- Email: [email protected]
Electroacupuncture Increases CB2 Receptor Expression on Keratinocytes and Infiltrating Inflammatory Cells in Inflamed Skin Tissues of Rats
The Journal of Pain, Vol 11, No 12 (December), 2010: pp 1250-1258 Available online at www.sciencedirect.com
Original Reports Electroacupuncture Increases CB2 Receptor Expression on Keratinocytes and Infiltrating Inflammatory Cells in Inflamed Skin Tissues of Rats Jing Zhang,* Lin Chen,* Tangfeng Su,* Fuyuan Cao,* Xianfang Meng,* Lei Pei,* Jing Shi,* Hui-Lin Pan,y and Man Li* * Department of Neurobiology, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, PR China. y Department of Anesthesiology and Pain Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas.
Abstract: Endogenous cannabinoids and peripheral cannabinoid CB2 receptors (CB2Rs) are involved in the antinociceptive effect of electroacupuncture (EA) on inflammatory pain. However, it remains unclear about how EA affects the expression and distribution patterns of peripheral CB2Rs in inflamed skin tissues. To study this, inflammatory pain was induced by local injection of complete Freund’s adjuvant into the hindpaw of rats. The mRNA and protein levels of CB2Rs were quantified by using RTPCR and Western blotting, respectively. The distribution of CB2Rs on keratinocytes and immune cells recruited to the inflamed skin tissues was determined by using double-immunofluorescence labeling. Induction of tissue inflammation significantly increased the mRNA and protein levels of CB2Rs in the skin tissue. Also, both 2 Hz and 100 Hz EA, applied to GB30 and GB34, significantly increased the mRNA and protein levels of CB2Rs in inflamed tissues compared to the sham EA group. CB2Rimmunoreactivities were mainly distributed in keratinocytes, macrophages, and T-lymphocytes in the epidermis and dermis of the inflamed skin tissue. Inflammation caused a significant increase in the number of CB2R-immunoreactive keratinocytes, macrophages, and T-lymphocytes. Furthermore, compared to the sham EA group, EA at 2 or 100 Hz significantly increased the number of keratinocytes, macrophages, and T-lymphocytes with CB2R-immunoreactivity in the inflamed skin tissue. Therefore, our findings suggest that EA is associated with upregulation of local CB2Rs in the inflamed skin tissue. EA primarily potentiates the expression of CB2Rs on keratinocytes and infiltrating inflammatory cells at the site of inflammation. Perspective: This study shows that electroacupuncture increases the CB2 receptor expression on keratinocytes and infiltrating inflammatory cells in inflammatory skin tissues. This finding provides new evidence showing the potential role of CB2 receptors in the analgesic effect of acupuncture on inflammatory pain. ª 2010 by the American Pain Society Key words: Electroacupuncture, analgesia, hyperalgesia, inflammatory pain, inflammatory cells, cannabinoid CB2 receptors. cupuncture is an ancient therapeutic technique and an important part of traditional Chinese medicine. Acupuncture analgesia is rapidly Received September 11, 2009; Revised December 9, 2009; Accepted February 16, 2010. gaining interest and recognition by the West and Supported by a grant from the National Natural Science Foundation of has been validated by several clinical studies.14,16 China No. 30600832 and funds from the Scientific Research Foundation Electroacupuncture (EA), a modified therapy method for the Returned Overseas Chinese Scholars of State Education Ministry No. 2008890. based on the theory of traditional manual acupuncture Address reprint requests to Dr Man Li, Department of Neurobiology, is effective in the treatment of pain.31 Our recent study Tongji Medical College of Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, HuBei, PR China. E-mail: suggests that the endocannabinoid system participates [email protected] in EA analgesia in a rat model of inflammatory pain. In 1526-5900/$36.00 this regard, EA increases the level of anandmide in inª 2010 by the American Pain Society flamed skin tissues.3 Also, the antinociceptive effect of doi:10.1016/j.jpain.2010.02.013
A
1250
Zhang et al
The Journal of Pain
1251
Figure 1. Effects of inflammation and electroacupuncture (EA) treatments on the mRNA level of CB2 receptors (CB2Rs) in the skin tissue of the rat hind paw. A, a representative gel image showing the CB2R mRNA level in the skin tissue from 5 groups of rats. b-actin was used as a loading control. M: marker; Lanes from left to right represent the naı¨ve control, complete Freund adjuvant (CFA) alone, CFA plus 2 Hz EA, CFA plus 100 Hz EA, and CFA plus sham EA group. B and C, summary graphs showing the ratio of CB2R/b-actin mRNA levels in the skin tissue of the naı¨ve control, CFA alone, CFA plus 2 Hz EA, CFA plus 100 Hz EA, and CFA plus sham EA groups. Data are expressed as mean 6 SEM (n = 6 rats in each group). *P < .05 compared with the naı¨ve control group; #P < .05 compared with the CFA plus sham EA group.
EA is attenuated by local treatment with cannabinoid CB2 receptor (CB2R) antagonists.3 Our finding is consistent with other studies showing that peripheral CB2Rs can modulate inflammatory pain.11,19
Figure 3. Effects of inflammation and electroacupuncture (EA)
Figure 2. Effects of inflammation and electroacupuncture (EA) treatments on the protein level of CB2 receptors (CB2Rs) in the skin tissue of the rat hind paw. A, Representative gel image showing the CB2R protein level in the skin tissue from 5 groups of rats. b-actin was used as a loading control. A strong immunopositive band at 45 kDa was detected in the skin samples of the naı¨ve control, complete Freund adjuvant (CFA) alone, CFA plus 2 Hz EA, CFA plus 100 Hz EA, and CFA plus sham EA group. B and C, summary graphs showing the ratio of CB2R/b-actin protein levels in the skin tissue of the naı¨ve control, CFA alone, CFA plus 2 Hz EA, CFA plus 100 Hz EA, and CFA plus sham EA groups. Data are expressed as mean 6 SEM (n = 6 rats in each group). * P < .05 compared with the naı¨ve control group; #P < .05 compared with the CFA plus sham EA group.
treatments on keratinocytes labeled with CB2 receptor (CB2R) immunoreactivity in the skin tissues. A, Representative confocal images showing pan cytokeratin positive-keratinocytes (green, left panels) and CB2R-immunoreactive cells (red, middle panels). The right panels are overlay images showing double-labeled CB2R and keratinocytes (yellow). Images include naı¨ve control (a through c), complete Freund adjuvant (CFA) alone (d through f), CFA plus 2 Hz EA (g through i), CFA plus 100 Hz EA (j through l), CFA plus sham EA (m through o). Panels p through r show highmagnification images from panels g through i. Scale bar, 50 mm. B and C, summary graphs show the percentage of doublelabeled keratinocytes with CB2R-immunoreactivity in the total of pan cytokeratin positive-cells in the skin tissue from the naı¨ve control, CFA alone, CFA plus 2 Hz EA, CFA plus 100 Hz EA, and CFA plus sham EA groups. Data are expressed as mean 6 SEM (n = 6 rats in each group). * P < .05 compared with the naı¨ve control group; #P < .05 compared with the CFA plus sham EA group.
1252
Electroacupuncture Increases CB2 Receptor Expression
The Journal of Pain
Effects of Inflammation and EA Treatments on the Number of Keratinocytes With CB2R Immunoreactivity in Inflamed Skin Tissues
Table 1.
NO .
NAI¨VE CONTROL
CFA ALONE
CFA 1 2 HZ
CFA 1 100 HZ
CFA 1 SHAM EA
No. of double-labeled cells No. of keratinocytes No. of cells with CB2Rs
5.17 6 0.48 38.33 6 1.20 36.83 6 1.51
40.17 6 1.01* 96.83 6 0.95* 86.67 6 1.02*
124.00 6 0.97*,y 132.67 6 0.67*,y 126.00 6 1.06*,y
121.33 6 1.23*,y 130.50 6 0.76*,y 123.17 6 0.83*,y
41.83 6 1.17* 99.00 6 0.97* 88.50 6 0.99*
Abbreviations: EA, electroacupuncture; CB2R, cannabinoid CB2 receptor; CFA, complete Freund adjuvant. NOTE. Data are presented as mean 6 SEM; n= 6 rats in each group. *P < .05 compared with the naı¨ve control group. yP < .05 compared with the sham EA group.
Cannabinoid receptors and their endogenous ligands constitute the endocannabinoid system. Cannabinoid CB1 receptors (CB1Rs) are localized primarily in the central nervous system and in the periphery.27 On the other hand, CB2Rs are mainly present on immune cells, including mast cells, macrophages, B-lymphocytes, T-lymphocytes, and natural killer cells.7,9 Activation of CB2Rs on immune cells can inhibit the release of nociceptive mediators such as nerve growth factor,21,36 interleukin1b, and tumor necrosis factor-a.28,35 Another cell type that might be involved in the antinociceptive actions of selective CB2R agonists is keratinocytes in the skin, because these cells express CB2Rs and contain endogenous opioid peptides.13 For example, activation of CB2Rs expressed on keratinocytes may result in the release of the endogenous opioid peptide b-EP, which then acts on primary afferent neurons to inhibit nociception.13 However, the CB2R-mediated antihyperalgesic effect may be independent of endogenous opioids.34 Although findings from recent studies clearly support an important role of CB2Rs in the modulation of inflammatory pain,6,26 it remains unclear about how EA affects the expression level and distribution pattern of peripheral CB2Rs in inflamed skin tissues. In the present study, we determined the effects of inflammation and EA on the mRNA and protein levels of CB2Rs in the inflamed skin tissue and the distribution of CB2Rs on keratinocytes and immune cells recruited to the inflammatory site in a rat model of inflammatory pain. Our study provides new information that EA increases CB2R expression on keratinocytes, macrophages, and T-lymphocytes in inflamed skin tissues. This mechanism of action may mediate the antinociceptive effect of EA on inflammatory pain.
Materials and Methods Animal Models Experiments were carried out on male adult SpragueDawley rats (180 to 200 g) purchased from Experimental Animal Center of Tongji Medical College of Huazhong University of Science and Technology. All procedures were approved by the Animal Care Committee at Huazhong University of Science and Technology and conformed to the ethical guidelines of the International Association for the Study of Pain.39 The rats were housed in cages individually with a 12-hour light/dark cycle and had free access to food and water.
Inflammation was induced by injecting 50 mL complete Freund adjuvant (CFA, Sigma, St. Louis, MO) subcutaneously into the dorsal surface of the left hind paw of rats using a 25-gauge hypodermic needle.3,38 The injections were carried out under light anesthesia by means of ether inhalation. The naı¨ve rats were used as the control group. It has been shown that the vehicle (mineral oil) injected into the dorsal surface of the hind paw does not cause tissue inflammation and hyperalgesia.4,25 We selected the dorsal surface of the hind paw as the CFA injection site in order to produce an inflammatory pain focus in Gallbladder Channel of Foot Shaoyang, where GB30 and GB34 are located, according to the meridian theory of traditional Chinese medicine and our previous study.3,37
Electroacupuncture Treatment In the EA treatment group, the rats received EA administration on the ipsilateral ‘‘Huantiao’’ (GB30) and ‘‘Yanglingquan’’ (GB34) once every other day, starting at the second day after CFA injection, for 7 days. EA was administered at 2 or 100 Hz (1 mA and 0.1 ms pulse width) for 30 min using a modified current-constant Han Acupoint Nerve Stimulator (LH202, Huawei Co Ltd, Beijing, China). GB30 and GB34 were chosen because EA at these sites reduces inflammatory pain in rats.3,38 Two acupuncture needles were inserted into 2 acupoints that correspond to GB30 and GB34 in humans. GB30 is located at the junction of the lateral one third and medial two thirds of the distance between the greater trochanter and the hiatus of the sacrum, and GB34 lies on the lateral aspect of the leg, in the depression anterior and inferior to the head of the fibula in rats.33 During EA treatment, the rat was placed under an inverted clear plastic chamber (approximately 4 cm4 cm11 cm) but was neither restrained nor given any anesthetics. The animals remained awake and still during treatment and showed no evident signs of distress. For the CFA plus sham EA control, acupuncture needles were inserted ipsilaterally into GB30 and GB34 without electrical stimulation or manual needle manipulation. For the following experiments, the skin tissues at the site of inflammation were removed on day 7 after CFA injection (ie, 1 day after the third EA treatment). Because our previous study showed that EA produced significant antinociceptive effects on day 7 after CFA, we harvested the skin tissue only at this time point to show the relationship between CB2R expression and EA analgesia.3
Zhang et al
The Journal of Pain
1253
RT-PCR Procedures The skin tissue was excised from rats immediately after the animals were anesthetized with an overdose of sodium pentobarbitone (120 mg /kg, i.p.) and decapitated. Total RNA was isolated from the skin tissue using RNArose reagent (Watsonbiot, Shanghai, China) and reverse transcribed into cDNA using ReverTra Ace-a-TM (Toyobo, Osaka, Japan). PCR was performed in a 25-mL reaction volume using 2Taq PCR Mastermix (Tiangen Biotech, Beijing, China). The reactions were carried out in an MJ Research thermocycler using individual programs as follows: CB2Rs (product size: 474 bp) with a forward primer (5’-TTTCATTGCCATCCTCTTT-3’) and a reverse primer (5’-CCCAACTCCTGCTTATCC-3’); programmed at 95 C for 4 minutes; 94 C for 30 seconds; 48 C for 45 seconds; and 72 C for 1 minute. A total of 40 cycles were run, and each program was ended with 10 minutes at 72 C. The final PCR products were analyzed by agarose gel electrophoresis. The housekeeping gene b-actin (product size: 206 bp) was used as a control with a forward primer (5’-CCTTCCTGGGCATGGAGTCCTG -3’) and a reverse primer (5’-GGAGCAATGATCTTGATCTTC-3’). The optical density of each band was then measured with a computer-assisted imaging analysis system and normalized by using the band density of b-actin. All the primers were synthesized by Invitrogen Biotech (Shanghai, China).
Western Blotting
Figure 4. Effects of inflammation and electroacupuncture (EA) treatments on ED1-positive macrophages labeled with CB2 receptor (CB2R) immunoreactivity in the skin tissues. A, Representative confocal images showing ED1 positive macrophages (green, left panels) and CB2R-immunoreactive cells (red, middle panels). The right panels are overlay images showing doublelabeled CB2R and macrophages (yellow). Images include naı¨ve control (a through c), complete Freund adjuvant (CFA) alone (d through f), CFA plus 2 Hz EA (g through i), CFA plus 100 Hz EA (j through l), CFA plus sham EA (m through o). Panels p through r show enlarged images from panels g through i. Scale bar, 50 mm. B and C, Summary graphs show the percentage of double-labeled macrophages with CB2R immunoreactivity in the total of ED1-positive cells in the skin tissue from the naı¨ve control, CFA alone, CFA plus 2 Hz EA, CFA plus 100 Hz EA, and CFA plus sham EA group. Data are expressed as mean 6 SEM (n = 6 rats in each group). * P < .05 compared with the naı¨ve control group; #P < .05 compared with the CFA plus sham EA group.
The skin tissues (10 mm8 mm2 mm) were removed as described above, minced with scissors, and homogenized in 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 0.1% Nonidet P40, 0.1% cholic acid, 2 mg/mL leupeptin, 1 mg/mL pepstatin, 2 mM phenylmethylsulfonyl fluoride, and 2 mM EDTA, and centrifuged at 15 000g for 10 minutes. The pellet was discarded and protein concentrations from the supernatant were determined using the method of Lowry et al.18 Sixty micrograms of proteins of each tissue was separated on a 12% SDSpolyacrylamide gel and transferred onto PVDF membrane, blocked for 1.5 hours in 5% nonfat dry milk in Tris-buffered saline (TBS) containing 0.1% Tween-20. The membrane was incubated with the rabbit antiCB2R antibody (1:500; Cayman, MI) for 1 hour. After 3 washes in 0.1% TBS-Tween 20, the membranes were then incubated with a peroxidase-conjugated goat anti-rabbit secondary antibody (1:8000; Jackson ImmunoResearch, Baltimore, MD) for 1 hour, and washed 3 times. The enhanced chemiluminescence method (ECL Plus Western blotting detection reagents; Amersham Biosciences, Little Chalfont, UK) was used to reveal the protein bands according to the manufacturer’s protocol. The optical density of each band was then measured with a computer-assisted imaging analysis system (Qauntity One, Bio-Rad, Hemel Hempstead, UK) and normalized with the housekeeping gene b-actin.
Double-Immunofluorescence Labeling Procedures Rats were deeply anesthetized with an overdose of sodium pentobarbitone (120 mg/kg, i.p.) and were
1254
Electroacupuncture Increases CB2 Receptor Expression
The Journal of Pain
Effects of Inflammation and EA Treatments on the Number of Macrophages With CB2R Immunoreactivity in Inflamed Skin Tissues
Table 2.
NO .
NAI¨VE CONTROL
CFA
CFA 1 2HZ
CFA 1 100HZ
CFA 1 SHAM EA
No. of double-labeled cells No. of macrophages No. of cells with CB2Rs
7.83 6 0.61 17.67 6 0.56 52.50 6 1.98
33.50 6 0.76* 43.50 6 0.99* 99.50 6 2.40*
94.33 6 1.05*,y 103.83 6 0.70*,y 156.67 6 0.71*,y
92.00 6 0.86*,y 101.83 6 1.01*,y 152.67 6 0.80*,y
31.67 6 1.12* 43.17 6 0.95* 97.17 6 1.25*
Abbreviations: EA, electroacupuncture; CB2R, cannabinoid CB2 receptor; CFA, complete Freund adjuvant. NOTE. Data are presented as mean 6 SEM; n = 6 rats in each group. *P < .05 compared with the naı¨ve control group. yP < .05 compared with the sham EA group.
transcardially perfused with normal saline and 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. The skin tissue with the injection point in the middle was harvested. Tissues were postfixed at 4 C in the perfusion fixative for 8 hours, cryoprotected in 20% sucrose overnight, and sectioned at 20 mm on a cryostat in a plane perpendicular to the skin surface and parallel to the long axis of the foot. The sections were mounted onto gelatin-coated slides, air dried overnight, and used for double-immunofluorescence labeling of CB2Rs with markers of keratinocytes, macrophages, or T-lymphocytes. Double immunolabeling was performed with rabbit anti-CB2R (1:200; Cayman, MI) and either mouse monoclonal anti-pan cytokeratin antibody (1:100; Abcam, Cambridge, UK) for identification of keratinocytes, mouse monoclonal anti-CD68 antibody (Clone ED1, 1:200; Serotec, Oxford, UK) for identification of macrophages, or mouse monoclonal anti-ab T cell receptor (TCR) antibody (Clone R73, 1:200; BD BiosciencesPharMingen, San Diego, CA) for identification of T-lymphocytes. The specificity of the CB2R antibody has been demonstrated by antigen preabsorption with the corresponding blocking peptides.2 The anti-pan cytokeratin antibody reacts specifically with the skin epidermal keratinocytes.17 The anti-CD68 antibody specifically labels macrophages in the tissues.5 The anti-TCR antibody is a specific marker for peripheral T-lymphocytes and does not react with gd TCR-bearing T cells.12 All sections were blocked for 30 minutes with 5% donkey serum and 0.2% Tween-20 in PBS, followed by incubation at 37 C for 1 hour then at 4 C overnight with the primary antibody diluted in PBS containing 5% bovine serum albumin (BSA). The sections were washed 4 times with 0.05% Tween-20 in PBS for 5 minutes, and incubated with a mixture of secondary antibodies: donkey anti-mouse IgG conjugated with Cy2 (1:100; Jackson ImmunoResearch) and donkey anti-rabbit IgG conjugated with Cy3 (1:400; Jackson ImmunoResearch). Sections were washed 4 times with 0.05% Tween-20 in PBS for 5 min, and then treated with the fluorescence-mounting medium to inhibit quenching of fluorescence before being coverslipped. Negative controls were included by omitting the primary antibodies and with primary antibodies preabsorbed with their specific blocking peptides in the above procedures, which resulted in no positive labeling in the skin tissues. Six rats per group were used for the double-immunolabeling and cell counting.
Imaging Acquisition and Analysis Digital confocal images were acquired using a laser scanning confocal microscope (LSM 510, Carl Zeiss, Jena, Germany). The sections were scanned using excitation at 488 nm (argon laser) for Cy2 and at 543 nm (helium neon laser) for Cy3. A total of 3-4 sections were imaged from the same skin tissue in each rat, and counting of single- and double-labeled cells was done on confocal images randomly taken from 3 view fields from each section. Cell counting was performed by an investigator in a blind fashion using NIH Image J software (Bethesda, MD).
Statistical Analysis Data are presented as mean 6 SEM and analyzed using either Student t test or 1-way analysis of variance followed by Dunnett post hoc multiple comparison test. A P value of <.05 was considered statistically significant.
Results EA Increased the mRNA Level of CB2Rs in Inflamed Skin Tissues A total of 5 groups of rats were used: naı¨ve control, CFA alone, CFA plus sham EA, CFA plus 2 Hz EA, and CFA plus 100 Hz EA. The CB2R mRNA was detected in the skin tissue of all the groups. The optical density of CB2R mRNA in the CFA group was significantly greater than that in the naı¨ve control group (P < .05, Fig 1, A and B). Furthermore, there was a significant increase in the optical density of the CB2R mRNA in the CFA plus 2 Hz and CFA plus 100 Hz EA groups than that in the CFA plus sham EA group (P < 0.05, Fig 1, A,C).
EA Increased the CB2R Protein Level in Inflamed Skin Tissues The specific CB2R protein band at 45 kDa was present in the skin tissue samples of all the groups. The optical density of the CB2R protein in the CFA group was significantly higher than that in the naı¨ve control group (P < .05, Fig 2, A and B). Also, the optical density of the CB2R protein band in the CFA plus 2 Hz and CFA plus 100 Hz EA groups was significantly increased compared with that in the CFA plus sham EA group (P < .05, Fig 2, A and C).
Zhang et al
The Journal of Pain
1255
EA Increased the Expression of CB2Rs on Keratinocytes in Inflamed Skin Tissues The epidermal layer of the skin tissues from 4 groups of rats receiving CFA injections became much thicker than that in the naı¨ve control rats (Fig 3A). The CB2R immunoreactivity was distributed in keratinocytes in the uppermost layer of the epidermis, including the stratum granulosum and the stratum spinasum. Intense CB2Rimmunoreactivity was present in keratinocytes throughout the epidermis in inflamed skin tissues of both CFA plus 2 Hz and CFA plus 100 Hz EA groups (Fig 3A). The number of pan cytokeratin-positive cells and CB2R-positive cells and the percentage of double labeled CB2R-immunoreactive cells in the total of pan cytokeratin-positive cells were significantly increased in the skin tissues in CFA-treated rats than in the naı¨ve rats (P < .05; Table 1 and Fig 3B). Furthermore, the number of pan cytokeratin-positive cells and CB2R-positive cells and the percentage of double labeled CB2Rimmunoreactive cells in the total of pan cytokeratinpositive cells in the inflamed skin were significantly increased in the CFA plus 2 Hz and CFA plus 100 Hz EA groups, compared with those in the CFA plus sham EA group (P < .05;Table 1 and Fig 3C).
EA Increased the Expression of CB2Rs on Macrophages in Inflamed Skin Tissues
Figure 5. Effects of inflammation and electroacupuncture (EA) treatments on T cell receptor (TCR) positive T-lymphocytes labeled with CB2 receptor (CB2R) immunoreactivity in the skin tissues. A, Representative confocal images showing TCR-positive T-lymphocytes (green, left panels) and CB2R-immunoreactive cells (red, middle panels). The right panels are overlay images showing double-labeled CB2R and T lymphocytes (yellow). Images include naı¨ve control (a through c), complete Freund adjuvant (CFA) alone (d through f), CFA plus 2 Hz EA (g through i), CFA plus 100 Hz EA (j through l), CFA plus sham EA (m through o). Panels p through r show enlarged images from panels g through i. Scale bar, 50 mm. B and C, Summary graphs show the percentage of double-labeled T-lymphocytes with CB2R immunoreactivity in the total of TCR-positive cells in the skin tissue from the naı¨ve control, CFA alone, CFA plus 2 Hz EA, CFA plus 100 Hz EA, and CFA plus sham EA group. Data are expressed as mean 6 SEM (n = 6 rats in each group). *P < .05 compared with the naı¨ve control group; #P < .05 compared with the CFA plus sham EA group.
In the naı¨ve control group, only a few ED1-positive macrophages were found in the dermis of the skin, and the sizes of these cells were much smaller than those in the other groups and were rarely labeled with CB2Rs. In the CFA group, CFA plus 2 Hz, CFA plus 100 Hz, and CFA plus sham EA groups, there were a large number of infiltrating macrophages in the inflamed skin. These cells were generally large in sizes and multivacuolated, and many of them were labeled with CB2R immunoreactivity (Fig 4A). The number of ED1-positive cells and CB2R-positive cells and the percentage of double labeled CB2Rimmunoreactive cells in the total of ED1-positive cells were significantly increased in the inflamed skin tissues than those in the control skin tissue (P < .05; Table 2 and Fig 4B). Furthermore, the number of ED1-positive cells and CB2R-positive cells and the percentage of double labeled CB2R-immunoreactive cells in the total of ED1-positive cells in the inflamed skin were significantly increased in the CFA plus 2 Hz and CFA plus 100 Hz EA groups, compared with those in the CFA plus sham EA group (P < .05; Table 2 and Fig 4C).
EA Increased the Expression of CB2Rs on T-Lymphocytes in Inflamed Skin Tissues TCR-positive cells were present in the dermis of the skin section in all CFA-treated groups and had the size and oval shape consisting with the typical characteristics of T-lymphocytes. However, these cells were rarely present in the rat skin tissue in the naı¨ve control group, and only few of them were labeled with CB2R immunoreactivity. In the skin tissues from the CFA alone, CFA plus
1256
Electroacupuncture Increases CB2 Receptor Expression
The Journal of Pain
Effects of Inflammation and EA Treatments on the Number of T-Lymphocytes With CB2R Immunoreactivity in Inflamed Skin Tissues
Table 3.
NO .
NAI¨VE CONTROL
CFA
CFA 1 2HZ
CFA 1 100HZ
CFA 1 SHAM EA
No. of double-labeled cells No. of T-lymphocytes No. of cells with CB2Rs
10.67 6 0.99 17.50 6 0.56 56.33 6 1.45
20.83 6 1.40* 30.50 6 0.76* 98.00 6 1.06*
39.17 6 0.91*,y 46.83 6 0.79*,y 165.50 6 0.76*,y
37.33 6 0.71*,y 45.33 6 0.71*,y 164.83 6 1.01*,y
18.33 6 0.76* 27.00 6 0.86* 99.17 6 1.64*
Abbreviations: EA, electroacupuncture; CB2R, cannabinoid CB2 receptor; CFA, complete Freund adjuvant. NOTE. Data are presented as mean 6 SEM; n = 6 rats in each group. *P < .05 compared with the naı¨ve control group. yP < .05 compared with the sham EA group.
2 Hz, CFA plus 100 Hz, and CFA plus sham EA groups, there was a marked increase in infiltrating T-lymphocytes and some of them were labeled with CB2R immunoreactivity (Fig 5A). The number of TCR-positive cells and CB2R-positive cells and the percentage of double labeled CB2Rimmunoreactive cells in the total of TCR-positive cells were significantly increased in the inflamed skin tissues than in the control skin tissue (P < .05; Table 3 and Fig 5B). Furthermore, the number of TCR-positive cells and CB2R-positive cells and the percentage of double labeled CB2R-immunoreactive cells in the total of TCR-positive cells in the inflamed skin were significantly increased in the CFA plus 2 Hz and CFA plus 100 Hz EA groups, compared with those in the CFA plus sham EA group (P < .05; Table 3 and Fig 5C).
Discussion Our study provides new information that EA treatment upregulates CB2R expression in the inflamed skin tissue. We found that EA, at 2 and 100 Hz, significantly increased the mRNA and protein levels of CB2Rs at the time when EA produces significant antinociceptive effects on inflammatory pain. Furthermore, the number of keratinocytes, macrophages, and T-lymphocytes with CB2R-immunoreactivity in the inflamed skin tissue was significantly increased by EA treatment. Thus, our findings provide new information that EA may reduce inflammatory pain by increasing the expression and synthesis of CB2Rs in the inflamed tissues. The endocannabinoid system consists of 2 known cannabinoid receptor subtypes, CB1Rs and CB2Rs,20 a number of endogenous ligands (including anandamide and 2-arachidonoyl glycerol (2-AG),22 a high-affinity reuptake transport system, and endocannabinoid synthesizing and metabolizing enzymes.1 The CB2R mRNA has been detected in inflamed tissues, and its level is 10 to 100 times higher than that of CB1R mRNA.9 CB2R mRNA is present mainly in immune cells with particularly high levels in the mast cells, polymorphonuclear neutrophils, monocytes, B cells, natural killer cells, T4 cells, and T8 cells.9 We have shown recently that both CFA plus 2 Hz and CFA plus 100 Hz EA significantly reduced allodynia and hyperalgesia and increases the endogenous anandmide level in inflamed skin tissues.3 Also, the antinociceptive actions of EA are mediated by CB2Rs at peripheral sites, because injection of the CB2R antagonist
AM630 into the inflamed paw blocks the antinociceptive actions of EA. 3 Thus, both CB2R upregulation and elevated anandmide levels in the skin tissues may be involved in EA-produced analgesic effects on inflammatory pain. The presence of CB2R mRNA and protein has been reported in the paw tissues of rats and mice.32 We found that the mRNA and protein levels of CB2Rs are markedly increased in the skin tissue after induction of inflammation with CFA. Also, EA treatment significantly increased the mRNA and protein levels of CB2Rs in inflamed skin tissue, which provides further evidence that the CB2Rs probably play an important role in the analgesic effects of EA on inflammatory pain. Another salient finding of our study is the identification of the cell types that express CB2Rs in the skin tissue after inflammation and EA treatment. The changes in the distribution of CB2Rs in different cell types caused by inflammation and EA have not been studied previously. Although an increased level of CB2R proteins in acute (detrusor) and chronic (detrusor and mucosa) bladder inflammation has been reported,23 little is know about the effect of inflammation on CB2R expression on immune cells in the inflamed skin tissues. In the present study, we found that CB2Rs were distributed on T-lymphocytes, macrophages, and keratinocytes in the epidermis and dermis of inflamed skin tissues. Our data are consistent with the previous studies showing that the CB2R is present on immune cells, including mast cells, macrophages, B-lymphocytes, T-lymphocytes, and keratinocytes.9,13,24 We found that CFA-induced inflammation significantly increased the number of keratinocytes, macrophages, and T-lymphocytes expressing CB2Rs. Increased CB2 expression in inflamed tissues can be explained by increased immune cells (eg, macrophages and T-lymphocytes) recruited to the inflammatory site and increased keratinocytes in inflamed skin tissues.30 Thus, increased expression of CB2Rs on these cells after inflammation may mediate the antinociceptive effects of CB2R agonists on inflammatory pain.10,34 Interestingly, both inflammation and EA treatments significantly increased CB2R expression in the skin tissues. We found that CFA plus 2 and 100 Hz EA produced a further increase in the number of keratinocytes, macrophages, and T-lymphocytes expressing CB2Rs in the inflamed skin compared with those in the CFA plus sham EA group. The mechanisms underlying the EA action on CB2Rs expression in inflamed skin tissues are not clear.
Zhang et al
The Journal of Pain
1257
Because the number of keratinocytes, macrophages, and T-lymphocytes was significantly increased after chronic EA treatment, increased CB2Rs expression could be due to the result of increased infiltration of macrophages and T-lymphocytes with CB2Rs to inflamed skin tissues. EA treatment can influence the expression level of CB2Rs in a number of biological processes, including the signal transduction and nociceptive signaling.15 Since the percentage of keratinocytes, macrophages, and T-lymphocytes with CB2R immunoreactivity was also significantly increased by EA, it appears that increased CB2 expression in the inflamed skin tissue by EA could be the result of increased synthesis of CB2Rs on resident keratinocytes and infiltrated macrophages and T-lymphocytes. Consistent with this notion, we found that CFA plus 2 Hz and CFA plus 100 Hz EA significantly increased the mRNA and protein levels of CB2Rs in inflamed skin tissues. Because this EA treatment protocol is sufficient to produce an increase in the level of endogenous anandmide in the skin tissue,3 increased CB2R expression can mediate and amplify the action of anandmide to produce the analgesic effects of EA. Thus, 2 Hz and 100 Hz may be the effective frequency of EA to upregulate the endocannabinoid system and the CB2R expression in the inflamed skin tissue. One type of cells that may mediate the analgesic actions of the CB2R agonists and EA treatment is keratinocytes, which are abundantly present in the skin and express CB2Rs and contain endogenous opioid peptides.13 The skin keratinocytes are where nociceptive stimuli are applied to test the antinociceptive effects of
the specific CB2R agonists and EA treatment. It has been reported that the CB2R-selective agonist AM1241 may result in the release of the endogenous opioid peptide b-EP, which then acts on primary afferent terminals to inhibit inflammatory pain.13 EA could suppress inflammatory pain by increasing opioid release, which, in turn, activates peripheral opioid receptors.29 However, others have shown that the antihyperalgesic effects of the CB2R-selective agonists are not dependent upon the release of endogenous opioids.34 Furthermore, stimulation of CB2Rs on macrophages, T-lymphocytes, and other inflammatory cells may produce analgesia by reducing the release of inflammatory mediators that sensitize the peripheral nociceptors, such as nerve growth factor,36 IL-1b, and TNF-a.28,35 In support of this possibility, it has been shown that EA produces antinociception by inhibiting the release and gene expression of proinflammatory cytokines or neurotrophins.8 Therefore, the mechanisms of EA analgesia may include activation of CB2Rs on macrophages, T-lymphocytes, and keratinocytes, and attenuation of the release of pronociceptive mediators from the infiltrating inflammatory cells.19 In summary, we found in present study that EA significantly increased the mRNA and protein levels of CB2Rs in inflamed skin tissues. Our data suggest that EA increases CB2R expression on keratinocytes and inflammatory cells present in the inflammatory skin tissue. This new information is important to our understanding of the mechanisms underlying the antinociceptive effects of EA on inflammatory pain.
References
7. Facci L, Dal Toso R, Romanello S, Buriani A, Skaper SD, Leon A: Mast cells express a peripheral cannabinoid receptor with differential sensitivity to anandamide and palmitoylethanolamide. Proc Natl Acad Sci U S A 92:3376-3380, 1995
1. Beltramo M, Stella N, Calignano A, Lin SY, Makriyannis A, Piomelli D: Functional role of high-affinity anandamide transport, as revealed by selective inhibition. Science 277: 1094-1097, 1997 2. Casanova ML, Blazquez C, Martinez-Palacio J, Villanueva C, Fernandez-Acenero MJ, Huffman JW, Jorcano JL, Guzman M: Inhibition of skin tumor growth and angiogenesis in vivo by activation of cannabinoid receptors. J Clin Invest 111:43-50, 2003 3. Chen L, Zhang J, Li F, Qiu Y, Wang L, Li YH, Shi J, Pan HL, Li M: Endogenous anandamide and cannabinoid receptor2 contribute to electroacupuncture analgesia in rats. J Pain 10:732-739, 2009 4. Cook CD, Moore KI: Effects of sex, hindpaw injection site and stimulus modality on nociceptive sensitivity in arthritic rats. Physiol Behav 87:552-562, 2006 5. Damoiseaux JG, Dopp EA, Calame W, Chao D, MacPherson GG, Dijkstra CD: Rat macrophage lysosomal membrane antigen recognized by monoclonal antibody ED1. Immunology 83:140-147, 1994 6. Elmes SJ, Jhaveri MD, Smart D, Kendall DA, Chapman V: Cannabinoid CB2 receptor activation inhibits mechanically evoked responses of wide dynamic range dorsal horn neurons in naive rats and in rat models of inflammatory and neuropathic pain. Eur J Neurosci 20:2311-2320, 2004
8. Fu X, Zhu ZH, Wang YQ, Wu GC: Regulation of proinflammatory cytokines gene expression by nociceptin/orphanin FQ in the spinal cord and the cultured astrocytes. Neuroscience 144:275-285, 2007 9. Galiegue S, Mary S, Marchand J, Dussossoy D, Carriere D, Carayon P, Bouaboula M, Shire D, Le Fur G, Casellas P: Expression of central and peripheral cannabinoid receptors in human immune tissues and leukocyte subpopulations. Eur J Biochem 232:54-61, 1995 10. Giblin GM, O’Shaughnessy CT, Naylor A, Mitchell WL, Eatherton AJ, Slingsby BP, Rawlings DA, Goldsmith P, Brown AJ, Haslam CP, Clayton NM, Wilson AW, Chessell IP, Wittington AR, Green R: Discovery of 2-[(2,4-dichlorophenyl)amino]-N-[(tetrahydro-2H-pyran-4-yl)methyl]-4-(trifluoromethyl)-5-pyrimidinecarboxamide, a selective CB2 receptor agonist for the treatment of inflammatory pain. J Med Chem 50:2597-2600, 2007 11. Hanus L, Breuer A, Tchilibon S, Shiloah S, Goldenberg D, Horowitz M, Pertwee RG, Ross RA, Mechoulam R, Fride E: HU-308: A specific agonist for CB(2), a peripheral cannabinoid receptor. Proc Natl Acad Sci U S A 96:14228-14233, 1999 12. Hunig T, Wallny HJ, Hartley JK, Lawetzky A, Tiefenthaler G: A monoclonal antibody to a constant determinant of the rat T cell antigen receptor that induces T cell
1258
The Journal of Pain
Electroacupuncture Increases CB2 Receptor Expression
activation: Differential reactivity with subsets of immature and mature T lymphocytes. J Exp Med 169:73-86. 1989
matory hyperalgesia by activation of peripheral CB2 cannabinoid receptors. Anesthesiology 99:955-960, 2003
13. Ibrahim MM, Porreca F, Lai J, Albrecht PJ, Rice FL, Khodorova A, Davar G, Makriyannis A, Vanderah TW, Mata HP, Malan TP Jr: CB2 cannabinoid receptor activation produces antinociception by stimulating peripheral release of endogenous opioids. Proc Natl Acad Sci U S A 102: 3093-3098, 2005
27. Rice AS, Farquhar-Smith WP, Nagy I: Endocannabinoids and pain: Spinal and peripheral analgesia in inflammation and neuropathy. Prostaglandins Leukot Essent Fatty Acids 66:243-256, 2002
14. Jubb RW, Tukmachi ES, Jones PW, Dempsey E, Waterhouse L, Brailsford S: A blinded randomised trial of acupuncture (manual and electroacupuncture) compared with a non-penetrating sham for the symptoms of osteoarthritis of the knee. Acupunct Med 26:69-78, 2008 15. Ko J, Na DS, Lee YH, Shin SY, Kim JH, Hwang BG, Min BI, Park DS: cDNA microarray analysis of the differential gene expression in the neuropathic pain and electroacupuncture treatment models. J Biochem Mol Biol 35:420-427, 2002 16. Kong J, Fufa DT, Gerber AJ, Rosman IS, Vangel MG, Gracely RH, Gollub RL: Psychophysical outcomes from a randomized pilot study of manual, electro, and sham acupuncture treatment on experimentally induced thermal pain. J Pain 6:55-64, 2005 17. Lane EB, Rugg EL, Navsaria H, Leigh IM, Heagerty AH, Ishida-Yamamoto A, Eady RA: A mutation in the conserved helix termination peptide of keratin 5 in hereditary skin blistering. Nature 356:244-246, 1992 18. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275, 1951 19. Malan TP Jr., Ibrahim MM, Vanderah TW, Makriyannis A, Porreca F: Inhibition of pain responses by activation of CB(2) cannabinoid receptors. Chem Phys Lipids 121:191-200, 2002 20. Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner TI: Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 346:561-564, 1990 21. McMahon SB, Bennett DL, Priestley JV, Shelton DL: The biological effects of endogenous nerve growth factor on adult sensory neurons revealed by a trkA-IgG fusion molecule. Nat Med 1:774-780, 1995 22. Mechoulam R, Fride E, Di Marzo V: Endocannabinoids. Eur J Pharmacol 359:1-18, 1998 23. Merriam FV, Wang ZY, Guerios SD, Bjorling DE: Cannabinoid receptor 2 is increased in acutely and chronically inflamed bladder of rats. Neurosci Lett 445:130-134, 2008 24. Munro S, Thomas KL, Abu-Shaar M: Molecular characterization of a peripheral receptor for cannabinoids. Nature 365:61-65, 1993 25. Oliveira PG, Brenol CV, Edelweiss MI, Brenol JC, Petronilho F, Roesler R, Dal-Pizzol F, Schwartsmann G, Xavier RM: Effects of an antagonist of the bombesin/ gastrin-releasing peptide receptor on complete Freund’s adjuvant-induced arthritis in rats. Peptides 29:1726-1731, 2008 26. Quartilho A, Mata HP, Ibrahim MM, Vanderah TW, Porreca F, Makriyannis A, Malan TP Jr.: Inhibition of inflam-
28. Safieh-Garabedian B, Poole S, Allchorne A, Winter J, Woolf CJ: Contribution of interleukin-1 beta to the inflammation-induced increase in nerve growth factor levels and inflammatory hyperalgesia. Br J Pharmacol 115: 1265-1275, 1995 29. Sekido R, Ishimaru K, Sakita M: Differences of electroacupuncture-induced analgesic effect in normal and inflammatory conditions in rats. Am J Chin Med 31: 955-965, 2003 30. Tewari-Singh N, Rana S, Gu M, Pal A, Orlicky DJ, White CW, Agarwal R: Inflammatory biomarkers of sulfur mustard analog 2-chloroethyl ethyl sulfide-induced skin injury in SKH-1 hairless mice. Toxicol Sci 108:194-206, 2009 31. Tsui P, Leung MC: Comparison of the effectiveness between manual acupuncture and electro-acupuncture on patients with tennis elbow. Acupunct Electrother Res 27: 107-117, 2002 32. Walczak JS, Pichette V, Leblond F, Desbiens K, Beaulieu P: Behavioral, pharmacological and molecular characterization of the saphenous nerve partial ligation: A new model of neuropathic pain. Neuroscience 132: 1093-1102, 2005 33. Wang L, Zhang Y, Dai J, Yang J, Gang S: Electroacupuncture (EA) modulates the expression of NMDA receptors in primary sensory neurons in relation to hyperalgesia in rats. Brain Res 1120:46-53, 2006 34. Whiteside GT, Gottshall SL, Boulet JM, Chaffer SM, Harrison JE, Pearson MS, Turchin PI, Mark L, Garrison AE, Valenzano KJ: A role for cannabinoid receptors, but not endogenous opioids, in the antinociceptive activity of the CB2selective agonist, GW405833. Eur J Pharmacol 528:65-72, 2005 35. Woolf CJ, Allchorne A, Safieh-Garabedian B, Poole S: Cytokines, nerve growth factor and inflammatory hyperalgesia: The contribution of tumour necrosis factor alpha. Br J Pharmacol 121:417-424, 1997 36. Woolf CJ, Safieh-Garabedian B, Ma QP, Crilly P, Winter J: Nerve growth factor contributes to the generation of inflammatory sensory hypersensitivity. Neuroscience 62: 327-331, 1994 37. Yung KT: Birdcage model for the Chinese meridian system, VI: Meridians as the primary regulatory system. Am J Chin Med 33:759-766, 2005 38. Zhang RX, Lao L, Wang L, Liu B, Wang X, Ren K, Berman BM: Involvement of opioid receptors in electroacupuncture-produced anti-hyperalgesia in rats with peripheral inflammation. Brain Res 1020:12-17, 2004 39. Zimmermann M: Ethical guidelines for investigations of experimental pain in conscious animals. Pain 16:109-110, 1983
Original Reports Electroacupuncture Increases CB2 Receptor Expression on Keratinocytes and Infiltrating Inflammatory Cells in Inflamed Skin Tissues of Rats Jing Zhang,* Lin Chen,* Tangfeng Su,* Fuyuan Cao,* Xianfang Meng,* Lei Pei,* Jing Shi,* Hui-Lin Pan,y and Man Li* * Department of Neurobiology, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, PR China. y Department of Anesthesiology and Pain Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas.
Abstract: Endogenous cannabinoids and peripheral cannabinoid CB2 receptors (CB2Rs) are involved in the antinociceptive effect of electroacupuncture (EA) on inflammatory pain. However, it remains unclear about how EA affects the expression and distribution patterns of peripheral CB2Rs in inflamed skin tissues. To study this, inflammatory pain was induced by local injection of complete Freund’s adjuvant into the hindpaw of rats. The mRNA and protein levels of CB2Rs were quantified by using RTPCR and Western blotting, respectively. The distribution of CB2Rs on keratinocytes and immune cells recruited to the inflamed skin tissues was determined by using double-immunofluorescence labeling. Induction of tissue inflammation significantly increased the mRNA and protein levels of CB2Rs in the skin tissue. Also, both 2 Hz and 100 Hz EA, applied to GB30 and GB34, significantly increased the mRNA and protein levels of CB2Rs in inflamed tissues compared to the sham EA group. CB2Rimmunoreactivities were mainly distributed in keratinocytes, macrophages, and T-lymphocytes in the epidermis and dermis of the inflamed skin tissue. Inflammation caused a significant increase in the number of CB2R-immunoreactive keratinocytes, macrophages, and T-lymphocytes. Furthermore, compared to the sham EA group, EA at 2 or 100 Hz significantly increased the number of keratinocytes, macrophages, and T-lymphocytes with CB2R-immunoreactivity in the inflamed skin tissue. Therefore, our findings suggest that EA is associated with upregulation of local CB2Rs in the inflamed skin tissue. EA primarily potentiates the expression of CB2Rs on keratinocytes and infiltrating inflammatory cells at the site of inflammation. Perspective: This study shows that electroacupuncture increases the CB2 receptor expression on keratinocytes and infiltrating inflammatory cells in inflammatory skin tissues. This finding provides new evidence showing the potential role of CB2 receptors in the analgesic effect of acupuncture on inflammatory pain. ª 2010 by the American Pain Society Key words: Electroacupuncture, analgesia, hyperalgesia, inflammatory pain, inflammatory cells, cannabinoid CB2 receptors. cupuncture is an ancient therapeutic technique and an important part of traditional Chinese medicine. Acupuncture analgesia is rapidly Received September 11, 2009; Revised December 9, 2009; Accepted February 16, 2010. gaining interest and recognition by the West and Supported by a grant from the National Natural Science Foundation of has been validated by several clinical studies.14,16 China No. 30600832 and funds from the Scientific Research Foundation Electroacupuncture (EA), a modified therapy method for the Returned Overseas Chinese Scholars of State Education Ministry No. 2008890. based on the theory of traditional manual acupuncture Address reprint requests to Dr Man Li, Department of Neurobiology, is effective in the treatment of pain.31 Our recent study Tongji Medical College of Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, HuBei, PR China. E-mail: suggests that the endocannabinoid system participates [email protected] in EA analgesia in a rat model of inflammatory pain. In 1526-5900/$36.00 this regard, EA increases the level of anandmide in inª 2010 by the American Pain Society flamed skin tissues.3 Also, the antinociceptive effect of doi:10.1016/j.jpain.2010.02.013
A
1250
Zhang et al
The Journal of Pain
1251
Figure 1. Effects of inflammation and electroacupuncture (EA) treatments on the mRNA level of CB2 receptors (CB2Rs) in the skin tissue of the rat hind paw. A, a representative gel image showing the CB2R mRNA level in the skin tissue from 5 groups of rats. b-actin was used as a loading control. M: marker; Lanes from left to right represent the naı¨ve control, complete Freund adjuvant (CFA) alone, CFA plus 2 Hz EA, CFA plus 100 Hz EA, and CFA plus sham EA group. B and C, summary graphs showing the ratio of CB2R/b-actin mRNA levels in the skin tissue of the naı¨ve control, CFA alone, CFA plus 2 Hz EA, CFA plus 100 Hz EA, and CFA plus sham EA groups. Data are expressed as mean 6 SEM (n = 6 rats in each group). *P < .05 compared with the naı¨ve control group; #P < .05 compared with the CFA plus sham EA group.
EA is attenuated by local treatment with cannabinoid CB2 receptor (CB2R) antagonists.3 Our finding is consistent with other studies showing that peripheral CB2Rs can modulate inflammatory pain.11,19
Figure 3. Effects of inflammation and electroacupuncture (EA)
Figure 2. Effects of inflammation and electroacupuncture (EA) treatments on the protein level of CB2 receptors (CB2Rs) in the skin tissue of the rat hind paw. A, Representative gel image showing the CB2R protein level in the skin tissue from 5 groups of rats. b-actin was used as a loading control. A strong immunopositive band at 45 kDa was detected in the skin samples of the naı¨ve control, complete Freund adjuvant (CFA) alone, CFA plus 2 Hz EA, CFA plus 100 Hz EA, and CFA plus sham EA group. B and C, summary graphs showing the ratio of CB2R/b-actin protein levels in the skin tissue of the naı¨ve control, CFA alone, CFA plus 2 Hz EA, CFA plus 100 Hz EA, and CFA plus sham EA groups. Data are expressed as mean 6 SEM (n = 6 rats in each group). * P < .05 compared with the naı¨ve control group; #P < .05 compared with the CFA plus sham EA group.
treatments on keratinocytes labeled with CB2 receptor (CB2R) immunoreactivity in the skin tissues. A, Representative confocal images showing pan cytokeratin positive-keratinocytes (green, left panels) and CB2R-immunoreactive cells (red, middle panels). The right panels are overlay images showing double-labeled CB2R and keratinocytes (yellow). Images include naı¨ve control (a through c), complete Freund adjuvant (CFA) alone (d through f), CFA plus 2 Hz EA (g through i), CFA plus 100 Hz EA (j through l), CFA plus sham EA (m through o). Panels p through r show highmagnification images from panels g through i. Scale bar, 50 mm. B and C, summary graphs show the percentage of doublelabeled keratinocytes with CB2R-immunoreactivity in the total of pan cytokeratin positive-cells in the skin tissue from the naı¨ve control, CFA alone, CFA plus 2 Hz EA, CFA plus 100 Hz EA, and CFA plus sham EA groups. Data are expressed as mean 6 SEM (n = 6 rats in each group). * P < .05 compared with the naı¨ve control group; #P < .05 compared with the CFA plus sham EA group.
1252
Electroacupuncture Increases CB2 Receptor Expression
The Journal of Pain
Effects of Inflammation and EA Treatments on the Number of Keratinocytes With CB2R Immunoreactivity in Inflamed Skin Tissues
Table 1.
NO .
NAI¨VE CONTROL
CFA ALONE
CFA 1 2 HZ
CFA 1 100 HZ
CFA 1 SHAM EA
No. of double-labeled cells No. of keratinocytes No. of cells with CB2Rs
5.17 6 0.48 38.33 6 1.20 36.83 6 1.51
40.17 6 1.01* 96.83 6 0.95* 86.67 6 1.02*
124.00 6 0.97*,y 132.67 6 0.67*,y 126.00 6 1.06*,y
121.33 6 1.23*,y 130.50 6 0.76*,y 123.17 6 0.83*,y
41.83 6 1.17* 99.00 6 0.97* 88.50 6 0.99*
Abbreviations: EA, electroacupuncture; CB2R, cannabinoid CB2 receptor; CFA, complete Freund adjuvant. NOTE. Data are presented as mean 6 SEM; n= 6 rats in each group. *P < .05 compared with the naı¨ve control group. yP < .05 compared with the sham EA group.
Cannabinoid receptors and their endogenous ligands constitute the endocannabinoid system. Cannabinoid CB1 receptors (CB1Rs) are localized primarily in the central nervous system and in the periphery.27 On the other hand, CB2Rs are mainly present on immune cells, including mast cells, macrophages, B-lymphocytes, T-lymphocytes, and natural killer cells.7,9 Activation of CB2Rs on immune cells can inhibit the release of nociceptive mediators such as nerve growth factor,21,36 interleukin1b, and tumor necrosis factor-a.28,35 Another cell type that might be involved in the antinociceptive actions of selective CB2R agonists is keratinocytes in the skin, because these cells express CB2Rs and contain endogenous opioid peptides.13 For example, activation of CB2Rs expressed on keratinocytes may result in the release of the endogenous opioid peptide b-EP, which then acts on primary afferent neurons to inhibit nociception.13 However, the CB2R-mediated antihyperalgesic effect may be independent of endogenous opioids.34 Although findings from recent studies clearly support an important role of CB2Rs in the modulation of inflammatory pain,6,26 it remains unclear about how EA affects the expression level and distribution pattern of peripheral CB2Rs in inflamed skin tissues. In the present study, we determined the effects of inflammation and EA on the mRNA and protein levels of CB2Rs in the inflamed skin tissue and the distribution of CB2Rs on keratinocytes and immune cells recruited to the inflammatory site in a rat model of inflammatory pain. Our study provides new information that EA increases CB2R expression on keratinocytes, macrophages, and T-lymphocytes in inflamed skin tissues. This mechanism of action may mediate the antinociceptive effect of EA on inflammatory pain.
Materials and Methods Animal Models Experiments were carried out on male adult SpragueDawley rats (180 to 200 g) purchased from Experimental Animal Center of Tongji Medical College of Huazhong University of Science and Technology. All procedures were approved by the Animal Care Committee at Huazhong University of Science and Technology and conformed to the ethical guidelines of the International Association for the Study of Pain.39 The rats were housed in cages individually with a 12-hour light/dark cycle and had free access to food and water.
Inflammation was induced by injecting 50 mL complete Freund adjuvant (CFA, Sigma, St. Louis, MO) subcutaneously into the dorsal surface of the left hind paw of rats using a 25-gauge hypodermic needle.3,38 The injections were carried out under light anesthesia by means of ether inhalation. The naı¨ve rats were used as the control group. It has been shown that the vehicle (mineral oil) injected into the dorsal surface of the hind paw does not cause tissue inflammation and hyperalgesia.4,25 We selected the dorsal surface of the hind paw as the CFA injection site in order to produce an inflammatory pain focus in Gallbladder Channel of Foot Shaoyang, where GB30 and GB34 are located, according to the meridian theory of traditional Chinese medicine and our previous study.3,37
Electroacupuncture Treatment In the EA treatment group, the rats received EA administration on the ipsilateral ‘‘Huantiao’’ (GB30) and ‘‘Yanglingquan’’ (GB34) once every other day, starting at the second day after CFA injection, for 7 days. EA was administered at 2 or 100 Hz (1 mA and 0.1 ms pulse width) for 30 min using a modified current-constant Han Acupoint Nerve Stimulator (LH202, Huawei Co Ltd, Beijing, China). GB30 and GB34 were chosen because EA at these sites reduces inflammatory pain in rats.3,38 Two acupuncture needles were inserted into 2 acupoints that correspond to GB30 and GB34 in humans. GB30 is located at the junction of the lateral one third and medial two thirds of the distance between the greater trochanter and the hiatus of the sacrum, and GB34 lies on the lateral aspect of the leg, in the depression anterior and inferior to the head of the fibula in rats.33 During EA treatment, the rat was placed under an inverted clear plastic chamber (approximately 4 cm4 cm11 cm) but was neither restrained nor given any anesthetics. The animals remained awake and still during treatment and showed no evident signs of distress. For the CFA plus sham EA control, acupuncture needles were inserted ipsilaterally into GB30 and GB34 without electrical stimulation or manual needle manipulation. For the following experiments, the skin tissues at the site of inflammation were removed on day 7 after CFA injection (ie, 1 day after the third EA treatment). Because our previous study showed that EA produced significant antinociceptive effects on day 7 after CFA, we harvested the skin tissue only at this time point to show the relationship between CB2R expression and EA analgesia.3
Zhang et al
The Journal of Pain
1253
RT-PCR Procedures The skin tissue was excised from rats immediately after the animals were anesthetized with an overdose of sodium pentobarbitone (120 mg /kg, i.p.) and decapitated. Total RNA was isolated from the skin tissue using RNArose reagent (Watsonbiot, Shanghai, China) and reverse transcribed into cDNA using ReverTra Ace-a-TM (Toyobo, Osaka, Japan). PCR was performed in a 25-mL reaction volume using 2Taq PCR Mastermix (Tiangen Biotech, Beijing, China). The reactions were carried out in an MJ Research thermocycler using individual programs as follows: CB2Rs (product size: 474 bp) with a forward primer (5’-TTTCATTGCCATCCTCTTT-3’) and a reverse primer (5’-CCCAACTCCTGCTTATCC-3’); programmed at 95 C for 4 minutes; 94 C for 30 seconds; 48 C for 45 seconds; and 72 C for 1 minute. A total of 40 cycles were run, and each program was ended with 10 minutes at 72 C. The final PCR products were analyzed by agarose gel electrophoresis. The housekeeping gene b-actin (product size: 206 bp) was used as a control with a forward primer (5’-CCTTCCTGGGCATGGAGTCCTG -3’) and a reverse primer (5’-GGAGCAATGATCTTGATCTTC-3’). The optical density of each band was then measured with a computer-assisted imaging analysis system and normalized by using the band density of b-actin. All the primers were synthesized by Invitrogen Biotech (Shanghai, China).
Western Blotting
Figure 4. Effects of inflammation and electroacupuncture (EA) treatments on ED1-positive macrophages labeled with CB2 receptor (CB2R) immunoreactivity in the skin tissues. A, Representative confocal images showing ED1 positive macrophages (green, left panels) and CB2R-immunoreactive cells (red, middle panels). The right panels are overlay images showing doublelabeled CB2R and macrophages (yellow). Images include naı¨ve control (a through c), complete Freund adjuvant (CFA) alone (d through f), CFA plus 2 Hz EA (g through i), CFA plus 100 Hz EA (j through l), CFA plus sham EA (m through o). Panels p through r show enlarged images from panels g through i. Scale bar, 50 mm. B and C, Summary graphs show the percentage of double-labeled macrophages with CB2R immunoreactivity in the total of ED1-positive cells in the skin tissue from the naı¨ve control, CFA alone, CFA plus 2 Hz EA, CFA plus 100 Hz EA, and CFA plus sham EA group. Data are expressed as mean 6 SEM (n = 6 rats in each group). * P < .05 compared with the naı¨ve control group; #P < .05 compared with the CFA plus sham EA group.
The skin tissues (10 mm8 mm2 mm) were removed as described above, minced with scissors, and homogenized in 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 0.1% Nonidet P40, 0.1% cholic acid, 2 mg/mL leupeptin, 1 mg/mL pepstatin, 2 mM phenylmethylsulfonyl fluoride, and 2 mM EDTA, and centrifuged at 15 000g for 10 minutes. The pellet was discarded and protein concentrations from the supernatant were determined using the method of Lowry et al.18 Sixty micrograms of proteins of each tissue was separated on a 12% SDSpolyacrylamide gel and transferred onto PVDF membrane, blocked for 1.5 hours in 5% nonfat dry milk in Tris-buffered saline (TBS) containing 0.1% Tween-20. The membrane was incubated with the rabbit antiCB2R antibody (1:500; Cayman, MI) for 1 hour. After 3 washes in 0.1% TBS-Tween 20, the membranes were then incubated with a peroxidase-conjugated goat anti-rabbit secondary antibody (1:8000; Jackson ImmunoResearch, Baltimore, MD) for 1 hour, and washed 3 times. The enhanced chemiluminescence method (ECL Plus Western blotting detection reagents; Amersham Biosciences, Little Chalfont, UK) was used to reveal the protein bands according to the manufacturer’s protocol. The optical density of each band was then measured with a computer-assisted imaging analysis system (Qauntity One, Bio-Rad, Hemel Hempstead, UK) and normalized with the housekeeping gene b-actin.
Double-Immunofluorescence Labeling Procedures Rats were deeply anesthetized with an overdose of sodium pentobarbitone (120 mg/kg, i.p.) and were
1254
Electroacupuncture Increases CB2 Receptor Expression
The Journal of Pain
Effects of Inflammation and EA Treatments on the Number of Macrophages With CB2R Immunoreactivity in Inflamed Skin Tissues
Table 2.
NO .
NAI¨VE CONTROL
CFA
CFA 1 2HZ
CFA 1 100HZ
CFA 1 SHAM EA
No. of double-labeled cells No. of macrophages No. of cells with CB2Rs
7.83 6 0.61 17.67 6 0.56 52.50 6 1.98
33.50 6 0.76* 43.50 6 0.99* 99.50 6 2.40*
94.33 6 1.05*,y 103.83 6 0.70*,y 156.67 6 0.71*,y
92.00 6 0.86*,y 101.83 6 1.01*,y 152.67 6 0.80*,y
31.67 6 1.12* 43.17 6 0.95* 97.17 6 1.25*
Abbreviations: EA, electroacupuncture; CB2R, cannabinoid CB2 receptor; CFA, complete Freund adjuvant. NOTE. Data are presented as mean 6 SEM; n = 6 rats in each group. *P < .05 compared with the naı¨ve control group. yP < .05 compared with the sham EA group.
transcardially perfused with normal saline and 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. The skin tissue with the injection point in the middle was harvested. Tissues were postfixed at 4 C in the perfusion fixative for 8 hours, cryoprotected in 20% sucrose overnight, and sectioned at 20 mm on a cryostat in a plane perpendicular to the skin surface and parallel to the long axis of the foot. The sections were mounted onto gelatin-coated slides, air dried overnight, and used for double-immunofluorescence labeling of CB2Rs with markers of keratinocytes, macrophages, or T-lymphocytes. Double immunolabeling was performed with rabbit anti-CB2R (1:200; Cayman, MI) and either mouse monoclonal anti-pan cytokeratin antibody (1:100; Abcam, Cambridge, UK) for identification of keratinocytes, mouse monoclonal anti-CD68 antibody (Clone ED1, 1:200; Serotec, Oxford, UK) for identification of macrophages, or mouse monoclonal anti-ab T cell receptor (TCR) antibody (Clone R73, 1:200; BD BiosciencesPharMingen, San Diego, CA) for identification of T-lymphocytes. The specificity of the CB2R antibody has been demonstrated by antigen preabsorption with the corresponding blocking peptides.2 The anti-pan cytokeratin antibody reacts specifically with the skin epidermal keratinocytes.17 The anti-CD68 antibody specifically labels macrophages in the tissues.5 The anti-TCR antibody is a specific marker for peripheral T-lymphocytes and does not react with gd TCR-bearing T cells.12 All sections were blocked for 30 minutes with 5% donkey serum and 0.2% Tween-20 in PBS, followed by incubation at 37 C for 1 hour then at 4 C overnight with the primary antibody diluted in PBS containing 5% bovine serum albumin (BSA). The sections were washed 4 times with 0.05% Tween-20 in PBS for 5 minutes, and incubated with a mixture of secondary antibodies: donkey anti-mouse IgG conjugated with Cy2 (1:100; Jackson ImmunoResearch) and donkey anti-rabbit IgG conjugated with Cy3 (1:400; Jackson ImmunoResearch). Sections were washed 4 times with 0.05% Tween-20 in PBS for 5 min, and then treated with the fluorescence-mounting medium to inhibit quenching of fluorescence before being coverslipped. Negative controls were included by omitting the primary antibodies and with primary antibodies preabsorbed with their specific blocking peptides in the above procedures, which resulted in no positive labeling in the skin tissues. Six rats per group were used for the double-immunolabeling and cell counting.
Imaging Acquisition and Analysis Digital confocal images were acquired using a laser scanning confocal microscope (LSM 510, Carl Zeiss, Jena, Germany). The sections were scanned using excitation at 488 nm (argon laser) for Cy2 and at 543 nm (helium neon laser) for Cy3. A total of 3-4 sections were imaged from the same skin tissue in each rat, and counting of single- and double-labeled cells was done on confocal images randomly taken from 3 view fields from each section. Cell counting was performed by an investigator in a blind fashion using NIH Image J software (Bethesda, MD).
Statistical Analysis Data are presented as mean 6 SEM and analyzed using either Student t test or 1-way analysis of variance followed by Dunnett post hoc multiple comparison test. A P value of <.05 was considered statistically significant.
Results EA Increased the mRNA Level of CB2Rs in Inflamed Skin Tissues A total of 5 groups of rats were used: naı¨ve control, CFA alone, CFA plus sham EA, CFA plus 2 Hz EA, and CFA plus 100 Hz EA. The CB2R mRNA was detected in the skin tissue of all the groups. The optical density of CB2R mRNA in the CFA group was significantly greater than that in the naı¨ve control group (P < .05, Fig 1, A and B). Furthermore, there was a significant increase in the optical density of the CB2R mRNA in the CFA plus 2 Hz and CFA plus 100 Hz EA groups than that in the CFA plus sham EA group (P < 0.05, Fig 1, A,C).
EA Increased the CB2R Protein Level in Inflamed Skin Tissues The specific CB2R protein band at 45 kDa was present in the skin tissue samples of all the groups. The optical density of the CB2R protein in the CFA group was significantly higher than that in the naı¨ve control group (P < .05, Fig 2, A and B). Also, the optical density of the CB2R protein band in the CFA plus 2 Hz and CFA plus 100 Hz EA groups was significantly increased compared with that in the CFA plus sham EA group (P < .05, Fig 2, A and C).
Zhang et al
The Journal of Pain
1255
EA Increased the Expression of CB2Rs on Keratinocytes in Inflamed Skin Tissues The epidermal layer of the skin tissues from 4 groups of rats receiving CFA injections became much thicker than that in the naı¨ve control rats (Fig 3A). The CB2R immunoreactivity was distributed in keratinocytes in the uppermost layer of the epidermis, including the stratum granulosum and the stratum spinasum. Intense CB2Rimmunoreactivity was present in keratinocytes throughout the epidermis in inflamed skin tissues of both CFA plus 2 Hz and CFA plus 100 Hz EA groups (Fig 3A). The number of pan cytokeratin-positive cells and CB2R-positive cells and the percentage of double labeled CB2R-immunoreactive cells in the total of pan cytokeratin-positive cells were significantly increased in the skin tissues in CFA-treated rats than in the naı¨ve rats (P < .05; Table 1 and Fig 3B). Furthermore, the number of pan cytokeratin-positive cells and CB2R-positive cells and the percentage of double labeled CB2Rimmunoreactive cells in the total of pan cytokeratinpositive cells in the inflamed skin were significantly increased in the CFA plus 2 Hz and CFA plus 100 Hz EA groups, compared with those in the CFA plus sham EA group (P < .05;Table 1 and Fig 3C).
EA Increased the Expression of CB2Rs on Macrophages in Inflamed Skin Tissues
Figure 5. Effects of inflammation and electroacupuncture (EA) treatments on T cell receptor (TCR) positive T-lymphocytes labeled with CB2 receptor (CB2R) immunoreactivity in the skin tissues. A, Representative confocal images showing TCR-positive T-lymphocytes (green, left panels) and CB2R-immunoreactive cells (red, middle panels). The right panels are overlay images showing double-labeled CB2R and T lymphocytes (yellow). Images include naı¨ve control (a through c), complete Freund adjuvant (CFA) alone (d through f), CFA plus 2 Hz EA (g through i), CFA plus 100 Hz EA (j through l), CFA plus sham EA (m through o). Panels p through r show enlarged images from panels g through i. Scale bar, 50 mm. B and C, Summary graphs show the percentage of double-labeled T-lymphocytes with CB2R immunoreactivity in the total of TCR-positive cells in the skin tissue from the naı¨ve control, CFA alone, CFA plus 2 Hz EA, CFA plus 100 Hz EA, and CFA plus sham EA group. Data are expressed as mean 6 SEM (n = 6 rats in each group). *P < .05 compared with the naı¨ve control group; #P < .05 compared with the CFA plus sham EA group.
In the naı¨ve control group, only a few ED1-positive macrophages were found in the dermis of the skin, and the sizes of these cells were much smaller than those in the other groups and were rarely labeled with CB2Rs. In the CFA group, CFA plus 2 Hz, CFA plus 100 Hz, and CFA plus sham EA groups, there were a large number of infiltrating macrophages in the inflamed skin. These cells were generally large in sizes and multivacuolated, and many of them were labeled with CB2R immunoreactivity (Fig 4A). The number of ED1-positive cells and CB2R-positive cells and the percentage of double labeled CB2Rimmunoreactive cells in the total of ED1-positive cells were significantly increased in the inflamed skin tissues than those in the control skin tissue (P < .05; Table 2 and Fig 4B). Furthermore, the number of ED1-positive cells and CB2R-positive cells and the percentage of double labeled CB2R-immunoreactive cells in the total of ED1-positive cells in the inflamed skin were significantly increased in the CFA plus 2 Hz and CFA plus 100 Hz EA groups, compared with those in the CFA plus sham EA group (P < .05; Table 2 and Fig 4C).
EA Increased the Expression of CB2Rs on T-Lymphocytes in Inflamed Skin Tissues TCR-positive cells were present in the dermis of the skin section in all CFA-treated groups and had the size and oval shape consisting with the typical characteristics of T-lymphocytes. However, these cells were rarely present in the rat skin tissue in the naı¨ve control group, and only few of them were labeled with CB2R immunoreactivity. In the skin tissues from the CFA alone, CFA plus
1256
Electroacupuncture Increases CB2 Receptor Expression
The Journal of Pain
Effects of Inflammation and EA Treatments on the Number of T-Lymphocytes With CB2R Immunoreactivity in Inflamed Skin Tissues
Table 3.
NO .
NAI¨VE CONTROL
CFA
CFA 1 2HZ
CFA 1 100HZ
CFA 1 SHAM EA
No. of double-labeled cells No. of T-lymphocytes No. of cells with CB2Rs
10.67 6 0.99 17.50 6 0.56 56.33 6 1.45
20.83 6 1.40* 30.50 6 0.76* 98.00 6 1.06*
39.17 6 0.91*,y 46.83 6 0.79*,y 165.50 6 0.76*,y
37.33 6 0.71*,y 45.33 6 0.71*,y 164.83 6 1.01*,y
18.33 6 0.76* 27.00 6 0.86* 99.17 6 1.64*
Abbreviations: EA, electroacupuncture; CB2R, cannabinoid CB2 receptor; CFA, complete Freund adjuvant. NOTE. Data are presented as mean 6 SEM; n = 6 rats in each group. *P < .05 compared with the naı¨ve control group. yP < .05 compared with the sham EA group.
2 Hz, CFA plus 100 Hz, and CFA plus sham EA groups, there was a marked increase in infiltrating T-lymphocytes and some of them were labeled with CB2R immunoreactivity (Fig 5A). The number of TCR-positive cells and CB2R-positive cells and the percentage of double labeled CB2Rimmunoreactive cells in the total of TCR-positive cells were significantly increased in the inflamed skin tissues than in the control skin tissue (P < .05; Table 3 and Fig 5B). Furthermore, the number of TCR-positive cells and CB2R-positive cells and the percentage of double labeled CB2R-immunoreactive cells in the total of TCR-positive cells in the inflamed skin were significantly increased in the CFA plus 2 Hz and CFA plus 100 Hz EA groups, compared with those in the CFA plus sham EA group (P < .05; Table 3 and Fig 5C).
Discussion Our study provides new information that EA treatment upregulates CB2R expression in the inflamed skin tissue. We found that EA, at 2 and 100 Hz, significantly increased the mRNA and protein levels of CB2Rs at the time when EA produces significant antinociceptive effects on inflammatory pain. Furthermore, the number of keratinocytes, macrophages, and T-lymphocytes with CB2R-immunoreactivity in the inflamed skin tissue was significantly increased by EA treatment. Thus, our findings provide new information that EA may reduce inflammatory pain by increasing the expression and synthesis of CB2Rs in the inflamed tissues. The endocannabinoid system consists of 2 known cannabinoid receptor subtypes, CB1Rs and CB2Rs,20 a number of endogenous ligands (including anandamide and 2-arachidonoyl glycerol (2-AG),22 a high-affinity reuptake transport system, and endocannabinoid synthesizing and metabolizing enzymes.1 The CB2R mRNA has been detected in inflamed tissues, and its level is 10 to 100 times higher than that of CB1R mRNA.9 CB2R mRNA is present mainly in immune cells with particularly high levels in the mast cells, polymorphonuclear neutrophils, monocytes, B cells, natural killer cells, T4 cells, and T8 cells.9 We have shown recently that both CFA plus 2 Hz and CFA plus 100 Hz EA significantly reduced allodynia and hyperalgesia and increases the endogenous anandmide level in inflamed skin tissues.3 Also, the antinociceptive actions of EA are mediated by CB2Rs at peripheral sites, because injection of the CB2R antagonist
AM630 into the inflamed paw blocks the antinociceptive actions of EA. 3 Thus, both CB2R upregulation and elevated anandmide levels in the skin tissues may be involved in EA-produced analgesic effects on inflammatory pain. The presence of CB2R mRNA and protein has been reported in the paw tissues of rats and mice.32 We found that the mRNA and protein levels of CB2Rs are markedly increased in the skin tissue after induction of inflammation with CFA. Also, EA treatment significantly increased the mRNA and protein levels of CB2Rs in inflamed skin tissue, which provides further evidence that the CB2Rs probably play an important role in the analgesic effects of EA on inflammatory pain. Another salient finding of our study is the identification of the cell types that express CB2Rs in the skin tissue after inflammation and EA treatment. The changes in the distribution of CB2Rs in different cell types caused by inflammation and EA have not been studied previously. Although an increased level of CB2R proteins in acute (detrusor) and chronic (detrusor and mucosa) bladder inflammation has been reported,23 little is know about the effect of inflammation on CB2R expression on immune cells in the inflamed skin tissues. In the present study, we found that CB2Rs were distributed on T-lymphocytes, macrophages, and keratinocytes in the epidermis and dermis of inflamed skin tissues. Our data are consistent with the previous studies showing that the CB2R is present on immune cells, including mast cells, macrophages, B-lymphocytes, T-lymphocytes, and keratinocytes.9,13,24 We found that CFA-induced inflammation significantly increased the number of keratinocytes, macrophages, and T-lymphocytes expressing CB2Rs. Increased CB2 expression in inflamed tissues can be explained by increased immune cells (eg, macrophages and T-lymphocytes) recruited to the inflammatory site and increased keratinocytes in inflamed skin tissues.30 Thus, increased expression of CB2Rs on these cells after inflammation may mediate the antinociceptive effects of CB2R agonists on inflammatory pain.10,34 Interestingly, both inflammation and EA treatments significantly increased CB2R expression in the skin tissues. We found that CFA plus 2 and 100 Hz EA produced a further increase in the number of keratinocytes, macrophages, and T-lymphocytes expressing CB2Rs in the inflamed skin compared with those in the CFA plus sham EA group. The mechanisms underlying the EA action on CB2Rs expression in inflamed skin tissues are not clear.
Zhang et al
The Journal of Pain
1257
Because the number of keratinocytes, macrophages, and T-lymphocytes was significantly increased after chronic EA treatment, increased CB2Rs expression could be due to the result of increased infiltration of macrophages and T-lymphocytes with CB2Rs to inflamed skin tissues. EA treatment can influence the expression level of CB2Rs in a number of biological processes, including the signal transduction and nociceptive signaling.15 Since the percentage of keratinocytes, macrophages, and T-lymphocytes with CB2R immunoreactivity was also significantly increased by EA, it appears that increased CB2 expression in the inflamed skin tissue by EA could be the result of increased synthesis of CB2Rs on resident keratinocytes and infiltrated macrophages and T-lymphocytes. Consistent with this notion, we found that CFA plus 2 Hz and CFA plus 100 Hz EA significantly increased the mRNA and protein levels of CB2Rs in inflamed skin tissues. Because this EA treatment protocol is sufficient to produce an increase in the level of endogenous anandmide in the skin tissue,3 increased CB2R expression can mediate and amplify the action of anandmide to produce the analgesic effects of EA. Thus, 2 Hz and 100 Hz may be the effective frequency of EA to upregulate the endocannabinoid system and the CB2R expression in the inflamed skin tissue. One type of cells that may mediate the analgesic actions of the CB2R agonists and EA treatment is keratinocytes, which are abundantly present in the skin and express CB2Rs and contain endogenous opioid peptides.13 The skin keratinocytes are where nociceptive stimuli are applied to test the antinociceptive effects of
the specific CB2R agonists and EA treatment. It has been reported that the CB2R-selective agonist AM1241 may result in the release of the endogenous opioid peptide b-EP, which then acts on primary afferent terminals to inhibit inflammatory pain.13 EA could suppress inflammatory pain by increasing opioid release, which, in turn, activates peripheral opioid receptors.29 However, others have shown that the antihyperalgesic effects of the CB2R-selective agonists are not dependent upon the release of endogenous opioids.34 Furthermore, stimulation of CB2Rs on macrophages, T-lymphocytes, and other inflammatory cells may produce analgesia by reducing the release of inflammatory mediators that sensitize the peripheral nociceptors, such as nerve growth factor,36 IL-1b, and TNF-a.28,35 In support of this possibility, it has been shown that EA produces antinociception by inhibiting the release and gene expression of proinflammatory cytokines or neurotrophins.8 Therefore, the mechanisms of EA analgesia may include activation of CB2Rs on macrophages, T-lymphocytes, and keratinocytes, and attenuation of the release of pronociceptive mediators from the infiltrating inflammatory cells.19 In summary, we found in present study that EA significantly increased the mRNA and protein levels of CB2Rs in inflamed skin tissues. Our data suggest that EA increases CB2R expression on keratinocytes and inflammatory cells present in the inflammatory skin tissue. This new information is important to our understanding of the mechanisms underlying the antinociceptive effects of EA on inflammatory pain.
References
7. Facci L, Dal Toso R, Romanello S, Buriani A, Skaper SD, Leon A: Mast cells express a peripheral cannabinoid receptor with differential sensitivity to anandamide and palmitoylethanolamide. Proc Natl Acad Sci U S A 92:3376-3380, 1995
1. Beltramo M, Stella N, Calignano A, Lin SY, Makriyannis A, Piomelli D: Functional role of high-affinity anandamide transport, as revealed by selective inhibition. Science 277: 1094-1097, 1997 2. Casanova ML, Blazquez C, Martinez-Palacio J, Villanueva C, Fernandez-Acenero MJ, Huffman JW, Jorcano JL, Guzman M: Inhibition of skin tumor growth and angiogenesis in vivo by activation of cannabinoid receptors. J Clin Invest 111:43-50, 2003 3. Chen L, Zhang J, Li F, Qiu Y, Wang L, Li YH, Shi J, Pan HL, Li M: Endogenous anandamide and cannabinoid receptor2 contribute to electroacupuncture analgesia in rats. J Pain 10:732-739, 2009 4. Cook CD, Moore KI: Effects of sex, hindpaw injection site and stimulus modality on nociceptive sensitivity in arthritic rats. Physiol Behav 87:552-562, 2006 5. Damoiseaux JG, Dopp EA, Calame W, Chao D, MacPherson GG, Dijkstra CD: Rat macrophage lysosomal membrane antigen recognized by monoclonal antibody ED1. Immunology 83:140-147, 1994 6. Elmes SJ, Jhaveri MD, Smart D, Kendall DA, Chapman V: Cannabinoid CB2 receptor activation inhibits mechanically evoked responses of wide dynamic range dorsal horn neurons in naive rats and in rat models of inflammatory and neuropathic pain. Eur J Neurosci 20:2311-2320, 2004
8. Fu X, Zhu ZH, Wang YQ, Wu GC: Regulation of proinflammatory cytokines gene expression by nociceptin/orphanin FQ in the spinal cord and the cultured astrocytes. Neuroscience 144:275-285, 2007 9. Galiegue S, Mary S, Marchand J, Dussossoy D, Carriere D, Carayon P, Bouaboula M, Shire D, Le Fur G, Casellas P: Expression of central and peripheral cannabinoid receptors in human immune tissues and leukocyte subpopulations. Eur J Biochem 232:54-61, 1995 10. Giblin GM, O’Shaughnessy CT, Naylor A, Mitchell WL, Eatherton AJ, Slingsby BP, Rawlings DA, Goldsmith P, Brown AJ, Haslam CP, Clayton NM, Wilson AW, Chessell IP, Wittington AR, Green R: Discovery of 2-[(2,4-dichlorophenyl)amino]-N-[(tetrahydro-2H-pyran-4-yl)methyl]-4-(trifluoromethyl)-5-pyrimidinecarboxamide, a selective CB2 receptor agonist for the treatment of inflammatory pain. J Med Chem 50:2597-2600, 2007 11. Hanus L, Breuer A, Tchilibon S, Shiloah S, Goldenberg D, Horowitz M, Pertwee RG, Ross RA, Mechoulam R, Fride E: HU-308: A specific agonist for CB(2), a peripheral cannabinoid receptor. Proc Natl Acad Sci U S A 96:14228-14233, 1999 12. Hunig T, Wallny HJ, Hartley JK, Lawetzky A, Tiefenthaler G: A monoclonal antibody to a constant determinant of the rat T cell antigen receptor that induces T cell
1258
The Journal of Pain
Electroacupuncture Increases CB2 Receptor Expression
activation: Differential reactivity with subsets of immature and mature T lymphocytes. J Exp Med 169:73-86. 1989
matory hyperalgesia by activation of peripheral CB2 cannabinoid receptors. Anesthesiology 99:955-960, 2003
13. Ibrahim MM, Porreca F, Lai J, Albrecht PJ, Rice FL, Khodorova A, Davar G, Makriyannis A, Vanderah TW, Mata HP, Malan TP Jr: CB2 cannabinoid receptor activation produces antinociception by stimulating peripheral release of endogenous opioids. Proc Natl Acad Sci U S A 102: 3093-3098, 2005
27. Rice AS, Farquhar-Smith WP, Nagy I: Endocannabinoids and pain: Spinal and peripheral analgesia in inflammation and neuropathy. Prostaglandins Leukot Essent Fatty Acids 66:243-256, 2002
14. Jubb RW, Tukmachi ES, Jones PW, Dempsey E, Waterhouse L, Brailsford S: A blinded randomised trial of acupuncture (manual and electroacupuncture) compared with a non-penetrating sham for the symptoms of osteoarthritis of the knee. Acupunct Med 26:69-78, 2008 15. Ko J, Na DS, Lee YH, Shin SY, Kim JH, Hwang BG, Min BI, Park DS: cDNA microarray analysis of the differential gene expression in the neuropathic pain and electroacupuncture treatment models. J Biochem Mol Biol 35:420-427, 2002 16. Kong J, Fufa DT, Gerber AJ, Rosman IS, Vangel MG, Gracely RH, Gollub RL: Psychophysical outcomes from a randomized pilot study of manual, electro, and sham acupuncture treatment on experimentally induced thermal pain. J Pain 6:55-64, 2005 17. Lane EB, Rugg EL, Navsaria H, Leigh IM, Heagerty AH, Ishida-Yamamoto A, Eady RA: A mutation in the conserved helix termination peptide of keratin 5 in hereditary skin blistering. Nature 356:244-246, 1992 18. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275, 1951 19. Malan TP Jr., Ibrahim MM, Vanderah TW, Makriyannis A, Porreca F: Inhibition of pain responses by activation of CB(2) cannabinoid receptors. Chem Phys Lipids 121:191-200, 2002 20. Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner TI: Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 346:561-564, 1990 21. McMahon SB, Bennett DL, Priestley JV, Shelton DL: The biological effects of endogenous nerve growth factor on adult sensory neurons revealed by a trkA-IgG fusion molecule. Nat Med 1:774-780, 1995 22. Mechoulam R, Fride E, Di Marzo V: Endocannabinoids. Eur J Pharmacol 359:1-18, 1998 23. Merriam FV, Wang ZY, Guerios SD, Bjorling DE: Cannabinoid receptor 2 is increased in acutely and chronically inflamed bladder of rats. Neurosci Lett 445:130-134, 2008 24. Munro S, Thomas KL, Abu-Shaar M: Molecular characterization of a peripheral receptor for cannabinoids. Nature 365:61-65, 1993 25. Oliveira PG, Brenol CV, Edelweiss MI, Brenol JC, Petronilho F, Roesler R, Dal-Pizzol F, Schwartsmann G, Xavier RM: Effects of an antagonist of the bombesin/ gastrin-releasing peptide receptor on complete Freund’s adjuvant-induced arthritis in rats. Peptides 29:1726-1731, 2008 26. Quartilho A, Mata HP, Ibrahim MM, Vanderah TW, Porreca F, Makriyannis A, Malan TP Jr.: Inhibition of inflam-
28. Safieh-Garabedian B, Poole S, Allchorne A, Winter J, Woolf CJ: Contribution of interleukin-1 beta to the inflammation-induced increase in nerve growth factor levels and inflammatory hyperalgesia. Br J Pharmacol 115: 1265-1275, 1995 29. Sekido R, Ishimaru K, Sakita M: Differences of electroacupuncture-induced analgesic effect in normal and inflammatory conditions in rats. Am J Chin Med 31: 955-965, 2003 30. Tewari-Singh N, Rana S, Gu M, Pal A, Orlicky DJ, White CW, Agarwal R: Inflammatory biomarkers of sulfur mustard analog 2-chloroethyl ethyl sulfide-induced skin injury in SKH-1 hairless mice. Toxicol Sci 108:194-206, 2009 31. Tsui P, Leung MC: Comparison of the effectiveness between manual acupuncture and electro-acupuncture on patients with tennis elbow. Acupunct Electrother Res 27: 107-117, 2002 32. Walczak JS, Pichette V, Leblond F, Desbiens K, Beaulieu P: Behavioral, pharmacological and molecular characterization of the saphenous nerve partial ligation: A new model of neuropathic pain. Neuroscience 132: 1093-1102, 2005 33. Wang L, Zhang Y, Dai J, Yang J, Gang S: Electroacupuncture (EA) modulates the expression of NMDA receptors in primary sensory neurons in relation to hyperalgesia in rats. Brain Res 1120:46-53, 2006 34. Whiteside GT, Gottshall SL, Boulet JM, Chaffer SM, Harrison JE, Pearson MS, Turchin PI, Mark L, Garrison AE, Valenzano KJ: A role for cannabinoid receptors, but not endogenous opioids, in the antinociceptive activity of the CB2selective agonist, GW405833. Eur J Pharmacol 528:65-72, 2005 35. Woolf CJ, Allchorne A, Safieh-Garabedian B, Poole S: Cytokines, nerve growth factor and inflammatory hyperalgesia: The contribution of tumour necrosis factor alpha. Br J Pharmacol 121:417-424, 1997 36. Woolf CJ, Safieh-Garabedian B, Ma QP, Crilly P, Winter J: Nerve growth factor contributes to the generation of inflammatory sensory hypersensitivity. Neuroscience 62: 327-331, 1994 37. Yung KT: Birdcage model for the Chinese meridian system, VI: Meridians as the primary regulatory system. Am J Chin Med 33:759-766, 2005 38. Zhang RX, Lao L, Wang L, Liu B, Wang X, Ren K, Berman BM: Involvement of opioid receptors in electroacupuncture-produced anti-hyperalgesia in rats with peripheral inflammation. Brain Res 1020:12-17, 2004 39. Zimmermann M: Ethical guidelines for investigations of experimental pain in conscious animals. Pain 16:109-110, 1983