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

Macrophages play important roles in host defence, wound healing, immune regulation and muscle regeneration. In particular, the central role of the macrophage in removing necrotic cellular debris, shaping the inflammatory microenvironment and their key role in the pathogenesis of viral infection have led to an intense interest in furthering understanding of the alveolar macrophage within the field of respiratory research.

Chronic obstructive pulmonary disorder (COPD) is one of the most common respiratory diseases and represents a major public health problem. The World Health Organisation estimates that COPD was the fifth leading cause of death worldwide in 2002 and by 2030 will become the third leading cause of death. The disease is typically progressive and characterised by chronic airflow limitation caused by a mixture of small airway disease (obstructive bronchiolitis) and parenchymal destruction (emphysema). Key pathological changes in COPD include a marked increase in macrophages within the airways, lung parenchyma and pulmonary vasculature[1]. Macrophages are the predominant inflammatory cell within the lung where they secrete a wide range of inflammatory mediators and proteases. Combined with the phagocytic properties of a macrophage the large diversity of their biological functions can account for most of the known features of COPD[2]. Consequently, the in-vitro study of macrophage biology is a key activity within the COPD research area.

Macrophages have long been known to be produced by the differentiation of monocytes following their recruitment from blood-vessels into surrounding tissues[3]. Primary macrophages isolated from resident tissues cannot be readily expanded for prolonged culture, which limits their routine use for biological studies and drug discovery; therefore alternative cellular models more amenable to cell culture are frequently used as a surrogate for the investigation of macrophage biology. Commonly used macrophage surrogates include the circulating monocytic precursor cell, monocyte derived macrophages (MDMs), in-vitro differentiated promonocytic THP-1 and U937 cells or promyelocytic HL-60 leukaemia cells. It is unclear how well these surrogate systems recapitulate the biology of the primary macrophage for which they are intended to model; indeed, major phenotypic differences such as altered chemokine release and phagocytic behaviour have been shown to separate the primary macrophage and commonly used surrogate cell types[4]. Enhancing our understanding of these differences will help enable the choice of appropriate macrophage model systems for investigating macrophage biology, modelling disease pathology in-vitro and for establishing appropriate cell based screening assays for drug discovery.

G-protein coupled receptors (GPCRs) and ion channels are large families of cell surface proteins and together comprise the molecular target of approximately 40% of all FDA approved drugs[5]. Integral membrane proteins such as receptors and ion channels are key regulators of cellular function and unsurprisingly are dynamically regulated at the transcriptional level with differentiated cell types displaying marked differences in expression[6]. As is typical for membrane bound signalling proteins, the amount of GPCR and ion channel transcript is often low within the cell and at the limit of detection for DNA microarray technologies[7, 8]. Quantitative PCR techniques such as TaqMan offer a higher sensitivity for detecting low-abundance transcript, but until recently have been limited to small scale gene expression studies due to their low throughput. Only recently with the advent of high density microfluid-based TaqMan arrays has the large scale quantitative analysis of whole, low-abundance gene families become feasible.

The aim of this study was to investigate how the expression of GPCR and ion channel genes differ between cell systems commonly used to model macrophage function. In this study we report for the first time a quantitative TaqMan-based transcriptional profile of entire families of integral membrane proteins, across several macrophage-like cell types, providing insights into their distinct gene signatures and identifying novel macrophage markers and potential future drug targets.

In this study the application of high throughput PCR has enabled the quantification of transcript expression of entire target families across multiple cell types. The GPCR and ion channel transcriptome of primary AMs was compared with several cell types commonly used to model macrophage function. Given the dynamic transcriptional control of GPCR and ion channel families and their important role in modulating cellular function, the changes in gene expression quantified in this study should provide a good measure of functional similarities or differences between different macrophage-like cell types[6, 14].

The data in this study suggests that the widely used AM models are poor substitutes for primary AMs isolated from BAL. Surprisingly, we found that the GPCR and ion channel expression profile of commonly used PMA-differentiated promonocytic and promyelocytic surrogates was less primary AM-like than non-treated peripheral blood monocytes. GM-CSF differentiated monocyte-derived macrophages appear to be most similar to AMs with respect to GPCR and ion channel expression but nonetheless demonstrate many significant differences in the expression of genes known to play critical roles in regulating AM function.

It is over 50 years since the first report described the isolation of macrophages from BAL fluid, yet no method has since been established for the culturing and expansion of these cells[15]. Consequently, surrogate cell types continue to be widely employed to interrogate the biology of the macrophage and to establish high throughput screening assays for drug discovery. An improved understanding of the GPCR and ion channel expression profiles of AMs and AM surrogates will provide valuable information on the design and interpretation of experiments utilising macrophage surrogates.

Exacerbations remain a major cause of hospitalization, mortality, and morbidity in COPD patients, with viral or bacterial infections representing a principle aetiological factor[16]. Alveolar macrophage dysfunction in COPD patients is thought to contribute to an increased susceptibility to exacerbations through impaired phagocytosis, chemotaxis, and cytokine production leading to an impaired host defence[2, 17, 18]. Many cell surface receptors including GPCRs and ion channels participate in the regulation of this process and may thus play in important role in regulating host immunity[9, 19, 20]. The elucidation of differential GPCR and ion channel expression in primary AMs versus macrophage surrogates will help to refine the choice of appropriate models for investigating macrophage biology. For example, the high-affinity CCL1 receptor, CCR8, has previously been shown to be highly expressed on AMs and its expression has been associated with phagocytic macrophages[21]. Furthermore, CCR8 is upregulated on AMs from COPD patients and is reported to impact macrophage adhesion, chemotaxis, ROS generation and cytokine production[22]. In this study we report that although CCR8 is highly expressed on primary AMs, it is essentially absent on all evaluated macrophage surrogates.

Macrophages play a crucial role in the pathogenesis of viral infections, are often among the first cells to be infected and can act as a reservoir for further spread to other cell types[23, 24]. Numerous chemokine GPCRs have been previously shown to be expressed in AMs[25], furthermore, many viruses utilise chemokine GPCR co-receptors to mediate membrane fusion and promote viral entry into target cells; for example CCR5 and CXCR4 are the principle HIV-1 co-receptors mediating T cell and macrophage tropism with T-cell tropic viruses using CXCR4[26], whereas macrophage tropic HIV-1 viruses primarily utilise CCR5 as a co-receptor[27]. In this study we found that CCR5 and several other chemokine receptors which support viral infection, such as CCR8 and CXCR6 are either absent or demonstrate markedly different expression levels in macrophage-like cell lines compared with primary AMs. Both CCR8 and CXCR6 are highly expressed in primary AMs but are not expressed in PMA-differentiated surrogates and are expressed at below levels of quantification in both MDMs and primary monocytes. CCR5 is highly expressed in AMs but has >200 fold lower expression in PMA-differentiated THP-1 and U937 cells. These findings are of particular interest as macrophage surrogates are widely used to model changes in cellular tropism of viral variants[28], however in contrast to primary isolated macrophages viral isolates often lack the capacity to infect and replicate in phorbol ester-differentiated promonocytic and promyelocytic cells[29, 30]. The importance of chemokine receptor signalling in regulating macrophage function and the stark differences in chemokine expression from cell types described in this study highlights the importance of using primary cells to investigate macrophage biology wherever possible. A better understanding of the GPCR and ion channel expression profiles of primary AMs and surrogate cell models will likely inform the use of appropriate macrophage models for studying different aspects of macrophage biology. The application of high-throughput TaqMan arrays to better understand changes in gene expression in diseased tissues is also likely to further our understanding of disease aetiology and progression and may lead to the identification of novel drug targets.

Several different protocols may be used to drive promonocytic or promyelocytic cell lines into macrophage-like cells in order to generate model systems for studying macrophage biology and the protocols used can impact the resulting cellular phenotype[4, 31]. Furthermore, it is well established that primary macrophages display marked phenotypic heterogeneity covering a spectrum of classically and alternatively activated phenotypes[32]. It should also be remembered that this study has only compared GPCR and ion channel expression and not the entire transcriptome. Furthermore, macrophages from different tissues and even within the same tissue can show considerable phenotypic heterogeneity[33]. Additionally, it is unclear to what extent primary AMs isolated from BAL fluid are representative of the AM population resident within the alveoli[34]. Further studies will be required to better understand what impact the different protocols used to establish macrophage surrogates may have on the resulting gene expression profiles and perhaps better understand how the resulting surrogates reflect the many primary macrophage phenotypes observed within physiological and pathophysiological settings.

Macrophages play an important role in regulating many biological processes including host defence and muscle regeneration. In this study we have validated the use of high-throughput microfluid-based TaqMan arrays for detecting quantitative changes in gene expression profiles and applied this technology to the quantitative analysis of macrophage gene expression. To our knowledge this is the first study to describe the expression of the entire non-sensory GPCR and ion channel families in AMs and macrophage surrogates. We have identified multiple genes whose expression were hitherto not described in human alveolar macrophages and provide a platform for future studies to further explore the impact that GPCRs and ion channels may have on the macrophage phenotype. These data may also provide researchers with novel macrophage markers and potential future drug targets.

Without access to a plentiful supply of primary macrophages, surrogates continue to be widely used to model macrophage function and will likely continue to form a cornerstone of the in-vitro methodologies employed to investigate macrophage biology. This study provides insight into the observed phenotypic characteristics of surrogates and informs on the use of most appropriate models. However, the differences observed in GPCR and ion channel expression described in this study highlights the importance, wherever possible, of using primary cells for the in-vitro modelling of alveolar macrophage behaviour.