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PNAS 109 (16): 6325-6330

Copyright © 2012 by the National Academy of Sciences.

Morphine activates neuroinflammation in a manner parallel to endotoxin

Xiaohui Wanga, Lisa C. Loramb,c, Khara Ramosb,c, Armando J. de Jesusa, Jacob Thomasd, Kui Chenga, Anireddy Reddyb,c, Andrew A. Somogyid, Mark R. Hutchinsone, Linda R. Watkinsb,c, and Hang Yina,c,f,1

aDepartment of Chemistry and Biochemistry, bDepartment of Psychology and Neuroscience, cCenter for Neuroscience, and fBiofrontiers Institute, University of Colorado at Boulder, Boulder, CO 80309; and dDiscipline of Pharmacology and eDiscipline of Physiology, School of Medical Sciences, University of Adelaide, Adelaide, South Australia, 5005, Australia


Figure 01
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Fig. 1. Biophysical characterizations of morphine binding to human MD-2. (A) Different concentrations of biotin-morphine were coated onto streptavidin-precoated plate as the probe. Human MD-2 (10 μg/mL, protein A tagged) was added, and the morphine-bound MD-2 was detected by IgG-HRP conjugate. Absorbance with 40 μM biotin-morphine was set as 100%. (B) Biotin-morphine (10 μM) was coated onto streptavidin precoated plate. Different concentrations of human MD-2 were added, and the morphine-bound MD-2 was detected by IgG-HRP conjugate. Protein A was used as the negative control. Absorbance with 40 μg/mL MD-2 was set as 100%. (C), 10 μM of biotin-morphine was coated onto streptavidin-precoated plate. Human MD-2 (40 μg/mL) and different concentrations of LPS were added. The morphine-bound MD-2 was detected by IgG-HRP conjugate. Absorbance with 0 μg/mL LPS was set as 100%. (D) Human MD-2 (10 μg/mL) or BSA (used as the control) was coated onto the plate. Different concentrations of biotin-morphine were added, and the bound morphine was detected by streptavidin conjugated with HRP. Absorbance with MD-2 in the presence of 2 μM biotin-morphine was set as 100%. (E) Human MD-2 (10 μg/mL) was coated onto the plate. Biotin-morphine (4.0 μM) and different concentrations of LPS were added, and the bound morphine was detected by streptavidin conjugated with HPR. Absorbance with 0 μg/mL LPS was set as 100%. (F) MD-2 capturing antibody (2 μg/mL) was coated onto the plate. MD-2 (5 μg/mL; protein A tag was removed) and different concentrations of morphine or roxithromycin (used as the control) were added. Anti-MD-2 antibody 9B4 (0.1 μg/mL), which specifically recognizes apoMD-2, was added. MD-2 without morphine bound was detected by HRP-coupled secondary antibody. Absorbance with 0 μg/mL morphine or roxithromycin was set as 100%. *P < 0.05 vs. roxithromycin control group. (G) Biotin-LPS (0.1 μg/mL) was coated onto streptavidin-precoated plate as the probe. Human MD-2 (10 μg/mL) and different concentrations of morphine were added. The LPS-bound MD-2 was detected by IgG-HRP conjugate. Absorbance with 0 μg/mL morphine or roxithromycin was set as 100%. (H) Morphine replaced curcumin binding to MD-2. Different concentrations of morphine or M6G were titrated into MD-2 (0.5 μM). Curcumin (0.5 μM) was added as the extrinsic fluorescence probe. The excitation wavelength was set at 430 nm, and the fluorescence signal at 493 nm was plotted against the titrated morphine concentration. Fluorescence intensity of curcumin–MD-2 complex in the absence of morphine was set as 100%. Data fitting to a one-site competitive model gives a Ki of 4.3 ± 3.3 μM.

 

Figure 02
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Fig. 2. Morphine induces TLR4 oligomerization and activates TLR4 signaling. (A) Morphine bound to MD-2 in a cellular environment. HEK Blue hTLR4 cells, which overexpress the human TLR4 and MD-2, were treated with morphine (300 μM) for 12 h, and cell lysates were immunoprecipitated by morphine antibody and then detected by Western blotting by MD-2 antibody. GAPDH served as the cell lysates input control. (B) Morphine-induced TLR4 receptor oligomerization. Ba/F3 cells simultaneously overexpressing human TLR4-Flag, human TLR4-GFP, human CD14, and human MD-2 were stimulated with morphine (300 μM) for 72 h. Cells were then subjected to immunoprecipitation with anti-Flag antibody and immunoprobing with anti-GFP antibody (Upper) and anti-Flag antibody (Lower). (C and D) Structural comparison of apo- and ligand-bound MD-2. The ligands are shown in a ball-and-stick presentation. X-ray crystal structure of apoMD-2 (cyan; PDB ID 2E56) superimposed with (C) X-ray crystal structure of the LPS-bound MD-2 (pink; PDB ID 3FXI) or (D) morphine-bound MD-2 (yellow) derived from atomic molecular dynamics simulation.

 

Figure 03
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Fig. 3. Isolated primary CNS endothelial cells incubated with (+)-morphine induce proinflammatory mediator (IL-1β) and TLR (TLR4 and the coreceptor MD-2) mRNA up-regulation after 2-h, 4-h, and 24-h incubations. (A) (+)-Morphine (100 μM) increased IL-1β mRNA at 2 h with a gradual decline in IL-1β mRNA expression by 24 h. (B and C) Gradual increase in TLR4 and MD-2 mRNA expression over time with the 100 μM (+)-morphine compared with vehicle control. (+)-Morphine (100 μM) was coincubated for 24 h with 10 ng/mL LPS-RS, a competitive TLR-4 antagonist, 1 μM CLI-095, an intracellular TLR4 antagonist, and 1 μM BAY11-7082, an I{kappa}B-α inhibitor. LPS-RS significantly attenuated (+)-morphine-induced IL-1β (D) and TLR4 (E) mRNA. CLI-095 significantly attenuated TLR4 (E) and MD-2 (F) mRNA. BAY11-7082 significantly attenuated IL-1β (D), TLR4 (E), and MD-2 (F) mRNA. Experimental procedures are described in SI Appendix, SI Materials and Methods. n = 6 per group per time interval. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. One-way ANOVA compared with vehicle control, with Bonferroni post hoc comparisons.

 

Figure 04
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Fig. 4. TLR4/MD-2 antagonists abolish morphine-induced proinflammatory activation in vitro and in vivo. (A) A strategy of potentiating opioid analgesia by disrupting the nonneuronal TLR4/MD-2 complex. Upper: Opioids activate CNS immunocompetent cells by triggering a signal transduction cascade mediated by TLR4 (dimeric form in complex with MD-2). This results in the release of the cytokine intercellular mediator IL-1β and other proinflammatory factors, which suppresses the desired opioid-induced neuronal analgesic effect. Lower: In the presence of TLR4-signaling antagonists such as inhibitors of the critical TLR4/MD-2 interaction, CNS immunocompetent cells remain in the resting state. Opioids (red star) then show higher analgesic efficacy by binding solely to opioid receptors (orange hexagon) on neurons. (B) Competitive ELISA binding. Compound 1 competes against MD-2-I peptide binding to TLR4, and compound 2 competes against morphine binding to MD-2. Negative control compound 3, which shares similar structure with compound 1, showed no apparent binding to TLR4. (C) TLR4/MD-2 antagonists inhibited morphine-induced IL-1β overproduction in microglial BV-2 cells. BV-2 cells were incubated in the presence of morphine (200 μM), morphine (200 μM), and 10 μM of compound 1 or 2. Cell lysates from BV-2 cells were assayed for IL-1β protein by ELISA. (D and E), TLR4/MD-2 antagonists 1 (D) and 2 (E) potentiated the acute intrathecal morphine analgesia. After predrug (baseline) assessment of responsivity to radiant heat (Hargreaves test), rats received intrathecal morphine (1 μL, 15 mg/mL), 1/2 (1 μL, 30 mM), or the combination of morphine and 1/2 at same doses. Data are expressed as percent maximum potential effect (MPE). Data are means from six animals. (F and G) Selectivity of potentiation of morphine analgesia by TLR4/MD-2 antagonists 1 (F) and 2 (G) in wild-type vs. TLR4 knockout mice. It should be noted here that we chose a morphine dose that produced low analgesia in wild-type mice (has greater analgesia in TLR4 KO mice). Therefore, behavioral tests were not hampered by the ceiling cutoff. After predrug (baseline) assessment of responsivity to radiant heat (Hotplate test), mice received i.p. 1 (82 mg/kg) or 2 (153 mg/kg), followed 10 min later by i.p. morphine (2.5 mg/kg). n = 6 animals for morphine groups and n = 3 for small molecules alone. It should be note here that morphine used here is (-)-morphine.

 


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