- [Show abstract] [Hide abstract] ABSTRACT: Background: Non-psychotropic atypical cannabinoids have therapeutic potential in a variety of inflammatory conditions including those of the gastrointestinal tract. Here we examined the effects of the atypical cannabinoid abnormal cannabidiol (Abn-CBD) on wound healing, inflammatory cell recruitment and colitis in mice. Methods: Colitis was induced in CD1 mice by a single intrarectal administration of trinitrobenzene sulfonic acid (TNBS, 4 mg/100 μl in 30 % ethanol) and Abn-CBD and/or the antagonists O-1918 (Abd-CBD), AM251 (CB1 receptor) and AM630 (CB2 receptor), were administered intraperitoneally (all 5 mg/kg, twice daily for 3 days). The degree of colitis was assessed macro- and microscopically and tissue myeloperoxidase activity was determined. The effects of Abn-CBD on wound healing of endothelial and epithelial cells (LoVo) were assessed in a scratch injury assay. Human neutrophils were employed in Transwell assays or perfused over human umbilical vein endothelial cells (HUVEC) to study the effect of Abn-CBD on neutrophil accumulation and transmigration. Results: TNBS-induced colitis was attenuated by treatment with Abn-CBD. Histological, macroscopic colitis scores and tissue myeloperoxidase activity were significantly reduced. These effects were inhibited by O-1918, but not by AM630, and only in part by AM251. Wound healing of both HUVEC and LoVo cells was enhanced by Abn-CBD. Abn-CBD inhibited neutrophil migration towards IL-8, and dose-dependently inhibited accumulation of neutrophils on HUVEC. Conclusions: Abn-CBD is protective against TNBS-induced colitis, promotes wound healing of endothelial and epithelial cells and inhibits neutrophil accumulation on HUVEC monolayers. Thus, the atypical cannabinoid Abn-CBD represents a novel potential therapeutic in the treatment of intestinal inflammatory diseases.
- [Show abstract] [Hide abstract] ABSTRACT: Peloruside A is a novel antimitotic drug originally isolated from the marine sponge Mycale hentschieli. Previous studies showed that peloruside A stabilizes microtubules by binding to a site on tubulin distinct from paclitaxel, another microtubule stabilizing drug. Peloruside A blocks mitosis, but little is known about the effects on other cellular activities. Here we report that peloruside A is the most potent microtubule inhibitor yet tested for its ability to block endothelial cell migration. Quantitative analysis indicated that it inhibits microtubule dynamics and endothelial cell migration at 1/200 th of the concentration needed to inhibit cell division (the cytotoxic concentration), indicating that it could potentially have a large margin of safety when used to specifically target angiogenesis. By comparison, paclitaxel, a well-known cancer therapeutic drug, suppresses cell migration at 1/13 th of its cytotoxic concentration; and vinblastine suppresses cell migration at just slightly below its cytotoxic antimitotic concentration. Thus, different microtubule targeted drugs have varying relative potencies for inhibition of cell migration versus cell division. The results suggest that peloruside A may be an especially useful agent for anti-angiogenesis therapy and point to the likelihood that other antimitotic drugs might be found with an even larger potential margin of safety.
- [Show abstract] [Hide abstract] ABSTRACT: Focal adhesion kinase (FAK)-related nonkinase (FRNK) is a cytoskeletal regulatory protein recently shown to dampen lung fibrosis, yet its role in inflammation is unknown. Here we show for the first time that expressing FRNK negatively regulates IL-4 inflammation using a human model of eosinophil recruitment. Mechanistically, FRNK blocked eosinophil accumulation, firm adhesion and transmigration by preventing transcription and protein expression of VCAM-1 and CCL26. IL-4 activates STAT6 to induce VCAM-1 and CCL26 transcription. We now show IL-4 also increases GATA6 to induce VCAM-1 expression. FRNK blocked IL-4-induced GATA6 transcription, but had little effect on GATA6 protein, and had no effect on STAT6 activation. FRNK can block FAK or Pyk2 signaling, thus we down-regulated these proteins with siRNA to determine if signaling from either protein was involved in regulating VCAM-1 and CCL26. Knocking down FAK, Pyk2 or both had no effect on VCAM-1 or CCL26 expression suggesting that FRNK acts independently of FAK and Pyk2 signaling. Finally, we found that IL-4 induces the late expression of endogenous FRNK. In summary, FRNK represents a novel mechanism for negatively regulating IL-4 inflammation.
- [Show abstract] [Hide abstract] ABSTRACT: Drugs that target microtubules are potent inhibitors of angiogenesis but their mechanism of action is not well understood. To explore this, we treated human umbilical vein endothelial cells with paclitaxel, vinblastine, and colchicine and measured the effects on microtubule dynamics and cell motility. In general, lower drug concentrations suppressed microtubule dynamics and inhibited cell migration whereas higher concentrations were needed to inhibit cell division; but, surprisingly, large drug-dependent differences were seen in the relative concentrations needed to inhibit these two processes. Suppression of microtubule dynamics did not significantly affect excursions of lamellipodia away from the nucleus or prevent cells from elongating; but, it did inhibit retraction of the trailing edges that are normally enriched in dynamic microtubules, thereby limiting cell locomotion. Complete removal of microtubules with a high vinblastine concentration caused a loss of polarity that resulted in roundish rather than elongated cells, rapid but non-directional membrane activity, and little cell movement. The results are consistent with a model in which more static microtubules stabilize the leading edge of migrating cells while more dynamic microtubules locate to the rear where they can remodel and allow tail retraction. Suppressing microtubule dynamics interferes with tail retraction, but removal of microtubules destroys the asymmetry needed for cell elongation and directional motility. The prediction that suppressing microtubule dynamics might be sufficient to prevent angiogenesis was supported by showing that low concentrations of paclitaxel could prevent the formation of capillary-like structures in an in vitro tube formation assay.
- [Show abstract] [Hide abstract] ABSTRACT: Leukocyte recruitment from the vasculature occurs under conditions of haemodynamic shear stress. The parallel plate flow chamber apparatus is an in vitro system that is widely used to study leukocyte recruitment under shear conditions. The flow chamber is a versatile tool for examining adhesive interactions, as it can be used to study a variety of adhesive substrates, ranging from monolayers of primary cells to isolated adhesion molecules, and a variety of adhesive particles, ranging from leukocytes in whole blood to antibody-coated latex beads. We describe here methods for studying leukocyte recruitment to cytokine-stimulated, transfected or transduced endothelial cells using both whole blood and isolated leukocyte suspensions. These methods enable multiple parameters to be measured, including the total number of recruited leukocytes, the percentage of leukocytes that are rolling or firmly adherent, and the percentage of leukocytes that have transmigrated. Although these methods are described for interactions between leukocytes and endothelial cells, they are broadly applicable to the study of interactions between many combinations of adhesive substrates and adhesive particles.
- [Show abstract] [Hide abstract] ABSTRACT: Although microtubules have long been implicated in cell locomotion, the mechanism of their involvement remains controversial. Most studies have concluded that microtubules play a positive role by regulating actin polymerization, transporting membrane vesicles to the leading edge, and/or facilitating the turnover of adhesion plaques. Here we used wild-type and mutant CHO cell lines with alterations in tubulin to demonstrate that microtubules can also act to restrain cell motility. Tubulin mutations or low concentrations of drugs that suppress microtubule dynamics without affecting the amount of microtubule polymer inhibited the rate of migration by preventing microtubule reorganization in the trailing portion of the cells where the more dynamic microtubules are normally found. Under these conditions, cells along the edge of a wound still extended lamellipodia and elongated toward the wound but were inhibited in their ability to retract their tails, thus retarding forward progress. The idea that microtubules normally act to restrain cell locomotion was confirmed by treating cells with high concentrations of nocodazole to depolymerize the microtubule network. In the absence of microtubules, wild-type CHO and HeLa cells could still move at near normal speeds but the movement became more random. We conclude that microtubules act both to restrain cell movement and establish directionality.
- [Show abstract] [Hide abstract] ABSTRACT: Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes. This method focuses on neutrophil transmigration across human endothelial cells. One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC). Neutrophil isolation has been described visually elsewhere; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-α results in increased E-selectin and IL-8 expression. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature. The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass. Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi μslide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.
- [Show abstract] [Hide abstract] ABSTRACT: During an inflammatory response, endothelial cells undergo morphological changes to allow for the passage of neutrophils from the blood vessel to the site of injury or infection. Although endothelial cell junctions and the cytoskeleton undergo reorganization during inflammation, little is known about another class of cellular structures, the focal adhesions. In this study, we examined several focal adhesion proteins during an inflammatory response. We found that there was selective loss of paxillin and focal adhesion kinase (FAK) from focal adhesions in proximity to transmigrating neutrophils; in contrast the levels of the focal adhesion proteins β1-integrin and vinculin were unaffected. Paxillin was lost from focal adhesions during neutrophil transmigration both under static and flow conditions. Down-regulating endothelial paxillin with siRNA blocked neutrophil transmigration while having no effect on rolling or adhesion. As paxillin dynamics are regulated partly by FAK, the role of FAK in neutrophil transmigration was examined using two complementary methods. siRNA was used to down-regulate total FAK protein while dominant-negative, kinase-deficient FAK was expressed to block FAK signaling. Disruption of the FAK protein or FAK signaling decreased neutrophil transmigration. Collectively, these findings reveal a novel role for endothelial focal adhesion proteins paxillin and FAK in regulating neutrophil transmigration.
- [Show abstract] [Hide abstract] ABSTRACT: The endothelium actively participates in neutrophil migration out of the vasculature via dynamic, cytoskeleton-dependent rearrangements leading to the formation of transmigratory cups in vitro, and to domes that completely surround the leukocyte in vivo. Leukocyte-specific protein 1 (LSP1), an F-actin-binding protein recently shown to be in the endothelium, is critical for effective transmigration, although the mechanism has remained elusive. Herein we show that endothelial LSP1 is expressed in the nucleus and cytosol of resting endothelial cells and associates with the cytoskeleton upon endothelial activation. Two-photon microscopy revealed that endothelial LSP1 was crucial for the formation of endothelial domes in vivo in response to neutrophil chemokine keratinocyte-derived chemokine (KC) as well as in response to endogenously produced chemokines stimulated by cytokines (tumor necrosis factor α [TNFα] or interleukin-1β [IL-1β]). Endothelial domes were significantly reduced in Lsp1(-/-) compared with wild-type (WT) mice. Lsp1(-/-) animals not only showed impaired neutrophil emigration after KC and TNFα stimulation, but also had disproportionate increases in vascular permeability. We demonstrate that endothelial LSP1 is recruited to the cytoskeleton in inflammation and plays an important role in forming endothelial domes thereby regulating neutrophil transendothelial migration. The permeability data may underscore the physiologic relevance of domes and the role for LSP1 in endothelial barrier integrity.
- [Show abstract] [Hide abstract] ABSTRACT: Eotaxin-3/CCL26 is an agonist for chemokine receptor 3 (CCR3) and a natural antagonist for CCR1, CCR2 and CCR5. CCL26 expression by non-haematopoietic cells has been well documented; however, no studies to date have demonstrated CCL26 expression by leucocytes. In this study, we investigated the ability of human monocytic cells to produce CCL26 in response to cytokines. We found that interleukin-4 (IL-4) increased the expression of CCL26 messenger RNA (mRNA) and protein in U937 cells, in human monocytes and in human monocyte-derived macrophages. Tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) alone did not induce CCL26 expression, yet these pro-inflammatory cytokines synergized with IL-4 to increase CCL26 protein expression. Signal transducer and activator of transcription 6 (STAT6) was not affected by costimulation with TNF-alpha, suggesting that the synergy between IL-4 and TNF-alpha occurs at a step downstream of STAT6 activation. Co-incubation of interferon-gamma (IFN-gamma) with IL-4 had no effect on CCL26 protein release. By contrast, pretreatment with IFN-gamma decreased total STAT6 protein, blocked IL-4-mediated STAT6 phosphorylation and decreased IL-4-mediated CCL26 mRNA expression and protein release. These data show that IL-4 and pro-inflammatory cytokines such as TNF-alpha, IL-1beta and IFN-gamma regulate CCL26 synthesis in human monocytic cells, which may be important in regulating monocyte inflammatory responses.
- [Show abstract] [Hide abstract] ABSTRACT: Marijuana has been used for thousands of years to affect human health. Dissecting the peripheral effects from the central psychotropic effects has revealed a complex interplay between cannabinoids, endocannabinoids and their receptors. This review examines recent advances in understanding the expression, regulation and utilization of the CB(2) receptor. Here we highlight the molecular aspects of the CB(2) receptor, CB(2) receptor signaling and new ligands for this receptor. We focus in the rest of the review on recent findings in the immune system, the gastrointestinal tract and liver, the brain and the cardiovascular system and airways as examples of areas where new developments in our understanding of the CB(2) receptor have occurred. Early studies focused on expression of this receptor under baseline physiologic conditions; however, perturbations such as those that occur during inflammation, ischemia/reperfusion injury and cancer are revealing a critical role for the CB(2) receptor in regulating these disease processes amongst others. As a result, the CB(2) receptor is an appealing therapeutic target as well as a useful tool for shedding new light on physiological regulatory processes throughout the body.
- [Show abstract] [Hide abstract] ABSTRACT: Activation of cannabinoid (CB)(1) receptors results in attenuation of experimental colitis. Our aim was to examine the role of CB(2) receptors in experimental colitis using agonists (JWH133, AM1241) and an antagonist (AM630) in trinitrobenzene sulfonic acid (TNBS)-induced colitis in wildtype and CB(2) receptor-deficient (CB(2) (-/-)) mice. Mice were treated with TNBS to induce colitis and then given intraperitoneal injections of the CB(2) receptor agonists JWH133, AM1241, or the CB(2) receptor antagonist AM630. Additionally, CB(2) (-/-) mice were treated with TNBS and injected with JWH133 or AM1241. Animals were examined 3 days after the induction of colitis. The colons were removed for macroscopic and microscopic evaluation, as well as the determination of myeloperoxidase activity. Quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) for CB(2) receptor was also performed in animals with TNBS and dextran sodium sulfate colitis. Intracolonic installation of TNBS caused severe colitis. CB(2) mRNA expression was significantly increased during the course of experimental colitis. Three-day treatment with JWH133 or AM1241 significantly reduced colitis; AM630 exacerbated colitis. The effect of JWH133 was abolished when animals were pretreated with AM630. Neither JWH133 nor AM1241 had effects in CB(2) (-/-) mice. We show that activation of the CB(2) receptor protects against experimental colitis in mice. Increased expression of CB(2) receptor mRNA and aggravation of colitis by AM630 suggests a role for this receptor in normally limiting the development of colitis. These results support the idea that the CB(2) receptor may be a possible novel therapeutic target in inflammatory bowel disease.
- [Show abstract] [Hide abstract] ABSTRACT: The endogenous cannabinoid system plays an important role in the regulation of gastrointestinal function in health and disease. Endocannabinoid levels are regulated by catabolic enzymes. Here, we describe the presence and localization of monoacylglycerol lipase (MGL), the major enzyme responsible for the degradation of 2-arachidonoylglycerol. We used molecular, biochemical, immunohistochemical, and functional assays to characterize the distribution and activity of MGL. MGL mRNA was present in rat ileum throughout the wall of the gut. MGL protein was distributed in the muscle and mucosal layers of the ileum and in the duodenum, proximal colon, and distal colon. We observed MGL expression in nerve cell bodies and nerve fibers of the enteric nervous system. There was extensive colocalization of MGL with PGP 9.5 and calretinin-immunoreactive neurons, but not with nitric oxide synthase. MGL was also present in the epithelium and was highly expressed in the small intestine. Enzyme activity levels were highest in the duodenum and decreased along the gut with lowest levels in the distal colon. We observed both soluble and membrane-associated enzyme activities. The MGL inhibitor URB602 significantly inhibited whole gut transit in mice, an action that was abolished in cannabinoid 1 receptor-deficient mice. In conclusion, MGL is localized in the enteric nervous system where endocannabinoids regulate intestinal motility. MGL is highly expressed in the epithelium, where this enzyme may have digestive or other functions yet to be determined.
- [Show abstract] [Hide abstract] ABSTRACT: The endocannabinoid (EC) system mediates protection against intestinal inflammation. In this study, we investigated the effects of blocking EC degradation or cellular reuptake in experimental colitis in mice. Mice were treated with trinitrobenzene-sulfonic acid in presence and absence of the fatty acid amide hydrolase (FAAH) blocker URB597, the EC membrane transport inhibitor VDM11, and combinations of both. Inflammation was significantly reduced in the presence of URB597, VDM11, or both as evaluated by macroscopic damage score, myeloperoxidase levels, and colon length. These effects were abolished in CB1- and CB2-receptor-gene-deficient mice. Quantitative reverse transcription polymerase chain reaction after induction of experimental colitis by different pathways showed that expression of FAAH messenger RNA (mRNA) is significantly reduced in different models of inflammation early in the expression of colitis, and these return to control levels as the disease progresses. Genomic DNA from 202 patients with Crohn’s disease (CD) and 206 healthy controls was analyzed for the C385A polymorphism in the FAAH gene to address a possible role in humans. In our groups, the C385A polymorphism was equally distributed in patients with CD and healthy controls. In conclusion, drugs targeting EC degradation offer therapeutic potential in the treatment of inflammatory bowel diseases. Furthermore, reduction of FAAH mRNA expression is involved in the pathophysiological response to colitis.
- [Show abstract] [Hide abstract] ABSTRACT: Enhanced intestinal transit due to lipopolysaccharide (LPS) is reversed by cannabinoid (CB)2 receptor agonists in vivo, but the site and mechanism of action are unknown. We have tested the hypothesis that CB2 receptors are expressed in the enteric nervous system and are activated in pathophysiological conditions. Tissues from either saline- or LPS-treated (2 h; 65 microg/kg ip) rats were processed for RT-PCR, Western blotting, and immunohistochemistry or were mounted in organ baths where electrical field stimulation was applied in the presence or absence of CB receptor agonists. Whereas the CB2 receptor agonist JWH133 did not affect the electrically evoked twitch response of the ileum under basal conditions, in the LPS-treated tissues JWH133 was able to reduce the enhanced contractile response in a concentration-dependent manner. Rat ileum expressed CB2 receptor mRNA and protein under physiological conditions, and this expression was not affected by LPS treatment. In the myenteric plexus, CB2 receptors were expressed on the majority of neurons, although not on those expressing nitric oxide synthase. LPS did not alter the distribution of CB2 receptor expression in the myenteric plexus. In vivo LPS treatment significantly increased Fos expression in both enteric glia and neurons. This enhanced expression was significantly attenuated by JWH133, whose action was reversed by the CB2 receptor antagonist AM630. Taking these facts together, we conclude that activation of CB2 receptors in the enteric nervous system of the gastrointestinal tract dampens endotoxin-induced enhanced intestinal contractility.
- [Show abstract] [Hide abstract] ABSTRACT: Objective: The objective of this study was to examine the role of protein kinase C zeta (PKCzeta) in interleukin (IL)-8-mediated activation of Mac-1 (CD11b/CD18) in human neutrophils. Materials and methods: Neutrophils were stimulated with IL-8 in the presence or absence of pharmacologic inhibitors of PKC or a myristoylated PKCzeta pseudosubstrate. The resulting changes in Mac-1 surface expression, affinity, and avidity, as measured by clustering, were determined by using a combination of flow cytometry and immunofluorescence (IF). Colocalization of Mac-1 with PKCzeta was also probed using IE Finally, neutrophil adhesion to matrix proteins was examined under static conditions and adhesion to tumor necrosis factor-alpha-stimulated human umbilical vein endothelial cells was examined under flow conditions, using a parallel-plate flow chamber Results: PKCzeta and Mac-1 colocalized following stimulation with IL-8. Blocking PKCzeta prevented IL-8-induced Mac-1 clustering while simultaneously increasing Mac-1 affinity. To determine the relative contribution of affinity versus avidity in neutrophil adhesion, we examined adhesion under both static and flow conditions, and found that blocking PKCzeta prevented neutrophil adhesion, despite increased affinity of Mac-1. Conclusions: These data suggest that PKCzeta is a negative regulator of Mac-1 affinity and a positive regulator of Mac-1 avidity. Further, Mac-1 avidity is more important than increased affinity alone in regulating neutrophil firm adhesion.
- [Show abstract] [Hide abstract] ABSTRACT: This study investigated the ongoing effects of participation in a mindfulness-based stress reduction (MBSR) program on quality of life (QL), symptoms of stress, mood and endocrine, immune and autonomic parameters in early stage breast and prostate cancer patients. Forty-nine patients with breast cancer and 10 with prostate cancer enrolled in an eight-week MBSR program that incorporated relaxation, meditation, gentle yoga and daily home practice. Demographic and health behaviors, QL, mood, stress symptoms, salivary cortisol levels, immune cell counts, intracellular cytokine production, blood pressure (BP) and heart rate (HR) were assessed pre- and post-intervention, and at 6- and 12-month follow-up. Fifty-nine, 51, 47 and 41 patients were assessed pre- and post-intervention and at 6- and 12-month follow-up, respectively, although not all participants provided data on all outcomes at each time point. Linear mixed modeling showed significant improvements in overall symptoms of stress which were maintained over the follow-up period. Cortisol levels decreased systematically over the course of the follow-up. Immune patterns over the year supported a continued reduction in Th1 (pro-inflammatory) cytokines. Systolic blood pressure (SBP) decreased from pre- to post-intervention and HR was positively associated with self-reported symptoms of stress. MBSR program participation was associated with enhanced quality of life and decreased stress symptoms, altered cortisol and immune patterns consistent with less stress and mood disturbance, and decreased blood pressure. These pilot data represent a preliminary investigation of the longer-term relationships between MBSR program participation and a range of potentially important biomarkers.
- [Show abstract] [Hide abstract] ABSTRACT: It has been known for many years that neutrophils and platelets participate in the pathogenesis of severe sepsis, but the inter-relationship between these players is completely unknown. We report several cellular events that led to enhanced trapping of bacteria in blood vessels: platelet TLR4 detected TLR4 ligands in blood and induced platelet binding to adherent neutrophils. This led to robust neutrophil activation and formation of neutrophil extracellular traps (NETs). Plasma from severely septic humans also induced TLR4-dependent platelet-neutrophil interactions, leading to the production of NETs. The NETs retained their integrity under flow conditions and ensnared bacteria within the vasculature. The entire event occurred primarily in the liver sinusoids and pulmonary capillaries, where NETs have the greatest capacity for bacterial trapping. We propose that platelet TLR4 is a threshold switch for this new bacterial trapping mechanism in severe sepsis.
The University of Calgary
Calgary, Alberta, Canada
- Immunology Research Group (IRG)