Expression analysis of G Protein-Coupled Receptors in mouse macrophages

Macrophages are key cellular mediators of acute and chronic inflammation. They can be defined on the basis of morphology, function (e.g. non-specific uptake of particles) and expression of specific cell surface markers (e.g. EMR1 that is detected by the F4/80 antibody). EMR1 belongs to the GPCR super-family [1], and it is clear that macrophages express a diverse repertoire of other GPCRs [2]. A large number of GPCRs are yet to be deorphanized and hence, there is great potential for this family in both the discovery of gene function and in drug development. In this study we performed a systematic micro-array analysis of GPCR expression in primary mouse macrophages and identified family members that are either enriched in macrophages compared to a panel of other cell types, or are regulated by the pro-inflammatory stimulus, bacterial LPS. Some of these GPCRs are likely to provide future targets for drug discovery in inflammatory disease settings.

With an understanding of the GPCRs that are (a) highly expressed by the mononuclear phagocyte system, (b) selectively expressed by certain macrophage populations, or (c) regulated by the pro-inflammatory stimulus LPS, one can make some inferences about the role of specific GPCRs in macrophage biology. One of the most obvious findings from our analysis was that many of the P2Y purinergic receptors, which detect purine and pyrimidine nucleotides, were expressed in a restricted and/or regulated fashion in macrophages. Mechanical stress, cellular injury, inflammatory stimuli such as LPS, degranulation of mast cells and hypoxia can all increase extracellular concentrations of ATP and UTP [17, 18, 19, 20]. Consequently, these receptors are very likely to impact upon macrophage-mediated inflammatory responses.

Of the P2Y family, perhaps most striking in its expression pattern was P2ry6, which was essentially restricted to cells of the myeloid lineage. P2ry6 mRNA levels were greatly elevated in BMM, TEPM, RAW264 cells, microglia, osteoclasts and dendritic cells, compared to all other cell types (Figure 1b). P2ry6 expression in osteoclasts, which are a closely related lineage to macrophages, has been confirmed by others [21]. Whilst no previous studies have indicated that P2ry6 expression is restricted to the monocyte/macrophage lineage, there is evidence to suggest that this receptor does regulate the function of macrophages. UDP, which is reportedly a selective P2RY6 agonist [22, 23], triggered interleukin (IL)-8 release from the human monocyte-like cell line, THP-1 [24]. Conversely, the P2RY6 receptor antagonists, anthraquinone-sulfonic acid derivative reactive blue 2 and suramin, inhibited LPS-induced IL-8 production by THP-1 cells [24]. In microglia, P2ry6 mRNA expression was up-regulated in response to neuronal damage and UDP promoted phagocytosis by microglia, thus implying a role for P2RY6 in the clearance of damaged or dead neuronal cells [23]. Our observation that this receptor is widely expressed in the mononuclear phagocyte system suggests that this receptor may have a broader function in sensing and engulfing damaged cells throughout the body, as well as promoting pro-inflammatory responses in response to LPS and/or UDP.

This laboratory previously reported that P2ry5 mRNA expression was dramatically repressed by LPS in human and mouse macrophages [2], a finding supported by micro-array data presented here. This receptor was originally classified as a member of the P2Y receptor family on the basis of sequence identity and its ability to bind the nucleotides ATP and ADP [25]. Nucleotides failed to initiate downstream signaling from this receptor however [26], and it is therefore possible that P2RY5 acts as an antagonist of other P2Y receptors and restricts the macrophage response to extracellular nucleotides. Down-regulation of P2RY5 in response to a macrophage activating stimulus such as LPS might remove this inhibitory effect, thus allowing activated macrophages to respond to nucleotides. Alternatively, it is possible that the initial assignment of P2RY5 as a P2 receptor was incorrect. Cross-genome phylogenetic analysis showed that it clustered with a heterogenous group of receptors including the protease-activated receptors, as well as lysophosphatidylcholine (LPA) and sphingosylphosphorylcholine (S1P) receptors, rather than with other P2Y receptors [27]. In this case, LPS-mediated macrophage activation would presumably render cells unresponsive to the true ligand for this receptor.

Emerging evidence also implicates other P2Y receptors in macrophage-mediated inflammatory responses. For example, extracellular ATP, an agonist of P2RY2, induced IL-6 transcription in human monocyte-derived macrophages [28] and chemotaxis in human neutrophils [29], while the P2RY1 agonist 2-methylthio ATP increased IL-6 secretion from mouse splenocytes [30]. In support of a role for P2Y ATP receptors in macrophage function, and consistent with our data, P2ry1 and P2ry2 mRNAs were expressed at high levels in macrophages and LPS-stimulated monocytes [31]. Apart from these P2Y family members, P2RY12 was also reported to be selectively expressed in microglia in the rat brain [32]. Our analysis also showed that this receptor was highly enriched in microglia, as well as BMM, plasmacytoid dendritic cells, osteoclasts and a variety of lymphoid and neural tissues [see additional file 2a]. Finally, the P2Y-like receptor, P2ry14 (Gpr105), which is a receptor for UDP-conjugated sugars, mediated chemotaxis of bone marrow hematopoietic stem cells [33]. Despite the emerging literature that suggests a role for P2Y receptors in macrophage-mediated migration and inflammatory responses, a clear understanding of the selective agonists of these receptors and their interplay with each other and non-P2Y receptors is lacking. Nonetheless, our demonstration of the restricted and regulated expression of this family in macrophages identifies them as promising candidates for further analysis.

Other GPCRs that had a relatively restricted expression pattern and were strongly expressed by macrophages include Edg5, Gpr85 and Gpr84 (Table 2, Figure 1a and 1c, Additional file 2b). Edg5 is one of eight members of the endothelial differentiation gene (EDG) family of GPCRs that recognize LPA and S1P [34]. Several studies have documented potent effects of the EDG5 ligand, S1P on macrophages. Rat alveolar macrophages responded to S1P by producing O₂ ^- at levels comparable to those induced by LPS or formyl-Methionyl-Leucyl-Phenylalanine [35]. Not surprisingly, given this finding, anti-microbial effects of S1P have been reported. In human monocyte-derived macrophages or THP-1 cells infected with M. smegmatis or M. tuberculosis, S1P reduced intracellular bacterial loads in a dose-dependent manner. Similar effects of S1P were also apparent in mice infected with M. smegmatis or M. tuberculosis [36]. Apart from activating anti-microbial responses, S1P also provided a survival signal to both mouse and human macrophage cell lines upon subsequent exposure to an apoptosis-inducing signal [37]. EDG1 was implicated in this response, but given the high expression of Edg5 reported here, this receptor may also be involved in macrophage responses to S1P.

Expression of the SREB (super conserved receptor expressed in brain) family member Gpr85 (Sreb2) was restricted to macrophages and neuronal tissues (Figure 1a). Whilst the expression of Gpr85 in the central nervous system was previously reported [38, 39, 40], a function has yet to be identified. The co-regulated expression of receptors such as Gpr85 in neural tissues and leukocyte populations may provide a mechanism for the neuro-immune cross-talk that has been observed in several studies [41, 42].

Gpr84 is an orphan GPCR that was strikingly restricted to BMM and microglia in an unstimulated state (Table 2, Figure 1c) and was strongly up-regulated by LPS in both BMM and TEPM (Figure 3a, Table 3). This up-regulation is consistent with elevated Gpr84 expression in microglia and tissue macrophages in an endotoxin shock model [43]. Microglia also expressed high levels of Gpr84 in the experimental autoimmune encephalomyelitis model of multiple sclerosis [43]. These data imply that GPR84 has a role in neuroinflammation, but the exact function is difficult to predict without any knowledge of a ligand. Nonetheless, given the highly restricted expression pattern of Gpr84 mRNA, antibodies against this receptor may provide useful tools for tracking macrophage populations in vivo.

As with Gpr84, mRNA for several other GPCRs, particularly chemokine receptors, were differentially expressed between various unstimulated macrophage populations. TEPM expressed elevated levels of mRNA for Cxcr4, Ccr1, Ccrl2, whilst BMM expressed higher levels of Ccr2, Cx3cr1 and Cxcr3. The fractalkine receptor CX3CR1 has been identified as a marker of patrolling monocytes that survey the endothelial surface [44] and its down-regulation on macrophages that have already trafficked to an inflammatory site is not surprising. Conversely, CXCR4 has a well-characterized function in targeting leukocytes and their progenitors away from the circulation [45], consistent with its enhanced expression in TEPM. Apart from chemokine receptors, the expression of a number of other GPCRs was different between BMM and TEPM. For example, the thrombin-related receptor, Ebi2 [46], was strongly expressed in BMM but not TEPM, whilst Gprc5b was enriched in TEPM but not BMM. Such differences are likely to contribute to functional divergence between tissue macrophage and inflammatory macrophage populations.

Many of the GPCRs that were highly expressed by macrophages in an unstimulated state (described above), were also regulated by LPS. Several other GPCRs were weakly expressed by unstimulated macrophages but were strongly regulated by LPS. One such example was the frizzled family member, Fzd1. Frizzleds (Fzd) represent the cell membrane receptors for a family of secreted glycoproteins called wingless-related proteins (WNTs). Wnts play essential roles in development, including cell fate determination, adhesion, polarity, migration and proliferation [47, 48, 49, 50]. The WNT family has also been implicated in immune regulation. For example, the WNT family member, WNTD, acted as a negative feedback regulator of Toll signaling in Drosophila [51]. In human macrophages, WNT5A expression was up-regulated by TLR ligands, as well as challenge with M. tuberculosis and M. avium, and the WNT5A/Frizzled-5 (FZD5) pathway promoted IL-12 production from peripheral blood mononuclear cells [52]. The striking up-regulation of Fzd1 in BMM (17-fold at 6 h LPS) implies that this FZD family member shares similar functions in regulating macrophage inflammatory responses.

We also identified several GPCRs that were differentially regulated by LPS in BMM and TEPM. In BMM, Prokr1 was transiently induced by LPS (37-fold at 6 h) but repressed in TEPM (20-fold at 7 h). PROKR1 is a receptor for prokineticin 1 (PK1), a peptide that regulates monocyte differentiation, as well as macrophage activation and migration. PK1 triggered the differentiation of both murine and human bone marrow progenitor cells into the monocyte/macrophage lineage [53], and reprogrammed the response of human monocytes to LPS by amplifying IL-12 and tumor necrosis factor alpha (TNF) production, but inhibiting IL-10 release [53]. The differential regulation of Prokr1 by LPS in BMM versus TEPM implies that this receptor has distinct functions during activation of tissue versus inflammatory macrophage populations.

Gpr18 was induced 52-fold in BMM following 6 h LPS exposure, while it remained unchanged in TEPM. This receptor was also highly expressed in other immune cell populations including B cells, T cells and DC cells [see additional file 2c]. N-arachidonylglycine (NAGly), a conjugate of arachidonic acid and glycine, is an endogenous ligand for GPR18 [54] that was reported to suppress inflammatory pain and have analgesic properties [55, 56]. Given the potent regulation of Gpr18 expression in BMM by LPS, NAGly is also likely to regulate macrophage function. The adenosine receptor, Adora2b was also strongly induced by LPS in BMM (191-fold at 6 h) yet was only modestly regulated (3-fold at 7 h) in TEPM. Adenosine-mediated activation of this receptor augmented IL-10 production by RAW264 cells in response to LPS [57]. In contrast, adenosine attenuated LPS-induced production of the pro-inflammatory cytokines TNF [58, 59, 60, 61] and IL-12 [60, 62] in mouse and human macrophages. Inducible Adora2b expression may therefore contribute to feedback regulation of macrophage inflammatory responses.

In this study, we have documented several GPCRs that are detectably expressed in primary mouse macrophages (BMM, TEPM), enriched in either or both BMM and TEPM, or regulated in activated macrophages (LPS-stimulated BMM and/or TEPM). For some of these genes, constitutive or regulated expression in macrophages has not previously been described. Future studies on such GPCRs and their agonists are likely to provide important insights into macrophage biology, as well as novel inflammatory pathways which could be future targets for drug discovery.