Karen L. Wright

Cannabinoid CB2 receptors in the gastrointestinal tract: a regulatory system in states of inflammation

The emerging potential for the cannabinoid (CB) system in modulating gastrointestinal inflammation has gained momentum over the last few years. Traditional and anecdotal use of marijuana for gastrointestinal disorders, such as diarrhoea and abdominal cramps is recognized, but the therapeutic benefit of cannabinoids in the 21st century is overshadowed by the psychoactive problems associated with CB1 receptor activation. However, the presence and function of the CB2 receptor in the GI tract, whilst not yet well characterized, holds great promise due to its immunomodulatory roles in inflammatory systems and its lack of psychotropic effects. This review of our current knowledge of CB2 receptors in the gastrointestinal tract highlights its role in regulating abnormal motility, modulating intestinal inflammation and limiting visceral sensitivity and pain. CB2 receptors represent a braking system and a pathophysiological mechanism for the resolution of inflammation and many of its symptoms. CB2 receptor activation therefore represents a very promising therapeutic target in gastrointestinal inflammatory states where there is immune activation and motility dysfunction.

Evidence that SHIP-1 contributes to phosphatidylinositol 3,4,5-trisphosphate metabolism in T lymphocytes and can regulate novel phosphoinositide 3-kinase effectors

The leukemic T cell line Jurkat is deficient in protein expression of the lipid phosphatases Src homology 2 domain containing inositol polyphosphate phosphatase (SHIP) and phosphatase and tensin homolog deleted on chromosome ten (PTEN). We examined whether the lack of expression of SHIP-1 and PTEN is shared by other leukemic T cell lines and PBLs. Analysis of a range of cell lines and PBLs revealed that unlike Jurkat cells, two other well-characterized T cell lines, namely CEM and MOLT-4 cells, expressed the 5′-phosphatase SHIP at the protein level. However, the 3-phosphatase PTEN was not expressed by CEM or MOLT-4 cells or Jurkat cells. The HUT78 cell line and PBLs expressed both SHIP and PTEN. Jurkat cells exhibited high basal levels of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3; the lipid substrate for both SHIP and PTEN) as well as saturated protein kinase B (PKB) phosphorylation. Lower levels of PI(3,4,5)P3 and higher levels of phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2) as well as unsaturated constitutive phosphorylation of PKB were observed in CEM and MOLT-4 cells compared with Jurkat cells. In PBLs and HUT78 cells which express both PTEN and SHIP-1, there was no constitutive PI(3,4,5)P3 or PKB phosphorylation, and receptor stimuli were able to elicit robust phosphorylation of PKB. Expression of a constitutively active SHIP-1 protein in Jurkat cells was sufficient to reduce both constitutive PKB membrane localization and PKB phosphorylation. Together, these data indicate important differences between T leukemic cells as well as PBLs, regarding expression of key lipid phosphatases. This study provides the first evidence that SHIP-1 can influence the constitutive levels of PI(3,4,5)P3 and the activity of downstream phosphoinositide 3-kinase effectors in T lymphocytes.

Cytokine-induced Apoptosis in Epithelial HT-29 Cells Is Independent of Nitric Oxide Formation EVIDENCE FOR AN INTERLEUKIN-13-DRIVEN PHOSPHATIDYLINOSITOL 3-KINASE-DEPENDENT SURVIVAL MECHANISM

A combination of the pro-inflammatory cytokines interleukin (IL)-1α, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α induces nitric oxide synthase mRNA expression and nitric oxide (NO) generation in the human colon carcinoma cell line HT-29. This can be inhibited by pretreatment with IL-13 via a phosphatidylinositol (PI) 3-kinase-dependent mechanism (Wright, K., Ward, S. G., Kolios, G., and Westwick, J. (1997)J. Biol. Chem. 272, 12626–12633). Since NO has been implicated in regulating mechanisms leading to cell death, while activation of PI 3-kinase-dependent signaling cascades are thought to be involved with promoting cell survival events, we have investigated the outcome of these cytokine treatments on apoptosis and cell survival of HT-29 cells. Initiation of apoptosis can be achieved by the combinations of IFN-γ/TNF-α, IFN-γ/CD95, IL-1α/IFN-γ, and IL-1α/IFN-γ/TNF-α to varying extents. Induction of apoptotic markers by HT-29 cells in response to cytokine treatment is not dependent on NO production. Pretreatment with IL-13 protects against IL-1α/IFN-γ/TNF-α- and IFN-γ/TNF-α- as well as IFN-γ/CD95-induced (but not IL-1α/IFN-γ-induced) cell death. In addition, IFN-γ/TNF-α and IL-1α/IFN-γ/TNF-α stimulate activation of caspase-8 and caspase-3, which IL-13 pretreatment was able to partially inhibit and delay. IL-13 also stimulates activation of the major PI 3-kinase effector, protein kinase B. The PI 3-kinase inhibitors wortmannin and LY294002 inhibit IL-13 stimulation of protein kinase B as well as the cell survival effects of IL-13. These data demonstrate that cytokine-induced apoptosis of HT-29 cells is NO-independent and that the activation of a PI 3-kinase-dependent signaling cascade by IL-13 is a key signal responsible for the inhibition of apoptosis.

Optimal Chemotactic Responses of Leukemic T Cells to Stromal Cell-Derived Factor-1 Requires the Activation of Both Class IA and IB Phosphoinositide 3-Kinases

Stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 are a multifunctional chemokine/receptor system with essential roles in the development of the immune system and other aspects of embryogenesis, including vascularization and organ development. SDF-1 is also a potent chemoattractant for T cells and has roles in both inflammation and immune homeostasis. Our group has previously demonstrated that phosphoinositide 3-kinase (PI 3-kinase) is activated in SDF-1-stimulated T cells and is indeed required for SDF-1-mediated chemotaxis. In this study Jurkat clones were established, stably expressing dominant negative constructs of class IA and class IB PI 3-kinases under the control of the tetracycline off inducible gene system, to determine the relative roles of these PI 3-kinases in SDF-1 signaling. Our results show that expression of either kinase-dead PI3Kγ (KD-PI3Kγ) or Δp85 (a construct unable to bind class IA p110α, -β, or -δ) leads to a partial inhibition of SDF-1-stimulated protein kinase B phosphorylation, but had no effect on SDF-1-induced phosphorylation of the mitogen-activated protein kinase ERK1/2. Functional studies demonstrated that expression of KD-PI3Kγ markedly inhibited SDF-1-mediated chemotaxis, typically eliciting 40–60% inhibition. Interestingly, the expression of Δp85 also leads to inhibition of the SDF-1-mediated chemotactic response, albeit to a much lesser extent than achieved with the KD-PI3Kγ mutant, typically in the range of 20–40% inhibition. Furthermore, the inhibition of chemotaxis by the expression of dominant negative class IA or class IB PI 3-kinases could be enhanced by the presence of the PI 3-kinase inhibitor LY294002. Together, these results demonstrate that optimal chemotactic response of leukemic T cells to SDF-1 requires the activation of both class IA and class IB PI 3-kinases.

C-X-C and C-C chemokine expression and secretion by the human colonic epithelial cell line, HT-29: differential effect of T lymphocyte-derived cytokines

Differential chemokine production by colonic epithelial cells is thought to contribute to the characteristic increased infiltration of selected population of leukocytes cells in inflammatory bowel disease. We have previously demonstrated that IL‐13 enhances IL‐1α‐induced IL‐8 secretion by the colonic epithelial cell line HT‐29. We have now explored the C‐C chemokine expression and modulation in this system. The combination of TNF‐α and IFN‐γ was the minimal stimulation required for regulated on activation, normal T cell expressed and secreted (RANTES) and monocyte chemoattractant protein (MCP‐1) mRNA expression and secretion by HT‐29 cells. The same stimulation induced a stronger IL‐8 mRNA expression and secretion. Pretreatment with IL‐13 or IL‐4, reduced significantly the RANTES, and MCP‐1, but not IL‐8 mRNA expression and secretion. In contrast, IL‐10 had no effect on either MCP‐1, or RANTES, or IL‐8 generation. Pretreatment of HT‐29 cells with wortmannin suggested that the IL‐13‐induced inhibition of C‐C chemokine expression is via activation of a wortmannin‐sensitive phosphatidylinositol 3‐kinase. These data demonstrate that colonic epithelial cell chemokine production can be differentially regulated by T cell‐derived cytokines and suggest an interplay between epithelial cells and T lymphocytes potentially important in the intestinal inflammation.

The Chemokines CXCL9, CXCL10, and CXCL11 Differentially Stimulate Gαi-Independent Signaling and Actin Responses in Human Intestinal Myofibroblasts

Intestinal myofibroblasts have been implicated in the pathogenesis of chronic inflammatory conditions such as Crohn’s disease via interactions with an elaborate network of cytokines, growth factors, and other inflammatory mediators. CXCR3 is a Gαi protein-coupled receptor that binds the proinflammatory chemokines CXCL9, CXCL10, and CXCL11, which are released from the intestinal epithelium. The three CXCR3 ligands shared the ability to activate biochemical (e.g., PI3K and MAPK activation) and functional events (actin reorganization) in intestinal myofibroblasts. However, CXCL11 is unique in its ability to elevate intracellular calcium. Surprisingly, although CXCR3 mRNA is detectable in these myofibroblasts, there is no detectable surface expression of CXCR3. Furthermore, the biochemical responses and actin reorganization stimulated by the CXCR3 ligands in intestinal myofibroblasts are insensitive to the Gαi inhibitor, pertussis toxin. This suggests either the existence of differential receptor coupling mechanisms in myofibroblasts for CXCR3 that are distinct from those observed in PBLs and/or that these cells express a modified or variant CXCR3 compared with the CXCR3 expressed on PBLs.

Cannabinoids mediate opposing effects on inflammation‐induced intestinal permeability

BACKGROUND AND PURPOSE Activation of cannabinoid receptors decreases emesis, inflammation, gastric acid secretion and intestinal motility. The ability to modulate intestinal permeability in inflammation may be important in therapy aimed at maintaining epithelial barrier integrity. The aim of the present study was to determine whether cannabinoids modulate the increased permeability associated with inflammation in vitro.

Pharmacological Effects of Cannabinoids on the Caco-2 Cell Culture Model of Intestinal Permeability

Activation of cannabinoid receptors decreases emesis, inflammation, gastric acid secretion, and intestinal motility. However, the effects of cannabinoids on intestinal permeability have not yet been established. The aim of the present study is to examine the effects of cannabinoids on intestinal permeability in an in vitro model. Caco-2 cells were grown until fully confluent on inserts in 12-well plates. Transepithelial electrical resistance (TEER) measurements were made as a measure of permeability. EDTA (50 μM) was applied to reversibly increase permeability (reduce TEER). The effects of cannabinoids on permeability in combination with EDTA, or alone, were assessed. Potential target sites of action were investigated using antagonists of the cannabinoid (CB)1 receptor, CB2 receptor, transient receptor potential vanilloid subtype 1 (TRPV1), peroxisome proliferator-activated receptor (PPAR)γ, PPARα, and a proposed cannabinoid receptor. When applied to the apical or basolateral membrane of Caco-2 cells, Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) enhanced the speed of recovery of EDTA-induced increased permeability. This effect was sensitive to cannabinoid CB1 receptor antagonism only. Apical application of endocannabinoids caused increased permeability, sensitive to cannabinoid CB1 receptor antagonism. By contrast, when endocannabinoids were applied basolaterally, they enhanced the recovery of EDTA-induced increased permeability, and this involved additional activation of TRPV1. All cannabinoids tested increased the mRNA of the tight junction protein zona occludens-1, but only endocannabinoids also decreased the mRNA of claudin-1. These findings suggest that endocannabinoids may play a role in modulating intestinal permeability and that plant-derived cannabinoids, such as THC and CBD, may have therapeutic potential in conditions associated with abnormally permeable intestinal epithelium.

Differential regulation of prostaglandin E biosynthesis by interferon-γ in colonic epithelial cells

Cyclooxygenase (COX)‐2 expression and activity in response to pro‐inflammatory cytokines TNFα and IFNγ was evaluated in the colonic epithelial cell line HT29 and the airway epithelial cell line A549. TNFα induced concentration‐ and time‐dependent upregulation of COX‐2 mRNA, protein and prostaglandin (PG)E2 synthesis. Co‐stimulation of TNFα with IFNγ resulted in reduced COX‐2 mRNA and protein expression. IFNγ had no effect on the stability of TNFα‐induced COX‐2 mRNA. TNFα‐induced PGE2 biosynthesis was significantly enhanced by the simultaneous addition of IFNγ and was COX‐2 dependent. The combination of IFNγ and TNFα induced the microsomal prostaglandin E synthase (mPGES), comensurate with the enhanced PGE2 synthesis. These results suggest that, in terms of PGE2 biosynthesis, IFNγ plays a negative regulatory role at the level of COX‐2 expression and a positive regulatory role at the level of mPGES expression. This may have important implications for the clinical use of IFNγ in inflammatory diseases.

Long term cannabinoid receptor (CB1) blockade in obesity: implications for the development of colorectal cancer.

Dear Sir, An important detail in Greenhough et al. is the response of colorectal cancer cell lines to the CB1 antagonist/inverse agonist, AM251. In this study, the authors provide evidence that the plant-derived cannabinoid, delta(9)-tetrahydrocannabinol (THC), inhibits survival and induces apoptosis in these cells through the CB1 receptor. This endorses the results from Ligresti et al., and Patsos et al., that endogenous and synthetic cannabinoids inhibit colorectal cancer cell growth. The launch of Acomplia (rimonabant) in the UK as an antiobesity treatment has led to much debate regarding the benefits of pharmacotherapy for this condition. Rimonabant is a cannabinoid (CB)-1 receptor antagonist/inverse agonist, which acts both centrally and peripherally to inhibit food intake and regulate metabolic functions at multiple cellular pathways. One major side effect of daily consumption of rimonabant (20 mg) is diarrhea, and one mechanism for this effect is through CB1 antagonism/inverse agonism on enteric nerves, leading to an increase in gut motility. We have reported that the CB1 receptor is normally expressed in the cytoplasm and luminal membrane of human intestinal epithelium, and CB1 agonists have been shown to ameliorate disease severity in animal models of intestinal inflammation. In our studies investigating the mechanism of CB1 and CB2 involvement in human Inflammatory Bowel Diseases (IBD), we have utilised a cell line derived from normal human colonic epithelium, NCM460 and the synthetic CB1 antagonist/inverse agonist, AM251 (Tocris, UK). NCM460 cells have a nontransformed phenotype and may more accurately model the intestinal epithelium than cell lines derived from colorectal tumours. AM251 is structurally and pharmacologically similar to rimonabant and has also been shown to reduce food intake and increase gut motility. In standard proliferation experiments, the daily addition of AM251 had a profound, concentration-dependent antiproliferative effect on NCM460 cells, even at optimal growth conditions in medium complete with serum and growth factors (EC50 5 5.08 lM, Figure 1¤). This effect was more pronounced in low serum conditions (EC50 5 1.84 lM, Figure 1n). Concentrations of AM251 greater than 1 lM have effects that may not be CB1-specific, and the concentration of rimonabant that reaches the intestinal tract in humans is unknown, but this data is in contrast to the published antiproliferative effect of CB1 agonists in colorectal tumour cell lines. Indeed, Greenhough et al., state that AM251 reversed the apoptotic effects of THC in these cells. This highlights the differential effects of cannabinoids on normal versus cancer cell types, but more importantly, we suggest that long term daily consumption of CB1 antagonists during antiobesity treatment could have a negative impact on the integrity and maintenance of the intestinal mucosa, with unknown consequences. Persistent mucosal damage might trigger persistent regenerative responses that predispose susceptible individuals to increased cancer risk, as seen in IBD. Furthermore, the risk of developing colorectal cancer is increased in obesity and sustained CB1 blockade could support the survival of altered intestinal epithelial cells that progress through the adenoma-carcinoma sequence. Equally, it could be argued that unlike bona fide primary intestinal epithelium, NCM460 cells can be cultured in vitro, and thus, could be at an early stage of transformation. Therefore, the point at which CB1 antagonists/inverse agonists switch function is important because they could either prevent the development of aberrant epithelial cells, or promote their survival. Careful analysis and interpretation of CB1-mediated functions, in the FIGURE 1 – Colonic epithelial proliferation in the presence of CB1 antagonist/inverse agonist. About 10 NCM460 cells (¤) were cultured in M3 Base medium (INCELL, San Antonio, TX) with either 10% (n) or 1% (n) serum, and maintained at 37 C/5% CO2 (8). AM251 was added daily at the concentrations indicated. After 96 hr, equating to 3 doubling times, viable cells were counted and numbers expressed as a % of vehicle control cells, n5 6.