A novel synthetic, nonpsychoactive cannabinoid acid (HU-320) with antiinflammatory properties in murine collagen-induced arthritis

OBJECTIVE: To explore the antiarthritic potential of a novel synthetic cannabinoid acid, Hebrew University-320 (HU-320), in the DBA/1 mouse model of arthritis, and to investigate in vitro antiinflammatory and immunosuppressive effects of HU-320 on macrophages and lymphocytes. METHODS: DBA/1 mice were immunized with bovine type II collagen (CII) to induce arthritis and then injected intraperitoneally daily with HU-320. The effects of treatment on arthritic changes in hind feet were assessed clinically and histologically, and draining lymph node responses to CII were assayed. Murine splenic and human blood lymphocytes were cultured to study the effect of HU-320 on polyclonal mitogenic stimulation. Macrophage cultures were set up to evaluate in vitro effects of HU-320 on production of tumor necrosis factor alpha (TNF alpha) and reactive oxygen intermediates (ROIs). The effect of HU-320 administration on lipopolysaccharide-induced serum TNF levels was assayed using C57BL/6 mice. Bioactive TNF production was measured using BALB/c clone 7 target cells. Evaluation of HU-320 psychoactivity was performed using established laboratory tests on Sabra mice. RESULTS: Systemic daily administration of 1 and 2 mg/kg HU-320 ameliorated established CII-induced arthritis. Hind foot joints of treated mice were protected from pathologic damage. CII-specific and polyclonal responses of murine and human lymphocytes were down-modulated. HU-320 inhibited production of TNF from mouse macrophages and of ROIs from RAW 264.7 cells and suppressed the rise in serum TNF level following endotoxin challenge. HU-320 administration yielded no adverse psychotropic effects in mice. CONCLUSION: Our studies show that the novel synthetic cannabinoid acid HU-320 has strong antiinflammatory and immunosuppressive properties while demonstrating no psychoactive effects. The profound suppressive effects on cellular immune responses and on the production of proinflammatory mediators all indicate its usefulness as a novel nonpsychoactive, synthetic antiinflammatory product.     

P-selectin glycoprotein ligand 1 therapy ameliorates established collagen-induced arthritis in DBA/1 mice partly through the suppression of tumour necrosis factor

We investigated the therapeutic potential of P-selectin glycoprotein ligand (PSGL)-1 in established collagen-induced arthritis (CIA) in DBA/1 mice. PSGL-1 is the high-affinity specific ligand for P-selectin and is thus important in cell recruitment to inflammatory sites. I-316 PSGL-1 or rPSGL-1Ig fusion protein were administered to mice after the onset of clinical arthritis for 10 days, and the effect of treatment on both clinical and histopathological progression of disease was studied. It was found that both PSGL-1 biologicals effectively suppressed progression of clinical arthritis, and this was accompanied by protection against damage of joint tissues. We sought to investigate a mechanism underlying the effect of rPSGL-1Ig on the reduction of clinical arthritis. Blockade of PSGL-1/P-selectin interaction blocks recruitment of leucocytes, thus we observed a notable reduction in viable cell numbers of synoviocytes from rPSGL-1Ig treated mice. In view of this finding we suspected an effect of treatment on the production of pro-inflammatory mediators such as bioactive tumour necrosis factor-alpha (TNF) in synovial membrane ex vivo cell cultures. Production of TNF was reduced in arthritic mice that had been treated with rPSGL-1Ig. To further investigate the mechanism of rPSGL-1Ig, we explored the possibility that PSGL-1 might also have a direct signalling effect on TNF release from inflammatory cells. Thus synoviocyte cultures from arthritic mice were incubated with rPSGL-1Ig. A significant reduction in the spontaneous bioactive TNF release from these cultures was noted. We therefore confirmed these surprising findings using cultures of a mouse macrophage like cell line RAW 264.7, stimulated by LPS. Our results indicate that both forms of PSGL-1 have significant therapeutic effects in CIA murine model of RA. The mechanism of action involves reduced cellularity of synovium as anticipated, along with a reduction in TNF production from inflammatory cells in the synovium. The latter mechanism needs further mechanistic analysis.     

Blockade of NKG2D ameliorates disease in mice with collagen‐induced arthritis: A potential pathogenic role in chronic inflammatory arthritis

Objective

To assess the role of the activating receptor NKG2D in arthritis.

Methods

Levels of NKG2D and its ligands were determined by fluorescence‐activated cell sorting, real‐time polymerase chain reaction, and immunohistochemistry in rheumatoid arthritis (RA) synovial membrane tissue and in paw tissue from arthritic mice. Arthritis was induced in DBA/1 mice by immunization with type II collagen, and mice were treated intraperitoneally with a blocking anti‐NKG2D antibody (CX5) on days 1, 5, and 8 after clinical onset and were monitored for 10 days.

Results

We demonstrated expression of NKG2D and its ligands on human RA synovial cells and extended this finding to the paws of arthritic mice. Expression of messenger RNA for the NKG2D ligand Rae‐1 was up‐regulated, and NKG2D was present predominantly on natural killer (NK) and CD4+ T cells, in arthritic paw cell isolates. NKG2D was down‐modulated during the progression of collagen‐induced arthritis (CIA). NKG2D expression in arthritic paws was demonstrated by immunohistochemistry. Blockade of NKG2D ameliorated established CIA, with significant reductions in clinical scores and paw swelling. Histologic analysis of arthritic joints from anti‐NKG2D–treated mice demonstrated significant joint protection, compared with control mice. Moreover, anti‐NKG2D treatment significantly reduced both interleukin‐17 production from CD4+ T cells in arthritic paws and splenic NK cell cytotoxic effector functions in vivo and in vitro.

Conclusion

Our findings indicate that blockade of NKG2D in a murine model and in human explants has beneficial therapeutic potential that merits further investigation in RA.

     

Suppression of tumour necrosis factor production from mononuclear cells by a novel synthetic compound, CLX-090717

Objectives. To evaluate the clinical efficacy of a novel syntheticperoxisome proliferator-activated receptor gamma (PPAR-) agonist,CLX-090717, in several in vitro cell culture systems and murineCIA, an experimental model of RA. Methods. Peripheral blood monocytes purified by elutriation,and rheumatoid synovial cells isolated from clinical tissuewere cultured with CLX-090717 and TNF- release was measured.Molecular mechanism of action was analysed by western blottingand electrophoretic mobility shift assay. Thioglycollate-elicitedmurine peritoneal macrophages were cultured with CLX-090717and lipopolysaccharide (LPS)-induced TNF- release was assayed.Therapeutic studies were done in mice with established arthritisby evaluating clinical parameters and histology. In addition,type II collagen response of lymphocytes from mice with CIAwas examined. Results. CLX-090717 significantly inhibited spontaneous TNF-release by RA synovial membrane cells, as well as LPS-inducedTNF- release from human and murine monocytic cells. Inhibitionof TNF- in monocytes was mediated partially through a nuclearfactor-B (NF-B)-dependent pathway, as judged by sustained levelsof IB in cytosolic extracts and a reduced level of LPS-inducedNF-B activity in nuclear extracts. CLX-090717 reduced clinicalsigns of arthritis and damage to joint architecture when administeredtherapeutically to arthritic mice. Mechanisms of action in CIAinvolved the reduction in proliferation of arthritic lymphocytesto antigen in vitro as well as reduced TNF- release. Conclusions. Our data suggest that the synthetic compound CLX-090717has potential as a small molecular weight anti-inflammatorytherapeutic for chronic inflammatory conditions. KEY WORDS: Arthritis, Collagen, Cytokines, Monocytes, Synovium, Inflammation     

A novel synthetic,nonpsychoactive cannabinoid acid (HU‐320) with antiinflammatory properties in murine collagen‐induced arthritis

Objective

To explore the antiarthritic potential of a novel synthetic cannabinoid acid, Hebrew University–320 (HU‐320), in the DBA/1 mouse model of arthritis, and to investigate in vitro antiinflammatory and immunosuppressive effects of HU‐320 on macrophages and lymphocytes.

Methods

DBA/1 mice were immunized with bovine type II collagen (CII) to induce arthritis and then injected intraperitoneally daily with HU‐320. The effects of treatment on arthritic changes in hind feet were assessed clinically and histologically, and draining lymph node responses to CII were assayed. Murine splenic and human blood lymphocytes were cultured to study the effect of HU‐320 on polyclonal mitogenic stimulation. Macrophage cultures were set up to evaluate in vitro effects of HU‐320 on production of tumor necrosis factor α (TNFα) and reactive oxygen intermediates (ROIs). The effect of HU‐320 administration on lipopolysaccharide‐induced serum TNF levels was assayed using C57BL/6 mice. Bioactive TNF production was measured using BALB/c clone 7 target cells. Evaluation of HU‐320 psychoactivity was performed using established laboratory tests on Sabra mice.

Results

Systemic daily administration of 1 and 2 mg/kg HU‐320 ameliorated established CII‐induced arthritis. Hind foot joints of treated mice were protected from pathologic damage. CII‐specific and polyclonal responses of murine and human lymphocytes were down‐modulated. HU‐320 inhibited production of TNF from mouse macrophages and of ROIs from RAW 264.7 cells and suppressed the rise in serum TNF level following endotoxin challenge. HU‐320 administration yielded no adverse psychotropic effects in mice.

Conclusion

Our studies show that the novel synthetic cannabinoid acid HU‐320 has strong antiinflammatory and immunosuppressive properties while demonstrating no psychoactive effects. The profound suppressive effects on cellular immune responses and on the production of proinflammatory mediators all indicate its usefulness as a novel nonpsychoactive, synthetic antiinflammatory product.

     

Antagonism of the human epidermal growth factor receptor family controls disease severity in murine collagen-induced arthritis

OBJECTIVE: To evaluate the therapeutic potential of the human epidermal growth factor receptor (HER) family inhibitor, herstatin, in an animal model of arthritis. METHODS: Constructs of herstatin and modified tissue plasminogen activator (tPA)-herstatin were expressed in HEK 293T cells, and secreted protein was analyzed by Western blotting. Tissue PA-herstatin adenovirus (Ad-tPA-Her) was prepared, and titers established. Gene expression of Ad-tPA-Her was determined by polymerase chain reaction using HeLa cells. Pharmacokinetics of gene and protein expression in vivo in liver tissue and serum samples were confirmed via intravenous administration of Ad-tPA-Her. Clinical signs of disease were monitored in arthritic DBA/1 mice after therapeutic administration of Ad-tPA-Her, and histologic analysis of hind foot specimens was performed. RESULTS: Native herstatin was not secreted in supernatants, while modified tPA-herstatin was detected in abundance. HeLa cells stably expressed the tPA-herstatin gene when infected with virus. Additionally, tPA-herstatin gene and protein expression was observed over time in mice treated with virus. Importantly, Ad-tPA-Her, when administered therapeutically to arthritic mice, controlled clinical and histologic signs of disease and reduced the number of joints with severe damage. CONCLUSION: Our results support the notion that the human epidermal growth factor receptor family has a role in the progression of collagen-induced arthritis. The novel tPA-herstatin fusion protein could be used as an effective therapeutic tool for control of inflammatory disorders involving an angiogenic component.     

The nonpsychoactive cannabis constituent cannabidiol is an oral anti-arthritic therapeutic in murine collagen-induced arthritis

The therapeutic potential of cannabidiol (CBD), the major nonpsychoactive component of cannabis, was explored in murine collagen-induced arthritis (CIA). CIA was elicited by immunizing DBA/1 mice with type II collagen (CII) in complete Freund's adjuvant. The CII used was either bovine or murine, resulting in classical acute CIA or in chronic relapsing CIA, respectively. CBD was administered after onset of clinical symptoms, and in both models of arthritis the treatment effectively blocked progression of arthritis. CBD was equally effective when administered i.p. or orally. The dose dependency showed a bell-shaped curve, with an optimal effect at 5 mg/kg per day i.p. or 25 mg/kg per day orally. Clinical improvement was associated with protection of the joints against severe damage. Ex vivo, draining lymph node cells from CBD-treated mice showed a diminished CII-specific proliferation and IFN-gamma production, as well as a decreased release of tumor necrosis factor by knee synovial cells. In vitro effects of CBD included a dose-dependent suppression of lymphocyte proliferation, both mitogen-stimulated and antigen-specific, and the blockade of the Zymosan-triggered reactive oxygen burst by peritoneal granulocytes. It also was found that CBD administration was capable of blocking the lipopolysaccharide-induced rise in serum tumor necrosis factor in C57/BL mice. Taken together, these data show that CBD, through its combined immunosuppressive and anti-inflammatory actions, has a potent anti-arthritic effect in CIA.     

Analysing the effect of novel therapies on cytokine expression in experimental arthritis

Type II collagen-induced arthritis (CIA) is an animal model of rheumatoid arthritis that has been used extensively to address questions of disease pathogenesis and to validate novel therapeutic targets. Susceptibility to CIA is strongly associated with major histocompatibility complex class II genes, and the development of arthritis is accompanied by a robust T- and B-cell response to type II collagen. The main pathological features of CIA include proliferative synovitis with infiltration of inflammatory cells, pannus formation, cartilage degradation, erosion of bone and fibrosis. Pro-inflammatory cytokines, such as tumour necrosis factor alpha and interleukin-1beta, are expressed in the arthritic joints in both murine CIA and human rheumatoid arthritis, and blockade of these molecules results in amelioration of disease. Hence, there is a great deal of interest in the development of small-molecular-weight inhibitors of pro-inflammatory cytokines. There is also interest in the development and testing of drugs with the capacity to modulate the immune pathways involved in driving the inflammatory response in arthritis. For these reasons, there is a need to monitor the effect of novel treatments on cytokine expression in vivo. In this review, we outline the various techniques used to detect cytokines in experimental arthritis and describe how these techniques have been used to quantify changes in cytokine expression following therapeutic intervention.