. (Blood. 2014;123(25):3963-3971)

show abstract

“…Quantification of leukocyte subsets in the brains of infected animals was performed as described previously (65,66), with minor modifications. Briefly, brains were mashed between frosted glass slides in RPMI 1640, digested with collagenase/DNase for 20 min at room temperature, and triturated before a second 20-min collagenase/DNase step.…”

Section: Flow Cytometrymentioning

confidence: 99%

Inflammasome-Dependent IFN-γ Drives Pathogenesis inStreptococcus pneumoniaeMeningitis

Mitchell

Yau

McQuillan

et al. 2012

The Journal of Immunology

Self Cite

View full text Add to dashboard Cite

The pathology associated with Streptococcus pneumoniae meningitis results largely from activation of immune-associated pathways. We systematically investigated the production of IFN subtypes, as well as their influence on pathology, in a mouse model of S. pneumoniae meningitis. Despite the occurrence of a mixed IFN type I/II gene signature, no evidence for production or involvement of type I IFNs in disease progression was found. In contrast, type II IFN (IFN-γ) was strongly induced, and IFN-γ−/− mice were significantly protected from severe disease. Using intracellular cytokine staining and targeted cell-depletion approaches, NK cells were found to be the dominant source of IFN-γ. Furthermore, production of IFN-γ was found to be dependent upon ASC and IL-18, indicating that an ASC-dependent inflammasome pathway was responsible for mediating IFN-γ induction. The influence of IFN-γ gene deletion on a range of processes known to be involved in bacterial meningitis pathogenesis was examined. Although neutrophil numbers in the brain were similar in infected wild-type and IFN-γ−/− mice, both monocyte recruitment and CCL2 production were less in infected IFN-γ−/− mice compared with infected wild-type controls. Additionally, gene expression of NO synthase was strongly diminished in infected IFN-γ−/− mice compared with infected controls. Finally, bacterial clearance was enhanced in IFN-γ−/− mice, although the underlying mechanism remains unclear. Together, these data suggest that inflammasome-dependent IFN-γ contributes via multiple pathways to pathology during S. pneumoniae meningitis.

show abstract

Technical Advance: Autofluorescence as a tool for myeloid cell analysis

Cited by 61 publications

References 34 publications

Flow Cytometric Analysis of Macrophages and Dendritic Cell Subsets in the Mouse Lung

Flow Cytometric Analysis of Macrophages and Dendritic Cell Subsets in the Mouse Lung

MerTK-mediated engulfment of pyrenocytes by central macrophages in erythroblastic islands

Inflammasome-Dependent IFN-γ Drives Pathogenesis inStreptococcus pneumoniaeMeningitis

Contact Info

Product

Resources

About