extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry Medicine

text:
Monoacylglycerol Lipase (MAGL) Inhibition Attenuates Acute Lung Injury in Mice

Endocannabinoid signaling is terminated by enzymatic hydrolysis, a process that, for 2-Arachidonoylglycerol (2-AG), is mediated by monoacylglycerol lipase (MAGL). The piperidine carbamate, 4-nitrophenyl- 4-(dibenzo[d] [1,3]dioxol-5-yl (hydroxy) methyl) piperidine- 1-carboxylate (JZL184), is a drug that inhibits MAGL and presents high potency and selectivity. Thus, JZL184 increases the levels of 2-AG, an endocannabinoid that acts on the CB1 and CB2 cannabinoid receptors. Here, we investigated the effects of MAGL inhibition, with a single dose (16 mg/kg, intraperitoneally (i.p.)) of JZL184, in a murine model of lipopolysaccharide (LPS) -induced acute lung injury (ALI) 6, 24 and 48 hours after the inflammatory insult. Treatment with JZL184 decreased the leukocyte migration into the lungs as well as the vascular permeability measured through the bronchoalveolar lavage fluid (BAL) and histological analysis. JZL184 also reduced the cytokine and chemokine levels in the BAL and adhesion molecule expression in the blood and BAL. The CB1 and CB2 receptors were considered involved in the anti-inflammatory effects of JZL184 because the AM281 selective CB1 receptor antagonist (1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide) and the AM630 selective CB2 receptor antagonist ([6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)-methanone) blocked the anti-inflammatory effects previously described for JZL184. It was concluded that MAGL inhibition, and consequently the increase in 2-AG levels, produced anti-inflammatory effects in a murine model of LPS-induced ALI, a finding that was considered a consequence of the activation of the CB1 and CB2 receptors.

Introduction
The discovery of the CB 1 and CB 2 cannabinoid receptors and their major ligands N-arachidonoylethanolamide (anandamide) and 2-arachidonoylglycerol (2-AG) led to the characterization of the endocannabinoid system [1] [2][3] [4]. This system has become a subject of great interest in pharmacology due to its remarkable distribution in mammals and its capacity to play a modulating role in diverse physiological functions including immunomodulation and inflammation [5]. 2-AG and anandamide are regulated by the catabolic enzymes monoacylglycerol lipase (MAGL) [4] and fatty acid amide hydrolase (FAAH) [6], respectively.
To investigate the MAGL role in endocannabinoid metabolism and signaling in vivo,   [7] described in mice a potent and selective inhibitor for this enzyme termed 4-nitrophenyl-4-(dibenzo [d] [1,3] dioxol-5-yl (hydroxy) methyl ) piperidine-1-carboxylate (JZL184) that decreases the 2-AG hydrolysis in both the Central Nervous System (CNS) and in the peripheral tissues, including in the lungs [8]. JZL184 has also been shown to have potent immunosuppressive and anti-inflammatory properties in some rodent models of inflammation [9] [10]. Employing this drug (16 mg/kg, given systemically), a remarkable variety of CB 1dependent effects such as analgesia, hypomotility and hypothermia were shown in mice [7]. Using JZL184 (8 mg/kg i.p.), Busquets-Garcia et al. (2011) [11] highlighted the anxiolytic effects of this drug in mice and suggested that the observed effects might have been induced by 2-AG actions on the CB 2 receptors; an antinociceptive effect related to the CB 1 and the CB 2 receptor activation has also been reported in mice [11].
Acute lung injury (ALI) and its most severe form, acute respiratory distress syndrome (ARDS) in humans, is a lung disease with an acute onset that is characterized by bilateral pulmonary infiltrates with neutrophil accumulation [12] [13]. ALI has been shown to result in persistent respiratory failure and prolonged dependence on mechanical ventilation, increased susceptibility to multi-organ dysfunction and mortality [14]. Despite numerous innovations in intensive care medicine, the ALI-related mortality rate has been reported to vary by approximately 40% [14] [15].
Although the search for the effects of endocannabinoids such as anandamide and 2-AG on immunity has received considerable research attention, the effects of MAGL inhibition on inflammation models are still unclear. This work was designed to investigate the potential anti-inflammatory effect of MAGL inhibition in a murine model of LPS-induced ALI using JZL184 as a pharmacological tool. Elucidating the roles of CB 1 and CB 2 cannabinoid receptor activation on JZL184 effects was also an objective of this work.

Animals
Male C57BL/6 mice from our own colony, weighing 22-28 g and approximately 60 days old, were used. The animals were housed under conditions of controlled temperature (22-26 °C) and artificial light (12-h light/12-h dark, lights on at 7:00 a.m.), with free access to rodent chow and water. The experiments were performed in a different room at the same temperature as the animal colony. The animals were transferred and maintained in their home cages 7 days before the beginning of the experiments. This study protocol was approved by the Bioethical Committee of Care and Use of Laboratory Animal Resource of the School of Veterinary Medicine, University of São Paulo, Brazil (permit number: 2255/2011); the guidelines are similar to the guidelines of the National Institutes of Health (NIH), USA. All surgery was performed under ketamine and xylazine anesthesia, and all efforts were made to minimize suffering.

Experimental protocol and group formation
Two experiments were conducted in accordance with the Good Laboratory Practice (GLP) protocols and quality assurance methods. The first experiment was performed to investigate the possible effects of JZL184 on ALI. For that purpose, 61 mice were randomly assigned into four groups: control (C1 and C2) and experimental (E1 and E2) groups. The JZL184 was intraperitoneally (i.p.) administered at a single dose of 16 mg/kg (0.1 mL/10 g weight) to the animals of the experimental groups. The control groups received a similar volume of JZL184 vehicle alone. Sixty minutes after treatment, ALI was induced, as described below. Mice were analyzed 6, 24 and 48 hours after the LPS (C2 and E2 groups) or the saline (C1 and E1 groups) intranasal (i.n.) instillation.
The participation of the CB 1 or the CB 2 receptors in the JZL184-induced effects on ALI was assessed in the second experiment, which used 99 mice. As in the first experiment, the animals were divided into two experimental (E1 and E2) and two control (C1 and C2) groups that received i.p. JZL184 (16 mg/kg) or a similar volume of its vehicle (0.1 mL/10 g). Within each of these 4 groups, the animals were again divided into 3 groups that received one of the following treatments i.p. 30 minutes before JZL184 or the JZL184 vehicle: AM281 (2.5 mg/ kg), AM630 (2.5 and 5.0 mg/kg) or AM281 and AM630 vehicle (0.1 mL/10 g). The ALI was induced 60 minutes after the JZL184 or JZL184 vehicle treatment, and the mice were analyzed 6, 24 and 48 hours later.

Acute lung injury (ALI)
ALI was performed according to previous work from our laboratory [13]. The mice were anesthetized with ketamine and xylazine injected i.p. (100 and 10 mg/kg, respectively) before intranasal instillation of LPS. Escherichia coli LPS (O55:B5 L2880, Sigma-Aldrich, St. Louis, USA) at a concentration of 100 μg/mL or sterile 0.9% saline were intranasal instilled into the mice (1 μL/g of mouse body weight). Six, twenty-four and forty-eight hours after the induction of inflammation, mice were anesthetized and killed by exsanguination through the inferior vena cava for the bronchoalveolar lavage fluid (BAL), blood and bone marrow collected and analysis.

Leukocyte trafficking
Blood. Blood studies were performed as described by Ligeiro-Oliveira et al. (2004) [19]. The mice were anesthetized as described above, and the samples of the blood were taken from the abdominal vena cava with plastic syringes containing 10 µL of 10% EDTA and subsequently diluted 1:20 in Turk´s fluid (3% acetic acid).The total number of cells present in the blood was counted with the aid of a light microscope in Neubauer chambers. Differential leukocyte counts were performed on smears stained with Rosenfeld's dye using standard morphological criteria.
Bronchoalveolar lavage fluid (BAL). BAL was performed according to Tavares de Lima, Sirois and Jancar (1992) [20]. After exsanguination, the BAL was collected. The lungs were flushed with 1.5 mL of phosphate-buffered saline (PBS) solution through the use of a cannula inserted by tracheostomy. After collection, the BAL fluid was centrifuged at 250 x g for 5 min. The supernatant was stored, and the remaining cell pellet was resuspended in 1 mL of PBS solution. Total leukocyte counts were performed by adding 10 µL of crystal violet to 90 µL of the cell suspension. Neubauer chambers were used for cell counting under a light microscope. The differential cell count was carried out on the cytocentrifuged (250 x g for 5 min) (FANEM, São Paulo, Brazil) cell suspension (100 µL) stained with Rosenfeld's dye using standard morphological criteria.
Bone marrow. The percentage of granulocytes in the bone marrow was quantified from the femoral marrow lavage fluid that was obtained as described by Ligeiro-Oliveira (2004) [19]. Briefly, mice had their femurs removed and both epiphyses were cut off. A needle connected to a plastic syringe containing PBS (5 mL) was inserted into each femoral marrow to allow cell removal by flushing. The cell suspension was centrifuged at 250 x g for 5 min; the cell pellet obtained was resuspended for the total leukocyte count as describe above for the BAL. To analyze the percentage of granulocytes in the bone marrow, the cells were adjusted to 1 x 10 6 and were incubated with FITC-conjugated anti-mouse Ly6G, clone 1A8 (Granulocytes, Biolegend, CA, USA), according to the manufacturer's instructions. A flow cytometer (FACSCalibur, Becton Dickinson Immunocytometry System, San Jose, CA, USA) was used to analyze the granulocyte expression. FlowJo® software (Tree Star, Inc., Ashland, OR, USA) was used to analyze the data.

Histological analysis
Histological analysis was performed according to [21,22] modified. The left lung was prepared, cut into 5 µm and hematoxylin and eosin (H.E.) stained as previously described [23] modified. From each sample four representative photos were taken (magnification x200). Five high power fields were randomly assigned to each photo. Subsequently, alveolar wall thickness was analyzed by ImageJ software (ImageJ, U. S.National Institutes of Health, Bethesda, Maryland, USA). For each high power fields, a modified ALI score was used to determine the degree of lung damage: In brief, (a) thickness of the alveolar walls, (b) infiltration or aggregation of inflammatory cells, (c) hemorrhage and (d) alveolar congestion were rated in a blinded classification. Each sample was graded according to the point scale: 0: minimal damage, 1: mild damage, 2: moderate damage, 3: severe damage, 4: maximal damage. The samples were evaluated by two professional and the degree of ALI was assessed by the sum of scores, ranging from 0 to 16. The average of the sum of each reading was compared among groups.

Cytokine and chemokine analysis
A BD TM Cytometric Bead Array (CBA) Mouse Inflammation Kit (BD Biosciences, San Jose, USA) was used to measure the IL-6, IL-10, MCP-1, IFN-γ, TNF-α, and IL-12p70 in the animals' BAL supernatant. The assays were performed according to the manufacturer's instructions.

Protein analysis in the BAL
The BAL supernatant aliquots were placed in a 96-well ELISA plate (10 μL/well), and 250 μL of Bradford reagent (Sigma-Aldrich, St. Louis, USA) was added to each well; after a 30 min incubation period, the absorbance was measured at 595 nm. A standard curve using bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, USA) was obtained by plotting the net absorbance vs. the protein concentration (1.500 -100 µg/mL) to determine the protein concentration in the samples.

Statistical analysis
GraphPad Prism version 5.0 (GraphPad Software, Inc.) was used for statistical analysis. Parametric data were analyzed by one-way ANOVA followed by a Tukey-Kramer test for multiple comparisons. Non-parametric data were analyzed by a Mann Whitney U test for two-group comparisons or by a Kruskal-Wallis followed by Dunn´s tests for multiple comparisons. The BAL protein concentration data were first analyzed by normalizing to the percentage of control (vehicle + saline) and were then compared to the C2 (vehicle + LPS) and the E2 (JZL184+LPS) groups using the non-parametric tests. In all experiments, p ≤ 0.05 was considered significant. Data are presented as the mean ± S.D.

Results
In both experiments, no differences were found in mice that received intranasal saline, whether they were treated (E1) or not (C1) with JZL184 (data not shown), i.e., JZL184 induced no effects in the absence of LPS-induced ALI.
No differences were found for the monocyte counts in the blood in all periods evaluated ( Figure 1B), as well as for the total and differential leukocyte counts in the blood taken 24 hours after the LPS intranasal instillation ( Figure 1B).
Bone marrow. The mice in the C2 group exhibited a decreased expression of granulocytes in the bone marrow 6 hours after the LPS-induced ALI (F (2,17) = 6.212; p < 0.05) ( Figure 1C); this finding was not abrogated by the JZL184 treatment as observed in the mice of the E2 group (F (2,17) = 6.212; p < 0.05) ( Figure 1C). Further analysis showed that 48 hours after the LPS intranasal instillation, the expression of granulocytes in the bone marrow of the mice of the E2 group was smaller than the expression measured in the C2 group (F (2,17) = 3.553; p < 0.05) ( Figure 1C). However, no differences were found in the granulocyte expression in the bone marrow 24 hours after LPS intranasal instillation ( Figure 1C).

Treatment with JZL184 decreased lung damage
In the presented model of acute lung injury, H.E. staining of lung sections showed that, as compared to control condition

Treatment with JZL184 changed the adhesion molecule expression in the neutrophils of the bronchoalveolar lavage fluid and blood
We next investigated the expression of the adhesion molecules (L-selectin, β2-integrin and PECAM) in the leukocytes recovered from the blood and the BAL of mice treated or not with JZL184. In relation to the animals of the C2 group, the JZL184 treatment decreased the β2-integrin expression (U = 8.0; p < 0.05) ( Figure 4A); however, at the same time, this treatment increased the L-selectin expression (U = 9.0; p < 0.05) ( Figure 4B) in the neutrophils taken from the blood 6 hours after LPS-induced ALI. No differences were found for PECAM expression in the neutrophils of the blood taken in all periods analyzed (data not shown) or in β2-integrin and L-selectin expression at 24 and 48 hours after LPS intranasal instillation (Table 1). Furthermore, it was also observed that MAGL inhibition by JZL184 decreased the β2integrin expression (U = 7.0; p < 0.001) in neutrophils in the BAL 48 hours after LPS-induced ALI ( Figure 4C). However, significant difference were not found for both L-selectin (Table  1) and PECAM (data not shown) expression in the neutrophils taken from BAL in all of the periods analyzed.

Treatment with JZL184 decreased vascular permeability
Vascular permeability within the lungs was measured by the albumin concentration in the BAL. It was shown that JZL184 decreased the protein concentration in the BAL in relation to mice in the C2 group at 6 (U = 3.0; p < 0.05) and 48 (U = 4.0; p < 0.001) hours after LPS-induced ALI ( Figure 5).

Treatment with JZL184 decreased the production of pro-inflammatory cytokines/chemokines in the bronchoalveolar lavage fluid
In relation to mice in the C1 group, large increases in the TNF-α and IL-6 concentrations were observed in the C2 group 6 hours after the LPS-induced ALI ( Figure 6A-B). Gradual reductions in the TNF-α and IL-6 concentrations were detected at 24 and 48 hours after the LPS intranasal instillation ( Figure  6A-B). However, at the same time, a gradual increase in the MCP-1 concentration was observed in the BAL in animals of the C2 group in all of the evaluated periods ( Figure 6C). The JZL184 treatment decreased the TNF-α concentration at 24 (U = 3.0; p < 0.05) and 48 (U = 4.0; p < 0.001) hours after LPSinduced ALI (Figure 6A), as well as the IL-6 concentration at 6 (U = 2.0; p < 0.05), 24 (U = 6.0; p < 0.05) and 48 (U = 9.0; p < 0.05) hours after LPS-induced ALI ( Figure 6B). Further analysis showed that JZL184 treatment (E2 group) also exhibited a reduced MCP-1 concentration 6 (U = 4.0; p < 0.001) and 48 (U = 7.0; p < 0.05) hours after LPS-induced ALI ( Figure 6C). Statistically significant differences were not observed among the groups for the IL-10, IFN-γ and IL-12p70 concentrations measured in the BAL of mice taken at 6, 24 and 48 hours after the LPS intranasal instillation ( Table 2).

CB 1 and CB 2 receptor antagonism attenuated the antiinflammatory effects induced by JZL184 on ALI
The roles of the CB 1 and CB 2 cannabinoid receptors in the anti-inflammatory effects of JZL184 were next analyzed using AM281 (a CB 1 receptor antagonist) and AM630 (a CB 2 receptor antagonist) as pharmacological tools. The data showed that the AM281 and AM630 treatments partially abrogated the JZL184induced actions on leukocyte migration into the lungs 6 hours after LPS instillation (F (5,28) = 13.53) ( Figure 7A). However, only the AM630 treatment abrogated the JZL184-induced inhibition of leukocyte migration into the lungs at 24 (F (5,28) = 23.33; p < 0.05) and 48 (F (5,25) = 13.73; p < 0.001) hours after LPS intranasal instillation ( Figure 7A). Further analysis revealed that the AM281 and AM630 treatments partially reverted both the JZL184-induced actions on the leukocyte count in the blood (F (5,25) = 3.61) ( Figure 7B) and the percentage of granulocytes in the bone marrow (F (5,25) = 6.336) ( Figure 7D) at 48 hours after LPS-induced ALI. It was also shown that the JZL184-induced actions on the neutrophil count in the blood were partially reversed by the AM281 treatment (F (5,25) = 7.498) ( Figure 7C). The JZL184 effects in prevented alveolar wall tickening and the lung damage was reversed with AM630 (5mg/kg) treatment 6 hours after LPS intranasal instillation (F (5,29) = 61.23; p < 0.0001 and KW = 21.44; p < 0.05) ( Figure 8A-B).
Further analysis showed that the JZL184-induced actions on the expression of adhesion molecules in the blood and in the BAL were reduced or abrogated by the use of both AM281 and AM630. Thus, AM281 reduced the JZL184-induced inhibition of β2-integrin expression in the blood 6 hours after ALI (KW = 20.30) ( Figure 9A Figure 11C).

Discussion
JZL184 is commonly known as a highly efficacious and selective inhibitor of MAGL, a 2AG-degrading enzyme [7]; it has been reported that JZL184 has potent immunosuppressive and anti-inflammatory properties [9]. We are now reporting that a single dose of JZL184 (16 mg/kg, i.p.) is able to decrease several lung inflammation parameters during the course of a murine model of LPS-induced ALI, specifically leukocyte migration (neutrophils, lymphocytes and macrophages) to the lungs, adhesion molecule expression in the BAL and blood, vascular permeability and cytokine/ chemokine production in the BAL. We also showed that signaling through the CB 1 and CB 2 cannabinoid receptors might have a relevant role in the anti-inflammatory effects now being reported for JZL184.
Organ dysfunction and failure from inflammatory response continues to be the major problem after injury in many clinical conditions such as sepsis, acute pancreatitis and hemorrhagic shock [24]. In humans, the acute lung injury (ALI) that manifests clinically as acute respiratory distress syndrome (ARDS), a major component of multiple organ dysfunction syndrome, is described as "syndrome of inflammation and increased capillary endothelial permeability" [25,26]. Inflammatory mediators play a key role in the pathogenesis of ARDS, which is the primary cause of death in these conditions [24].
Neutrophil influx into the interstitium and the bronchoalveolar space is considered a keystone for the progression of ALI [12]. We found that JZL184 treatment decreased total leukocyte migration into the lungs of mice at 6, 24 and 48 hours after LPS-induced ALI, as shown by the BAL leukocyte count and ALI score. Specifically, we observed that the neutrophils and the lymphocytes were greatly reduced in the BAL taken from JZL184-treated mice in relation to the inflamed non-treated mice. Leukocyte migration into the site of inflammation is known to be important because the presence of high amounts  of inflammatory cells, especially neutrophils, might damage the lung tissue [27]. Thus, it appears feasible to suggest that a reduction in the inflammatory process, induced by JZL184 in mice, could decrease ALI lung damage.
In the presence of an inflammatory process, the blood cells are immediately recruited to the site of inflammation; the bone marrow produces the largest quantity of leukocytes that migrate to the bloodstream and are directed to the inflammatory focus [28].
The control mice treated with LPS exhibited an increased number of lymphocytes in their blood 6 hours after the LPS instillation; the JZL184-treated animals exhibited a decrease in this neutrophil number in the blood 48 hours after LPS-induced ALI. It is known that the inflammatory process begins at the microcirculation site, specifically by the formation of the intercellular spaces that are responsible for the increased microvascular permeability observed during the course of ALI [29]. Thus, it appears that a reduction in the epithelial cell barrier function might have facilitated the influx of a fluid rich in cells and macromolecules into the lung alveolar space. Indeed, we showed that MAGL inhibition by JZL184 reduced the vascular permeability to protein influx into the BAL at 6 and 48 hours after LPS instillation, a fact that might have contributed to the observed decrease in lung inflammation. In our experimental model, JZL184 also induced a decrease in the number of granulocytes produced in the bone marrow in relation to the inflamed control lung.
Neutrophil migration into the lungs is not sufficient to cause ALI; neutrophil activation and pro-inflamatory cytokine release are also required [12]. To initiate the inflammatory process, the immune system cells produce and release cytokines and  TNF-α is released during the first 30-90 min after exposure to LPS and in turn activate a second level of inflammatory cascades including cytokines, lipid mediators, and reactive oxygen species, as well as up-regulating cell adhesion molecules that result in the initiation of inflammatory cell migration into tissues [30]. TNF-α is present in the bronchoalveolar lavage fluid of patients at risk for ARDS and with established ARDS [31]. Interleukin 6 (IL-6) is produced by a wide range of cells including monocytes/macrophages, endothelial cells, fibroblasts, and smooth muscle cells in response to stimulation by endotoxin, IL-1β, and TNFα [30,[32][33][34]. Circulating levels of IL-6 have been shown to be excellent predictors of the severity of ARDS of different aetiologies such as sepsis [35] and acute pancreatitis [36].  Chemokines can be classified as constitutive (developmentally regulated) or inducible (inflammatory). Chemokines have chemotactic and activating effects on leukocyte subsets, which provide a key stimulus for directing leukocytes to areas of injury [24].
Treatment with JZL184 has been reported to block cytokine expression in the gut or blood in a gastric inflammation model [9] [10]. A reduction in pro-inflammatory cytokine (TNF-α and IL-6) and chemokine (MCP-1) production in JZL184-treated mice in relation to the inflamed control group was presently observed. These results appear to be extremely important because cytokines and chemokines are essential for both inflammation establishment and phagocyte activation. This action might explain the presently observed reduction in leukocyte migration into the lungs. MCP-1, also known as CCL2, is a molecule that attracts monocytes and lymphocytes as well as neutrophils to the site of inflammation [37]. Thus, it is reasonable to suggest that JZL184, by reducing MCP-1 in the BAL, decreased the total leukocyte count in the BAL.
Our data suggest that the inhibition of adhesion molecules by JZL184 might have been involved in the anti-inflammatory effects presently being reported. Leukocyte migration into inflamed tissues involves complex interactions of leukocytes with the endothelium through the regulated expression of surface adhesion molecules. In the present study, we showed that JZL184 treatment (1) decreased the cellular expression of β2-integrin in the blood 6 hours after the LPS-induced ALI (2), increased the cellular expression of L-selectin in the blood 6 hours after the LPS instillation, and (3) decreased the cellular expression of β2-integrin in the BAL 48 hours after the induction of inflammation. Together, these facts appear to justify the less intense inflammatory response observed in the mice of the JZL-treated group in relation to those of the inflamed control group. Indeed, β2-integrin and L-selectin are known to increase the leukocyte adhesion process [38] [39]. Time course studies of the β2-integrin and L-selectin expression during inflammation have shown an inverse relationship in the serum, where β2-integrins are upregulated and L-selectins are downregulated [38] [39]. It has been speculated that this inverse relationship is necessary to allow the adhesion of the neutrophils [40]. Taking this fact into account and also the changes observed here in the adhesion molecule expression, it seems possible to suggest that adhesion molecules might have accounted for the reduction in inflammatory cell adhesion in the JZL184-treated mice in the present experiment.
JZL184 is known to exert its effects by inhibiting MAGL, thereby increasing the levels of the endocannabinoid 2-AG [7]. 2-AG and anandamide are signaling lipids that bind to the cannabinoid receptors CB 1 [1] and CB 2 [3]. Activation of the CB 1 and CB 2 receptors has been shown to play an important role in inflammation [9]. Endogenous and exogenous cannabinoid receptor agonists are involved in leukocyte activation and cytokine production in different inflammatory conditions [41][42][43][44][45][46].
The 2-AG, mainly through the CB2 receptor, stimulates inflammatory reactions and allergic responses by inducing robust adhesion of leukocytes to vascular endothelial cells [47]. In addition, other non-hematopoietic cells, such as endothelial cells, can respond to ligands of CB receptors. The CB1 and CB2 receptors are expressed during inflammatory angiogenesis; CB1 receptor appeared to be strongly involved in leukocyte accumulation and cytokine production, CB2 receptor is mainly linked to a proangiogenic response, especially through early neutrophil activation. Double blockade of cannabinoid receptors favored slightly better inhibition of inflammatory angiogenesis [48]. In the skin samples of healthy dogs, CB1 and CB2 receptors immunoreactivity was detected in various types of cells in the epidermis and in cells in the dermis, including perivascular cells with mast cell morphology, fibroblasts, and endothelial cells; in the skin samples of dogs with atopic dermatitis, CB1 and CB2 immunoreactivity was stronger than it was in skin samples of healthy dogs [49].
Therefore, to evaluate the involvement of the CB 1 and CB 2 receptor activation in the JZL184-induced anti-inflammatory effects, we used the CB 1 (AM281) and CB 2 (AM630) antagonists before the JZL184 treatment. Remarkably, the AM281 or AM630 treatments reduced all of the antiinflammatory effects of JZL184 reported here, i.e., leukocyte migration into the lungs (from bone marrow to the blood and from the blood to the lungs), the lung damage, adhesion molecule expression, vascular permeability reduction and the production of pro-inflammatory cytokines/chemokines at 6, 24 or 48 hours after LPS-induced ALI. It has been reported that CB 1 and, mainly, CB 2 receptor activation decreased the inflammatory response during drug-induced gastric hemorrhages [10], as well as in models of colitis, a finding also observed during a systemic-induced inflammation in mice [9]. Furthermore, neutrophil migration in humans [50], cytokine/ chemokine production in mice [9], vascular permeability in guinea pigs [51] and adhesion molecule expression in mice [52] have also been reported to be reduced by CB 1 or CB 2 activation. Thus, it appears feasible to suggest that the abrogation of the anti-inflammatory effects induced by JZL184 in LPS-induced ALI might have involved CB 1 and CB 2 receptor activation. However, it should not be forgotten that cannabinoid actions can be related to other mechanisms besides CB 1 and CB 2 activation, such as adenosine receptor activation [13] or partial FAAH inhibition by JZL184 administration [7]. Furthermore, beyond the CB 1 and CB 2 receptor participation, other possibilities might also explain the anti-inflammatory effects of JZL184 demonstrated in this work. One possibility may be the release of arachidonic acid from 2-AG and/or anandamide hydrolysis, a fact that would lead to eicosanoid (leukotriene) formation in neutrophils. It has been shown that 2-AG in vitro activates human neutrophils, an effect attributable to the leukotriene (LT) B4 ligation to specific neutrophil receptors (LTB4 receptors) [53].
Taken together, the present findings showed that a single dose of JZL184, given prior to LPS-induced ALI, resulted in an anti-inflammatory effect. It was also shown that the JZL184 effects on leukocyte migration from the blood to the lungs and from the bone marrow to the blood in the presently used model of ALI relies on adhesion molecule expression and on cytokine/ chemokine participation. Finally, it was shown that the CB 1 and CB 2 cannabinoid receptors appear to be the most likely mechanism involved in the JZL184 anti-inflammatory effects in the LPS-induced ALI model. Although care should be taken when extrapolating the present data to patients. MAGL inhibition might become, in the future, a useful therapeutic tool for the treatment of inflammatory lung diseases, such as ALI and acute respiratory distress syndrome.


extfieldsofstudy: Medicine Chemistry

text:
Protective effect of ginseng against gamma-irradiation-induced oxidative stress and endothelial dysfunction in rats

This study investigated the potential protective effects of ginseng on gamma-irradiation-induced oxidative stress and endothelial dysfunction in rats. Twenty four male albino rats were divided into four groups. In the control group, rats were administered vehicle by tube for 7 consecutive days. The second group was administered ginseng extract (100 mg/kg, by gavage) for 7 consecutive days. Animals in the third group were administered vehicle by tube for 7 consecutive days, then exposed to single dose gamma-irradiation (6 Gy). The Fourth group received ginseng extract for 7 consecutive days, one hour later rats were exposed to gamma-irradiation. Oral administration of ginseng extract prior to irradiation produced a significant protection which was evidenced by a significant reduction in serum creatine kinase (CPK) and lactate dehydrogenase (LDH) activities and asymmetric dimethylarginine (ADMA), urea and creatinine levels with significant increase in serum total nitrate/nitrite (NO(x)) level. Moreover, ginseng significantly increased cardiac and renal superoxide dismutase (SOD) and glutathione peroxidase (GSHPx) activities, and reduced glutathione (GSH) content, associated with a significant depletion in malondialdehyde (MDA) and NO(x) levels compared to irradiated group. This study suggests that ginseng may serve as a potential protective agent against gamma-irradiation-induced cardio-nephrotoxicity via enhancing the antioxidant activity and inhibition of endothelial dysfunction.

INTRODUCTION
Radiotherapy is frequently used as a part of cancer treatment to achieve tumor control. Although radiotherapy treatment has been widely used as an effective tool to kill tumor cells, it might produce harmful effects to surrounding healthy tissues (Ostrau et al., 2009;Sezen et al., 2008). It is well known that ionizing radiations induce oxidative stress on target tissues, mainly through the generation of reactive oxygen species (ROS) resulting in imbalance of the pro-oxidant and antioxidant in the cells, attack diverse cellular macromolecules such as DNA, lipids, and proteins, eventually inducing cell death (Boerma and Hauer-Jensen, 2011;Lee et al., 2010a;Sathyasaikumar et al., 2007).
Several studies have shown that irradiation plays an important role in the development of endothelial dysfunction; in particular, reduced bioavailability of nitric oxide (NO) (Soucy et al., 2010). Asymmetric dimethylarginine (ADMA) is widely recognized as the major endogenous inhibitor of NO-synthase and is considered an emerging cardiovascular risk factor (Kiani et al., 2007). Plasma concentration of ADMA is markedly increased in patients with chronic renal failure, and in a variety of cardiovascular diseases and moderately increased in patients with many other diseases including hyperlipidemia, diabetes mellitus, arterial hypertension, hyperhomocysteinemia and heart failure, suggesting that ADMA is an early marker of atherosclerosis vascular disease (Beltowski and Kêdra, 2006).
Ginseng is one of the most highly valued natural dietary supplements (Yi et al., 2009;Wu et al., 2011). The term ginseng refers to the dried root of several species in the genus Panax of the Araliaceae family (Wang et al., 2007a) including two commonly used species, i.e., Panax ginseng C. A. Meyer (Asian ginseng) and Panax quinquefolius L (North American ginseng) (Lee and Lau, 2011). Ginseng contains many physiologically important constituents including saponin (ginsenosides), polysaccharides, peptides, polyacetylenes, alkaloids, nitrogen-containing compounds, fatty acids and phenolic compounds (Attele et al., 1999;Choi, 2008;Lee et al., 2010b). Ginseng has drawn attention worldwide for its invaluable medicinal potential including antidiabetic, anticarcinogenic (Xie et al., 2005;Wang et al., 2007b), analgesic, antipyretic, antistress, and antifatigue effects (Yi et al., 2009;Lee et al., 2005), as well as enhancement of immune-modulating capabilities (Hwanga et al., 2011). These multifold bioactive medicinal properties of ginseng have been closely linked to its antioxidative effects (Kang et al., 2007;Wang et al., 2007a). In addition, ginseng and its partially purified constituents have potential radioprotective properties (Lee et al., 2006;Syaifudin et al., 2008). Therefore, the purpose of this study was to investigate the protective effect of ginseng against heart and kidney damage induced by gamma-radiation in rats.

Chemicals
Panax ginseng was purchased from Eipico, Egypt. It was suspended in 1 % carboxy methyl cellulose (CMC) in water and administrated orally to rats at a dose of 100 mg/kg body weight for 7 consecutive days (Gadkariem et al., 2010).

Animals
Male Wistar rats (weighing 120-150 g) were obtained from the animal farm of the Egyptian Holding Company for Biological Products and Vaccines, Egypt. Upon arrival, the animals were allowed to acclimatize for 1 week before starting the experiment. Animals were kept under standard conditions and were allowed free access to a standard requirement diet and water ad libitum. Animals were kept under a controlled lighting condition (light: dark, 13 h-11 h). The animals' treatment protocol was approved by the Animal Care Committee of the National Center for Radiation Research and Technology (NCRRT), Cairo, Egypt.

Irradiation
Whole-body gamma-irradiation was performed at the National Centre for Radiation Research and Technology (NCRRT), Atomic Energy Authority, Cairo, Egypt, using (137 cesium) Gamma Cell-40 biological irradiator. Animals were irradiated at an acute single dose level of 6 Gy delivered at a dose rate of 0.012 Gy/s.

Experimental design
Male albino rats were divided into four groups, 6 rats in each. In the control group, rats were administered vehicle (0.5 ml of 1 % CMC suspension in water) by gavage for 7 consecutive days. The second group was administered ginseng (100 mg/kg, by gavage) for 7 consecutive days. Animals in the third group were administered vehicle by gavage for 7 consecutive days, then exposed to single dose γ-irradiation (6 Gy). The Fourth group received ginseng (100 mg/kg, by gavage) for 7 consecutive days, one hour later rats were exposed to single dose γ-irradiation (6 Gy) (Mansour and Hafez, 2012).

Biochemical assays
Twenty-four hours after the last dose of the specific treatment, animals were anesthetized with ether, and blood samples were obtained by heart puncture and serum was separated by centrifugation (Sorvall TC centrifuge, Hamburg, Germany) at 750 g at room temperature for 10 min. Serum urea nitrogen, creatinine were determined according to the methods of Hallet and Cook (1971) and Bonsenes and Taussky (1945), respectively. Serum creatine phosphokinase (CPK) and Lactate Dehydrogenase were determined according to the methods of Swanson and Wilkinson (1972) and IFCC (1980) respectively. Serum total nitrate/nitrite (NO(x)) was measured as stable end product, nitrite, according to the method of Miranda et al. (2001). ADMA was estimated using a standard enzyme linked immunosorbent assay (ELISA) method according to the manufacturer's instructions (Immundiagnostik AG, Bensheim/Germany).
Hearts and kidneys were quickly excised, washed with saline, blotted with a piece of filter paper and homogenized in ice-cold 0.15 MTris-KCl buffer (pH 7.4) to yield a 20 % (w/v) homogenate using a Branson sonifier (250, VWR Scientific, Danbury, CT, USA). The homogenates were used for the determination of malondialdehyde (MDA) level, glutathione peroxidase (GSHPx) and superoxide dismutase (SOD) activities, total glutathione (GSH) content, and total nitrate/nitrite (NO(x)). The homogenates were centrifuged at 800 g for 5 min at 4 ºC to separate the nuclear debris. The supernatant so obtained was centrifuged (Eppendorf AG, centrifuge 5804R, Hamburg, Germany) at 15000 g for 30 min at 4 ºC to get the post mitochondrial supernatant which was used to assay superoxide dismutase (SOD) activity.
Reduced glutathione (GSH) and malondialdehyde (MDA) levels in heart and kidney homogenates were determined spectrophotometrically using the methods of Ellman (1959) and Buege and Aust (1978), respectively. Total nitrate/nitrite (NO(x)) was measured as the stable end product, nitrite, according to the method of Miranda et al. (2001). The activities of GSHPx and SOD were determined according to the methods of Lawrence and Burk (1976) and Minami and Yoshikawa (1979), respectively.

Statistical analysis
Results were expressed as mean ± SEM. The intergroup variation was measured by one way analysis of variance (ANOVA) followed by Tukey's Multiple comparison test. Statistical significance was considered at p < 0.05. Table 1 shows the effects of ginseng, irradiation and their combination on serum creatine phosphokinase (CPK), lactate dehydrogenase (LDH), creatinine and urea. Gamma-irradiation (6 Gy) induced a significant increase in CPK and LDH activities and significant increase in the levels of serum urea nitrogen and serum creatinine compared to control (P < 0.001). Administration of ginseng for 7 consecutive days before irradiation significantly reduced the activities of CPK and LDH, and the levels of urea and creatinine in serum (P < 0.001) compared to the irradiated group (Table 1). Data are presented as mean ± SEM, n = 6. * and # indicate significant changes from control and IR respectively at p ≤ 0.05 using ANOVA followed by Tukey-Kramer as a post ANOVA test. Table 2 shows the effects of ginseng, gamma-irradiation and their combination on levels of GSH, MDA, and NO(x), the activity of SOD and GSHPx in kidney tissues. Gamma-irradiation exposure resulted in significant (54.9 %, 18 % and 40.3 %) decrease in SOD and GSHPx activities and GSH content and significant (67.7 % and 114.3 %) increase in MDA and NO(x), respectively, as compared to the control group. Treatment with ginseng for 7 consecutive days prior to irradiation resulted in a significant (100.4 %, 18.8 % and 60.7 %) increase in the activities of SOD and GSHPx and in GSH content, respectively, as compared with the irradiated group, and significant (29.6 % and 44.1 %) decrease in MDA and NO(x) levels ,respectively, compared to the irradiated group. Table 3 shows the effects of ginseng, gamma-irradiation and their combination on levels of GSH, MDA, and NO(x), the activity of SOD and GSHPx in cardiac tissues. Gamma-irradiation exposure resulted in significant (33.8 %, 52.4 % and 50 %) decrease in SOD and GSHPx activities and GSH content and significant (65.9 % and 191.7 %) increase in MDA and NO(x), respectively, as compared to the control group. Treatment with ginseng for 7 consecutive days prior to irradiation resulted in a significant (31.9 %, 117.8 % and 87.5 %) increase in the activities of SOD and GSHPx and in GSH content, respectively as compared with the irradiated group, and significant (37.9 % and 43.2 %) decrease in MDA and NO (x) levels, respectively, compared to the irradiated group. Data are presented as mean ± SEM, n = 6. * and # indicate significant changes from control and IR respectively at p ≤ 0.05 using ANOVA followed by Tukey-Kramer as a post ANOVA test. Data are presented as mean ± SEM, n = 6. * and # indicate significant changes from control and IR respectively at p ≤ 0.05 using ANOVA followed by Tukey-Kramer as a post ANOVA test. Figures 1 and 2 show the effects of ginseng, irradiation and their combination on serum NO(x) and ADMA. Gamma-irradiation exposure resulted in a significant increase in the level of serum ADMA and significant decrease in serum NO(x) level compared to control (P < 0.001). Administration of ginseng for 7 consecutive days before irradiation significantly reduced the level of serum ADMA and the increase in serum NO(x) level (P < 0.001) compared to the irradiated group. Data are presented as mean ± SEM, n = 6. * and # indicate significant changes from control and IR respectively at p ≤ 0.05 using ANOVA followed by Tukey-Kramer as a post ANOVA test. Data are presented as mean ± SEM, n = 6. * and # indicate significant changes from control and IR respectively at p ≤ 0.05 using ANOVA followed by Tukey-Kramer as a post ANOVA test.

DISCUSSION
Ionizing radiation is known to induce oxidative stress through generation of ROS in an imbalance in pro-oxidant, antioxidant status in the cells (Bhosle et al., 2005). In the present study, Gamma-irradiation caused a marked increase in serum activities of LDH and CPK, levels of creatinine, urea and ADMA in parallel with a significant decrease in NO(x) level. These data agree with that reported in previous studies, which reported that IRR caused a significant increase in CPK and LDH activities (Sridharan and Shyamaladevi, 2002) and significant increase in urea and creatinine (Barakat et al., 2011). The excessive production of free radicals and lipid peroxides might have caused the leakage of cytosolic enzymes such as lactate dehydrogenase, creatine kinase and phosphatases. Also, it could induce lipid peroxidation of cell membranes structure by oxygen derived free radicals leading to ionic leakage through cellular membranes and excessive calcium influx with ensuring cellular dysfunction and death from calcium overload (Ramadan et al., 1997). Increase in serum urea was due to increase in glutamate dehydrogenase enzyme as a result of irradiation and this may increase carbamoyl phosphate synthetase activity leading to increase in urea concentration (Ramadan et al., 2001).
Treatment with ginseng for 7 consecutive days prior to irradiation ameliorated the activites of serum CPK and LDH and the levels of serum creatinine and urea. This effect might be related to the antioxidative properties of ginseng, which protect the outer membrane of mammalian cells (Block and Mead, 2003). The antioxidative ability of ginseng is closely related to its ginsenoside content. Ginsenosides have the ability to intercalate into the plasma membrane, change its fluidity, and inhibit lipid peroxidation by chelating transition metals and scavenging ROS (Kang et al., 2007), ginsenosides thus affect membrane function, eliciting cellular responses to cytotoxic stresses (Attele, 1999).
In the present study, the γ-irradiated rats showed a significant increase in serum ADMA concomitantly with a significant decrease in NO(x) level. The effect is probably mediated by oxidative stress (Maas et al., 2007). In agreement with our results, previous studies of Schnabel et al. (2005), Busch et al. (2006), Ueda et al. (2007) and Wilcox (2012) have reported elevated ADMA levels and decreased NO(x) levels in states of cardiovascular diseases and chronic kidney disease in human and rat and also in response of endothelial cells to ionizing radiation (Lanza et al., 2007). Elevated levels of ADMA inhibit NO synthesis and therefore impair endothelial function (Sibal et al., 2010). Reduction of NO(x) levels might be due to both decreased pro-duction and increased consumption, with possible endothelial dysfunction and vascular impairment (Soloviev et al., 2003).
Consistent with previous studies (Kim and Lee, 2010;Pan et al., 2012), the present study showed that the endothelial NO(x) and ADMA levels were ameliorated by the administration of ginseng. Pan et al. (2012) reported that, ginseng reduced ROS production and increased NO(x) levels, thus ameliorating endothelial dysfunction. Panax ginseng is known to enhance the release of nitric oxide (NO) from endothelial cells of the rat aorta and kidney, and to protect the heart from injury via up-regulation of endothelial NO synthase (eNOS) expression (Wu et al., 2011;Razavi et al., 2005;Hare and Stamler, 2005;Han and Kim, 1996), resulting in Ca(2+) channel inhibition, activation of cardiac potassium channels and protection against ischemia-reperfusion injury (Wang et al., 2008;Szelid et al., 2010) and this protection is caused by reducing oxidative stress (Kim and Lee, 2010). These findings suggest that some of the observed effects of ginseng are possibly mediated through its antioxidant property.
Consistent with previous studies (Mansour and Hafez, 2012;Pradeep et al., 2012), the present study showed a significant depletion in the antioxidant system accompanied by enhancement of lipid peroxides and NO(x) levels in cardiac and renal tissues after whole body gamma-irradiation. Ionizing radiation is known to induce oxidative stress through generation of ROS in an imbalance in pro-oxidant, antioxidant status in the cells (Bhosle et al., 2005). The increase in lipid peroxidation levels in γirradiated rats might be due to the interaction of free radicals with polyunsaturated fatty acids in the phospholipids portion of cellular membranes (Prasad et al., 2005;Spitz et al., 2004). The decrease in the activities of SOD and GSHPx and the decreased level of GSH might be due to their utilization by the enhanced production of ROS, which interacts with the enzyme molecules causing their denaturation and partial inactivation (Kregel and Zhang, 2007;Maurya et al., 2006). Under normal conditions the inherent defense system, including glutathione and the antioxidant enzymes, protects against oxidative damage. GSH, a well-known antioxidant, provides major protection in oxidative injury by participating in the cellular system of defense against oxidative damage (Sener et al., 2006). It was reported that tissue GSH levels and the activities of glutathione reductase and glutathione peroxidase, which are critical constituents of GSH redox cycle, were significantly reduced due to oxidative stress, permitting enhanced free radical-induced tissue damage (Reiter et al., 2001).
Our results show that whole body gamma-irradiation of rats at 6 Gy enhanced the formation of cardiac and renal NO(x). Similar results have been reported by Gorbunov et al. (2000). Gamma-irradiation may enhance endogenous NO biosynthesis in liver, intestine, lung, kidney, brain, spleen or heart of the animals, presumably by facilitating the entry of Ca 2+ ions into the membrane as well as the cytosol of NOproducing cells through irradiation-induced membrane lesions. The enhancement of NO production following exposure to a high dose (6 Gy) of gamma rays was attributed to high levels of expression of the inducible nitric oxide synthase (Ibuki and Goto, 1997).
Consistent with previous studies (Abdel-Wahhab and Ahmed, 2004; Kim et al., 2011;Park et al., 2011;Ramesh et al., 2012), administration of ginseng markedly elevated the levels of antioxidant enzymes, indicating the antioxidant potential of the ginseng. This might be due to metabolized ginsenosides, which protect the outer membrane of mammalian cells (Block and Mead, 2003;Lee et al., 2009), scavenge free radicals, restore the GSH level and inhibit NO production (Zhu et al., 2009;Zhang et al., 2010). Panax ginseng extract has been shown to inhibit lipid peroxidation through transition metal chelation and scavenging of hydroxyl and superoxide radicals (Kitts et al., 2000). It has been also reported that Panax ginseng administration increased the activity of the antioxidant enzymes SOD and GSHPx in rats (Han et al., 2005;Sena et al., 2012;Sun, 2011). Kumar et al. (2003) found that administration of panax ginseng root extract before irradiation significantly decreased lipid peroxidation levels and reduced the radiation damage in mice testes.
The results from the present investigation indicate that ginseng pretreatment protects against radiation damage by inhibiting radiation-induced oxidative stress and endothelial dysfunction by decreasing serum ADMA, increasing serum NO(x) and ameliorating the antioxidant system in cardiac and renal tissues. Kim TH, Lee SM. The effects of ginseng total saponin, panaxadiol and panaxatriol on ischemia/reperfusion injury in isolated rat heart. Food Chem Toxicol 2010;48: 1516-20.


extfieldsofstudy: Biology Medicine

text:
The role of the cytoskeleton at the immunological synapse

It has been over 30 years since the reorganization of both the microtubule network and a ‘peculiar actin polarization’ was reported at the contact area of cytotoxic T lymphocytes interacting with target cells. Since that time, hundreds of studies have been published in an effort to elucidate the structure and function of the microtubule network and the actin cytoskeleton in T-cell activation, migration, and effector function at the interface between a T cell and its cognate antigen-presenting cell or target cell. This interface has become known as the immunological synapse, and this review examines some of the roles played by the cytoskeleton at the synapse.

Introduction
The unusual reorganization of both the microtubule network and actin cytoskeleton was noted 30 years ago (1)(2)(3). Both actin and microtubule networks take up characteristic localizations both in migratory T cells and when the immunological synapse, referred to throughout this review as 'synapse', forms. In migrating T cells, actin accumulates at the leading edge, as in other migratory cell types. However, T cells show a distinctive positioning of the centrosome [which is the microtubule organizing center (MTOC) in T cells], which is localized in the uropod (4) in migrating T cells, in contrast to other cell types where the centrosome polarizes toward the leading edge (5,6). The position of the centrosome changes dramatically upon target cell recognition, with the centrosome translocating from the rear of the cell all the way to the leading edge where the synapse forms (7). A mature synapse is characterized by centrosome contact with the plasma membrane at the center of the bull's-eye behind the central supramolecular activation cluster (cSMAC), with actin accumulating around the edge of the T-cell/target interface in the distal SMAC (dSMAC) (Fig. 1). The same organization is found not only in the synapses formed between cytotoxic T lymphocytes (CTLs) and target cells but also in other cytolytic cells, including natural killer (NK) and invariant NKT cells (8), as well as in CD4 + T cells where the centrosome also docks within the center of the synapse (9) and actin accumulates toward the edge of the cell.

Actin in T-cell activation
Actin dynamics and T-cell signaling are very closely linked. Not only does T-cell receptor (TCR) ligation initiate a robust actin polymerization response, but actin dynamics are also required for effective TCR signaling as inhibitors of actin polymerization disrupt T-cell activation (10)(11)(12). This is thought to be due to the fact that actin plays a role in the formation of TCR signaling clusters. TCR signaling initiates in numerous microclusters at the periphery of the synapse (13)(14)(15), which migrate toward the center where they coalesce to form the cSMAC. Actin retrograde flow has been shown to promote the centripetal movement of TCR microclusters to the cSMAC, as treatment of T cells with latrunculin, an inhibitor of actin polymerization, halts transport of TCR microclusters to the cSMAC and abrogates formation of new signaling assemblies (15,16). However, the mechanism required for this actin retrograde flow is not well understood. The actin nucleation promotion factors Wiskott-Aldrich syndrome protein (WASp), WASp family verprolin homologous protein 2, and HS1 are thought to cooperate with Arp2/3 to polymerize filamentous actin (F-actin) from the plasma membrane triggering centripetal inward movement toward the F-actin-poor cSMAC, where subsequent depolymerization is thought to occur (17,18). Myosin IIA has also been implicated in TCR microcluster translocation to the cSMAC and maintenance of synapse architecture, and this has been comprehensively reviewed recently (19).
Studies by a number of different groups have explored the role of myosin IIA in T-cell signaling as well as synapse organization and maintenance. Jacobelli et al. (20) demonstrated that myosin IIA accumulates at the synapse, but provided evidence that it is not required for TCR recruitment to the cSMAC. The authors conclude that myosin IIA is critical for T-cell motility, but entirely dispensable in the generation and maintenance of synapse architecture (20). Perplexingly, in 2009 a separate study presented data that completely contradicted the previous work by Jacobelli. Ilani et al. (21) showed that inhibition of myosin IIA activity with blebbistatin completely halts TCR microcluster movement and also that both blebbistatin and myosin IIA short-interfering RNA (siRNA) knockdown abrogates TCR signaling, measured by imaging calcium flux and staining with antibodies against phosphorylated versions of signaling proteins downstream of the TCR (21).
Recent studies exploring the role of myosin IIA in T-cell activation have not entirely ameliorated the conflict. Studies using total internal reflection fluorescence microscopy to track movement of individual SH2 domain-containing protein of 76 kDa (Slp76) or TCR microclusters after myosin IIA inhibition in T cells activated on either anti-CD3e-coated coverslips (22) or either anti-CD3e- (21,23) or pMHCloaded (24,25) bilayers present a variety of results, from complete inhibition (21) or retarded microcluster movement and increased meandering (23,24) to no effect on TCR microcluster movement at all (25). Yu et al. (26) report that both blebbistatin (a myosin II inhibitor) and ML7 (a myosin heavy-chain inhibitor) slow the initial phase of TCR movement, whereas later phases remain unaffected. The diversity of results from these studies most likely reflects the variety of experimental systems used by different groups to test the function of myosin IIA in T cells. Differences in cell type (Jurkat versus primary T cells), T-cell activation molecule (anti-CD3e antibody or pMHC), or substrate (coverslip versus supported lipid bilayer), as well as variation in the method of myosin IIA inhibition (pharmacological inhibition versus siRNA-mediated protein depletion) could all contribute to disparities between different studies.
Two studies show that myosin IIA inhibition reduces phosphorylation of CasL, a protein involved in mechanosensation The immunological synapse consists of a series of supramolecular activation clusters (SMACs) forming a bull'seye-like ring when observed in cross section. The central SMAC (cSMAC) consists of the T-cell receptor (TCR) and associated kinases, the peripheral SMAC (pSMAC) of integrins, and the distal (dSMAC) of excluded phosphatases, such as CD45, as well as actin and actininteracting proteins, e.g. IQGAP-1. The centrosome docks by the cSMAC and determines the point of secretory granule exocytosis in cytotoxic T lymphocytes (CTLs). (24,27). These studies implicate myosin IIA in the generation of force at the synapse, which may be detected through integrins. If myosin IIA activity does generate force at the synapse, the capacity of myosin IIA to affect signaling through integrins may be linked to substrate mobility. It has become apparent that stimulation of T cells using glass-or lipid bilayer-bound antigens and adhesion molecules can produce vastly different outcomes with respect to synapse formation and T-cell signaling due to differences in ligand mobility between these two systems. Ligand mobility can also be heterogeneous among different lipid bilayer systems, as variations in lipid composition can affect mobility of molecules within a lipid bilayer (28). To compensate for differences in activation parameters between the various studies examining the role of myosin IIA in T cells, it may be advantageous to adopt a live T-cell/antigen-presenting cell (APC) system. This more physiological method of T-cell stimulation may permit a more accurate assessment of how myosin IIA may cooperate with actin flow to organize the synapse, regulate signaling through integrins, or affect calcium flux into the cell.

The immunological synapse and migratory T cells
Parallels have been drawn between the actin dynamics and molecular makeup between the leading edge of a migratory cell and the two actin-rich outer segments of the synapse, leading to the proposal that the organization of the synapse is essentially that of a radially symmetric migratory cell (29). The dSMAC, like the lamellipodium (LP), is characterized by protrusive actin polymerization that pushes out the T-cell membrane at the edge of the T-cell/target contact site (18,30,31). The lamellum (LM) is the region of a migratory cell most associated with the formation of new focal adhesions, and like the peripheral SMAC (pSMAC) is enriched in the aLb2 integrin (leukocyte function-associated antigen-1) with integrin-associated talin used as a marker of both pSMAC and LM (31)(32)(33). Recent studies support the analogy of structures at the synapse with those found at the leading edge. Work by Yi et al. verifies the identity of the dSMAC as the LP and the pSMAC as the LM by staining endogenous proteins in fixed Jurkat T cells interacting with activating supported bilayers and showing that endogenous Arp2/3 and myosin IIA decorate the dSMAC and pSMAC, respectively, as they do in the LP and LM (23,32). This corroborates previous work showing cofilin and Arp3 enrichment in the dSMAC/LP and tropomyosin recruitment to the pSMAC/LM (33,34). Complementary work on anti-TCR-coated glass also demonstrates myosin IIA recruit-ment to the pSMAC, with some overlap in the dSMAC (22). Importantly, Yi et al. (23) show that myosin IIA in the pSMAC decorates concentric, bundled actin arc structures that lie just inside the dSMAC. These actomyosin contractile arcs are reminiscent of structures previously described in the LM of migrating neuronal growth cones and kidney epithelial cells (35,36). The observation that bona fide actomyosin contractile arc-like structures are present in the pSMAC of the synapse drives home the structural similarities between actin organization at the leading edge and at the synapse, and invites the possibility that the large body of work describing the structure and dynamics of actin at the leading edge might be used to inform T-cell biology.
Recent studies clarifying the relationship between the LP and the LM shed light on how actin in the dSMAC and pSMAC may cooperate to organize the synapse. Burnette et al. (35) have convincingly shown that actin arc structures in the LM are formed from bundling of branched actin that is generated in the LP. The data suggest that myosin IIA molecules bind to branched F-actin at the peak protrusion of the lamellipodium, compressing the actin into bundles of fibers, which migrate toward the interior of the cell. The majority of myosin in the kidney epithelial cells used in the study is found on these actin arc structures. Both Babich et al. (22) and Yi et al. (23) describe abundant myosin localization just inside and slightly overlapping with the actin signal in the dSMAC. Intriguingly, Yi et al. (23) demonstrate that TCR microclusters migrate within the pSMAC at the same rate as the actin arc-like structures, suggesting that the arcs may facilitate centripetal translocation of signaling components at the synapse, and Babich et al. found that SLP-76 microcluster centralization and subsequent T-cell activation were inhibited when myosin IIA and F-actin turnover was inhibited, supporting an important role for actin flow in T-cell activation. It will be interesting to see how work on the leading edge of migratory cells and the dSMAC/pSMAC of activated T cells will complement one another, as mechanisms for actin polymerization and organization are further defined in these systems.

Control of centrosome polarization
Although the polarization of the centrosome was first observed many years ago (1, 2), it is still not entirely clear how the centrosome is directed to such a specific location within the CTL synapse, nor how the centrosome interacts with the plasma membrane. What is clear is that in T cells the centrosome polarizes in response to TCR activation (7,(37)(38)(39)(40)(41)(42). Several downstream signaling proteins involved in transmission of the intracellular signaling from TCR have been implicated in the control of centrosome polarization. Lck is the proximal tyrosine kinase associated with CD4 + or CD8 + T-cell coreceptors, and initial studies in Jurkat cells lacking Lck expression implicated Lck in centrosome polarization (43). However, as Jurkat cells lacking either Lck or Zap70 are able to trigger both Ca ++ fluxes and extracellular signal-regulated kinase (Erk) activation in response to TCR cross-linking or superantigen stimulation (44), it is suggested that there are linker for activation of T cells (LAT)independent pathways in these cells, which are not seen in primary T cells. As T cells do not develop in the absence of Lck, an inducible Lck Off mouse model was needed to examine the role of Lck in centrosome polarization in primary CTLs (45). Interestingly, the centrosome is able to polarize around the nucleus toward the synapse when Lck is turned off. However, the centrosome does not reach the plasma membrane and is unable to dock in Lck Off CTLs, and consequently, target cell killing is ablated (45). Fyn appears to play a role in the polarization of the centrosome toward the synapse, as in CTL deficient in both Fyn and Lck of the centrosome was unable to polarize and remained in the uropod of the T cell (45). This effect seems to be dependent on the combined loss of both Fyn and Lck, as Fyn À/À CTLs (46) and NK cells (47) kill targets as effectively as wildtype cells, suggesting that the centrosome polarization is uncompromised by loss of Fyn alone. These results also reveal that Fyn cannot compensate for loss of Lck, as loss of Lck alone disrupted centrosome polarization. The role of LAT is not entirely clear, as although LAT-deficient Jurkat cells show decreased centrosome polarization (48), only a modest reduction in CTL killing is observed in LAT-deficient CTLs (49). Studies on Zap70, the upstream kinase which phosphorylates LAT, are clearer with early studies on Zap70-deficient Jurkat (48) or with use of dominant negative (43) or kinase dead forms of Zap-70 (50), showing some defects in centrosome polarization. A more recent mouse model expressing an analog-sensitive mutant form of Zap70 kinase allowed Zap70 kinase activity to be rapidly and reversibly inhibited, demonstrating defective killing activity when Zap70 catalytic activity is inhibited (51).
The centrosome is very sensitive to TCR signaling, polarizing to the synapse in response to weak signals, which are insufficient to trigger the associated granule polarization (52). This demonstrates how centrosome docking at the CTL plasma membrane allows the cell to prime itself for target cell killing without actually committing, thus ensuring execution of an appropriate immune response. Intriguingly, NK cells are able to polarize both centrosome and secretory granules to the synapse in response to either inhibitory receptor signals (53) or integrin activation alone (54,55), suggesting that the signaling for these events can be separated and that the control of centrosome and granule polarization may differ in CTLs and NK cells. There also appear to be some differences in signaling between mouse and human NK cells, with activating receptor engagement required to trigger granule polarization in mouse NK cells (56) but not in human NK cells (53).
The role of calcium signaling in control of centrosome polarization T-cell activation upon TCR engagement involves calcium signaling, but the role of calcium signaling in centrosome polarization toward target cells has been queried recently. Most published work focuses on the role of calcium signaling in mediating granule polarization and release, with little work focusing only on centrosome movement, potentially as it can be difficult to temporally separate these events. In some of the original work by Kupfer and colleagues, the accumulation of talin at the contact site between immortalized helper T cells and APCs was unaffected, but centrosome polarization toward the target cell dramatically increased by supply of exogenous calcium (38). Many years later, experiments on Jurkat cells supported this claim, as very little centrosome polarization toward anti-CD3-coated slides was observed in the absence of extracellular calcium (48). However, the authors were unable to determine how polarization of the centrosome depended on calcium: Inhibition of either calcineurin-or calcium/calmodulin-dependent kinase had no negative impact on the ability of the centrosome to polarize (48). These studies did not assess changes in intracellular calcium levels upon TCR engagement, but recent experiments have shown that TCR stimulation of CTLs carried out in medium lacking extracellular calcium resulted in an intracellular calcium flux with a similar magnitude to that which occurred in medium with calcium present (57). Although this study assessed the extent to which granule polarization relied on calcium flux, its role in centrosome polarization control was not examined (57). This area of research has been further complicated by studies carried out on primary helper T cells binding to peptide-MHC-coated glass coverslips: both intracellular and extracellular calcium were chelated, but centrosome polarization was unaffected (58). Furthermore, the cSMAC accumulating protein kinase C-h (PKC-h) (59), which is activated by diacylglycerol (DAG) but not calcium, has been implicated in control of centrosome polarization (60), and NK cells from patients mutated in the plasma membrane calcium channel protein ORAI1 are able to polarize their centrosome and granules normally when conjugated with target cells in the absence of any calcium flux (61).
It is difficult to explain these contrasting findings. However, each of these studies uses different cell types, and it is possible that differences in calcium dependence for centrosome polarization may exist between NK cells and T cells, as well as between immortalized cell lines and primary cells. Furthermore, recent data have revealed that intracellular calcium released from the secretory granules themselves within CTLs controls granule secretion from primary human CTLs (62), raising the possibility that relatively small and localized fluxes in calcium might be sufficient to trigger granule and presumably centrosome polarization, which may fall below the threshold of detection in some experimental situations.

Role of the motor protein dynein
Investigations into how the force required to move the centrosome could be generated have looked at microtubule motors. Interference with plus end-directed movement along microtubules fails to interfere with centrosome polarization (7), but studies on dynein suggest that perhaps it is the minus end-directed action of this motor protein that pulls the centrosome toward the synapse. Concomitantly, dynein accumulates at the synapse, relying on the earlier recruitment of DAG (58), as well as segregation of the SLP-76 adapter protein adhesion-and degranulation-promoting adapter protein (ADAP) into the pSMAC (63). Depletion of dynein heavy chain in Jurkat cells have been reported to cause defects in centrosome polarization toward the synapse (64), and ADAP depletion prevents both dynein accumulation and centrosome polarization to the synapse in human T cells, although not in mouse T cells (63). Because ADAP interacts with dynein and microtubules, it has been proposed that clearance of ADAP to the pSMAC could cause the microtubule tension required for dynein to pull the centrosome toward the synapse (63). This is supported by data in which microtubules are seen anchored at the pSMAC, but not the cSMAC, and the centrosome drawn to the synapse by a microtubule sliding mechanism (65). However, as ADAP may not have a role in centrosome polarization in mouse cells (63) and as a more recent study on primary mouse T cells showed that neither a dynein inhibitor nor depletion of dynein heavy chain affects centrosome polarization (66), the role of dynein was unclear. More recently, shRNA has been used to deplete dynein heavy chain (Dy-nHC) in T cells, and while DynHC depletion or blebbistatin treatment reduce centrosome polarization modestly, the effect is more pronounced when cells are both depleted of DynHC and treated with blebbistatin, suggesting that myosin IIA and dynein might act together in centrosome polarization (67).
Another unusual aspect of centrosome movement in CTLs is the ability of the centrosome to dissociate from its tight association with the nuclear envelope. Both in the uropod in migrating cells and when the synapse is formed, the centrosome and nucleus can appear to be some distance apart (4,45). The ability to dissociate from the nucleus does not seem to be a factor in controlling centrosome polarization, as the centrosome can still polarize to the synapse efficiently and CTL cytotoxicity remains the same as in control CTLs when the centrosome is artificially tethered to the nucleus (68).

Cytoskeletal regulators of microtubules and actin at the synapse
Actin reorganization correlates with the polarization and docking of the centrosome at the synapse (Fig. 2), suggesting a mechanistic link between these two events (7). A number of studies have examined the role of proteins that link to microtubules or the actin cytoskeleton in centrosome polarization. The microtubule destabilizing protein stathmin (69) has a role in controlling microtubule dynamics in migrating T cells (70) and localizes to the synapse when phosphorylated (71). In stathmin knockout mice, small but statistically significant decreases in centrosome polarization and CTL-mediated killing were documented compared to wildtype cells, with a defect in PKC-h accumulation at the synapse but normal TCR signaling (71). This is particularly interesting in the light of studies implicating PKC-h in centrosome polarization control (60). These studies suggest that stathmin may play a small but significant role in centrosome polarization which could feasibly be linked to its capability to destabilize microtubules (69), especially as microtubule dynamics were slower in stathmin knockout compared to wildtype cells (71).
Other proteins that could affect centrosome polarization via actin have also been studied. Ezrin is an adaptor protein that can link membrane components with the actin cytoskeleton (72). Ezrin localizes with actin in the periphery of the Jurkat cell synapse in response to TCR signaling (73) and also interacts with and recruits Zap70 to the synapse (74). More recent studies find silencing of ezrin expression in T cells to impair centrosome polarization, particularly its close apposition to the stimulatory surface, with microtubules not stably attaching at the pSMAC (75). Another candidate that has been implicated in this pathway is discs large 1 (Dlg1), which has previously been linked to lymphocyte activation (76) and interaction with Lck (77). Dlg1 colocalizes in the periphery of the synapse with ezrin but not if ezrin is depleted (75). Depletion of Dlg1 itself produces similar phenotypes to those observed upon ezrin depletion (75). The authors suggest that these proteins coordinate cytoskeletal control at the synapse by ezrin providing a link between cortical actin and Dlg1, which is posited to interact with microtubules via a complex of proteins at the pSMAC (75).
IQGAP-1 can also provide a link between microtubule plus ends and the actin cytoskeleton (78), leading to the speculation that it could have a role in the mechanism of centrosome polarization to the synapse. Interestingly, although IQGAP-1 clears to the dSMAC and colocalizes with actin at the CTL synapse (7), CTLs from an IQGAP-1 knockout mouse show IQGAP-1 to be dispensable for CTL centrosome polarization and target cell killing (79), even though it is required for NK-cell centrosome polarization and cytotoxicity (80) and is claimed to be required for centrosome polarization in Jurkat cells (18). As TCR activation is shown to be impaired in the IQGAP1 knockout mouse, it is possible that compensatory mechanisms during T-cell development might mask a role for IQGAP1 in CTLs.
IQGAP-1 is a target molecule of Cdc42 and Rac1 (81). Overexpression of dominant negative forms of Cdc42 prevents centrosome polarization, but actin polymerization is unaffected (82). In addition, a defect in centrosome polarization caused by shRNA-mediated depletion of Cdc42 or Rac1 in Jurkat cells can be rescued by expression of the FH2 domain of the INF2 formin, which regulates the stability of microtubules, and if depleted by minor amounts, it appears to cause a drastic reduction in centrosome polarization (83). The role of Cdc42 in control of centrosome polarization Fig. 2. Centrosome polarization brings lytic granules to the synapse. The actin cortex of a migrating cell is continuous and intact, which provides a barrier to restrict unwarranted secretion of lytic granules that lie along microtubules. Upon target recognition, the centrosome polarizes to the synapse, bringing with it lytic granules that cluster at the centrosome. Close apposition of the centrosome with the plasma membrane brings microtubules in close proximity to the membrane at the synapse. Because actin cortical density is low at the synapse, granules that come in close contact with the plasma membrane are able to tether and fuse independent of myosin Va, releasing their cytolytic contents toward the target cell. remains somewhat controversial with differing results in primary human CD4 + T cells (84) and Jurkat cells (18), where Cdc42 depletion does not cause defects. In fact, instead these studies suggested that it is Rac1 that plays a role in centrosome polarization (18). Interestingly, Vav1, the guanine nucleotide exchange factor (GEF) for Rho GTPases such as Cdc42 and Rac1, has been implicated in centrosome polarization control in T cells (85), but apparently its GEF activity is dispensable for its role in linking TCR signaling to actin polymerization (86), questioning the link between Vav1 and Rho GTPases in coordinated control of centrosome polarization.
Another study convincingly associates formins with centrosome polarization control: when either or both of the formins Dia1 or FMNL1 are depleted from primary human CD8 + T cells, the centrosome does not polarize effectively and these CTLs do not kill target cells as efficiently as control CTLs (18). FMNL1 and Dia1 localize in rings surrounding the centrosome and also with microtubules in primary T cells (18). IQGAP-1 is linked to Dia1, as it interacts with and is required for its localization in migrating fibroblasts (87). Dia1 regulates microtubules and binds microtubule tip proteins in fibroblast cells (88). If such interactions also exist in T cells, this strengthens the argument for a role of Dia1 in centrosome control in these cells.
These data strongly implicate the formin family of cytoskeletal regulators in control of centrosome polarization to the synapse; however, the coordination of the roles of Rho GTPases and IQGAP-1 in control of formins and any subsequent action on centrosome polarization requires further elucidation. As INF2 formin is implicated in microtubule stabilization in addition to a role in centrosome polarization control these two processes could potentially be linked: modest depletion of INF2 means microtubules are no longer detyrosinated, causing them to become unstable and the centrosome is unable to polarize, but both phenotypes are rescued by expression of only the FH2 domain of INF2. Impaired centrosome polarization caused by overexpression of the histone deacetylase HDAC6 is rescued by administration of a deacetylase inhibitor to CD4 + T cells (89), suggesting that microtubule cytoskeleton modifications are important for centrosome reorientation.

Centrosome proteins
A large number of proteins reside at the centrosome (90), and so it seems feasible that some of these proteins may actually control centrosome polarization. Support for this concept comes from a recent study on the centrosome-local-ized protein casein kinase 1 d (CK1d) (91). CK1d depletion from Jurkat cells abrogates centrosome polarization toward the synapse, but intriguingly, CK1d itself is not observed at the synapse, suggesting that the protein's localization could be quite dynamic. Further investigation into the role of CK1d found it to associate with the plus-end microtubulebinding protein EB1, and, upon CK1d depletion, microtubule dynamics decrease (91), providing a potential link between the cytoskeleton and regulation of centrosome movement in T cells. The formin Dia1 also interacts with EB1 (88), thus indicating possible involvement in the CK1d centrosome polarization mechanism.
Paxillin also localizes at the centrosome (92,93), and is a cytoskeletal adapter protein involved at focal adhesions (94) that appears to have a role in centrosome polarization to the synapse, as a dominant negative effect is observed upon expression of only its centrosome localizing domains (95). Curiously, paxillin also localizes to the pSMAC of the synapse and so must dissociate from the centrosome to elicit its centrosome polarization function (95), as was also discovered for CK1d (91).
The development of inducible knockout models, as used for the TCR signaling component Lck (45), will help to elucidate which proteins contribute to centrosome polarization in effector T cells. Such models will permit T cells to develop without problems that may arise due to lack of important cytoskeletal regulators.

Involvement of the actin cortex in regulated secretion
There is much work describing the role of the actin cytoskeleton in CTL activation and signaling, but the role of the actin cytoskeleton in the cytolytic effector function of CTLs, the secretion of lytic granules, is less well understood. Due to the cytolytic nature of the secretory granule components in CTLs, precise delivery of these granules in a polarized fashion toward the target cell is important. To facilitate directed secretion of lytic granules toward a target cell, CTLs appear to utilize a mechanism of secretion that is different from secretory cells derived from the other cells which engage in regulated secretion. When a CTL encounters a target cell, it is the centrosome docking at the plasma membrane that focuses secretion at the synapse (7,96). Granules move in a dynein-mediated, minus end-directed fashion toward the centrosome, which brings them very close the plasma membrane. The role cortical actin may play in facilitating secretion in CTLs once the granules are in proximity of the PM is not clear. Actin has been shown to clear toward the periphery (dSMAC) of the synapse formed by a CTL engaging a target cell (7,97) (Fig. 2). Although inability to clear actin at the synapse has been correlated with defective cytotoxicity (45,98), no study has directly examined the relevance of actin clearance to secretion in CTL.
The role of actin in exocytosis has been well studied in other specialized secretory cells. In cells that specialize in regulated exocytosis such as adipocytes, mast cells, pancreatic b cells, and adrenomedullary chromaffin cells, the dense network of F-actin known as the actin cortex is critical in controlling secretion. Cortical actin appears to play contradictory roles in the regulation of secretion in many cell types. One model suggests that the actin cortex acts as a physical barrier, preventing vesicles from coming close enough to the plasma membrane to dock and fuse in unstimulated cells, whereas other study models suggest that cortical actin actually facilitates trafficking of vesicles to the plasma membrane for fusion (99). The barrier model was supported by evidence that disruption of the actin cytoskeleton of pancreatic b cells with cytochalasin B induces secretion of insulin-containing granules (100). Furthermore, actin filaments lying directly beneath the plasma membrane of chromaffin cells serve to imprison secretory vesicles and prevent them from reaching the plasma membrane in the resting state, thus impeding secretion (101). Live cell imaging of stimulated secretion in these cells shows that localized actin depolymerization permits secretory vesicles to access the plasma membrane (102). Perplexingly, the same cortical actin that seems to block secretion in chromaffin cells also facilitates vesicle transport to the plasma membrane when the cells are stimulated, promoting secretion (101).
A new model that may illuminate the 'carrier' versus 'barrier' paradox of the function of cortical actin in secretion has recently been proposed with regards to exocytosis of dense-core granules in mast cells. In an exquisite study, Wollman and Meyer showed that antigen-mediated activation of mast cells results in oscillating calcium waves. These calcium waves perpetuate a periodic thickening and thinning of the actin cortex mediated by the actin nucleation promoting factor, N-WASp. The oscillation of the density of the actin cortex was shown to be critical for efficient secretion, as disruption of the actin density fluctuations or the obliteration of the actin cortex reduced the efficiency of secretion (103).
For many cells that specialize in secretion, interaction of vesicles with the actin cortex is thought to be critical for their transport to the plasma membrane. In many cases, this interaction is facilitated by myosin Va, which is known to associate with secretory vesicles in adipocytes, chromaffin cells, and pancreatic b cells (104). Secretory granules in these systems travel toward the plus ends of microtubules in a kinesin-dependent manner that brings them close to the cell periphery where they interact with the actin cortex through myosin Va. Here, fluctuations in the actin cortical density are thought to bring the vesicles close enough to the plasma membrane to mediate interaction of membranebound tethering factors and soluble NSF attachment protein receptor (SNARE) proteins which mediate fusion. The result is a relatively non-polarized, omnidirectional secretion of granule components. In T cells, the mode of delivery to the plasma membrane differs, as secretion needs to be focused precisely at the synapse. This is particularly important for cytolytic cells such as CTLs and NK cells, where target cell killing needs to be accurately controlled to prevent unwanted damage during an immune response. In this case, the movement of secretory granules along microtubules is reversed as granules move in a minus-end direction, thus the centrosome, the point at which microtubule minus ends are anchored in T cells, acts to define a very precise point for secretion (7).
Interestingly, myosin Va, which is important in mediating secretion in other cell types, is not required for secretion in human or mouse CTLs (105), which casts doubt on whether granule interaction with cortical actin is important in facilitating secretion in CTLs, as it is in other professional secretory cells (103,104,106,107). Thus, the current data support a model in which lytic granule movement along microtubules toward the docked centrosome brings them close enough to the plasma membrane to engage tethering and fusion-promoting factors in the two membranes to drive secretion of the granules. Interestingly, Rab27a, a protein which has been shown to interact with and recruit myosin Va to melanosomes in melanocytes (108,109), interacts with the fusion-priming factor Munc13-4, which is required for granule secretion in CTLs (110). These data point to Rab27 as a bridge between these two secretory systems: one in professional secretory cells that is dependent on plus-end microtubule transport and secretory granule interaction with the actin cortex and the other in cytolytic cells, which is dependent on minus-end microtubule transport and plasma membrane exposure to deploy secretory granules.
Two recent studies have shed light on the relationship between lytic granule exocytosis and cortical actin density in another professional cytolytic cell: the NK cell. Complementary studies using super-resolution techniques of structured illumination microscopy and stimulated emission depletion microscopy demonstrate that actin does not entirely clear from the site of NK-cell activation/degranulation (96,111). In both studies, NK cells were activated on glass coverslips coated with activating ligands or non-activating ligands, fixed after a period of time, and stained for actin and other markers. Both studies demonstrate that the density of the actin cortex at the interface of NK cells interacting with activating ligands is reduced compared to NK cells plated on inhibitory ligands and that actin is not entirely clear from the plasma membrane at the interface. A loose meshwork of actin remains, and the authors show that lytic granules preferentially localize (112) and secrete (111) at areas of actin 'hypodensity'. Whether this pervasive actin network across the synapse remains after antigen recognition in CTLs and whether this represents a difference between CTLs and NK cells remains to be seen.

Concluding comments
Thirty years after the unusual reorganization of both the microtubule and actin cytoskeletons was noted (1-3), much has been learned about how important these changes are for immune cell function. With the advent of new molecular and imaging technologies, the molecular events controlling the cytoskeleton and the details of the reorganization are emerging. But as with any biological system, the more we learn, the more intriguing questions are raised. It is clear that this will remain an exciting area of research in immunology with links to other biological systems for some time.