Quantitative GPCR and ion channel transcriptomics in primary alveolar macrophages and macrophage surrogates

Abstract

BackgroundAlveolar macrophages are one of the first lines of defence against invading pathogens and play a central role in modulating both the innate and acquired immune systems. By responding to endogenous stimuli within the lung, alveolar macrophages contribute towards the regulation of the local inflammatory microenvironment, the initiation of wound healing and the pathogenesis of viral and bacterial infections. Despite the availability of protocols for isolating primary alveolar macrophages from the lung these cells remain recalcitrant to expansion in-vitro and therefore surrogate cell types, such as monocyte derived macrophages and phorbol ester-differentiated cell lines (e.g. U937, THP-1, HL60) are frequently used to model macrophage function.MethodsThe availability of high throughput gene expression technologies for accurate quantification of transcript levels enables the re-evaluation of these surrogate cell types for use as cellular models of the alveolar macrophage. Utilising high-throughput TaqMan arrays and focussing on dynamically regulated families of integral membrane proteins, we explore the similarities and differences in G-protein coupled receptor (GPCR) and ion channel expression in alveolar macrophages and their widely used surrogates.ResultsThe complete non-sensory GPCR and ion channel transcriptome is described for primary alveolar macrophages and macrophage surrogates. The expression of numerous GPCRs and ion channels whose expression were hitherto not described in human alveolar macrophages are compared across primary macrophages and commonly used macrophage cell models. Several membrane proteins known to have critical roles in regulating macrophage function, including CXCR6, CCR8 and TRPV4, were found to be highly expressed in macrophages but not expressed in PMA-differentiated surrogates.ConclusionsThe data described in this report provides insight into the appropriate choice of cell models for investigating macrophage biology and highlights the importance of confirming experimental data in primary alveolar macrophages.

TRPV4 inhibition counteracts edema and inflammation and improves pulmonary function and oxygen saturation in chemically induced acute lung injury

“…TRPV4 is highly expressed in the pulmonary epithelium and vasculature, and also in alveolar macrophages, where it was found to be essential for the production of proinflammatory oxidative mediators and cytokines in the ventilator-induced lung injury model (15,16,26,33). In our present study we observed that treatment of chlorine-and acid-exposed mice with TRPV4 inhibitors strongly reduced BALF macrophage numbers, thereby diminishing inflammation.…”

“…Ventilator-induced lung injury is associated with subsequent inflammation driven by macrophages, and it was suggested that macrophage-expressed TRPV4 channels play a crucial role in activating these cells (16). Although previous studies have shown that TRPV4 transcripts and protein are highly expressed in lung epithelial and vascular cells and in alveolar macrophages; it has remained unclear whether TRPV4 is also expressed in neutrophils (15,16,26,33). In our present study we observed that TRPV4 inhibition, or genetic deletion, strongly reduced neutrophil numbers in the BALF, suggesting that, similar to macrophages, TRPV4 plays a critical role in cell recruitment to the injured lung.…”

TRPC6 channel translocation into phagosomal membrane augments phagosomal function

“…As shown in The activation of an OAG-sensitive current, TRPC6, at both the plasma and phagosomal membranes in the presence of OAG and (R)-roscovitine would predict that (R)-roscovitine might be acting indirectly through a GPCR of unknown identity. Such receptors are plentiful on the macrophage surface and include formyl peptide, adenosine, and purinergic, as well as chemokine receptors (33)(34)(35)(36). If (R)-roscovitine acts through a GPCR, our prediction would be that we should see measureable DAG generation.…”

The emerging role of transient receptor potential channels in chronic lung disease

“…TRPV4 is expressed across a wide range of non-neuronal human cell types in the airways, including structural cells such as airway smooth muscle, epithelial cells, fibroblasts and pulmonary vessels and inflammatory cells including macrophages (but not highly differentiated surrogates: the circulating monocytic precursor cell, monocyte derived macrophages, in vitro differentiated promonocytic THP-1 and U937 cells or promyelocytic HL-60 leukaemia cells), neutrophils and T-cells [99,107,111,112,114,[128][129][130][131][132]. However, in the respiratory system, the function of TRPV4 is probably best understood in relation to its role in acute lung injury, which is not covered in this review [133].…”