Julie Gardener
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The cannabinoid receptor CB2 exerts antifibrotic effects in experimental dermal fibrosis
Alfiya Akhmetshina1, Clara Dees1, Nicole Busch1, Jürgen Beer1, Kerstin Sarter1, Jochen Zwerina1, Andreas Zimmer2, Oliver Distler3, Georg Schett1, Jörg H. W. Distler1,*
Article first published online: 30 MAR 2009
DOI: 10.1002/art.24395
Abstract
Objective
The cannabinoid receptor CB2 is predominantly expressed in non-neuronal tissue and exerts potent immunomodulatory effects. This study was undertaken to evaluate the role of CB2 in the pathogenesis of dermal fibrosis.
Methods
Mice deficient in CB2 (CB2−/− mice) and their wild-type littermates (CB2+/+ mice) were injected with bleomycin to induce experimental fibrosis. Mice were treated with selective agonists and antagonists of CB2. Lesional skin was evaluated for dermal thickness and numbers of infiltrating leukocytes. Bone marrow transplantation experiments were performed.
Results
CB2−/− mice were more sensitive to bleomycin-induced dermal fibrosis than were CB2+/+ mice, and showed increased dermal thickness. Leukocyte counts were significantly higher in the lesional skin of CB2+/+ mice. Increased dermal fibrosis was also observed upon treatment with the CB2 antagonist AM-630. In contrast, the selective CB2 agonist JWH-133 reduced leukocyte infiltration and dermal thickening. The phenotype of CB2−/− mice was mimicked by transplantation of CB2−/− bone marrow into CB2+/+ mice, whereas CB2−/− mice transplanted with bone marrow from CB2+/+ mice did not display an increased sensitivity to bleomycin-induced fibrosis, indicating that leukocyte expression of CB2 critically influences experimental fibrosis.
Conclusion
Our findings indicate that CB2 limits leukocyte infiltration and tissue fibrosis in experimental dermal fibrosis. Since selective CB2 agonists are available and well tolerated, CB2 might be an interesting molecular target for the treatment of early inflammatory stages of systemic sclerosis.
Systemic sclerosis (SSc) is a connective tissue disease of unknown etiology that affects the skin and a variety of internal organs. In early stages, inflammatory infiltrates are a histopathologic hallmark of SSc (1). The inflammatory infiltrates are dominated by monocytes and activated T cells. Later stages of the disease are characterized by an excessive accumulation of extracellular matrix components. The resulting fibrosis frequently leads to dysfunction of the affected organs, which is a major cause of death in SSc patients. The overproduction of extracellular matrix components in SSc patients is mediated by activated fibroblasts. The mechanisms leading to the activation of fibroblasts in SSc, in particular how leukocytes regulate this process, are poorly understood so far (1). The observation that activated fibroblasts that release excessive amounts of collagen are mainly localized adjacent to inflammatory infiltrates supports the notion that leukocytes are involved in the initiation of fibrosis in early stages of SSc (2).
In the early 1990s, 2 different receptors for the marijuana component Δ9-tetrahydrocannabinol were identified and named CB1 and CB2 (3, 4). Both receptors are heterotrimeric GTP binding protein—coupled receptors. CB1 is primarily expressed in the central nervous system, whereas CB2 is predominantly expressed in peripheral tissue. The ligands for CB1 and CB2, the so-called "cannabinoids," can be subdivided into 3 different groups according to their origin. The family of cannabinoids includes plant-derived cannabinoids, synthetic cannabinoids, and endogenous cannabinoids (endocannabinoids), which are synthesized within the human body (5). Besides their effects on the central nervous system, endocannabinoids regulate physiologic and pathophysiologic processes in non-neuronal tissue.
Cannabinoids exert anti-tumor effects, with inhibition of tumor cell proliferation and induction of cell cycle arrest in transformed cells. Endocannabinoids also orchestrate immune responses by regulating cytokine release, chemotaxis, and proliferation and activation of leukocytes. Furthermore, cannabinoids play a central role in bone metabolism and turnover, since they control proliferation and activation of osteoblasts as well as differentiation of mononuclear precursor cells into osteoclasts (5, 6). Endocannabinoids might also play a role in fibrotic diseases. Recent data have demonstrated that cannabinoids regulate the activation of hepatic stellate cells, which contribute to the pathogenesis of liver fibrosis (7). Stimulation of CB2 reduced the proliferation of hepatic stellate cells and reduced oxidative stress in preclinical models (7). Expression of CB2 has also been found in the skin, suggesting that CB2 signaling might play a role in dermal fibrosis (8—10).
The broad implications of the endocannabinoid system for different diseases have resulted in considerable interest from pharmaceutical companies and stimulated the development of a number of synthetic small molecules that target the CB2 receptor. Several of these compounds are currently being evaluated in clinical trials, e.g., selective CB2 agonists for immunomodulation in multiple sclerosis (6). The initial results of these trials indicate that potent inhibition of the cannabinoid receptors can be achieved in humans and that the rate of severe adverse events associated with this class of drugs is low.
The aim of the present study was to investigate the role of the peripheral cannabinoid receptor CB2 in experimental dermal fibrosis. We demonstrate that inhibition of CB2 signaling, either by gene silencing or by treatment with small-molecule inhibitors, increases susceptibility to fibrosis. Moreover, activation of CB2 signaling reduces leukocyte infiltration into lesional skin and prevents the development of experimental fibrosis. Thus, stimulation of CB2 might be an interesting antifibrotic approach in the early inflammatory stages of SSc.
MATERIALS AND METHODS
Bleomycin-induced dermal fibrosis in CB2-deficient mice.
Mice deficient in CB2 (CB2−/− mice) have been described previously (11). CB2−/− mice were backcrossed onto a C57BL/6 background for at least 6 generations. Matched wild-type C57BL/6 mice expressing CB2 (CB2+/+ mice) from the same breedings were used as controls. Skin fibrosis was induced in 6-week-old mice by local injections of bleomycin for 4 weeks, as previously described (12). Briefly, 100 μl of bleomycin dissolved in 0.9% NaCl at a concentration of 0.5 mg/ml was administered every other day by subcutaneous injection into defined areas of 1 cm2 in the upper back. Subcutaneous injections of 100 μl of 0.9% NaCl were used as controls. Four different groups, 2 groups of CB2−/− mice and 2 groups of CB2+/+ mice, were analyzed. One group of CB2−/− mice and 1 group of CB2+/+ mice were challenged with bleomycin, and the remaining 2 groups were injected with NaCl. Each group consisted of 8 mice. After 4 weeks, animals were killed by cervical dislocation. All animal experiments were approved by the local ethics committee.
Analysis of the effects of CB2 antagonists and agonists on experimental fibrosis.
To confirm the increased susceptibility of CB2−/− mice to fibrosis using a pharmacologic approach, C57BL/6 mice challenged with bleomycin were treated with selective CB2 agonists or antagonists. AM-630 (6-Iodo-2-methyl-1-[2-(4-orpholinyl)ethyl]-1H-indol-3-yl] (4-methoxy-phenyl)methanone) is a selective antagonist of CB2 with a Ki value of 31.2 nM. Other receptors, including CB1, are not affected by AM-630 in pharmacologically relevant concentrations. JWH-133 ([6aR,10aR]-3-[1,1-dimethylbutyl]-6a,7,10,10a-tetrahydro-6,6,9-tri-methyl-6H-di-benzo[b,d] pyran) selectively activates CB2 with a Ki value of 3.4 nM and a 200-fold selectivity compared with CB1. Both compounds were purchased from Biozol (Eiching, Germany). AM-630 and JWH-133 were dissolved in DMSO at a concentration of 10 mg/ml. The working solutions were prepared fresh on the day of the experiments by diluting the stock solutions in NaCl. Mice injected with equal volumes of the solvent were used as controls. Treatment with AM-630 and JWH-133 was started in parallel to bleomycin challenge. AM-630 and JWH-133 were administered by daily intraperitoneal injections at concentrations of 2.5 mg/kg/day in a total volume of 100 μl for 4 weeks. Each treatment group consisted of 6 mice.
Bone marrow transplantation.
To investigate the relative contributions of fibroblasts and other mesenchymal cells and bone marrow—derived cells to the phenotype of CB2−/−, bone marrow transplantation experiments were performed. Female CB2−/− and CB2+/+ mice were used as donors for bone marrow. For isolation of unfractioned bone marrow cells, tibial and femur bones were prepared under sterile conditions. Bone marrow cells were flushed from the bone marrow cavities with phosphate buffered saline (PBS) and subsequently filtered through 70-μm nylon meshes (BD Biosciences, Heidelberg, Germany). Erythrocytes were hemolyzed, and the remaining bone marrow cells were kept on ice until transplantation. All isolated bone marrow cells were transplanted together without further purification or in vitro expansion of a particular subset of cells. Male CB2−/− or CB2+/+ mice received bone marrow transplants at age 4 weeks. Sixteen hours before transplantation, recipient CB2−/− or CB2+/+ mice underwent whole body irradiation with 11 Gy. For transplantation, 2.0 × 106 bone marrow cells from donor mice were resuspended in 0.2 ml of PBS and injected via the tail veins. To exclude sublethal irradiation and reconstitution by the old bone marrow, additional groups of CB2−/− and CB2+/+ mice were irradiated, but did not receive bone marrow transplants. Two weeks after bone marrow transplantation and after confirmation of a complete reconstitution of the hematopoiesis by the transplanted cells, mice were challenged with bleomycin for 4 weeks as described above. All groups of mice consisted of 6 animals each.
Histologic analysis.
Lesional skin areas were excised, fixed in 4% formalin, and embedded in paraffin. Five-micrometer sections were stained with hematoxylin and eosin. Dermal thickness was analyzed at 100-fold magnification by measuring the distance between the epidermal—dermal junction and the dermal—subcutaneous fat junction at 3 sites in lesional skin in each mouse (13). Collagen fibers were visualized by Masson's trichrome staining, according to the recommendations of the manufacturer (Sigma-Aldrich, Munich, Germany), and analyzed at 1,000-fold magnification. Infiltrating leukocytes in the lesional skin of CB2−/− mice, CB2+/+ mice, and CB2+/+ mice treated with AM-630 or JWH-133 were quantified on hematoxylin and eosin—stained sections. Twenty-five different high-power fields from different tissue sites from each mouse were evaluated for polymononuclear cells at 200-fold magnification by an experienced examiner (JHWD) who was blinded with regard to treatment group. Images were captured with a Nikon Eclipse 80i microscope (Badhoevedorp, The Netherlands) equipped with a DSP 3-CCD camera (Sony, Tokyo, Japan).
Statistical analysis.
Data are expressed as the mean ± SEM. The Mann-Whitney U test was used for statistical analyses. P values less than 0.05 were considered significant.
RESULTS
Increased sensitivity of CB2−/− mice to bleomycin- induced dermal fibrosis.
To evaluate whether CB2 plays a role in the development of dermal fibrosis, CB2−/− mice and CB2+/+ mice (wild-type mice) were challenged with bleomycin. Skin architecture and dermal thickness did not differ between CB2−/− mice and CB2+/+ mice injected with NaCl, suggesting that the skin phenotype is not altered in CB2−/− mice under nonfibrotic conditions (Figures 1 and 2). Upon injection of bleomycin, dermal thickness increased in both CB2−/− and CB2+/+ mice (Figures 1 and 2). However, CB2−/− mice were significantly more susceptible to the induction of fibrosis by bleomycin than were CB2+/+ mice. In CB2−/− mice, the mean ± SEM increase in dermal thickness was 72 ± 6%, as compared with 45 ± 6% in CB2+/+ mice (P = 0.028) (Figure 2). These data suggest that CB2−/− mice might be more susceptible to bleomycin-induced fibrosis.
Figure 1. Increase in dermal thickness in CB2−/− mice upon challenge with bleomycin. Representative sections from a CB2+/+ mouse injected with NaCl, a CB2−/− mouse injected with NaCl, a CB2+/+ mouse injected with bleomycin, and a CB2−/− mouse injected with bleomycin are shown. No differences in dermal thickness were observed between CB2−/− mice injected with NaCl and CB2+/+ mice injected with NaCl. However, challenge with bleomycin resulted in significantly greater dermal thickening in CB2−/− mice than in CB2+/+ mice. (Original magnification × 100.)
Figure 2. Increased sensitivity of CB2−/− mice to dermal fibrosis upon challenge with bleomycin. Bars show the mean and SEM fold increase in dermal thickness compared with CB2+/+ mice injected with NaCl (n = 8 mice per group).
Effect of pharmacologic modification of CB2 signaling on the outcome of dermal fibrosis.
To confirm our findings in CB2−/− mice by a pharmacologic approach, CB2+/+ mice were treated with AM-630, a specific inhibitor of CB2. Treatment of mice with AM-630 during bleomycin challenge lead to a significantly more pronounced increase in dermal thickness than did injection with bleomycin alone (mean ± SEM increase compared with controls 128 ± 8% versus 68 ± 3%; P = 0.004) (Figures 3 and 4). Thus, inhibition of CB2, either by gene silencing or by treatment with small-molecule inhibitors, results in increased sensitivity to bleomycin-induced dermal fibrosis.
Figure 3. Increased sensitivity to bleomycin-induced fibrosis in mice treated with the CB2 antagonist AM-630 and amelioration of experimental fibrosis in mice treated with the selective CB2 agonist JWH-133. Representative sections of lesional skin from a CB2+/+ mouse treated with NaCl, a CB2+/+ mouse treated with bleomycin alone, a CB2+/+ mouse treated with bleomycin and AM-630, and a CB2+/+ mouse treated with bleomycin and JWH-133 are shown. Treatment of CB2+/+ mice with AM-630 significantly increased dermal thickness upon challenge with bleomycin, resembling the phenotype of CB2−/− mice. In contrast, treatment with the selective CB2 agonist JWH-133 prevented the profibrotic effects of bleomycin. (Original magnification × 100.)
Figure 4. Exacerbation of bleomycin-induced fibrosis after pharmacologic inhibition of CB2 by AM-630 and reduction in dermal thickening after activation of CB2 by JHW-133 in CB2+/+ mice. Bars show the mean and SEM fold increase in dermal thickness compared with mice injected with NaCl (n = 6 mice per group).
To evaluate whether activation of CB2 exerts antifibrotic effects, CB2+/+ mice were treated with the selective CB2 agonist JWH-133 in pharmacologically relevant concentrations during bleomycin challenge. Treatment with JWH-133 ameliorated the profibrotic effects of bleomycin and significantly reduced dermal thickening, by 35 ± 3% (P = 0.004) (Figures 3 and 4). Thus, activation of CB2 signaling exerts antifibrotic effects in vivo.
CB2 regulation of the infiltration of leukocytes into lesional skin.
Accumulation of leukocytes in lesional skin is characteristic of the mouse model of bleomycin-induced dermal fibrosis and of early stages of SSc. To analyze whether CB2 affects leukocyte infiltration in experimental fibrosis, the numbers of leukocytes in lesional skin were quantified. The numbers of infiltrating leukocytes were significantly increased in CB2−/− mice compared with CB2+/+ mice. The mean ± SEM number of leukocytes detected per high-power field was 38.2 ± 3.1 in sections from the lesional skin of CB2−/− mice, compared with 27.7 ± 3.5 in CB2+/+ mice (P = 0.016). Consistent with these results, greater numbers of leukocytes were also detected in CB2+/+ mice upon treatment with the CB2 antagonist AM-630 (mean ± SEM 37.1 ± 1.6; P = 0.047 versus CB2+/+ mice treated with bleomycin alone). However, the infiltration of leukocytes into lesional skin was strongly reduced in CB2+/+ mice treated with the CB2 agonist JWH-133 (mean ± SEM 17.7 ± 1.4; P = 0.009 versus CB2+/+ mice treated with bleomycin alone).
Mediation of the phenotype of CB2−/− mice by bone marrow—derived cells.
To investigate whether the increased susceptibility of CB2−/− mice to fibrosis is based on lack of CB2 on bone marrow—derived cells or on fibroblasts and other mesenchymal cells, bone marrow transplantation experiments were performed, and the resulting chimeric mice were challenged with bleomycin. Transplantation of CB2−/− mice with bone marrow from CB2+/+ mice and subsequent challenge with bleomycin resulted in comparable increases in dermal thickness as in CB2+/+ mice with CB2+/+ bone marrow, with mean ± SEM increases of 53 ± 3% and 45 ± 6%, respectively, as compared with NaCl-treated mice (Figures 5 and 6). In contrast, transplantation of CB2+/+ mice with bone marrow cells from CB2−/− mice fully resembled the phenotype of CB2−/− mice. Bleomycin challenge of CB2+/+ mice with bone marrow from CB2−/− mice resulted in a mean ± SEM increase in dermal thickness of 77 ± 7%. This increase was significantly higher than in CB2+/+ mice with CB2+/+ bone marrow (P = 0.03), but did not differ significantly from the increase observed in CB2−/− mice (P = 0.67) (Figures 5 and 6). Taken together, these data demonstrate that CB2 deficiency in bone marrow—derived cells mediates the increased susceptibility of CB2−/− mice to experimental fibrosis.
Figure 5. Mediation of the increased susceptibility of CB2−/− mice to experimental fibrosis by bone marrow (BM)—derived cells. CB2−/− mice were transplanted with bone marrow from CB2+/+ mice and vice versa after lethal irradiation, and subsequently challenged with bleomycin. The dermal thickening in these chimeric mice was compared with that in CB2+/+ mice with CB2+/+ bone marrow and CB2−/− mice with CB2−/− bone marrow. The phenotype of CB2−/− mice was completely mimicked by transplantation of CB2−/− bone marrow into CB2+/+ mice, and CB2−/− mice with CB2+/+ bone marrow did not display increased sensitivity to fibrosis. (Original magnification × 100.)
Figure 6. Dermal thickness in CB+/+ mice with CD+/+ bone marrow (BM), CB−/− mice with CB+/+ bone marrow, CB+/+ mice with CB−/− bone marrow, and CB−/− mice with CB−/− bone marrow after treatment with bleomycin. Bone marrow transplantation experiments revealed that the increased sensitivity of CB2−/− mice to bleomycin-induced fibrosis is mediated by bone marrow—derived cells. Bars show the mean and SEM percent increase in dermal thickness compared with CB2+/+ mice with CB2+/+ bone marrow injected with NaCl (n = 6 mice per group).
DISCUSSION
In the present study, we demonstrated that CB2 exerts antifibrotic effects in vivo. Inhibition of CB2 signaling by either gene silencing or treatment with small-molecule inhibitors increased susceptibility to bleomycin-induced dermal fibrosis. Accumulation of thickened collagen fibers and dermal thickening upon challenge with bleomycin were more pronounced in CB2−/− mice and in mice treated with the CB2 antagonist AM-630. Conversely, activation of CB2 signaling by the selective agonist JWH-133 prevented the profibrotic effects of bleomycin and significantly reduced dermal thickening at pharmacologically relevant doses. Consistent with our results, antifibrotic effects of CB2 have previously been observed in the liver and pancreas (7, 14).
Based on these preclinical data, activation of CB2 by selective agonists might be a novel approach for the treatment of fibrosis. In SSc, this approach might be particularly promising for patients with early inflammatory stages of the disease. Of note, CB2 agonists are currently being evaluated in clinical trials for multiple sclerosis (5, 6). In these clinical trials, CB2 agonists have been well tolerated. Major adverse events such as dizziness, drowsiness, or headache were due to incomplete selectivity of these first compounds, which resulted in coactivation of the CB1 receptor. Thus, our study could have direct clinical impact, since CB2 agonists are well tolerated and already available for clinical trials in patients with fibrotic disorders such as SSc.
We have shown that CB2 mediates its antifibrotic effects in vivo by inhibiting the infiltration of leukocytes into lesional skin in preclinical models of SSc. Inhibition of CB2 signaling by either genetic knockout or treatment with the chemical inhibitor AM-630 increased the accumulation of leukocytes in bleomycin-injected areas of the skin. Consistent with our findings in experimental fibrosis, exacerbation of inflammation in mice deficient in CB1 and CB2 (CB1−/−CB2−/− mice) was previously observed in experimental contact dermatitis (9). Infiltration of leukocytes into lesional skin is a common feature in the early stages of SSc and in the mouse model of bleomycin-induced dermal fibrosis. Infiltrating leukocytes activate the collagen synthesis of resident fibroblasts by the release of a profibrotic mediator. Thus, CB2 signaling might affect the outcome of fibrosis indirectly by orchestrating the infiltration of leukocytes into lesional skin rather than by direct effects on the collagen synthesis of fibroblasts.
Consistent with this hypothesis, the increased susceptibility of CB2−/− mice to experimental fibrosis was fully replicated by transplantation of CB2-deficient bone marrow cells into CB2+/+ mice. In contrast, the phenotype of CB2−/− mice transplanted with CB2+/+ bone marrow did not differ from that of CB2+/+ mice with CB2+/+ bone marrow, evidence against a direct effect of CB2 signaling on fibroblasts. This hypothesis was further supported by the results of studies of cultured fibroblasts. Neither activation nor inhibition of CB2 altered the release of collagen or other major extracellular matrix proteins in cultured fibroblasts from SSc patients or healthy individuals (results not shown). Therefore, we propose that activation of CB2 inhibits leukocyte activation and migration into lesional skin in fibrotic diseases. The reduced infiltration of leukocytes and the decreased release of profibrotic mediators subsequently results in a less pronounced stimulation of resident fibroblasts and in protection against fibrosis.
On the molecular level, CB2 might exert its effects by regulating the expression of chemokines and affecting chemotaxis of leukocytes (6, 15). In particular, regulation of the expression of monocyte chemotactic protein 1 (MCP-1; CCL2) and its receptor CCR2 might be important in fibrotic diseases. MCP-1 exerts potent chemotactic effects on inflammatory cells via activation of CCR2. MCP-1 is up-regulated in the skin of SSc patients and has been implicated in the pathogenesis of the disease (16). MCP-1 might contribute to the development of fibrosis either directly by effecting fibroblasts or indirectly by stimulating the release of interleukin-4 from T cells (17, 18). A previous study demonstrated that activation of CB2 reduced the expression of MCP-1 and its receptor CCR2 in myeloid progenitor cells, whereas inhibition of CB2 stimulated their expression (15).
The mouse model of bleomycin-induced dermal fibrosis has limitations. The effect of bleomycin is not irreversible in this model, and dermal fibrosis spontaneously resolves over time. However, we recently demonstrated that dermal thickening progresses during the first 6 weeks of challenge with bleomycin and that the fibrotic changes persist for at least 3 weeks after the last bleomycin injection (19). Thus, we performed our analyses during a dynamic phase of progressive dermal fibrosis. Another limitation of the mouse model of bleomycin-induced dermal fibrosis is that this model mimics early, inflammatory stages of SSc, but is less suitable as a model of later stages of SSc, where inflammatory infiltrates are rarely observed. Therefore, our findings in the mouse model of bleomycin-induced fibrosis indicate a potential role of CB2 in the early stages of SSc, but the results should not be extrapolated to later stages of SSc. Moreover, additional studies are necessary to investigate the role of CB2 in the development of fibrosis in other organ systems, such as the lung.
In summary, we have demonstrated that CB2 exerts antifibrotic effects by limiting leukocyte infiltration and subsequent fibroblast activation in lesional skin. These data suggest that activation of CB2 might be a promising approach for therapy for the early inflammatory stages of SSc. Since CB2 agonists are available and seem to be well tolerated, these findings might lead to clinical trials in patients with SSc and other fibrotic diseases.
AUTHOR CONTRIBUTIONS
Dr. Jörg H. W. Distler had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study design. Akhmetshina, Oliver Distler, Schett, Jörg H. W. Distler.
Acquisition of data. Akhmetshina, Dees, Busch, Beer, Sarter, Zwerina, Jörg H. W. Distler.
Analysis and interpretation of data. Akhmetshina, Dees, Oliver Distler, Schett, Jörg H. W. Distler.
Manuscript preparation. Akhmetshina, Oliver Distler, Schett, Jörg H. W. Distler.
Statistical analysis. Jörg H. W. Distler.
Provision of materials. Zimmer.
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Source: The cannabinoid receptor CB2 exerts antifibrotic effects in experimental dermal fibrosis - Akhmetshina - 2009 - Arthritis & Rheumatism - Wiley Online Library
Alfiya Akhmetshina1, Clara Dees1, Nicole Busch1, Jürgen Beer1, Kerstin Sarter1, Jochen Zwerina1, Andreas Zimmer2, Oliver Distler3, Georg Schett1, Jörg H. W. Distler1,*
Article first published online: 30 MAR 2009
DOI: 10.1002/art.24395
Abstract
Objective
The cannabinoid receptor CB2 is predominantly expressed in non-neuronal tissue and exerts potent immunomodulatory effects. This study was undertaken to evaluate the role of CB2 in the pathogenesis of dermal fibrosis.
Methods
Mice deficient in CB2 (CB2−/− mice) and their wild-type littermates (CB2+/+ mice) were injected with bleomycin to induce experimental fibrosis. Mice were treated with selective agonists and antagonists of CB2. Lesional skin was evaluated for dermal thickness and numbers of infiltrating leukocytes. Bone marrow transplantation experiments were performed.
Results
CB2−/− mice were more sensitive to bleomycin-induced dermal fibrosis than were CB2+/+ mice, and showed increased dermal thickness. Leukocyte counts were significantly higher in the lesional skin of CB2+/+ mice. Increased dermal fibrosis was also observed upon treatment with the CB2 antagonist AM-630. In contrast, the selective CB2 agonist JWH-133 reduced leukocyte infiltration and dermal thickening. The phenotype of CB2−/− mice was mimicked by transplantation of CB2−/− bone marrow into CB2+/+ mice, whereas CB2−/− mice transplanted with bone marrow from CB2+/+ mice did not display an increased sensitivity to bleomycin-induced fibrosis, indicating that leukocyte expression of CB2 critically influences experimental fibrosis.
Conclusion
Our findings indicate that CB2 limits leukocyte infiltration and tissue fibrosis in experimental dermal fibrosis. Since selective CB2 agonists are available and well tolerated, CB2 might be an interesting molecular target for the treatment of early inflammatory stages of systemic sclerosis.
Systemic sclerosis (SSc) is a connective tissue disease of unknown etiology that affects the skin and a variety of internal organs. In early stages, inflammatory infiltrates are a histopathologic hallmark of SSc (1). The inflammatory infiltrates are dominated by monocytes and activated T cells. Later stages of the disease are characterized by an excessive accumulation of extracellular matrix components. The resulting fibrosis frequently leads to dysfunction of the affected organs, which is a major cause of death in SSc patients. The overproduction of extracellular matrix components in SSc patients is mediated by activated fibroblasts. The mechanisms leading to the activation of fibroblasts in SSc, in particular how leukocytes regulate this process, are poorly understood so far (1). The observation that activated fibroblasts that release excessive amounts of collagen are mainly localized adjacent to inflammatory infiltrates supports the notion that leukocytes are involved in the initiation of fibrosis in early stages of SSc (2).
In the early 1990s, 2 different receptors for the marijuana component Δ9-tetrahydrocannabinol were identified and named CB1 and CB2 (3, 4). Both receptors are heterotrimeric GTP binding protein—coupled receptors. CB1 is primarily expressed in the central nervous system, whereas CB2 is predominantly expressed in peripheral tissue. The ligands for CB1 and CB2, the so-called "cannabinoids," can be subdivided into 3 different groups according to their origin. The family of cannabinoids includes plant-derived cannabinoids, synthetic cannabinoids, and endogenous cannabinoids (endocannabinoids), which are synthesized within the human body (5). Besides their effects on the central nervous system, endocannabinoids regulate physiologic and pathophysiologic processes in non-neuronal tissue.
Cannabinoids exert anti-tumor effects, with inhibition of tumor cell proliferation and induction of cell cycle arrest in transformed cells. Endocannabinoids also orchestrate immune responses by regulating cytokine release, chemotaxis, and proliferation and activation of leukocytes. Furthermore, cannabinoids play a central role in bone metabolism and turnover, since they control proliferation and activation of osteoblasts as well as differentiation of mononuclear precursor cells into osteoclasts (5, 6). Endocannabinoids might also play a role in fibrotic diseases. Recent data have demonstrated that cannabinoids regulate the activation of hepatic stellate cells, which contribute to the pathogenesis of liver fibrosis (7). Stimulation of CB2 reduced the proliferation of hepatic stellate cells and reduced oxidative stress in preclinical models (7). Expression of CB2 has also been found in the skin, suggesting that CB2 signaling might play a role in dermal fibrosis (8—10).
The broad implications of the endocannabinoid system for different diseases have resulted in considerable interest from pharmaceutical companies and stimulated the development of a number of synthetic small molecules that target the CB2 receptor. Several of these compounds are currently being evaluated in clinical trials, e.g., selective CB2 agonists for immunomodulation in multiple sclerosis (6). The initial results of these trials indicate that potent inhibition of the cannabinoid receptors can be achieved in humans and that the rate of severe adverse events associated with this class of drugs is low.
The aim of the present study was to investigate the role of the peripheral cannabinoid receptor CB2 in experimental dermal fibrosis. We demonstrate that inhibition of CB2 signaling, either by gene silencing or by treatment with small-molecule inhibitors, increases susceptibility to fibrosis. Moreover, activation of CB2 signaling reduces leukocyte infiltration into lesional skin and prevents the development of experimental fibrosis. Thus, stimulation of CB2 might be an interesting antifibrotic approach in the early inflammatory stages of SSc.
MATERIALS AND METHODS
Bleomycin-induced dermal fibrosis in CB2-deficient mice.
Mice deficient in CB2 (CB2−/− mice) have been described previously (11). CB2−/− mice were backcrossed onto a C57BL/6 background for at least 6 generations. Matched wild-type C57BL/6 mice expressing CB2 (CB2+/+ mice) from the same breedings were used as controls. Skin fibrosis was induced in 6-week-old mice by local injections of bleomycin for 4 weeks, as previously described (12). Briefly, 100 μl of bleomycin dissolved in 0.9% NaCl at a concentration of 0.5 mg/ml was administered every other day by subcutaneous injection into defined areas of 1 cm2 in the upper back. Subcutaneous injections of 100 μl of 0.9% NaCl were used as controls. Four different groups, 2 groups of CB2−/− mice and 2 groups of CB2+/+ mice, were analyzed. One group of CB2−/− mice and 1 group of CB2+/+ mice were challenged with bleomycin, and the remaining 2 groups were injected with NaCl. Each group consisted of 8 mice. After 4 weeks, animals were killed by cervical dislocation. All animal experiments were approved by the local ethics committee.
Analysis of the effects of CB2 antagonists and agonists on experimental fibrosis.
To confirm the increased susceptibility of CB2−/− mice to fibrosis using a pharmacologic approach, C57BL/6 mice challenged with bleomycin were treated with selective CB2 agonists or antagonists. AM-630 (6-Iodo-2-methyl-1-[2-(4-orpholinyl)ethyl]-1H-indol-3-yl] (4-methoxy-phenyl)methanone) is a selective antagonist of CB2 with a Ki value of 31.2 nM. Other receptors, including CB1, are not affected by AM-630 in pharmacologically relevant concentrations. JWH-133 ([6aR,10aR]-3-[1,1-dimethylbutyl]-6a,7,10,10a-tetrahydro-6,6,9-tri-methyl-6H-di-benzo[b,d] pyran) selectively activates CB2 with a Ki value of 3.4 nM and a 200-fold selectivity compared with CB1. Both compounds were purchased from Biozol (Eiching, Germany). AM-630 and JWH-133 were dissolved in DMSO at a concentration of 10 mg/ml. The working solutions were prepared fresh on the day of the experiments by diluting the stock solutions in NaCl. Mice injected with equal volumes of the solvent were used as controls. Treatment with AM-630 and JWH-133 was started in parallel to bleomycin challenge. AM-630 and JWH-133 were administered by daily intraperitoneal injections at concentrations of 2.5 mg/kg/day in a total volume of 100 μl for 4 weeks. Each treatment group consisted of 6 mice.
Bone marrow transplantation.
To investigate the relative contributions of fibroblasts and other mesenchymal cells and bone marrow—derived cells to the phenotype of CB2−/−, bone marrow transplantation experiments were performed. Female CB2−/− and CB2+/+ mice were used as donors for bone marrow. For isolation of unfractioned bone marrow cells, tibial and femur bones were prepared under sterile conditions. Bone marrow cells were flushed from the bone marrow cavities with phosphate buffered saline (PBS) and subsequently filtered through 70-μm nylon meshes (BD Biosciences, Heidelberg, Germany). Erythrocytes were hemolyzed, and the remaining bone marrow cells were kept on ice until transplantation. All isolated bone marrow cells were transplanted together without further purification or in vitro expansion of a particular subset of cells. Male CB2−/− or CB2+/+ mice received bone marrow transplants at age 4 weeks. Sixteen hours before transplantation, recipient CB2−/− or CB2+/+ mice underwent whole body irradiation with 11 Gy. For transplantation, 2.0 × 106 bone marrow cells from donor mice were resuspended in 0.2 ml of PBS and injected via the tail veins. To exclude sublethal irradiation and reconstitution by the old bone marrow, additional groups of CB2−/− and CB2+/+ mice were irradiated, but did not receive bone marrow transplants. Two weeks after bone marrow transplantation and after confirmation of a complete reconstitution of the hematopoiesis by the transplanted cells, mice were challenged with bleomycin for 4 weeks as described above. All groups of mice consisted of 6 animals each.
Histologic analysis.
Lesional skin areas were excised, fixed in 4% formalin, and embedded in paraffin. Five-micrometer sections were stained with hematoxylin and eosin. Dermal thickness was analyzed at 100-fold magnification by measuring the distance between the epidermal—dermal junction and the dermal—subcutaneous fat junction at 3 sites in lesional skin in each mouse (13). Collagen fibers were visualized by Masson's trichrome staining, according to the recommendations of the manufacturer (Sigma-Aldrich, Munich, Germany), and analyzed at 1,000-fold magnification. Infiltrating leukocytes in the lesional skin of CB2−/− mice, CB2+/+ mice, and CB2+/+ mice treated with AM-630 or JWH-133 were quantified on hematoxylin and eosin—stained sections. Twenty-five different high-power fields from different tissue sites from each mouse were evaluated for polymononuclear cells at 200-fold magnification by an experienced examiner (JHWD) who was blinded with regard to treatment group. Images were captured with a Nikon Eclipse 80i microscope (Badhoevedorp, The Netherlands) equipped with a DSP 3-CCD camera (Sony, Tokyo, Japan).
Statistical analysis.
Data are expressed as the mean ± SEM. The Mann-Whitney U test was used for statistical analyses. P values less than 0.05 were considered significant.
RESULTS
Increased sensitivity of CB2−/− mice to bleomycin- induced dermal fibrosis.
To evaluate whether CB2 plays a role in the development of dermal fibrosis, CB2−/− mice and CB2+/+ mice (wild-type mice) were challenged with bleomycin. Skin architecture and dermal thickness did not differ between CB2−/− mice and CB2+/+ mice injected with NaCl, suggesting that the skin phenotype is not altered in CB2−/− mice under nonfibrotic conditions (Figures 1 and 2). Upon injection of bleomycin, dermal thickness increased in both CB2−/− and CB2+/+ mice (Figures 1 and 2). However, CB2−/− mice were significantly more susceptible to the induction of fibrosis by bleomycin than were CB2+/+ mice. In CB2−/− mice, the mean ± SEM increase in dermal thickness was 72 ± 6%, as compared with 45 ± 6% in CB2+/+ mice (P = 0.028) (Figure 2). These data suggest that CB2−/− mice might be more susceptible to bleomycin-induced fibrosis.
Figure 1. Increase in dermal thickness in CB2−/− mice upon challenge with bleomycin. Representative sections from a CB2+/+ mouse injected with NaCl, a CB2−/− mouse injected with NaCl, a CB2+/+ mouse injected with bleomycin, and a CB2−/− mouse injected with bleomycin are shown. No differences in dermal thickness were observed between CB2−/− mice injected with NaCl and CB2+/+ mice injected with NaCl. However, challenge with bleomycin resulted in significantly greater dermal thickening in CB2−/− mice than in CB2+/+ mice. (Original magnification × 100.)
Figure 2. Increased sensitivity of CB2−/− mice to dermal fibrosis upon challenge with bleomycin. Bars show the mean and SEM fold increase in dermal thickness compared with CB2+/+ mice injected with NaCl (n = 8 mice per group).
Effect of pharmacologic modification of CB2 signaling on the outcome of dermal fibrosis.
To confirm our findings in CB2−/− mice by a pharmacologic approach, CB2+/+ mice were treated with AM-630, a specific inhibitor of CB2. Treatment of mice with AM-630 during bleomycin challenge lead to a significantly more pronounced increase in dermal thickness than did injection with bleomycin alone (mean ± SEM increase compared with controls 128 ± 8% versus 68 ± 3%; P = 0.004) (Figures 3 and 4). Thus, inhibition of CB2, either by gene silencing or by treatment with small-molecule inhibitors, results in increased sensitivity to bleomycin-induced dermal fibrosis.
Figure 3. Increased sensitivity to bleomycin-induced fibrosis in mice treated with the CB2 antagonist AM-630 and amelioration of experimental fibrosis in mice treated with the selective CB2 agonist JWH-133. Representative sections of lesional skin from a CB2+/+ mouse treated with NaCl, a CB2+/+ mouse treated with bleomycin alone, a CB2+/+ mouse treated with bleomycin and AM-630, and a CB2+/+ mouse treated with bleomycin and JWH-133 are shown. Treatment of CB2+/+ mice with AM-630 significantly increased dermal thickness upon challenge with bleomycin, resembling the phenotype of CB2−/− mice. In contrast, treatment with the selective CB2 agonist JWH-133 prevented the profibrotic effects of bleomycin. (Original magnification × 100.)
Figure 4. Exacerbation of bleomycin-induced fibrosis after pharmacologic inhibition of CB2 by AM-630 and reduction in dermal thickening after activation of CB2 by JHW-133 in CB2+/+ mice. Bars show the mean and SEM fold increase in dermal thickness compared with mice injected with NaCl (n = 6 mice per group).
To evaluate whether activation of CB2 exerts antifibrotic effects, CB2+/+ mice were treated with the selective CB2 agonist JWH-133 in pharmacologically relevant concentrations during bleomycin challenge. Treatment with JWH-133 ameliorated the profibrotic effects of bleomycin and significantly reduced dermal thickening, by 35 ± 3% (P = 0.004) (Figures 3 and 4). Thus, activation of CB2 signaling exerts antifibrotic effects in vivo.
CB2 regulation of the infiltration of leukocytes into lesional skin.
Accumulation of leukocytes in lesional skin is characteristic of the mouse model of bleomycin-induced dermal fibrosis and of early stages of SSc. To analyze whether CB2 affects leukocyte infiltration in experimental fibrosis, the numbers of leukocytes in lesional skin were quantified. The numbers of infiltrating leukocytes were significantly increased in CB2−/− mice compared with CB2+/+ mice. The mean ± SEM number of leukocytes detected per high-power field was 38.2 ± 3.1 in sections from the lesional skin of CB2−/− mice, compared with 27.7 ± 3.5 in CB2+/+ mice (P = 0.016). Consistent with these results, greater numbers of leukocytes were also detected in CB2+/+ mice upon treatment with the CB2 antagonist AM-630 (mean ± SEM 37.1 ± 1.6; P = 0.047 versus CB2+/+ mice treated with bleomycin alone). However, the infiltration of leukocytes into lesional skin was strongly reduced in CB2+/+ mice treated with the CB2 agonist JWH-133 (mean ± SEM 17.7 ± 1.4; P = 0.009 versus CB2+/+ mice treated with bleomycin alone).
Mediation of the phenotype of CB2−/− mice by bone marrow—derived cells.
To investigate whether the increased susceptibility of CB2−/− mice to fibrosis is based on lack of CB2 on bone marrow—derived cells or on fibroblasts and other mesenchymal cells, bone marrow transplantation experiments were performed, and the resulting chimeric mice were challenged with bleomycin. Transplantation of CB2−/− mice with bone marrow from CB2+/+ mice and subsequent challenge with bleomycin resulted in comparable increases in dermal thickness as in CB2+/+ mice with CB2+/+ bone marrow, with mean ± SEM increases of 53 ± 3% and 45 ± 6%, respectively, as compared with NaCl-treated mice (Figures 5 and 6). In contrast, transplantation of CB2+/+ mice with bone marrow cells from CB2−/− mice fully resembled the phenotype of CB2−/− mice. Bleomycin challenge of CB2+/+ mice with bone marrow from CB2−/− mice resulted in a mean ± SEM increase in dermal thickness of 77 ± 7%. This increase was significantly higher than in CB2+/+ mice with CB2+/+ bone marrow (P = 0.03), but did not differ significantly from the increase observed in CB2−/− mice (P = 0.67) (Figures 5 and 6). Taken together, these data demonstrate that CB2 deficiency in bone marrow—derived cells mediates the increased susceptibility of CB2−/− mice to experimental fibrosis.
Figure 5. Mediation of the increased susceptibility of CB2−/− mice to experimental fibrosis by bone marrow (BM)—derived cells. CB2−/− mice were transplanted with bone marrow from CB2+/+ mice and vice versa after lethal irradiation, and subsequently challenged with bleomycin. The dermal thickening in these chimeric mice was compared with that in CB2+/+ mice with CB2+/+ bone marrow and CB2−/− mice with CB2−/− bone marrow. The phenotype of CB2−/− mice was completely mimicked by transplantation of CB2−/− bone marrow into CB2+/+ mice, and CB2−/− mice with CB2+/+ bone marrow did not display increased sensitivity to fibrosis. (Original magnification × 100.)
Figure 6. Dermal thickness in CB+/+ mice with CD+/+ bone marrow (BM), CB−/− mice with CB+/+ bone marrow, CB+/+ mice with CB−/− bone marrow, and CB−/− mice with CB−/− bone marrow after treatment with bleomycin. Bone marrow transplantation experiments revealed that the increased sensitivity of CB2−/− mice to bleomycin-induced fibrosis is mediated by bone marrow—derived cells. Bars show the mean and SEM percent increase in dermal thickness compared with CB2+/+ mice with CB2+/+ bone marrow injected with NaCl (n = 6 mice per group).
DISCUSSION
In the present study, we demonstrated that CB2 exerts antifibrotic effects in vivo. Inhibition of CB2 signaling by either gene silencing or treatment with small-molecule inhibitors increased susceptibility to bleomycin-induced dermal fibrosis. Accumulation of thickened collagen fibers and dermal thickening upon challenge with bleomycin were more pronounced in CB2−/− mice and in mice treated with the CB2 antagonist AM-630. Conversely, activation of CB2 signaling by the selective agonist JWH-133 prevented the profibrotic effects of bleomycin and significantly reduced dermal thickening at pharmacologically relevant doses. Consistent with our results, antifibrotic effects of CB2 have previously been observed in the liver and pancreas (7, 14).
Based on these preclinical data, activation of CB2 by selective agonists might be a novel approach for the treatment of fibrosis. In SSc, this approach might be particularly promising for patients with early inflammatory stages of the disease. Of note, CB2 agonists are currently being evaluated in clinical trials for multiple sclerosis (5, 6). In these clinical trials, CB2 agonists have been well tolerated. Major adverse events such as dizziness, drowsiness, or headache were due to incomplete selectivity of these first compounds, which resulted in coactivation of the CB1 receptor. Thus, our study could have direct clinical impact, since CB2 agonists are well tolerated and already available for clinical trials in patients with fibrotic disorders such as SSc.
We have shown that CB2 mediates its antifibrotic effects in vivo by inhibiting the infiltration of leukocytes into lesional skin in preclinical models of SSc. Inhibition of CB2 signaling by either genetic knockout or treatment with the chemical inhibitor AM-630 increased the accumulation of leukocytes in bleomycin-injected areas of the skin. Consistent with our findings in experimental fibrosis, exacerbation of inflammation in mice deficient in CB1 and CB2 (CB1−/−CB2−/− mice) was previously observed in experimental contact dermatitis (9). Infiltration of leukocytes into lesional skin is a common feature in the early stages of SSc and in the mouse model of bleomycin-induced dermal fibrosis. Infiltrating leukocytes activate the collagen synthesis of resident fibroblasts by the release of a profibrotic mediator. Thus, CB2 signaling might affect the outcome of fibrosis indirectly by orchestrating the infiltration of leukocytes into lesional skin rather than by direct effects on the collagen synthesis of fibroblasts.
Consistent with this hypothesis, the increased susceptibility of CB2−/− mice to experimental fibrosis was fully replicated by transplantation of CB2-deficient bone marrow cells into CB2+/+ mice. In contrast, the phenotype of CB2−/− mice transplanted with CB2+/+ bone marrow did not differ from that of CB2+/+ mice with CB2+/+ bone marrow, evidence against a direct effect of CB2 signaling on fibroblasts. This hypothesis was further supported by the results of studies of cultured fibroblasts. Neither activation nor inhibition of CB2 altered the release of collagen or other major extracellular matrix proteins in cultured fibroblasts from SSc patients or healthy individuals (results not shown). Therefore, we propose that activation of CB2 inhibits leukocyte activation and migration into lesional skin in fibrotic diseases. The reduced infiltration of leukocytes and the decreased release of profibrotic mediators subsequently results in a less pronounced stimulation of resident fibroblasts and in protection against fibrosis.
On the molecular level, CB2 might exert its effects by regulating the expression of chemokines and affecting chemotaxis of leukocytes (6, 15). In particular, regulation of the expression of monocyte chemotactic protein 1 (MCP-1; CCL2) and its receptor CCR2 might be important in fibrotic diseases. MCP-1 exerts potent chemotactic effects on inflammatory cells via activation of CCR2. MCP-1 is up-regulated in the skin of SSc patients and has been implicated in the pathogenesis of the disease (16). MCP-1 might contribute to the development of fibrosis either directly by effecting fibroblasts or indirectly by stimulating the release of interleukin-4 from T cells (17, 18). A previous study demonstrated that activation of CB2 reduced the expression of MCP-1 and its receptor CCR2 in myeloid progenitor cells, whereas inhibition of CB2 stimulated their expression (15).
The mouse model of bleomycin-induced dermal fibrosis has limitations. The effect of bleomycin is not irreversible in this model, and dermal fibrosis spontaneously resolves over time. However, we recently demonstrated that dermal thickening progresses during the first 6 weeks of challenge with bleomycin and that the fibrotic changes persist for at least 3 weeks after the last bleomycin injection (19). Thus, we performed our analyses during a dynamic phase of progressive dermal fibrosis. Another limitation of the mouse model of bleomycin-induced dermal fibrosis is that this model mimics early, inflammatory stages of SSc, but is less suitable as a model of later stages of SSc, where inflammatory infiltrates are rarely observed. Therefore, our findings in the mouse model of bleomycin-induced fibrosis indicate a potential role of CB2 in the early stages of SSc, but the results should not be extrapolated to later stages of SSc. Moreover, additional studies are necessary to investigate the role of CB2 in the development of fibrosis in other organ systems, such as the lung.
In summary, we have demonstrated that CB2 exerts antifibrotic effects by limiting leukocyte infiltration and subsequent fibroblast activation in lesional skin. These data suggest that activation of CB2 might be a promising approach for therapy for the early inflammatory stages of SSc. Since CB2 agonists are available and seem to be well tolerated, these findings might lead to clinical trials in patients with SSc and other fibrotic diseases.
AUTHOR CONTRIBUTIONS
Dr. Jörg H. W. Distler had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study design. Akhmetshina, Oliver Distler, Schett, Jörg H. W. Distler.
Acquisition of data. Akhmetshina, Dees, Busch, Beer, Sarter, Zwerina, Jörg H. W. Distler.
Analysis and interpretation of data. Akhmetshina, Dees, Oliver Distler, Schett, Jörg H. W. Distler.
Manuscript preparation. Akhmetshina, Oliver Distler, Schett, Jörg H. W. Distler.
Statistical analysis. Jörg H. W. Distler.
Provision of materials. Zimmer.
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Source: The cannabinoid receptor CB2 exerts antifibrotic effects in experimental dermal fibrosis - Akhmetshina - 2009 - Arthritis & Rheumatism - Wiley Online Library
