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The small protein Bv8, isolated from the amphibian skin, belongs to a novel family of secreted proteins linked to several biological effects. We describe the expression of Bv8/prokineticins and their receptors in mouse macrophages, and characterize their proinflammatory activities.
The rodent analogue of Bv8, prokineticin-2, is expressed by macrophages, as well as its G-protein-coupled receptor prokineticin receptor (PKR-1 and PKR-2). PKR-1 is expressed more abundantly.
Bv8 induces potent chemotaxis of macrophages at concentrations as low as 10−12 M.
It stimulates lipopolysaccharide-induced production of the proinflammatory cytokines IL-1 and IL-12, reducing that of the anti-inflammatory cytokine IL-10. The effects are observed starting at the very low concentration of 10−11 M.
Effects on chemotaxis and cytokine are not pertussis-toxin sensitive, but are completely prevented by addition of the phospholipase inhibitor U73122, suggesting a Gq protein is involved in the Bv8-induced effects.
Studies in PKR-1 knockout mice indicate that all the activities exerted by Bv8 on macrophages are mediated by the PKR-1 receptor.
In conclusion, Bv8 appears to be able to induce the macrophage to migrate and to acquire a proinflammatory phenotype.
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The small 77-amino-acid (aa) protein Bv8, isolated from the amphibian skin (Mollay et al., 1999) belongs to a novel family of secreted proteins whose homologues have been found in snakes (VPRA, MIT1) (Joubert & Strydom, 1980; Schweitz et al., 1999), rodents (mouse Bv8 or prokineticin (PK) 1 and 2) (Melchiorri et al., 2001) and humans (PK 1 or EG-VEGF and PK 2) (Masuda et al., 2002). This peptide family has structural shared motives, such as the 20-aa terminal sequence and a pattern of cysteine sequence that folds the molecules into a globular form.
The mRNA of murine Bv8-like protein, PK 1 and 2 (PK-1 and PK-2), has been detected in the brain, spinal cord, dorsal root ganglia, gastrointestinal tract, endocrine glands, spleen and circulating leukocytes of mice, rats and humans (Li et al., 2001; Melchiorri et al., 2001; Masuda et al., 2002; Le Couter et al., 2004). Two receptors for this family of secretory proteins have been identified in humans, rats and mice (Lin et al., 2002; Masuda et al., 2002). These receptors, PKR-1 and PKR-2, belong to the G-protein-coupled receptor and share approximately 85% aa identity. They are distributed in the brain and peripheral organs (Melchiorri et al., 2001; Negri et al., 2002), including the spleen and leukocytes (Le Couter et al., 2004).
The list of biological activities associated with Bv8/PK peptides is rapidly growing. They seem to influence complex behaviors such as feeding and drinking (Negri et al., 2004), and circadian rhythms (Cheng et al., 2002), and are involved in neuronal survival (Melchiorri et al., 2001), angiogenesis (Le Couter et al., 2001; Le Couter & Ferrara, 2003), and the reproductive cycle (Wechselberger et al., 1999). Bv8 and EG-VEGF are also related to regulation of hematopoiesis and hematopoietic cell mobilization (Le Couter et al., 2004). Moreover, an hyperalgesic activity of Bv8 has been clearly demonstrated. When injected intravenously or subcutaneously in rats, Bv8 induces intense systemic nociceptive sensitization to mechanical and thermal stimuli applied to the tail and paws (Mollay et al., 1999; Negri et al., 2002).
The cytokines interleukin (IL-)1β and tumor necrosis factor alpha (TNF-α) are closely involved in peripheral nerve and neural hyperexcitability, leading to the development of persistent hyperalgesia (Watkins & Maier, 1999; 2002). However, anti-inflammatory cytokines have been reported to limit the hyperalgesic responses induced by inflammatory stimuli and by IL-1β and TNF (Poole et al., 1995).
Considering that lymphoid organs, circulating leukocytes and hematopoietic cells all express high levels of Bv8-like protein (Le Couter et al., 2004), we assessed whether Bv8 could influence the physiology of the macrophage, the main cell involved in inflammatory responses. Macrophages play a central role in both innate and adaptive immunity (Schiffmann, 1982; Beutler, 2004). They are fundamental to the innate immune response and their ability to be chemotactically attracted to the site of initial microbial invasion or to an inflammatory focus is crucial for full activation of the immune/inflammatory response that follows (Beutler, 2004). They are the main producers of the proinflammatory cytokines IL-1β and TNF and the major anti-inflammatory cytokine IL-10 (Moore et al., 1993; Beutler, 2004; Hoebe et al., 2004). Macrophages also synthesize and release IL-12, the critical factor driving the development of T helper (Th)-1 cells (Moore et al., 1993; Trincheri, 1995; Mosman & Sad, 1996; Hoebe et al., 2004), linked to cellular immune responses and tissue injury.
Therefore, considering the importance of macrophages in all aspects of the inflammatory response, we evaluated the effect of Bv8 in vitro on several macrophage functions.
First of all we investigated whether murine peritoneal macrophages expressed PK receptors and PK 1/2. We then analyzed how Bv8 affected macrophage migration and the production of the pro-inflammatory cytokines IL-1β, TNF and IL-12 and the anti-inflammatory cytokine IL-10. In order to identify the type of receptor involved, we designed some experiments with macrophages from PKR-1 knockout (KO) mice, and tried to characterize the type of G-protein signaling in the effects observed.
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The macrophage is a fundamental effector cell of innate immune responses, but it also constitutes the interface between innate and adaptive immunity thanks to its ability to produce important regulatory cytokines such as IL-12 and IL-10 (Beutler, 2004; Hoebe et al., 2004). The present study found that Bv8 strongly influences all aspects of macrophage physiology, driving a proinflammatory macrophage phenotype.
The recruitment of macrophages and monocytes to sites of inflammation, injury and infection is a crucial step in inflammation and antimicrobial immune responses. The ability to migrate towards chemoattractants is a first, important event in macrophage physiology (Schiffmann, 1982; Beutler, 2004; Mantovani et al., 2004). Bv8 promotes substantial macrophage migration at very low concentrations indeed – as low as 10−12 M – with an effect similar to that of the classical chemoattractant fMLP.
High levels of Bv8 expression have been detected in infiltrating neutrophils at sites of inflammation (Le Couter et al., 2004). Neutrophils are the first cells to be recruited at sites of inflammation and injury, followed by macrophages. It is therefore possible that Bv8's potent chemotactic activity plays an important role in directing macrophage migration. In the same study, Le Couter et al. (2004) also showed that Bv8 can induce migration of human monocytes, indicating that the chemotactic properties are common to myeloid cells of different species.
However, besides attracting macrophages, Bv8 further stimulates them. The addition of the protein boosts the production of the proinflammatory cytokines IL-1β and IL-12 induced by LPS. Like chemotaxis, cytokine production is increased by Bv8 at low doses.
The observation that TNF production was not affected by Bv8 is unexpected, but it is known that in the proinflammatory cytokine cascade TNF is frequently the first to be synthesized and secreted, followed by IL-1β (Cavaillon et al., 2003; Cunha et al., 2005). It is therefore possible that Bv8 acts on specific mechanisms for IL-1β, downstream of TNF regulation, and not on common pathways (Beutler, 2004). Mirroring the Bv8-induced increase of IL-1β and IL-12, there was also a significant decrease in the anti-inflammatory cytokine IL-10. Bv8 therefore strongly skews the pro inflammatory macrophage profile by raising IL-1β and lowering IL-10.
IL-12, produced by antigen-presenting cells, links the innate and cell-mediated components of the immune response by inducing CD4+ T-cell differentiation to Th1-like cells, while IL-10 underlies the development of a Th2 profile (Mosman & Sad, 1996). Hypothetically, therefore, Bv8's effects on the macrophage might have an indirect impact on the Th1/Th2 balance, influencing the development of immune responses. This interesting aspect is being evaluated (Sacerdote et al., in preparation).
Bv8 and its mammalian analogues are ligands for two G-protein-coupled receptors, PKR-1 and PKR-2, which show distinct expression patterns in different organs. Although the PCR analysis did showed that macrophages express both PKR-1 and PKR-2 transcript, PKR-1 is expressed more abundantly. This observation is in agreement with other reports that PKR-2 is the form expressed mostly in the central nervous system (Melchiorri et al., 2001; Negri et al., 2002), while PKR-1 is largely present on immune tissue (Melchiorri et al., 2001; Le Couter et al., 2004).
In this study, we used, for the first time, recently generated PKR-1-deficient mice, clearly demonstrating that all the activities exerted by Bv8 on macrophages are completely mediated by the PKR-1 receptor. Bv8-induced chemotaxis and cytokine modulation were in fact completely abolished in these PKR-1 KO animals. We also characterized the early signal mechanisms activated immediately after Bv8 binds to its receptor. Pretreatment with PTX toxin did not affect either Bv8-induced chemotaxis or cytokine modulation. These results seem to rule out the coupling of macrophage PKR-1 receptors to a Gαi/0 protein. The complete blockade of Bv8 activity on macrophage with the phospholipase inhibitor U73122 suggests there is a Gq-coupled PKR-1 acting through the IP-3/phospholipase-C signaling pathways. The phospholipase-C isoenzymes hydrolyze PI 4,5-bisphosphate (PIP2) to produce DAG and inositol 1,4,5 triphosphate (IP3), a calcium mobilizer, second messenger. Mammalian phospholipase-C comprises four subtypes, β, δ, γ and ɛ. Classically, phospholipase C-β is considered to be regulated by G protein-coupled receptors through a Gαq protein (Rhee, 2001).
It also appears that different events in macrophage activation, such as chemotaxis and cytokine production, are governed by the same intracellular signaling, quite likely activated by Bv8 binding to PKR-1. The involvement of a G protein α subunit of the q type has been reported for other Bv8 effects too, such as the increase of [Ca2+] concentrations and the p42/p44 MAPK phosphorylation in CHO cells expressing PKRs and in the dorsal root ganglia cells, whereas PTX did not affect these phenomena (Negri et al., 2002). In contrast to our results, Le Couter et al. (2004) found that PTX blocked Bv8-induced migration of human monocytes. However, some explanations can be offered. First of all, the type of receptor involved in Bv8-induced human monocyte migration was not determined, and PKR-1 or PKR-2 could have different coupling systems. In addition, although monocytes and macrophages derive from a common myeloid lineage, tissue macrophages have different characteristics from monocytes.
It is also increasingly emerging that different macrophage subtypes might have different ‘inflammatory’ functions (Cook et al., 2003). In our study, however, we found similar effects on both chemotaxis and cytokine production when Bv8 was administered to thioglycollate-elicited inflammatory macrophages, or to resident peritoneal macrophages from naive animals, the sentinel cells that respond to injury and orchestrate the innate immune responses (Cailhier et al., 2005). Bv8's ability to induce an inflammatory macrophage phenotype seems therefore general in macrophages at different stages and states of maturation or activation. In a previous study, we found a similar response as regards the effect on cytokines, using either resident or elicited macrophages (Limiroli et al., 2002).
Besides PK-receptors, macrophages also express high levels of PK-2 mRNA, which was always present as a tissue-specific double-splice variant: the short form encodes for a protein similar to amphibian Bv8 while the long form encodes for an additional 21 aa domain, inserted in the center of the polypeptide chain, already described only in mouse and human testis (Wechselberger et al., 1999). The insert was sequenced and was rich in basic residues, arginine and lysine, containing several potential cleavage sites for prohormone convertases. Nothing is known about the function of the longer PK-2 variant. Bullock et al. (2004) produced a recombinant protein of this splice variant that is about 200 times less potent than PK-2, in an aequorin-based assay for [Ca2+] mobilization in PKR-transfected CHO cells.
We have found a similar PK-2 long variant in rat macrophages and granulocytes, in human circulating granulocytes and in the promyelocytic cell line HL-60 (Negri et al., in preparation). Interestingly, coexpression of the ligand and receptors of this family of proteins has been observed in most immune cell types (Le Couter et al., 2004).
Recently, evidence has emerged that cytokines link the immune and the nervous systems and may be involved in the generation of pain and hyperalgesia. IL-1β has been associated with hyperalgesic states, acting directly on sensory neurons to increase their susceptibility to noxious stimuli (Safie Garabedian et al., 1995; Sommer & Kress, 2004). In contrast, IL-10 lowers nociceptive thresholds in several pain models (Poole et al., 1995). In view of Bv8's potent hyperalgesic activity (Negri et al., 2002), it is conceivable that this might be related to its effects on cytokines.
In conclusion, both resident and inflammatory macrophages seem to be targets for the Bv8/PK protein family. As these cells have functional PKR-1 receptors and produce Bv8, this molecule might possibly have some paracrine/autocrine role in regulating macrophage function.