Published as doi: 10.1096/fj.06-6077fje.
(The FASEB Journal. 2006;20:2627-2629.)
© 2006 FASEB
Pain control by CXCR2 ligands through Ca2+-regulated release of opioid peptides from polymorphonuclear cells
Heike L. Rittner*,1,
Dominika Labuz*,
Michael Schaefer
,
Shaaban A. Mousa*,
Stefan Schulz
,
Michael Schäfer*,
Christoph Stein* and
Alexander Brack*
* Klinik für Anaesthesiologie und Operative Intensivmedizin and
Institut für Pharmakologie, Charité–Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany; and
Department of Pharmacology, Otto-von-Guericke-University, Magdeburg, Germany
1Correspondence: Klinik für Anaesthesiologie und Operative Intensivmedizin, Charité–Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, D-12200 Berlin, Germany. E-mail: heike.rittner{at}charite.de
SPECIFIC AIMS
Inflammatory pain can be effectively controlled by release of opioid peptides from infiltrating leukocytes binding to opioid receptors on peripheral sensory neurons. To elucidate the role of CXCR1/2 ligands in opioid peptide release from polymorphonuclear cells (PMN) in vitro and in the inhibition of inflammatory pain in vivo, we wanted 1) to inject CXCR2 ligands at the site of complete Freund’s adjuvans (CFA)-induced hindpaw inflammation and 2) to identify intracellular signaling pathways of CXCR1/2 ligand-induced opioid release from human and rat PMN in vitro. Finally, we tested the in vivo functional relevance of these mechanisms for pain control using local adoptive transfer of allogenic PMN that were pretreated with appropriate signaling pathway inhibitors ex vivo.
PRINCIPAL FINDINGS
1. Local CXCR2 but not CXCR4 ligand injection induces opioid receptor-mediated analgesia in inflammation
Injection of CFA into rat hindpaws resulted in a sustained hyperalgesia, as measured by a decrease in paw pressure threshold (PPT). Intraplantar application of CXCL2/3 (macrophage inflammatory protein-2), but not the CXCR4 ligand CXCL12 (stromal derived factor-1
) dose-dependently increased PPT (i.e., produced antinociception). CXCL2/3-induced antinociception was inhibited by local coadministration of the opioid antagonist naloxone indicating its mediation by opioid receptors.
2. Opioid peptide release from human and rat PMN after CXCR1/2 stimulation in vitro depends on intracellular Ca2+ and phosphoinositol-3-kinase (PI3K) activation
On the basis of the opioid-dependent antinociceptive effects of a CXCR2 ligand and the lack of effect of a CXCR4 ligand, we hypothesized that chemokines can release opioid peptides from PMN. Incubation of human PMN with the CXCR1/2 ligand CXCL8 stimulated a significant dose-dependent release of the opioid peptides ßbeta;-endorphin (END) and met-enkephalin (ENK). No opioid peptide release was observed after CXCL12 incubation.
To analyze the signaling pathways involved in opioid release, human PMN were incubated with the Ca2+ ionophore ionomycin and with thapsigargin. Thapsigargin blocks the sarcoendoplasmic reticulum Ca2+ ATPases resulting in passive Ca2+ leak from intracellular stores. Both treatments significantly stimulated opioid release. The removal of extracellular Ca2+ did not affect CXCL8-induced opioid peptide release. We then performed preincubation with thapsigargin in the absence of extracellular Ca2+ to deplete intracellular Ca2+ stores, or with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM, a membrane permeable intracellular Ca2+ chelator). Both treatments abrogated the CXCL8-induced release of opioid peptides. Taken together, although direct entry of extracellular Ca2+ elicited opioid release, CXCL8-induced release was independent of extracellular Ca2+ but required IP3R-triggered Ca2+ release from intracellular stores. To test the possible involvement of PI3K, we applied the two PI3K inhibitors wortmannin and LY294002 and found partial inhibition of CXCL8-stimulated END and ENK release.
In glycogen-elicited rat peritoneal PMN significant dose-dependent opioid peptide release was observed after stimulation with the CXCR2 ligand CXCL2/3 but not with the CXCR4 ligand CXCL12 (Fig. 1
A). CXCL2/3-induced END and ENK secretion from rat PMN was not altered in the absence of extracellular Ca2+ (Fig. 1B
), while chelating intracellular Ca2+ by BAPTA/AM abolished opioid peptide secretion (Fig. 1C
). Blocking PI3K by wortmannin (Fig. 1D
) or by LY294002 (data not shown) significantly but partially reduced opioid peptide release .
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Figure 1. Intracellular Ca2+- and phosphoinositol-3-kinase (PI3K)-dependency of CXCL2/3-induced opioid peptide release from rat PMN. A) Rat PMN were stimulated with the indicated concentrations of CXCL2/3 and CXCL12 for 7 min. Met-enkephalin (ENK, left) and ßbeta;-endorphin (END, right) release was measured in the supernatant by RIA (n=6–19). B) CXCL2/3 (1000 nM, cross-hatched bars in all experiments)-induced opioid peptide release was analyzed in the presence (open bars) and absence (gray bars) of extracellular Ca2+ (-[Ca2+]e, n=9). CXCL2/3-induced opioid peptide release was measured after preincubation of rat PMN (for 10 min) with the intracellular Ca2+ chelator BAPTA/AM (C), (100 µM, n=5–7) or the PI3K-inhibitor wortmannin (D) (100 nM, n=6–9, respectively). *P < 0.05 significant difference compared to respective controls (all one-way repeated-measures ANOVA, Student-Newman-Keuls method). Data are expressed as means ± SEM.
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3. Effect of PMN depletion, adoptive transfer of allogenic PMN, and intracellular Ca2+ depletion on CXCL2/3-induced antinociception in vivo
PMN depletion by an anti-PMN serum resulted in over 90% reduction of PMN (Fig. 2
A). Under these conditions, CXCL2/3-mediated antinociception was significantly attenuated (i.e., reduced rise in PPT) (Fig. 2B
).
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Figure 2. Effects of PMN depletion and reconstitution with adoptively transferred PMN on CXCL2/3-induced pain control. Rats were pretreated with i.v. anti-PMN serum (gray bars), control animals received nonimmune rabbit serum (open bars). Two hours after CFA the number of leukocytes in the paw (A) were quantified by flow cytometry. Likewise, paw pressure threshold (PTT) (B) was measured before (baseline) and after intraplantar injection of 100 ng CXCL2/3 (n=6, *P<0.05 t test). C) Different numbers of glycogen-elicited peritoneal PMN from allogenic animals were injected into the inflamed paws of PMN-depleted rats. PPT was obtained 15 min later and again after 100 ng i.p. CXCL2/3 (n=6, * P<0.05, one-way ANOVA, Dunnett method, open bar: before CXCL2/3 without PMN depletion; crosshatched bar: effect of intraplantar CXCL2/3 without PMN depletion, gray bars: PMN depletion, striped bars: PMN reconstitution). D) Effect of ex vivo BAPTA/AM (100 µM) or solvent pretreatment before allogenic PMN transfer. Rats were PMN-depleted and reconstituted as described in (C) using 1 x 106 PMN and CXCL2/3-induced PPT elevation was measured thereafter (n=6, *P<0.05, one-way ANOVA, Student-Newman-Keuls method). Data are expressed as means ± SEM
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Subsequent adoptive transfer of allogenic PMN by intraplantar injection dose-dependently reestablished CXCL2/3-induced antinociception in the inflamed paw (Fig. 2C
). To analyze dependency of CXCL2/3-induced antinociception on intracellular Ca2+ in vivo, allogenic PMN were pretreated ex vivo with BAPTA/AM. This treatment significantly reduced CXCL2/3-induced antinociception following PMN reconstitution (Fig. 2D
).
CONCLUSIONS AND SIGNIFICANCE
Chemokines like CXCL12 or CXCL1 (keratinocyte-derived chemokine) were previously shown to cause pain. However, these studies were performed in the absence of inflammation. In this study, we show that CXCL2/3, but not CXCL12, injected into inflamed paw tissue resulted in local opioid-dependent antinociception, and this was abolished by PMN depletion (Fig. 2A,B
).
In agreement with the effects in vivo, we showed that CXCR1/2 ligands trigger opioid peptide release from PMN in vitro (Fig. 1A
). We further examined signaling pathways for the secretion of opioid peptides (Fig. 3
) and showed dependency on intracellular Ca2+ release (Fig. 1C
) and PI3K activation (Fig. 1D
). In our experiments, CXCL2/3-induced opioid peptide release was independent of extracellular Ca2+ (Fig 1B
). Although stimulation of PMN with platelet-activating factor, formyl-Met-Leu-Phe or leukotriene B4 induces store-operated Ca2+ entry (i.e., prolonged elevation of [Ca2+]i due to entry of extracellular Ca2+), no store-operated Ca2+ entry has been found after CXCR1 stimulation with CXCL8 despite significant release of Ca2+ from intracellular stores. These results support our findings of independency of CXCL8-induced opioid peptide release from extracellular Ca2+.
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Figure 3. Schematic diagram. Opioid peptide-containing PMN migrate into inflamed tissue. These peptides are released and bind to opioid receptors on peripheral sensory neurons leading to analgesia. CXCL2/3 produces pain control by triggering opioid peptide release. CXCL2/3-induced opioid release requires elevation of intracellular Ca2+ through flux from the endoplasmic reticulum (ER). In addition, activation of phosphoinositol-3-kinase (PI3K) is involved. PLC, phospholipase C; IP3, inositol 1,4,5-triphosphate.
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In addition, we demonstrated that opioid peptide release was partially dependent on PI3K (Fig. 1D
). Other studies have shown that degranulation by CXCL8 or other mediators is also dependent on PI3K. However, the PI3K class and subclass involved in CXCL8-induced release still remains to be elucidated, as well as the pathways downstream from PI3K activation.
To confirm the relevance of intracellular Ca2+ for opioid peptide release in vivo, we employed an approach to avoid impairment of sensory nerve functioning by signaling cascade inhibitors. To this end, we established a model of ex vivo treatment and subsequent adoptive cell transfer. Rats were depleted of PMN, and local transfer of allogenic PMN was performed. This demonstrated dose-dependent reconstitution of CXCL2/3-induced antinociception (Fig. 2C
). Chelating intracellular Ca2+ of PMN before transfer significantly impaired CXCL2/3-induced antinociception (Fig. 2D
). Because we did not observe differences in baseline hyperalgesia, our approach apparently did not compromise sensory nerve function. This model provides a novel tool to selectively study the signaling pathway requirements of release from PMN in vivo.
Taken together, CXCR2 ligands have a dual antinociceptive role by mediating the recruitment of opioid-containing PMN, as previously shown, and by directly triggering opioid peptide release from PMN in inflammation.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6077fje
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Copyright © 2006 by The Federation of American Societies for Experimental Biology.
