CXCR1/2 ligands induce p38 MAPK-dependent translocation and release of opioid peptides from primary granules in vitro and in vivo☆
Introduction
Polymorphonuclear leukocytes (PMN)1 contain opioid peptides such as Met-enkephalin (ENK) and β-endorphin (END) (Vindrola et al., 1990, Mousa et al., 2004). In inflamed subcutaneous tissue these opioid peptides can be released, bind to μ (MOR) and δ (DOR) opioid receptors on peripheral sensory nerves and locally inhibit pain (reviewed in Stein et al., 2003). Human PMN express the chemokine receptors CXCR1 and 2, while murine and rat PMN only express CXCR2. We previously demonstrated that CXCR1/2 ligands (i.e. human CXCL8) and CXCR2 ligands (i.e. rat CXCL2/3, syn. macrophage inflammatory protein-2) trigger opioid peptide release from human and rat PMN in vitro and inhibit inflammatory pain in vivo (Rittner et al., 2006a).
PMN constitutively produce opioid peptides and store them in vesicular structures (Vindrola et al., 1990, Mousa et al., 2004), but the exact nature of the granules is unknown. Four distinct types of cytoplasmic granules have been described in human PMN: azurophil (primary), specific (secondary) and gelatinase (tertiary) granules as well as secretory vesicles (Borregaard and Cowland, 1997, Gullberg et al., 1997). Each of these have a well-characterized content: primary granules contain myeloperoxidase (MPO), elastase, and a wide array of lytic enzymes and proteins with bactericidal activities, which are released to eliminate potential pathogens. Secondary granules contain lactoferrin and several metalloproteinases including collagenase, and tertiary granules contain gelatinase. Upon PMN activation these granules are translocated to the cell periphery to fuse with the plasma membrane. Secretory vesicles are storage organelles for membrane receptors and secretable proteins such as albumin and alkaline phosphatase needed in the earliest phases of PMN-mediated inflammatory responses (Borregaard, 1988, Borregaard et al., 1993, Borregaard and Cowland, 1997). Exocytosis of the distinct granule populations may occur independently (Mollinedo et al., 1991, Sengelov et al., 1993, Mollinedo et al., 1997, Mollinedo et al., 2006). For example, a strict rank order of exocytosis of the four compartments was observed when cytosolic calcium was elevated by an ionophore in Fura-2-loaded cells (Sengelov et al., 1993). Secretory vesicles were discharged first, followed by gelatinase positive tertiary granules, secondary granules, and finally primary granules.
We previously demonstrated that CXCR1/2 ligands can induce opioid peptide release from PMN, that this release requires calcium mobilisation from intracellular but not extracellular stores and that it is partially dependent on phosphoinositol-3-kinase activation (Rittner et al., 2006a). Other groups have shown that CXCR1/2 ligands also activate members of the mitogen-activated protein kinase (MAPK) family, such as p38 and p42/44, by pathways that are distinct from calcium mobilisation and phosphoinositol-3-kinase activation (Takami et al., 2002, Chakrabarti and Patel, 2005, Fuhler et al., 2005). Furthermore, MAPK activation appears to play an important role in granule exocytosis induced by various chemoattractants including formyl-Met-Leu-Phe (fMLP) (Mocsai et al., 2000). In particular, fMLP-triggered release of primary and secondary granules, but not of secretory vesicles, was influenced by p38 but not by p42/44 MAPK activation (Mocsai et al., 2000).
In the present study, we set out to delineate which populations of granules contain opioid peptides and which types of MAPK are most relevant for opioid peptide release and subsequent pain inhibition. We sought to identify the granule subpopulations in human PMN that contain END and ENK by double immunofluorescence confocal microscopy with antibodies against CD63 and MPO (markers for primary granules), lactoferrin (secondary granules), MMP9 (tertiary granules) or albumin (secretory vesicles). We then examined whether CXCL8 stimulates translocation of END and ENK containing granules to the membrane and whether END/ENK release is influenced by the MAPK inhibitors SB203580 (p38) or PD98059 (p42/44). Finally, we tested whether END, ENK and MAPK are functionally relevant in vivo by investigating CXCL2/3-induced antinociception in rats with paw inflammation.
Section snippets
Antibodies and reagents
The following reagents and antibodies were used: human CXCL8 (R&D Systems, Minneapolis, MN, USA); rat CXCL2/3 (Peprotech, London, Great Britain); cytochalasin B (CyB), rabbit IgG, fentanyl and naloxone (Sigma–Aldrich Chemie, Deisenhofen, Germany); CFA, SB203580 and PD98059 (Calbiochem/Merck, Darmstadt, Germany); rabbit anti-rat END, rabbit anti-ENK, ENK and END (Bachem, Weil am Rhein, Germany); mouse monoclonal anti-MPO (Clone 8F4; Cell Sciences, Inc., Canton, MA, USA, or Clone MPO-7, Biomeda
Identification of granules containing opioid peptides
END and ENK were expressed in MPO- and CD63-positive primary granules of unstimulated PMN (Fig. 1, Fig. 2). These intensely double labelled granules were homogeneously distributed in all parts of the cytoplasm. In contrast, we found no overlap of END- or ENK-immunoreactivity with lactoferrin (marker for secondary granules), MMP9 (marker for tertiary granules), or albumin (marker for secretory vesicles) in unstimulated PMN (Fig. 1, Fig. 2). No difference in the subcellular localisation was found
Discussion
The major finding of this study is that END and ENK are mainly contained in primary granules of PMN and that the release of these opioid peptides, triggered by a CXCR1/2 ligand in vitro, is dependent on p38 MAPK but not p42/44 MAPK. Consistently, the in vivo application of a CXCR1/2 ligand inhibited inflammatory pain by p38 MAPK-dependent release of END and ENK. This is supported by several lines of evidence: (1) both END and ENK co-localized with primary granule markers (CD63 and MPO) within
Acknowledgments
This study was supported by the Deutsche Forschungsgemeinschaft (KFO 100). The authors thank Susanne Kotré, Katharina Hopp and Ute Oedekoven for expert technical assistance.
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