The difference in the expression

The difference in the expression of functional FFARs between neutrophils and monocytes/macrophages suggests that the role of FFA2R may be restricted to the very early phase of acute inflammation where neutrophils are dominating players called into action, whereas GPR84 may have a broader functional spectrum in modulating inflammatory processes where both cell types are involved. Our data obtained with both blood and tissue neutrophils indicate that GPR84 is a chemotactic receptor. This is in consistence with a previous study utilizing another GPR84 agonist (6-OAU, mentioned above) [39], but it is clear that the precise role of GPR84 or any of the other GPCRs shown to be up- or down-regulated during in vivo recruitment of neutrophils [18] is not yet understood in detail. Agonist binding to GPR84 triggers heterotrimeric G-protein mediated signaling transduction leading to an assembly of the superoxide generating NADPH-oxidase, and our Gαq and pertussis toxin sensitivity data show that GPR84 does not signal through Gαq but more likely through Gαi. Although pertussis toxin completely inhibited the ZQ16 response, we cannot exclude the possibility that other signaling proteins are involved due to the non-specific inhibition of pertussis toxin on Gαi [32]. GPCR-mediated release of ROS in neutrophils is rapidly terminated and the agonist-occupied receptors desensitized, a process critical for the limiting the harmful release of abundant and thereby harmful release of ROS by neutrophils in response to agonists. Generally, GPCR signaling termination and desensitization are processes involving β-arrestins that bind parts of the activated receptors exposed on the cytosolic side of the membrane [41,42]. The 92 3 cytoskeleton can, however, replace β-arrestin and terminate receptor signaling [5,29,30,43]. Desensitization and termination of responses mediated by FPRs is achieved through binding of the actin cytoskeleton to the G-protein binding part of the actively signaling receptors, a binding that physically separates the occupied receptors from the down-signaling components [30,43]. These cytoskeleton linked non-signaling/desensitized FPRs will be reactivated upon disruption of the cytoskeleton [5,29] and we have shown that Latrunculin A-induced reactivation of desensitized FPRs can occur even 20 minutes after agonist desensitization [44]. In contrast to FPRs, desensitized FFARs are not reactivated upon disruption of the cytoskeleton, i.e., the Latrunculin A effect has disappeared if it is added after an FFAR agonist, suggesting that that the responses induced by the FFARs are terminated through an actin cytoskeleton independent mechanism ([4] and this study). It is worth noting that reactivation of desensitized FPRs in addition to Latrunculin A, also can be triggered by PAF or ATP [45,46]. With respect to this, future studies should aim to investigate how GPCR cross-talk modulates GPR84 activation and reactivation in neutrophils. Although the precise domain(s) of the FPRs that are involved in actin cytoskeleton coupling for signaling termination is not clear, it is possible that different phosphorylation sites in cytosolic parts of the receptors have important roles [47]. An examination of the potential phosphorylation sites in the cytoplasmic tail of FPR1 (UniProt accession number P21462) and GPR84 (UniProt accession number Q9NQS5) reveals large differences between the two receptors in the number of phosphorylation sites and this may contribute to the differences in the desensitization process mediated by the actin cytoskeleton for the two receptors, but the precise mechanisms to limit and terminate signaling have to be further studied. The fact that Latrunculin A primes neutrophils activated by ZQ16, suggests that GPR84 signaling in naïve neutrophils is blocked by an actin cytoskeleton dependent mechanism, in a similar manner to that earlier described to regulate signaling by the ATP receptor P2Y2R [44]. It is not known whether this cytoskeleton regulated change in responsiveness is due to a direct conformational change leading to a switch of the receptor from a low affinity to a high affinity state or if it is due to changed access to downstream signaling partners [44]. In addition to Latrunculin A, we also show that pro-inflammatory cytokine TNFα significantly primes neutrophils in their response to ZQ16, but through a different mechanism. Our earlier studies have suggested that one possible mechanism underlying TNFα priming could be that the granule mobilization induced by TNFα results in an increased plasma membrane exposure of receptors present in the neutrophil storage granules [48,49]. This amplification mechanism affects many different agonist/receptor pairs including formyl peptides and FPRs [4,44,50]. Though, what opposes this as the only mechanism of TNFα priming is that TNFα also primes the response induced by IL8, despite the fact that no mobilizable receptors are available [50]. The precise subcellular localization of the free fatty acid receptors GPR84 and FFA2R and the degree of mobilization induced in neutrophils by TNFα using subcellular fractionation/western blotting is currently not possible to determine. We have made several attempts, using a number of commercially available antibodies from different sources, but specific antibodies for either of these receptors are still lacking.