The Journal of Neuroscience, July 1, 2002, 22(13):5588–5596 Opioid Control of Inflammatory Pain Regulated by Intercellular Adhesion Molecule-1 Halina Machelska, Shaaban A. Mousa, Alexander Brack, Julia K. Schopohl, Heike L. Rittner, Michael Scha¨ fer, and Christoph Stein Klinik fu¨ r Anaesthesiologie und operative Intensivmedizin, Klinikum Benjamin Franklin, Freie Universita¨ t Berlin, D-12200 Berlin, Germany Pain can be effectively controlled by endogenous mechanisms We found that ICAM-1 is upregulated on the vascular endothe- based on neuroimmune interactions. In inflamed tissue immune lium, simultaneously with an enhanced immigration of opioid- cell-derived opioid peptides activate opioid receptors on pe- containing immune cells into inflamed paw tissue. The intrave- ripheral sensory nerves leading to potent analgesia. This is nous administration of a monoclonal antibody against ICAM-1 brought about by a release of opioids from inflammatory cells markedly decreased the migration of opioid-containing leuko- after stimulation by stress or corticotropin-releasing hormone cytes and of granulocytes, monocytes–macrophages, and T (CRH). Immunocytes migrate from the circulation to inflamed cells to the inflamed tissue. At the same time, circulating im- tissue in multiple steps, including their rolling, adhesion, and munocytes increased in numbers, and macroscopic inflamma- transmigration through the vessel wall. This is orchestrated by tion (hyperalgesia, paw volume, and paw temperature) re- adhesion molecules on leukocytes and vascular endothelium. mained primarily unchanged. Most importantly, peripheral Intercellular adhesion molecule-1 [ICAM-1 (or CD54)] is ex- opioid analgesia elicited either by cold water swim stress or by pressed by endothelium and mediates adhesion and extrava- intraplantar administration of CRH was dramatically reduced. sation of leukocytes. The goal of this study was to show that Together, these findings indicate that ICAM-1 expressed on ICAM-1 regulates the homing of opioid-producing cells and the vascular endothelium recruits immunocytes containing opioids subsequent generation of analgesia within sites of painful in- to promote the local control of inflammatory pain. flammation. This was accomplished using immunofluores- Key words: adhesion molecules; intercellular adhesion mole- cence, flow cytometry, and behavioral (paw pressure) testing. cule-1; opioids; endogenous; stress; analgesia; pain; inflammation It is now recognized that pain and pain modulation is not solely The recruitment of leukocytes from the circulation to inflam- mediated by neurons but can involve neuroimmune interactions matory foci involves interactions with vascular endothelium, (Machelska and Stein, 2000; Watkins and Maier, 2000; DeLeo and which are orchestrated by adhesion molecules. Initially, selectins Yezierski, 2001). In inflammation, circulating immunocytes con- expressed on leukocytes and endothelial cells mediate leukocyte taining opioid peptides (predominantly ␤-endorphin) migrate to capture and rolling along the vessel wall. Subsequently, interac- the injured tissue, and, concurrently, opioid receptors are upregu- tions between integrins and Ig-like members arrest the rolling lated on peripheral endings of primary afferents (Cabot et al., cells and mediate firm adhesion, leading to their migration into 1997, 2001; Mousa et al., 2001; Rittner et al., 2001). During stress- sites of injury (Springer, 1990; Butcher and Picker, 1996; Petruz- ful stimulation (cold water swim or postoperative stress), these zelli et al., 1999). Intercellular adhesion molecule-1 [ICAM-1 (or cells locally secrete opioids to elicit potent and clinically relevant CD54)] is one of the major molecules required primarily for the opioid receptor-specific analgesia devoid of central side effects firm adhesion and diapedesis of leukocytes (Springer, 1990; (Stein et al., 1990a, 1993; Rittner et al., 2001). An important Butcher and Picker, 1996; Petruzzelli et al., 1999). In addition, it endogenous trigger for stress-induced analgesia is corticotropin- is the most extensively studied adhesion molecule in anti- releasing hormone (CRH) produced locally in inflammatory cells. inflammatory therapy. ICAM-1 is constitutively expressed by CRH activates its receptors on leukocytes, leading to release of endothelium, leukocytes, and synovial lining cells in animal mod- opioid peptides, which then activate peripheral opioid receptors to els and in patients with inflammatory diseases (Szekanecz et al., relieve pain (Scha¨fer et al., 1994, 1996; Cabot et al., 1997, 2001). 1994; Bennett et al., 1997; Salmi et al., 1997; Veihelmann et al., Immunosuppression abolishes stress- and CRH-induced analgesia, 1999). Blockade of ICAM-1 by monoclonal antibodies (mAbs), demonstrating the functional relevance of immunocytes (Stein et antisense oligonucleotides, or deletion of the ICAM-1 gene was al., 1990b; Przewlocki et al., 1992; Scha¨fer et al., 1994). shown to impair the migration of immunocytes to inflamed tissue and to decrease inflammation in animals and humans (Sligh et al., Received Sept. 24, 2001; revised Feb. 26, 2002; accepted March 26, 2002. 1993; Kavanaugh et al., 1994; Bullard et al., 1996; Bennett et al., This study was supported by the Deutsche Forschungsgemeinschaft (Sonderfor- 1997). More recently, we showed that adhesion mechanisms are schungsbereich 507/B8 and Klinische Forschergruppe 100/1) and by the Interna- tional Anesthesia Research Society. not exclusively involved in mounting an immune response to Correspondence should be addressed to Dr. Halina Machelska, Klinik fu¨r An- pathogens but also in pain control. Thus, interruption of rolling aesthesiologie und operative Intensivmedizin, Klinikum Benjamin Franklin, Freie by selectin blockade attenuated intrinsic opioid analgesia within Universita¨t Berlin, Hindenburgdamm 30, D-12200 Berlin, Germany. E-mail: [email protected]. injured tissue (Machelska et al., 1998). Copyright © 2002 Society for Neuroscience 0270-6474/02/225588-09$15.00/0 In this study, we sought to determine whether interfering with Machelska et al. • ICAM-1 and Opioid Pain Control J. Neurosci., July 1, 2002, 22(13):5588–5596 5589 later steps in the adhesion cascade, i.e., with ICAM-1-dependent The expression of ICAM-1 and ␤-endorphin was quantified by an observer blinded to the experimental protocol, using a Zeiss microscope adhesion and transendothelial migration of opioid-expressing leu- ϫ ϫ kocytes, will affect opioid-mediated pain inhibition. To this end, (objective, 20 ; eyepiece, 10 ). The mean number of ICAM-1- expressing blood vessels and ␤-endorphin-expressing cells in three sec- we investigated the cellular coexpression of ICAM-1 and tions per animal and five squares (384 mm 2 for ICAM-1; 384 ␮m 2 for ␤ -endorphin, and we evaluated the effects of an mAb against ␤-endorphin) per section was calculated. The percentage of ICAM-1- ICAM-1 (anti-ICAM-1) on the trafficking of opioid-containing stained vessels was determined according to the following formula: immune cells and on peripheral stress- and CRH-induced opioid number of ICAM-1-stained vessels/number of all vessels ϫ 100 (Mousa analgesia in a rat model of inflammatory pain. et al., 2000). Flow cytometry. To assess the contribution of ICAM-1 in trafficking of opioid-containing immune cells, rats (n ϭ 6 –12 per group) received intravenously anti-ICAM-1 or mouse IgG (both at 4 mg/kg in 0.6 ml of MATERIALS AND METHODS sterile water) under brief halothane anesthesia immediately before Animals. Experiments were performed on male Wistar rats (140–150 induction of inflammation. To evaluate the involvement of ICAM-1 in gm) (Freie Universita¨t, Berlin, Germany) in accordance with the Na- immune cell migration under normal conditions, rats received the tional Institutes of Health Guide for the Care and Use of Laboratory same intravenous treatment but without induction of inflammation. Six Animals and were approved by the local animal care committee. Rats hours after treatments, blood samples (1 ml) were obtained by direct were housed individually in cages and maintained on a 12 hr light/dark parasternal cardiac puncture under halothane anesthesia. Rats were schedule, with food pellets and water ad libitum. Room temperature was then killed, and subcutaneous tissue was dissected from inflamed paws. maintained at 22 Ϯ 0.5°C and a relative humidity between 60 and 65%. To obtain a single cell suspension, the tissue was cut into 1–2mm Antibodies. For immunofluorescence the following antibodies (Abs) pieces and digested (Rittner et al., 2001). Because no immune cells were used: polyclonal rabbit anti-rat ␤-endorphin (Peninsula Laborato- were found in noninflamed paws, these were not processed for flow ries, Belmont, CA), monoclonal mouse anti-rat ICAM-1 (clone number cytometry. 1A29; Seikagaku, Tokyo, Japan), and secondary Abs: goat anti-rabbit For surface staining, cells were incubated for 15 min at room temper- Texas Red-conjugated (anti-rabbit–Texas Red) and horse anti-mouse– ature with anti-CD3-PE (4 ␮g/ml) and anti-CD45-CyC (2 ␮g/ml), fluorescein isocyanate (FITC) (Vector Laboratories, Burlingame, CA). washed, and fixed with PBS containing 1% paraformaldehyde. Intracel- For flow cytometry, the following Abs were used: mouse IgG2a, rat lular staining was performed as described previously (Rittner et al., anti-mouse IgG2aϩb-phytoerythrin (PE), mouse anti-rat: CD3-PE (T cell 2001). Cells were permeabilized in PBS containing 0.5% saponin and marker), CD4-PE, CD11b-FITC (PharMingen/Becton Dickinson, Hei-