Introduction

Inflammation is characterized by the cardinal signs of rubor (redness), calor (heat), dolor (pain), tumor (swelling) and function laesa (loss of function). These signs reflect tissue response to inflammatory factors that are either external (eg, bacterial endotoxin) or host-derived (eg, TNFα). GPCRs contribute directly to these clinical manifestations due to their wide presence and diverse functions. Inflammation is shaped not only by leukocytes that accumulate at the site of inflammation, but also by cells in specific tissues and organs such as microglial cells in the brain and synovial fibroblasts in the joints. Endothelial cells in all tissues are actively involved in the process of inflammation and interact closely with leukocytes. GPCRs expressed in these cells play important roles in sensing the presence of chemoattractants, transducing signals that lead to the production of inflammatory cytokines, nociception, and regulation of intracellular and intercellular communications associated with increased blood flow and increased vascular endothelial permeability. Functions mediated by GPCRs can both exacerbate inflammation and promote its resolution.

GPCRs that mediate cell migration and phagocyte activation

It was long observed that phagocytes have the abilities to chase, capture and eventually eliminate invading bacteria. Based on observations made in the last century, it was reported that phagocytes could respond to small molecules derived from invading bacteria and fungi1. In addition, these cells also respond to substances produced by the host during the course of sterile inflammation. A number of small molecules was discovered in the 60's and 70's, including activated complement C5a and N-formylated peptides of bacterial origin2, 3, 4. Evidence that these “classic chemoattractants” act on GPCRs first came from the observation that pertussis toxin (PTX) could alter the binding affinity of chemotactic formyl peptides5, a characteristic feature of certain GPCR ligands6. Working independently, two laboratories reported that PTX could block formyl peptide (eg, fMet-Leu-Phe, fMLF)-induced neutrophil functions through ADP-ribosylation of the Gi class of heterotrimeric G proteins7, 8. These early studies demonstrate that the chemotactic peptide receptor functionally couples to a heterotrimeric G protein that is a substrate for ADP-ribosylation by PTX. Further characterization of the PTX substrate, also termed “islet-activating protein”, found it to be a member of the Gi family of G proteins9 that is critical to a variety of activities downstream of chemoattractant receptor signaling. The identity of the receptors for fMLF and C5a was first revealed, among all chemoattractant receptors, through molecular cloning of their cDNAs and analysis of the deduced protein sequence10, 11. It was confirmed that these receptors belong to the rhodopsin-like, 7-transmembrane (TM) receptor superfamily12, 13. Following these initial cloning efforts, other classic chemoattractant receptors including those for platelet-activating factor (PAF) and leukotriene B4 (LTB4), were sub-sequently identified as GPCRs14, 15.

Chemokines (chemotactic cytokines) are small proteins with cysteine residues located at fixed positions. A large number of chemokines have been identified in the mid-80's and early 90's. These chemokines bind to rhodopsin-like GPCRs, although not all of them are signaling receptors16, 17. Studies of patients with inflammatory disorders found induced expression of many chemokines, indicating that these small proteins play important roles in the development and progression of inflammatory diseases. Published reports have also shown that genetic deletion of selected chemokine receptors causes reduction in the severity of inflammation in various animal models. For instance, deletion of the CCR2 gene markedly reduced lesion in athrosclerosis-prone ApoE-null mice18. Atherosclerosis is an inflammatory disorder and the expression of CCL2, the ligand for CCR2, is upregulated in the atherosclerotic plaque and contributes to local accumulation of monocytes. In addition, CCR2 also contributes to the development of multiple sclerosis, rheumatoid arthritis, scleroderma and ischemia-reperfusion injury. Receptors for the CXCL class of chemokines are found in neutrophils. Among these GPCRs, CXCR1, and CXCR2 are involved in the pathology of myocardial infarction. CXCR1 and CXCR2 interact with CXCL1, CXCL2, and CXCL8, which are present during acute inflammation and acute injury. These chemokines are primarily responsible for the recruitment of neutrophils to the site of inflammation and tissue injury, where these professional phagocytes can affect granule release and reactive oxidant production.

Chemotaxis

All chemoattractant receptors have the ability to mediate cell migration. In inflammatory disorders such as rheumatoid arthritis and atherosclerosis, the presence of leukocytes is crucial to disease progression. FPR1 is also responsible for sensing mitochondrial N-formyl peptides released from damaged cells19, 20. Activation of the Gi family of G proteins is critical to chemotaxis, which involves a complex network of intracellular signaling and cytoskeleton reorganization. Phagocytes polarize upon chemoattractant stimulation, forming a leading edge and a trailing edge which is characteristic of a migrating cell. G protein signaling initiates at the leading edge, as evidenced by the production of PIP3 and translocation of proteins with the PIP3-binding PH domain such as Akt and guanine nucleotide exchange factors such as P-Rex121 (Figure 1). Cytoskeletal reorganization also requires activation of the small GTPase Rac, whereas RhoA, another small GTPase, is believed to play a role at the trailing edge of a migrating cell22. Not all chemotaxis-mediating receptors are 7-TM receptors; other potent chemoattractants such as TGF-β act on other receptors. However, the fact that all chemokines bind to 7-TM receptors illustrates the importance of this class of receptors in cell migration. Until recently, only Gi proteins were implicated in leukocyte chemotaxis. The fact that several chemoattractant receptors could also couple to Gq proteins prompted a study that identified its role in chemotaxis23. Using mice lacking Gq proteins, it was observed that chemotaxis of dendritic cells to selected chemokines requires both Gi and Gq proteins. This alternative pathway uses a CD38-dependent mechanism for regulation of chemotaxis. It is presently unclear why in these cells Gq is necessary for chemotaxis whereas in other cells Gi is sufficient, but studies of this sort make it clear that the study of chemotaxis will also expand the understanding of GPCR biology.

Figure 1
figure 1

Signaling pathways of GPCRs involved in inflammation. Signaling by GPCRs is initiated by a specific ligand that binds and activates the receptor inducing conformational changes in the receptor. A partial list of the relevant ligands is given in the top panel. All 4 classes of G proteins are involved in the regulation of events that lead to inflammatory cell activation or inactivation. Major effectors, including second messengers and other signaling molecules involved in the regulation of inflammation are shown. These include Ca2+, cAMP, protein kinases, lipid kinases, lipases, phosphoinositides, small GTPases and the relevant guanine nucleotide exchange factors, and transcriptional factors. The final output is manifested as nuclear, cytoplasmic and extracellular activities such as expression of proinflammatory genes, production of oxidants, and generation of tissue damaging and repairing factors.

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Degranulation

A large number of GPCR agonists, including chemotactic peptides, activated complement fragments and histamine, are able to stimulate granule release24. In neutrophils, binding of fMLF to the receptor FPR1 or C5a to C5aR, triggers strong degranulation. In fact, exogenous expression of FPR1 or C5aR in a rat basophilic leukemia cell line renders these cells capable of releasing β-hexosaminidase upon stimulation, demonstrates the sufficiency of these receptors to trigger degranulation25. Simultaneous activation of several signaling pathways is required for degranulation26. As with other secretory cells, fusion of intracellular granules or vesicles in phagocytes requires calcium influx, which is triggered by GPCR signaling (Figure 1). G protein signaling also leads to the activation of several protein kinases, including PKC, cGMP-dependent kinase, and the serine/threonine kinase Akt. Exactly how these kinases promote granule release remains incompletely understood, but it is reported that vesicular fusion-related proteins such as the SNAP proteins are phosphorylated upon cell stimulation and these phosphorylation events precede vesicular fusion27. In addition to the protein kinases, small GTPases are activated downstream of GPCRs and are required for fusion of intracellular vesicles.

Superoxide generation

Although the production of reactive oxygen species (ROS) is widespread among different types of cells, phagocytes can produce large amounts of ROS, a requirement for killing phagocytosed bacteria28. A number of chemoattractants, including fMLF and C5a, potently stimulate ROS production in neutrophils, although production is extracellular rather than intra-phagosome when induced by these soluble mediators. As a result, these oxygen radicals cause damage to endothelial cells that form the lining of vascular wall. Exposure to even low doses of these chemoattractants also “primes” the phagocytes for more robust oxidant production when stimulated with other inflammatory factors such as LPS29. Chemoattractant-induced ROS production requires Gi protein, as it is effectively blocked by PTX treatment. Research conducted thus far has found that chemoattractant-induced ROS production shares basic mechanisms with particle-induced ROS production, in that both require membrane translocation of cytosolic components and assembly of a functional NADPH oxidase at membrane. There are, however, major differences in the upstream signaling mechanisms involved. Whereas phagocytosis-induced oxidant production requires primarily tyrosine kinase activation, chemoattractant-induced ROS production relies mostly on the activation of serine/threonine kinases such as Akt and dual-specificity protein kinases such as p38 MAPK. These different signaling pathways then converge at activation of downstream targets such as PKC (Figure 1). Published reports show that upon activation of chemoattractant GPCRs, the released G protein βγ subunits triggers PI3K activation and the production of PIP330. The membrane-bound PIP3 is required for the subsequent activation of Akt and the small GTPase Rac, which is also required for the assembly of a functional NADPH oxidase complex31, 32. Thus, chemoattractant-induced ROS production requires simultaneous and sustained activation of multiple signaling pathways, which are also negatively regulated by phosphatases33. How chemoattractant GPCRs convert a single binding event to a cascade of signaling events remains incompletely understood. Recent studies have shown that both G protein-dependent and –independent pathways are involved in chemoattractant signaling.

GPCRs and inflammatory pain

Inflammation caused by trauma, infection and other forms of insult to the tissue is often accompanied by the uncomfortable sensation of pain. Inflammatory pain may result from enhanced nociception due to the interaction of inflammatory mediators with neurons and the resulting state of hypersensitivity. Inflammation may also cause damage to neurons and produce neuropathic pain34. Being the largest group of sensory receptors, GPCRs play important roles in inflammatory nociception. At the periphery, a number of GPCR agonists produced during inflammation, including bradykinin (BK) and selected prostaglandins (PGs), participate in inflammatory hyperalgesia. BK and kallidin both activate and sensitize primary afferent neurons. Two classes of BK receptors are present. Experimental data show that B1 BK receptor agonists produce pain only during inflammation, in accordance with the inducible nature of the B1 BK receptor. The B2 BK receptor is constitutively expressed and its blockade reduces inflammatory hyperalgesia in animal models35.

Prostaglandins (PGs) are lipid-derived autacoids generated through the sequential actions of cyclooxygenase and PG synthase. These metabolites of arachidonic acid include thromboxanes (TXA2), PGD2, PGE2, PGI2, and PGF2a. Collectively, they interact with 9 prostanoid receptors that couple to a variety of G proteins and are responsible for several features of inflammation including pain and edema (Table 1). Non-steroidal anti-inflammatory drugs (NSAIDs), including acetylsalicylic acid (aspirin) and the more selective COX-2 specific inhibitors, are effective anti-inflammatory and pain-relieving agents primarily because they block the synthesis of PGs36, 37. Among the various arachdonic acid metabolites, PGE2 interacts with several GPCRs. The 4 EP receptors are individual gene products but in the case of EP3, there are splice variants as well38. The 4 subclasses of EP receptors couple to various G proteins and are responsible for the variety of PGE2 effects. PGE2 is synthesized during the course of inflammation and contributes to tissue edema and hyperalgesia. In the nociceptive primary afferent nerve terminals, PGE2 modulates voltage-gated sodium currents39, a function mediated through the EP3 receptor40. PGE2 is also synthesized in the CNS during peripheral inflammation and contributes to increased pain hypersensitivity41.

Table 1 A partial list of GPCRs involved in inflammation.
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Prostacyclin (PGI), which acts on G protein-coupled IP receptor, is another arachdonic metabolite produced during inflammation that plays a central nociceptive role42. Mice lacking the IP receptor display altered pain perception as well as inflammatory responses43. Studies have shown that Gs-coupled PG receptors may enhance nociceptor sensitization by reducing the activation threshold for selected sodium channels through a cAMP and PKA-dependent mechanism44. GPCRs present in dorsal horn neurons are also involved in transmission of nociception and are responsible for the development of central sensitization. The NK1 receptor for tachykinin is a GPCR that responds to substance P and mediates PKC activation, leading to the phosphorylation and potentiation of N-methyl-D-aspartic acid (NMDA) receptors45. In addition to the NK1 receptor, the neuromedin U2 receptor (NMU2) plays a central role in nociception. NMU2 knockout mice display reduced sensitivity to pain induced by capsaicin and formalin46.

Besides functions of GPCRs in inflammatory nociception, there are instances in which GPCRs are antinociceptive. Loperamide, an opioid agonist developed for peripheral use, displays antinociceptive activity in experimental arthritis47. Cannabinoids, which are agonists for the CB1 and CB2 receptors, inhibit peripheral sensitization when used topically48. Exploration of the roles for GPCRs in inflammatory nociception is bound to provide novel therapeutics for effective control of pain associated with inflammatory diseases.

GPCRs and regulation of vascular endothelial permeability

Edema, an abnormal accumulation of interstitial fluid, often accompanies inflammation. The production of inflammatory factors, many of them GPCR agonists, is largely responsible for increased vascular endothelial permeability, which contributes to edema during inflammation. BK and PAF are well known for their roles in the regulation of vascular wall permeability. It has long been recognized that BK reproduces cardinal signs of inflammation, and the BK effect on vascular permeability is direct rather than dependent on histamine49. More recent studies have shown that both the B1 BK receptor and B2 BK receptor are involved in local edema during inflammation, for instance, paw edema and protein extravasation leading to joint swelling50. PAF profoundly affects microvascular permeability, allowing extravasation of plasma contents such as albumin51, 52. Like BK, PAF acts on endothelial cells directly, increasing gap formation between endothelial cells through the actions of eNOS53, tyrosine phosphorylation of VE-cadherin54, and the Rho family small GTPases55.

Histamine and thrombin are also major regulators of vascular permeability. Histamine is released by basophils and mast cells during the allergic response. It binds to one or more of the four G protein-coupled histamine receptors, stimulating vasodilation and increasing vascular permeability56. Histamine also participates in allergic inflammatory diseases such as asthma by inducing chemotaxis and bronchoconstriction57. Thrombin is one of the best studied regulators of vascular endothelial permeability58. The thrombin receptor was initially identified as a GPCR with a unique mechanism for activation. Upon binding of alpha thrombin, the receptor's N-terminus was cleaved, generating a tethered peptide that becomes an agonist for the receptor59. This receptor is the first member of the protease-activated receptor (PAR) subfamily, of which 3 of the 4 PARs bind thrombin (Table 1). Although initially known as a thrombotic agent that catalyzes the conversion of fibrinogen to fibrin, thrombin is also a potent agonist for platelets and its receptors are expressed in human endothelial cells as well as smooth muscle cells (SMC). Its vascular functions include cellular differentiation, migration and proliferation of SMC, angiogenesis and vascular development60. PAR1, which is widely studied in this subfamily of GPCRs, couples to multiple G proteins including Gi, Gq, and G12/13. The selectivity for activation of these G proteins depends on the agonists that bind to the receptor61. In endothelial cells, thrombin binding to PAR1 leads to activation of p115 RhoGEF, which provides a functional link between G13 and RhoA62. Activation of RhoA is responsible for stress fiber formation, and increased calcium flux triggers other signaling pathways which ultimately lead to myosin light chain-dependent contraction of endothelial cells63. This process is reversible, and focal adhesion kinase plays a role in the reversal of the increased vascular permeability64. Thrombin-induced increase in vascular endothelial permeability contributes to the edema seen in inflammatory disorders such as acute lung injury65, 66. Both the receptor and its downstream signaling pathways are targets for therapeutic intervention67, 68. In contrast to thrombin, another GPCR ligand sphingosine 1-phosphate (S1P) promotes stabilization of the endothelial barrier69. This effect of S1P is mediated through its receptor S1P1, which promotes adherens junction assembly in endothelial cells, thus fortifying vascular endothelial barrier70, 71.

GPCRs and regulation of inflammatory gene expression

A large number of GPCRs have been found to participate in transcriptional regulation72. G protein signaling leads to activation of transcription factors including CREB, c-Jun, NF-κB, and STAT3, among others (Figure 1). These transcription factors, particularly NF-κB, are closely associated with the expression of genes that encode inflammatory factors. It was first reported that the receptor for PAF could stimulate NF-κB activation through a PTX-insensitive pathway, suggesting that the Gq protein which couples to the PAF receptor is responsible for this function73. Subsequent studies identified both PTX-sensitive and PTX-insensitive mechanisms by which GPCRs activate NF-κB74, 75, 76, 77, 78. The Gi-dependent pathway requires the Gβγ subunits whereas Gq directly activates PLCβ, thus triggering PKC. In addition to PKC, protein kinases that are found to be involved in GPCR activation of NF-κB include the Ser/Thr kinase Akt77 and tyrosine kinase Pyk279. The β-arrestin pathway was also involved in regulating NF-κB activation. In resting cells, β-arrestins bind to IκBα and protect it from phosphorylation and proteosome degradation80, 81. Upon GPCR activation, β-arrestins participate in intracellular signaling leading to activation of multiple pathways that favor NF-κB activation82, 83, 84. Several intracellular signaling molecules that were initially identified for immune cell functions, including CARMA3 and Bcl10, were found to regulate GPCR signaling leading to NF-κB activation85, 86. These latter findings provide evidence for the complexity of signaling pathways downstream of GPCRs.

In addition to agonist-induced, GPCR-mediated NF-κB activation, a number of constitutively activated GPCRs are able to activate NF-κΒ even in the absence of agonists. Viruses such as KSHV, HHV8, and RCMV encode GPCRs that are constitutivelyactive and, when expressed in mammalian cells, activate NF-κB87, 88, 89, 90. Like agonist-induced GPCRs, these viral GPCRs couples to more than one G proteins for signaling88. Transcriptional activation by these GPCRs is not restricted to NF-κB87. These receptors are believed to contribute to their pathological functions including stimulation of angiogenesis in the case of KSHV GPCR, and are potential therapeutic targets91, 92.

Summary

The diverse actions of inflammatory factors are reflected in the diversity of receptors with which they interact. It is therefore not surprising that inflammatory factors of different nature and composition, including arachidonic acid metabolites, peptides, protein fragments and proteases, are found to partner with the 7-TM GPCRs. The diversity in ligand binding and transmembrane signaling by GPCRs are primarily responsible for the mediation of complex inflammatory (and anti-inflammatory) responses. There is no doubt that GPCRs play important roles in inflammation, as they do in other vital organ functions. A better understanding of these receptors as well as their signaling pathways will help to develop new therapeutic agents with higher specificity than traditional anti-inflammatory agents such as NSAIDs.