A δ2-Opioid Agonist Inhibits p38 MAPK and Suppresses Activation of Murine Macrophages

Journal of Surgical Research 128, 45– 49 (2005) doi:10.1016/j.jss.2005.04.003

A ␦ 2-Opioid Agonist Inhibits p38 MAPK and Suppresses Activation of Murine Macrophages Thomas L. Husted,* Meera Govindaswami,† Peter R. Oeltgen,† Steven M. Rudich,* and Alex B. Lentsch,*,1 *Laboratory of Trauma, Sepsis, and Inflammation Research, Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio; †Department of Pathology, University of Kentucky, Lexington, Kentucky Submitted for publication November 30, 2004

tation, trauma surgery, and hemorrhagic shock [1, 2]. Therapeutic modalities that reduce I/R injury in these scenarios may significantly improve organ function. Administration of ␦-opioid agonists is one potential strategy to limit I/R injury. There is mounting evidence to suggest that treatment with ␦-opioid agonists may protect against I/R injury. In an organ en-bloc preparation, perfusion with a nonspecific ␦-opioid agonist [D-Ala 2, D-Leu 5]-enkephalin (DADLE) significantly prolonged organ function and survival [3]. Similarly, in a model of lung preservation and transplantation, treatment with DADLE resulted in prolonged cold storage and subsequent successful transplantation [4]. More recently, DADLE and other nonspecific ␦-opioid agonists have been shown to protect against liver I/R injury both in situ and in vivo [5, 6]. The mechanism(s) by which ␦-opioid agonists protect against I/R injury is unknown. This may stem from an incomplete knowledge of the expression and function of specific ␦-opioid receptors. Activation of macrophages is a critical component of I/R injury. Macrophage activation during I/R results in the generation of reactive oxygen species (ROS) and proinflammatory cytokines [7]. ROS can directly damage tissues, and proinflammatory cytokines such as tumor necrosis factor (TNF) initiate a cytokine cascade that results in up-regulation of vascular cell adhesion molecules and leukocyte chemoattractants, called chemokines [1, 7]. The coordinated actions of adhesion molecules and chemokines result in neutrophil adhesion and transmigration into the postischemic tissue where they release oxidants and proteases that damage organ parenchymal cells [8]. Given the benefit of ␦-opioid receptor agonists in multiple models of I/R, we sought to determine whether signaling through ␦-opioid receptors (␦ 1 or ␦ 2) alters macrophage production of proinflammatory mediators. Lipo-

Background. ␦-Opioid agonists have been shown to attenuate ischemic organ injury in multiple models. The purpose of the present study was to determine if ␦-opioid agonists could inhibit proinflammatory cytokine production by macrophages. Material and methods. Murine macrophages (RAW 264.7) were pretreated for 4 h with media, a dose range (10 ⴚ4 to 10 ⴚ7 M) of DADLE ([D-Ala 2], D-Leu 5]-enkephalin, a nonspecific ␦-opioid receptor agonist), a dose range (10 ⴚ4 to 10 ⴚ7 M) of DPDPE ([D 2,5Pen]-enkephalin, a specific ␦ 1-opioid receptor agonist), or a dose range (10 ⴚ4 to 10 ⴚ7 M) of Deltorphin-D variant (a specific ␦ 2 opioid receptor agonist) and then incubated with 0.1 ␮g/ml lipopolysaccharide (LPS) for 1 or 4 h. Cytokine levels were measured by enzyme-linked immunosorbent assay. Activation of NF-␬B, AP-1, and p38 MAPK were determined by mobility shift assays and Western blot. Results. LPS induced significant increases in TNF␣ and MIP-2 production. Deltorphin-D variant, but not DADLE or DPDPE, dose-dependently reduced both TNF␣ and MIP-2 production. Deltorphin-D variant did not alter activation of the transcription factors NF-␬B or AP-1, but greatly reduced activation of p38 MAPK. Conclusions. The data show that ␦ 2- but not ␦ 1-opioid agonists suppress LPS-induced p38 MAPK activation and expression of TNF␣ and MIP-2. © 2005 Elsevier Inc. All rights reserved.

Key Words: inflammation; cytokines; NF-␬B; AP-1; p38 MAPK. INTRODUCTION

Ischemia reperfusion (I/R) injury is a primary complication of organ resection (such as liver) or transplan1 To whom correspondence and reprint requests should be addressed at University of Cincinnati, Department of Surgery, 231 Albert Sabin Way, Cincinnati, OH 45267-0558. E-mail: [email protected].

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0022-4804/05 $30.00 © 2005 Elsevier Inc. All rights reserved.

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polysaccharide (LPS) is a component of the cell wall of Gram-negative bacteria and is a potent stimulator of macrophage activation in a manner similar to that seen during I/R injury [9, 10]. Using LPS as a stimulus for macrophage activation, we examined the ability of select ␦-opioid receptor agonists to modulate proinflammatory cytokine production. MATERIALS AND METHODS Materials. RAW 264.7 cell lines were purchased from the American Type Culture Collection (Manassas, VA). ELISA reagents for murine TNF␣ and macrophage inflammatory protein 2 (MIP-2) were obtained from Peprotech (Rocky Hill, NJ). [D-Ala2, D-Leu5]-enkephalin (DADLE) and [D 2,5Pen]-enkephalin (DPDPE) were obtained from Sigma (St. Louis, MO). Lipopolysaccharide (from Escherichia coli 055:B5) was obtained from Sigma. The [␥- 32P]ATP was obtained from Amersham Biosciences (Arlington Heights, IL). Deltorphin D variant (var), a linear 17-amino-acid peptide with the following sequence: H-Tyr-D-AlaPhe-Ala-Asp-Val-Ala-Ser-Thr-Ile-Gly-Asp-Phe-Phe-His-Ser-Ile-NH2 [11], was custom synthesized by Bachem Biosciences Inc. (King of Prussia, PA). Macrophage cells and culture. The murine macrophage cell line RAW 264.7 was cultured in 5% CO 2/95% humidity at 37°C in Dulbecco’s modified Eagle’s medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose (90%), fetal bovine serum (10%), penicillin (100 U/ml), and streptomycin (100 ␮g/ml). For studies of chemokine production, 1 ⫻ 10 6 cells/well were seeded in 0.5 ml of medium in 24-well tissue-culture plates. Cells were allowed to grow to confluence (⬃24 h), and at that time the media were changed. Cells were then incubated with ␦-opioid agonists at varying concentrations for 4 h. The media were replaced with fresh media containing the same concentration of ␦-opioid agonist in addition to 0.1 ␮g/ml of LPS and allowed to incubate for another 4 h. Culture media was analyzed by ELISA for TNF␣ and MIP-2. For studies of signal transduction events, 1 ⫻ 10 6 cells/well were seeded in 0.5 ml of media in 24-well tissue-culture plates. Cells were allowed to grow to confluence (⬃24 h hours), and at that time the media were changed. Cells were then incubated with ␦-opioid agonists at the indicated concentrations for 4 h. The media were replaced with fresh media containing the same concentration of ␦-opioid agonist in addition to 0.1 ␮g/ml of LPS and allowed to incubate for 1 h. Cells were then harvested for cytoplasmic and nuclear protein isolation. Nuclear protein extraction and electrophoretic mobility shift assays (EMSA). Cytoplasmic and nuclear extracts were prepared using the methods described by Dignam et al. [12]. Protein concentrations were determined by bicinchoninic acid assay with TCA precipitation using BSA as a reference standard (Pierce). For EMSA, doublestranded consensus oligonucleotide to NF-␬B or AP-1 (Promega, Madison, WI) was end-labeled with [␥- 32P]ATP (3000 Ci/mmol at 10 mCi/ml). Binding reactions containing equal amounts of nuclear protein (30 ␮g) and 35 fmol (⬃50,000 cpm, Cherenkov counting) of oligonucleotide were incubated at room temperature for 30 min in binding buffer that contained 4% glycerol, 1 mM; MgCL 2, 0.5 mM EDTA, pH 8.0, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris-HCl (pH 7.6), and 50 ␮g/ml poly(dI/dC). For supershift analyses, antibodies to p50, p52 (NF-␬B2), p65, p68 (RelB), or p75 (c-Rel) (all antibodies were from Santa Cruz Biotechnology, Santa Cruz, CA) were added 15 min after the addition of radiolabeled NF-␬B oligonucleotide. Binding reaction products were separated in a 4% polyacrylamide gel and analyzed by autoradiography. Western blot. Cytoplasmic extracts and their protein concentrations were obtained as described above. Equal amounts of cytoplasmic protein (100 ␮g) were electrophoresed in a denaturing 10% polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. Nonspecific binding sites were blocked with TBS (40 mM

FIG. 1. Effects of ␦-opioid agonists on LPS-induced production of (A) TNF␣ and (B) MIP-2 in murine macrophages. Cells were treated with DADLE, DPDPE, or Deltorphin-D var for 4 h prior to the addition of LPS (0.1 ␮g/ml). Supernatants were harvested 4 h after the addition of LPS and analyzed by ELISA. Data represent mean ⫾ SEM with n ⫽ 4/group. *P ⬍ 0.05 compared to LPS group. Tris, pH 7.6, 300 mM NaCl) containing 5% nonfat dry milk for 1 h at room temperature. Membranes were then incubated in a 1:1000 dilution of rabbit polyclonal anti-mouse p38MAP kinase or rabbit polyclonal anti-mouse phospho-p38 MAP kinase (Cell Signaling, Beverly, MA) in TBS with 0.1% Tween 20 (TBST). After three washes in TBST, membranes were incubated in 1:2000 dilution of horseradish peroxidase conjugated anti-rabbit IgG (Cell Signaling). Immunoreactive proteins were detected by enhanced chemiluminescence. Statistical analysis. All data were expressed as mean ⫾ SEM. Data were analyzed with a one-way analysis of variance with subsequent Student–Newman–Keuls test. Differences were considered significant when P ⬍ 0.05.

RESULTS

␦-Opioid-Mediated Inhibition of Proinflammatory Cytokine Expression by Macrophages

To determine whether ␦-opioid receptor stimulation could modulate LPS-induced production of inflamma-

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FIG. 2. Effects of ␦-opioid agonists on LPS-induced NF-␬B activation in murine macrophages. (A) Cells were treated with 10 ⫺4 M DADLE, DPDPE, or Deltorphin-D var for 4 h prior to the addition of LPS (0.1 ␮g/ml). Nuclear extracts were obtained 1 h after the addition of LPS and analyzed by EMSA for NF-␬B DNA-binding capacity. (B) Nuclear extracts from LPS and LPS⫹DPDPE groups were used in supershift assays to determine NF-␬B subunit composition. This experiment was repeated three times.

tory cytokines in macrophages, we pretreated murine macrophages with DADLE, DPDPE, or Deltorphin-D var for 4 h prior to the addition of LPS. Supernatants were obtained 4 h after LPS and analyzed by ELISA for the cytokine, TNF␣, and the neutrophil-attracting chemokine, MIP-2. Stimulation with LPS, in the absence of any ␦-opioid receptor agonist, resulted in significant production of TNF␣ (Fig. 1A). Neither pretreatment with the nonspecific ␦-opioid receptor agonist, DADLE, nor the ␦ 1-specific agonist, DPDPE, had any effect on LPS-induced production of TNF␣. However, pretreatment with Deltorphin-D var significantly inhibited the production of TNF␣ at concentrations of 10 ⫺5 and 10 ⫺4 M (Fig. 1A). Stimulation of macrophages with LPS also resulted in marked production of MIP-2 (Fig. 1B). Pretreatment with DADLE had no significant effect on MIP-2 production. Treatment with DPDPE had a modest, but significant, effect on MIP-2 production at a concentration of 10 ⫺5 M (Fig. 1B). In contrast, pretreatment with Deltorphin-D var dose-dependently inhibited MIP-2 production with maximal inhibition at a concentration of 10 ⫺4 M (Fig. 1B).

Because activation of these signaling pathways occurs prior to protein expression, we examined the activation of AP-1, NF-␬B, and p38 MAPK 1 h after stimulation of macrophages with LPS. Treatment with LPS resulted

␦-Opioid Agonists Modulate Macrophage Signal Transduction Pathways Stimulated by LPS

To investigate the mechanism(s) by which ␦-opioid agonists alter TNF␣ and MIP-2 production in macrophages, we examined three signaling pathways known to be involved in the expression of these mediators. The transcription factors, AP-1 and NF-␬B, are known to coordinately regulate the expression of TNF␣ and MIP-2 in response to various inflammatory stimuli [13]. The p38 MAPK pathway also regulates the expression of TNF␣ and chemokines, such as MIP-2 [14].

FIG. 3. Effects of ␦-opioid agonists on LPS-induced activation p38 MAPK in murine macrophages. Cells were treated with 10 ⫺4 M DADLE, DPDPE, or Deltorphin-D var for 4 h prior to the addition of LPS (0.1 ␮g/ml). Cytoplasmic extracts were obtained 1 h after the addition of LPS and analyzed by Western blot for phospho-p38 and total p38. This experiment was repeated twice.

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in marked activation of AP-1 (data not shown). However, none of the ␦-opioid receptor agonists had any affect on AP-1 activation. Similarly, LPS treatment resulted in activation of NF-␬B (Fig. 2A). Pretreatment of macrophages with either DADLE or Deltorphin-D var had no effect on NF-␬B activation. Interestingly, treatment with DPDPE caused an alteration in migration of the NF-␬B band on the EMSA (Fig. 2A). To determine if this was due to a change in the composition of the NF-␬B dimer, we conducted supershift assays to identify NF-␬B components. These experiments identified the LPS-induced NF-␬B to comprise p50 and p65 subunits (Fig. 2B, left panel). Pretreatment with DPDPE resulted in a supershift of only p50, indicating that DPDPE altered the composition of the NF-␬B complex (Fig. 2B, right panel). These data may explain the modest reduction observed in MIP-2 production with DPDPE treatment (Fig. 1B). We next examined the activation of p38 MAPK. LPS treatment increased activation of p38 MAPK, as determined by increased phosphorylation of p38 (Fig. 3). Treatment with DADLE or DPDPE had no effect on LPS-induced activation of p38 (data not shown). However, treatment with Deltorphin-D var completely abrogated p38 activation (Fig. 3).

DISCUSSION

Our study is the first to demonstrate modulation of macrophage activation by ␦-opioid agonists. Our data show that stimulation of the ␦ 2-opioid receptor suppresses activation of p38 MAPK and reduces the production of proinflammatory mediators. We also show that a specific ␦ 1-opioid receptor agonist alters the dimer composition of NF-␬B, which was associated with modest inhibition of the production of MIP-2. These findings provide new clues to the potential mechanisms by which ␦-opioid agonists may protect against postischemic tissue injury. A major function of the MAPK p38 pathway is the regulation of proinflammatory gene expression, including TNF␣. Posttranscriptional effects of the p38 pathway are mediated by the MAPK-activated protein kinase-2 (MAPKAP-2) [15]. The importance of the p38 MAPK pathway in I/R injury has been demonstrated in models of both lung and liver tissues [16, 17]. Specific inhibitors of the p38 MAPK pathway (i.e., SB203580 which targets MAPKAP-2 and SB202190 which targets p38 MAPK) have been shown to prevent proinflammatory mediator expression and inflammation both in vitro and in vivo [16, 18, 19]. Our data provide evidence that ␦ 2-opioid agonists inhibit p38 MAPK activation in macrophages. This inhibition of p38 MAPK was associated with reduced macrophage production of TNF␣ and MIP-2. Thus, the published beneficial ef-

fects of ␦-opioid agonists in models of I/R injury [5, 6] may be related to reduced proinflammatory cytokine production. We also found that the ␦ 1-opioid receptor agonist, DPDPE, altered the composition of the transcription factor, NF-␬B, in activated macrophages. NF-␬B is a dimeric complex of proteins of the Rel family. Five members of this family have been identified (p50, p52, p65 [RelA], p68[RelB], and p75 [cRel]), which can form more than 12 dimers with various functions [20]. These functions are dependent upon which subunits compose the dimer; p50/p65, p50/p75, p65/p65, and p65/p75 heterodimers are transcriptionally active, while p50 and p52 homodimers are transcriptionally repressive [21]. Treatment of LPS-stimulated macrophages with DPDPE changed the subunit configuration of NF-␬B from a p50/p65 heterodimer to a p50/p50 homodimer. Because p50/p50 homodimers are not transcriptionally active, this alteration in NF-␬B complex structure may explain the attenuation of MIP-2 production observed with DPDPE treatment. Macrophage activation is a critical component to the inflammatory injury associated with I/R injury. Our findings provide the first evidence that ␦-opioid receptor agonists alter proinflammatory mediator production by macrophages. Based on the present data, it appears that agonism of ␦ 2-opioid receptors on macrophages results in robust inhibition of proinflammatory mediator production. Furthermore, our data show that ␦ 2-opioid receptor activation attenuates p38 MAPK activation. While a ␦ 1-opioid receptor agonist did modulate NF-␬B and slightly attenuate MIP-2 expression, this effect was very modest when compared to the effects of the ␦ 2 agonist. The present studies provide the first data to suggest that ␦-opioid agonists can suppress inflammatory activation of macrophages. Our results are consistent with other studies which have shown beneficial effects of ␦-opioid agonists in models of organ preservation and I/R injury [3– 6]. Continued research is warranted to determine the precise mechanisms by which ␦-opioid receptor stimulation interferes with inflammatory signal transduction pathways in macrophages. ACKNOWLEDGMENTS This work was supported by National Institutes of Health Grants DK56029 and HL72552 to A.B.L. and by the Office of Naval Research Grant N000-14-01-1-0494 to P.R.O.

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