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Oxidative burst and Dectin-1 triggered phagocytosis affected by Norepinephrine and Endocannabinoids - Implications for fungal clearance under stress

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Abstract

A prolonged stress burden is known to hamper the efficiency of both the innate and adaptive immune systems and to attenuate the stress responses by the catecholaminergic and endocannabinoid (EC) systems. Key mechanisms of innate immunity are the eradication of pathogens through phagocytosis and the respiratory burst. We tested the concentration-dependent, spontaneous and stimulated (via TNFα and fMLP) release of reactive oxygen species (ROS) by human PMNs in vitro in response to norepinephrine (NE) and AM1241, a pharmacological ligand for the endocannabinoid receptor CB2. We evaluated phagocytosis of Dectin-1 ligating zymosan particles and tested the cytokine response against Candida antigen in an in vitro cytokine release assay. Increasing concentrations of NE did not affect phagocytosis, yet stimulated ROS release was attenuated gradually reaching maximum suppression at 500nM. Adrenergic receptor (AR) mechanisms using non-AR-selective (labetalol) as well as specific α-(prazosin) and β-(propranolol) receptor antagonists were tested. Results show that only labetalol and propranolol were capable to recuperate cytotoxicity in the presence of NE, evidencing a β-receptor mediated effect. The CB2 agonist, AM1241, inhibited phagocytosis at 10μM and spontaneous peroxide release by PMNs. Use of the inverse CB2 receptor agonist SR144528 led to partial recuperation of ROS production, confirming the functional role of CB2. Additionally, AM1241 delayed early activation of monocytes and induced suppression of IL-2 and IL-6 levels in response to Candida via lower activity of mTOR. These findings provide new insights into key mechanisms of innate immunity under stressful conditions where ligands to the sympatho-adrenergic and endocannabinoid system are released.

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... Short-term exposure to altered gravity, such as during a parabolic flight, induces the increased release of compounds of the ECS such as anandamide/Narachidonylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG) in blood (Chouker et al., 2010). Recently, the capability of the ECS to alter immune function is studied intensively and it was shown that endocannabinoids (EC) can exert a strong suppression of innate and adaptive immune responses (Buchheim et al., 2018). The effects of long-term spaceflight on EC blood levels and their participation in the adaptation process aboard are unknown. ...
... The ECS is an important stress response system and has multiple roles in a myriad of physiological processes of stress (Chouker et al., 2010;Dlugos et al., 2012;Hauer et al., 2013;Neumeister et al., 2015), metabolism (Campolongo et al., 2009), sleep and activity patterns (Richard et al., 2009;Feuerecker et al., 2012). Most importantly, endocannabinoids are potent immune modulators (Sardinha et al., 2014;Buchheim et al., 2018). They act via the endocannabinoid receptors (CB) 1 and 2, which are not only expressed in the central and peripheral nervous systems (CB1), but also on the surface of many leukocytes (CB2). ...
... It appears that EC release is stimulated by conditions in space and not by the acute stress exerted during landing, which is well in line with the knowledge that acute and chronic stresses exert very different effects on the immune system (Moynihan, 2003). Correspondingly, our data show similarities to previously reported results, where the ECS was responsible for the modulation of constant baseline ROS release (without stimulus) in neutrophils and did not alter ROS production after stimulation with fMLP and TNF (Buchheim et al., 2018). in plasma, n = 9 on F+90, n = 11 on F+150 (C) Quantification of IL-10 in plasma, n = 9 on F+90, n = 11 on F+150, (D) Quantification of TGF-ß in plasma, n = 8 on F+90, n = 10 on F+150; all panels: data is expressed as median ± 95th and 5th percentile, * = P < 0.05 vs. L-25 control. ...
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... In murine macrophages, NE and E progress the development of a M2 regulatory phenotype in LPS co-culture via β-AR signaling, leading to an anti-inflammatory Th2 reaction (119). Since the ROS metabolism in response to different stimuli is key to survival, it is worth noting that stimulated H2O2 release in granulocytes was significantly inhibited by NE in a dose-dependent manner (120) and that β-AR activation by NE was the primary mechanism to inhibit the fLMP-induced respiratory burst response in vitro. Gene expression data indicates that β1 receptor subtypes might play a pivotal role. ...
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Mammalian tissues express at least two types of cannabinoid receptor, CB1 and CB2, both G protein coupled. CB1 receptors are expressed predominantly at nerve terminals where they mediate inhibition of transmitter release. CB2 receptors are found mainly on immune cells, one of their roles being to modulate cytokine release. Endogenous ligands for these receptors (endocannabinoids) also exist. These are all eicosanoids; prominent examples include arachidonoylethanolamide (anandamide) and 2-arachidonoyl glycerol. These discoveries have led to the development of CB1- and CB2-selective agonists and antagonists and of bioassays for characterizing such ligands. Cannabinoid receptor antagonists include the CB1-selective SR141716A, AM251, AM281 and LY320135, and the CB2-selective SR144528 and AM630. These all behave as inverse agonists, one indication that CB1 and CB2 receptors can exist in a constitutively active state. Neutral cannabinoid receptor antagonists that seem to lack inverse agonist properties have recently also been developed. As well as acting on CB1 and CB2 receptors, there is convincing evidence that anandamide can activate transient receptor potential vanilloid type 1 (TRPV1) receptors. Certain cannabinoids also appear to have non-CB1, non-CB2, non-TRPV1 targets, for example CB2-like receptors that can mediate antinociception and "abnormal-cannabidiol" receptors that mediate vasorelaxation and promote microglial cell migration. There is evidence too for TRPV1-like receptors on glutamatergic neurons, for alpha2-adrenoceptor-like (imidazoline) receptors at sympathetic nerve terminals, for novel G protein-coupled receptors for R-(+)-WIN55212 and anandamide in the brain and spinal cord, for novel receptors for delta9-tetrahydrocannabinol and cannabinol on perivascular sensory nerves and for novel anandamide receptors in the gastro-intestinal tract. The presence of allosteric sites for cannabinoids on various ion channels and non-cannabinoid receptors has also been proposed. In addition, more information is beginning to emerge about the pharmacological actions of the non-psychoactive plant cannabinoid, cannabidiol. These recent advances in cannabinoid pharmacology are all discussed in this review.
Article
PurposeTo describe concisely the current standards of care, major recent advances, common beliefs that have been contradicted by recent trials, areas of uncertainty, and clinical studies that need to be performed over the next decade and their expected outcomes with regard to Candida and Aspergillus infections in non-neutropenic patients in the ICU setting. MethodsA systematic review of the medical literature taking account of national and international guidelines and expert opinion. ResultsSevere invasive fungal infections (IFIs) are becoming increasingly frequent in critically ill patients. Approximately 80% of IFIs are due to Candida spp. and 0.3–19% to Aspergillus spp. Recent observations emphasize the necessity of building a worldwide sentinel network to monitor the emergence of new fungal species and changes in susceptibility. Robust data on the attributable mortality are essential for the design of clinical studies with mortality endpoints. Although early antifungal therapy for Candida has been recommended in patients with risk factors, sepsis of unknown cause, and positive Candida serum biomarkers [β-1 → 3-d-glucan (BDG) and Candida albicans germ tube antibody (CAGTA)], its usefulness and influence on outcome need to be confirmed. Future studies may specifically address the optimal diagnostic and therapeutic strategies for patients with abdominal candidiasis. Better knowledge of the pharmacokinetics of antifungal molecules and tissue penetration is a key issue for intensivists. Regarding invasive aspergillosis, further investigation is needed to determine its incidence in the ICU, its relationship with influenza outbreaks, the clinical impact of rapid diagnosis, and the significance of combination treatment. Conclusions Fundamental questions regarding IFI have to be addressed over the next decade. The clinical studies described in this research agenda should provide a template and set priorities for the clinical investigations that need to be performed.
Article
Mice were used to examine how the administration of adrenaline modulates the host defense system. The levels of granulocyte and lymphocyte subsets, including extrathymic T cells, were identified by immunofluorescence tests. When mice were continuously administered with adrenaline (400 μg/mouse/week) by a diffusion pump for 1 week, the numbers of mononuclear cells yielded by the liver, spleen, and bone marrow were relatively stable but that yielded by the thymus decreased on day 7. The proportion of granulocytes, especially in the bone marrow, and the proportion of CD3(int) cells (extrathymic T cells) in the liver and other organs increased. By a single intraperitoneal injection of adrenaline (20 μg/mouse), the acute response within 24 h was also examined. The number and proportion of granulocytes in the bone marrow decreased while those in the blood increased. It is presumed that the major result of the acute response is a migration of granulocytes from the bone marrow to the circulation. Concerning the similarity of responses between the administration of adrenaline and the previously reported results of excessive administrations of nonsteroidal anti-inflammatory drugs or calcitonin and from restraint stress, this raises the possibility that all these stimuli, which induce the activation of granulocytes and extrathymic T cells, are mediated via the production of catecholamines as a result of sympathetic nerve strain.
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Immune responses during fungal infections are predominately mediated by 5/15-lipoxygenases (LO)- or cyclooxygenase (COX)-2-catalysed bioactive eicosanoid metabolites like leukotrienes, lipoxins and prostaglandins. Although few host mediators of fungi-triggered eicosanoid production have been established, the molecular mechanism of expression and regulation of 5-LO, 15-LO and COX-2 are not well-defined. Here, we demonstrate that, macrophages infected with representative fungi Candida albicans, Aspergillus flavus or Aspergillus fumigatus or those treated with Curdlan, a selective agonist of pattern recognition receptor for fungi Dectin-1, displays increased expression of 5-LO, 15-LO and COX-2. Interestingly, Dectin-1-responsive Syk pathway activates mTOR-sonic hedgehog (SHH) signaling cascade to stimulate the expression of these lipid metabolizing enzymes. Loss-of-function analysis of the identified intermediaries indicates that while Syk-mTOR-SHH pathway-induced 5-LO and 15-LO suppressed the Dectin-1-responsive pro-inflammatory signature cytokines like TNF-α, IL-1β and IL-12, Syk-mTOR-SHH-induced COX-2 positively regulated these cytokines. Dectin-1-stimulated IL-6, however, is dependent on 5-LO, 15-LO and COX-2 activity. Together, the current study establishes Dectin-1-arbitrated host mediators that direct the differential regulation of immune responses during fungal infections and thus are potential candidates of therapeutic intervention.
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Influence of a relatively specific inhibitor cyclooxygenase (COX)-2, celecoxib, a relatively specific inhibitor of neuronal nitric oxide synthase (NOS), 7-Ni, and a relatively selective inhibitor of inducible NOS, L-NIL, on the action of a preferentially selective CB1 cannabinoid receptor agonist, Met-F-AEA and a selective CB2 cannabinoid receptor agonist, AM 1241 was investigated, in a streptozotocin (STZ)-induced neuropathy. Studies were performed on male Wistar rats. Changes in nociceptive thresholds were determined using mechanical stimuli - the modification of the classic paw withdrawal test described by Randall-Selitto. Diabetes was induced by a single administration of STZ. In a diabetic neuropathic pain model, pretreatment with celecoxib, L-NIL and 7-Ni, significantly increased the antihyperalgesic activity of both Met-F-AEA and AM 1241. The results of this study seemed to indicate that the interaction between cannabinoid, COX-2 and NOS(s) systems might exist. Concomitant administration of small doses of CB1 and/or CB2 receptor agonists and COX-2 or NOS inhibitors can be effective in the alleviation of diabetic neuropathic pain. Copyright © 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Article
Although the concept of stress has earned a bad reputation, it is important to recognize that the adaptive purpose of a physiological stress response is to promote survival during fight or flight. While long-term stress is generally harmful, short-term stress can be protective as it prepares the organism to deal with challenges. This review discusses the immune effects of biological stress responses that can be induced by psychological, physiological, or physical (including exercise) stressors. We have proposed that short-term stress is one of the nature's fundamental but under-appreciated survival mechanisms that could be clinically harnessed to enhance immunoprotection. Short-term (i.e., lasting for minutes to hours) stress experienced during immune activation enhances innate/primary and adaptive/secondary immune responses. Mechanisms of immuno-enhancement include changes in dendritic cell, neutrophil, macrophage, and lymphocyte trafficking, maturation, and function as well as local and systemic production of cytokines. In contrast, long-term stress suppresses or dysregulates innate and adaptive immune responses by altering the Type 1-Type 2 cytokine balance, inducing low-grade chronic inflammation, and suppressing numbers, trafficking, and function of immunoprotective cells. Chronic stress may also increase susceptibility to some types of cancer by suppressing Type 1 cytokines and protective T cells and increasing regulatory/suppressor T cell function. Here, we classify immune responses as being protective, pathological, or regulatory, and discuss "good" versus "bad" effects of stress on health. Thus, short-term stress can enhance the acquisition and/or expression of immunoprotective (wound healing, vaccination, anti-infectious agent, anti-tumor) or immuno-pathological (pro-inflammatory, autoimmune) responses. In contrast, chronic stress can suppress protective immune responses and/or exacerbate pathological immune responses. Studies such as the ones discussed here could provide mechanistic targets and conceptual frameworks for pharmacological and/or biobehavioral interventions designed to enhance the effects of "good" stress, minimize the effects of "bad" stress, and maximally promote health and healing.
Article
Aspergillus and Fusarium species are important causes of fungal infections worldwide. Airborne spores (conidia) of these filamentous fungi express a surface protein that confers hydrophobicity (hydrophobin) and covers cell wall components that would otherwise induce a host immune cell response. Using a mutant Aspergillus fumigatus strain (ΔrodA) that does not express the RodA hydrophobin, and Aspergillus and Fusarium conidia from clinical isolates that were treated with hydrofluoric acid (which removes the A. fumigatus RodA protein), we observed increased surface exposure of β1,3-glucan and α-mannose on Aspergillus and Fusarium conidia. We also found that ΔrodA and hydrofluoric acid-treated conidia stimulate significantly higher NF-κB p65 nuclear translocation and cytokine production by macrophages from C57BL/6, but not from Dectin-1(-/-) or Dectin-2(-/-) mice. Using a murine model of A. fumigatus corneal infection, we showed that ΔrodA conidia induced significantly higher cytokine production, neutrophil infiltration, and more rapid fungal clearance from C57BL/6 corneas compared with the parent G10 strain, which was dependent on Dectin-1 and Dectin-2. Together, these findings identify the hydrophobin RodA as a virulence factor that masks Dectin-1 and Dectin-2 recognition of conidia, resulting in impaired neutrophil recruitment to the cornea and increased fungal survival and clinical disease.
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Sickness behaviours are host defence adaptations that arise from integrated autonomic outputs in response to activation of the innate immune system. These behaviours include fever, anorexia, and hyperalgesia intended to promote survival of the host when encountering pathogens. Cannabinoid (CB) receptor activation can induce hypothermia and attenuate lipopolysaccharide (LPS)-evoked fever. The aim of the present study was to examine the role of CB1 receptors in the LPS-evoked febrile response. CB1 receptor-deficient (CB1(-/-)) mice did not display LPS-evoked fever; likewise pharmacological blockade of CB1 receptors in wild-type mice blocked LPS-evoked fever. This unresponsiveness is not limited to thermogenesis, as the animals were not hyperalgesic after LPS administration. A toll-like receptor (TLR)3 agonist and viral mimetic polyinosinic:polycytidylic acid evoked a robust fever in CB1(-/-) mice suggesting TLR3-mediated responses are functional. LPS-evoked c-Fos activation in areas of the brain associated with the febrile response was evident in wild-type mice but not in CB1(-/-) mice. Liver and spleen TLR4 mRNA were significantly lower in CB1(-/-) mice compared to wild-type, and peritoneal macrophages from CB1(-/-) mice did not release pro-inflammatory cytokines in response to LPS. These data indicate that CB1 receptors play a critical role in LPS-induced febrile responses through inhibiting TLR4-mediated cytokine production.
Article
The human immune system is orchestrated in a complex manner and protects the host against invading organisms and controls adequate immune responses to different antigen challenges in an endo-, auto- and paracrine-regulated fashion. The variety and intensity of immune responses are known to be dependent on stress-sensitive neural, humoral and metabolic pathways. The delayed-type hypersensitivity (DTH) skin test was a validated and standardized measure applied in clinical studies to monitor the integral function of cellular immune responses in vivo. The DTH skin test was, however, phased out in 2002. To obtain insight into the mechanisms of stress-sensitive immune reactions, we have developed an alternative in-vitro assay which allows the evaluation of antigen-dependent cellular immune responses triggered by T lymphocytes. The change in the concentration of proinflammatory cytokines in supernatant of the blood-antigen mixture is of particular interest to mirror the degree and adequacy of cellular immune responses. In this study we report that the proinflammatory cytokines interleukin (IL)-2, interferon (IFN)-γ and tumour necrosis factor (TNF)-α show a time-dependent increase upon ex-vivo bacterial, viral and fungal antigen stimulations. Furthermore, evidence is provided that this assay is sensitive to mirror stress hormone-mediated immune modulation in humans as shown either after hydrocortisone injection or after acute stress exposure during free fall in parabolic flight. This in-vitro test appears to be a suitable assay to sensitively mirror stress hormone-dependent inhibition of cellular immune responses in the human. Because of its standardization and relatively simple technical handling, it may also serve as an appropriate research tool in the field of psychoneuroendocrinology in clinical as in field studies.
Article
Aims: AM-1241, a novel, racemic cannabinoid-2 receptor (CB2) ligand, is the primary experimental agonist used to characterize the role of CB2-mediated lipid signaling in health and disease, including substance abuse disorders. In vivo pharmacological effects have been used as indirect proxies for AM-1241 biotransformation processes that could modulate CB2 activity. We report the initial pre-clinical characterization of AM-1241 biotransformation and in vivo distribution. Main methods: AM-1241 metabolism was characterized in a variety of predictive in vitro systems (Caco-2 cells; mouse, rat and human microsomes) and in the mouse in vivo. Liquid chromatography and mass spectrometry techniques were used to quantify AM-1241 tissue distribution and metabolic conversion. Key findings: AM-1241 bound extensively to plasma protein/albumin. A pharmacological AM-1241 dose (25mg/kg, i.v.) was administered to mice for direct determination of its plasma half-life (37 min), following which AM-1241 was quantified in brain, spleen, liver, and kidney. After p.o. administration, AM-1241 was detected in plasma, spleen, and kidney; its oral bioavailability was ~21%. From Caco-2 permeability studies and microsomal-based hepatic clearance estimates, in vivo AM-1241 absorption was moderate. Hepatic microsomal metabolism of AM-1241 in vitro generated hydroxylation and demethylation metabolites. Species-dependent differences were discovered in AM-1241's predicted hepatic clearance. Our data demonstrate that AM-1241 has the following characteristics: a) short plasma half-life; b) limited oral bioavailability; c) extensive plasma/albumin binding; d) metabolic substrate for hepatic hydroxylation and demethylation; e) moderate hepatic clearance. Significance: These results should help inform the design, optimization, and pre-clinical profiling of CB2 ligands as pharmacological tools and medicines.
Article
In experimental and clinical studies, expression of surface adhesion molecules such as ß2-integrine (CD11b) and L-selectin (CD62L) on polymorphonuclear leukocyte (PMNL) are investigated to assess certain crucial innate immune functions. Because the expression of CD11b and CD62L on PMNL can alter they cannot be quantified reliably when the time between blood draw and measurements is prolonged. Goals of this study were to test effects of cryopreservation on the expression of CD11b and CD62L on human PMNLs either under native conditions as well as after stimulation-dependant adhesion molecules´ expression pattern. CD11b and CD62L expression on PMNL can be cryopreserved with 10% of PEG-solution for at least one month at -60 degree C. This was observed in native, unstimulated as well as in stimulated cell-preparations. CD11b is very stable in contrast to CD62L expression which appears to be more susceptible to alteration due to freezing-thawing. However, the relative stimulus-dependant changes of activation can still be reflected.
Article
Based on previous findings that early growth response 1 (Egr-1) participates in leukocyte recruitment and cell proliferation in vitro, this study was designed to investigate its mode of action during arteriogenesis in vivo. In a model of peripheral arteriogenesis, Egr-1 was significantly upregulated in growing collaterals of wild-type (WT) mice, both on mRNA and protein level. Egr-1(-/-) mice demonstrated delayed arteriogenesis after femoral artery ligation. They further showed increased levels of monocytes and granulocytes in the circulation, but reduced levels in adductor muscles under baseline conditions. After femoral artery ligation, elevated numbers of macrophages were detected in the perivascular zone of collaterals in Egr-1(-/-) mice and mRNA of leukocyte recruitment mediators was upregulated. Other Egr family members (Egr-2 to -4) were significantly upregulated only in Egr-1(-/-) mice, suggesting a mechanism of counterbalancing Egr-1 deficiency. Moreover, splicing factor-1, downregulated in WT mice after femoral artery ligation in the process of increased vascular cell proliferation, was upregulated in Egr-1(-/-) mice. αSM-actin on the other hand, significantly downregulated in WT mice, showed no differential expression in Egr-1(-/-) mice. While cell cycle regulator cyclin E and cdc20 were upregulated in Egr-1(-/-) mice, cyclin D1 expression decreased below the detection limit in collaterals, and the proliferation marker ki67 was not differentially expressed. In conclusion, compensation for deficiency in Egr-1 function in leukocyte recruitment can presumably be mediated by other transcription factors; however, Egr-1 is indispensable for effective vascular cell cycle progression in arteriogenesis.
Article
The role of TLR4 in the recognition of Candida albicans has been brought into question. In order to assess whether discrepancies in the literature are due to differences in the recognition of various C. albicans strains, we selected 14 different isolates of C. albicans to evaluate their recognition by TLR4 and lectin receptors. We demonstrate that recognition of cell wall structures by lectin receptors is a consistent characteristic independent of the C. albicans strain selected, while recognition by TLR4 is a more variable feature. These data were corroborated by the increased susceptibility of TLR4-/- mice to a C. albicans strain recognized by TLR4, but not to a strain in which recognition has been shown to be independent of this receptor. This suggests a heavier reliance of in vivo antifungal host defense on lectin receptors than on TLRs, a notion compatible with the clinical picture in individuals deficient in MyD88/TLRs or dectin-1/CARD9.
Article
The pathophysiology underlying the high incidence of post-stroke infectious complications has not been fully understood. We measured the respiratory burst of neutrophils as an index of their bactericidal function in patients with haemorrhagic stroke, and we also measured the plasma concentrations of noradrenalin, cortisol and neutrophil function-related amino acids. Blood samples were obtained from patients who underwent emergency craniotomies for haemorrhagic stroke (n=11) or CT-guided stereotaxic aspiration of intracerebral haematomas (n=6). Neutrophils were isolated, and their chemiluminescence response to N-formyl-methionyl-leucyl-phenylalanine was measured. Healthy volunteers served as controls (n=15). In patients with stroke, the chemiluminescence response of the isolated neutrophils was significantly lower than in the controls, and it was significantly inversely correlated with the plasma noradrenalin concentration. By Day 30, this value approached control levels. Other parameters measured were not significantly correlated with the chemiluminescence response. Stroke-induced suppression of the neutrophil respiratory burst may be responsible for frequent post-stroke infectious complications.
Article
Preventive treatment with cannabinoid agonists has been reported to reduce the infarct size in a mouse model of myocardial ischemia/reperfusion. Here we investigated the possible cardioprotective effect of selective CB(2) cannabinoid receptor activation during ischemia. We performed left coronary artery ligature in C57Bl/6 mice for 30 min, followed by 24 h of reperfusion. Five minutes before reperfusion, mice received intraperitoneal injection of the CB(2) selective agonist JWH-133 (20 mg/kg) or vehicle. Infarct size was assessed histologically and by cardiac troponin I (cTnI) ELISA. Immunohistochemical analysis of leukocyte infiltration, oxidative stress in situ quantification, real-time RT-PCR analysis of inflammatory mediators as well as western blots for kinase phosphorylation was also performed. In addition, we studied chemotaxis and integrin expression of human neutrophils in vitro. JWH-133 significantly reduced the infarct size (I/area at risk: 19.27%+/-1.91) as compared to vehicle-treated mice (31.77%+/-2.7). This was associated with a reduction of oxidative stress and neutrophil infiltration in the infarcted myocardium, whereas activation of ERK 1/2 and STAT-3 was increased. Preinjection of PI3K inhibitor LY294002, MEK 1/2 inhibitor U0126 and JAK-2 inhibitor AG-490 partially abrogated the JWH-133 mediated infarct size reduction. No changes in cardiac CXCL1, CXCL2, CCL3, TNF-alpha, and ICAM-1 expression levels were found. Furthermore, JWH-133 inhibited the TNF-alpha induced chemotaxis and integrin CD18/CD11b (Mac-1) upregulation on human neutrophils. Our data suggest that JWH-133 administration during ischemia reduces the infarct size in a mouse model of myocardial ischemia/reperfusion through a direct cardioprotective activity on cardiomyocytes and neutrophils.
Article
The systemic response to injury is characterized by massive release of norepinephrine (NE) into the circulation as a result of global sympathetic activation. Multiple authors have demonstrated NE-mediated alterations in migration of circulating neutrophils to wounds. We hypothesized that NE further alters wound neutrophil phagocytic function through adrenergic signaling pathways. A standard subcutaneous sponge wound model was used. Murine wound neutrophils were harvested at 24 and 120 h after injury and treated with physiological (10(-9) M) and pharmacologic (10(-6) M) doses of NE. Phagocytosis of green fluorescent protein-labeled Escherichia coli was assayed by flow cytometry. The signaling pathways mediating NE modulation of phagocytosis by wound neutrophils were defined by pharmacologic manipulation of alpha- and beta-adrenoreceptors and protein kinase A. Pharmacologic-dose NE, but not-physiological-dose NE, suppressed the phagocytic efficiency of 120-h wound neutrophils. This alteration in phagocytic efficiency appears to be mediated through alpha- and beta- adrenoreceptors and downstream protein kinase A. Phagocytosis by 24-h wound neutrophils was not impacted by NE treatment. The present study is the first to demonstrate NE-mediated alterations in the process of phagocytosis by wound neutrophils. We conclude that NE plays a temporally and dose-defined immunomodulatory role in cutaneous wound healing through alterations in phagocytosis by wound neutrophils and may represent a target for therapeutic manipulation of the innate immune response.
Article
Although inhibition of polymorphonuclear leukocyte activation by beta-adrenoceptor agonists has been recognized for over a decade, effects have only been observed at high drug concentrations and in the presence of theophylline. In this study, catecholamine and prostaglandin modulation of the respiratory burst was evaluated with respect to the mechanism of polymorphonuclear leukocyte activation. Very low concentrations of isoproterenol and prostaglandin E2 inhibited the respiratory burst when induced by chemotactic peptide (N-formyl-methionyl-leucyl-phenylalanine) or calcium ionophore (A23187, ionomycin), but not when initiated by synthetic diacylglycerol. Because formyl-methionyl-leucyl-phenylalanine and ionophore mobilize calcium and arachidonic acid generation follows an increase in intracellular calcium, the arachidonic acid metabolite leukotriene B4 was studied. Isoproterenol at a very low (0.1 nM) concentration also rapidly inhibited leukotriene B4 generation. Since cyclic AMP was increased by isoproterenol regardless of the means of cell activation, modulation of intracellular calcium was evaluated with the fluorescent probe indo-1. A transient increase in calcium after formyl-methionyl-leucyl-phenylalanine or ionophore (but not oleoyl acetylglycerol) cell activation was inhibited by isoproterenol or prostaglandin E2. These results suggest that adrenergic agonists specifically modulate calcium-dependent polymorphonuclear leukocyte function. Because marked inhibition was observed at very low drug concentrations, cyclic AMP-dependent effects may be important in both homeostatic and therapeutic modulation of inflammatory response.
Article
CD69 is an early marker of lymphoid cell activation. The authors report on an up-regulation of CD69 in splenic B and T cells of C57Bl/6 mice after administration of lipopolysaccharide (LPS) or microbial immunosuppressive/mitogenic (ISM) proteins produced by C. albicans (p43) and African Swine Fever Virus (p36). This up-regulation of CD69 was observed 6 and 24 h after mitogenic treatments. The same pattern of increased CD69 expression was observed in the lymph nodes of mice treated with p43 or LPS, whereas p36 treatment failed to induce increased CD69 expression in this organ. Intracellular calcium mobilization was induced in splenic B and T lymphocytes after incubation of total spleen cells with LPS, p43 or p36. This increase was higher in B than in T cells. Increased calcium mobilization was also seen in lymph node B cells after incubation with p43 or p36 and in lymph node T cells after p43 stimulation. Up-regulation of CD69 expression on B and T cells was also observed after in vitro stimulation of spleen cells with the three mitogens used. Similar results were obtained with culture supernatants of macrophage/monocyte (M phi) cells activated with LPS (LPS/M phi CS). Stimulation of M phi cells with LPS or with the ISM proteins is demonstrated by the increased production of nitrites by these cells. The increased in vitro expression of CD69 was, however, not abolished by monoclonal antibodies to M phi cytokines such as IL-6, IL-10 or TNF alpha. No increased expression of CD69 was found in vitro on purified B or T cells, even when mixed upon stimulation with p43, p36, LPS or with LPS/M phi CS. However, an increase in the expression of CD69 was observed on B cells co-cultured with M phi cells after treatment with LPS or p36. All three mitogens failed to induce increased CD69 expression on cultured T cells mixed with M phi cells.
Article
Adenosine has been shown to inhibit the adhesion of polymorphonuclear leukocytes (PMNL) to the vascular endothelium. Because the underlying molecular mechanisms have not been fully understood, the present study characterizes the effect of adenosine on the expression of adhesion molecules of human PMNL. When PMNL were activated by N-formyl-methionyl-leucyl-phenylalanine the number of cell surface beta2 integrins increased fivefold, whereas L-selectin molecules were completely shed. Activation-dependent numerical up-regulation Of beta2 integrins and shedding of L-selectin were inhibited by exogenously applied adenosine receptor agonists in a concentration-dependent fashion. The rank order of potencies of adenosine receptor agonists, measured by the agonists' half-maximal inhibitory concentrations, revealed that adenosine inhibited the numerical up-regulation of beta2 integrins and shedding of L-selectin most likely via an A2(a) receptor site. When extracellular concentrations of endogenously formed adenosine were enhanced by the nucleoside uptake inhibitor dipyridamole, up-regulation of beta2 integrins, and shedding of L-selectin was again inhibited. Both effects were reversed by the enzyme adenosine deaminase, which degrades active adenosine to inactive inosine, suggesting that endogenously formed adenosine may play an important role in the regulation of beta2 integrins and L-selectin of human PMNL.
Article
In a porcine model of hyperdynamic endotoxemia, we studied the numerical expression of L-selectin and beta 2-integrins on circulating polymorphonuclear leukocytes (PMN). Functional changes of beta 2-integrins were determined by the adhesion of PMN to C3-coated zymosan particles. Anesthesized pigs received a continuous infusion of Salmonella abortus-equi endotoxin (5 micrograms.kg body wt-1.h-1) for 270 min (endotoxin group; n = 7). A control group received 0.9% NaCl (n = 6). L-selectin had decreased 30 min after the induction of endotoxemia [59.1 +/- 11.9 vs. 91.6 +/- 15.5 relative fluorescence units (RFU) at baseline; P < 0.05], reaching minimal values after 150 min (23.9 +/- 3.9 RFU in endotoxin group vs. 95.2 +/- 30.4 RFU in control group; P < 0.05). PMN adhesion to C3-coated zymosan increased at 30 min (41.3 +/- 9.9% in endotoxin group vs. 2.4 +/- 1.1% in control group; P < 0.05) and remained significantly elevated thereafter. In contrast to the rapid shedding of L-selectin and functional upregulation of beta 2-integrins, the numerical expression of beta 2-integrins remained unchanged until 60 min (44.8 +/- 2.8 vs. 32.2 +/- 1.7 RFU at baseline; P < 0.05); compared with the control group, significantly elevated values were observed 150 min after the start of endotoxin (48.9 +/- 2.4 RFU in endotoxin group vs. 36.5 +/- 2.7 RFU in control group; P < 0.05). We conclude that numerical and functional expressions of beta 2-integrins are dissociated during endotoxemia. Although upregulation of beta 2-integrins might render PMN more adhesive to the vascular endothelium, the presence of activated PMN in the circulation suggests that low expression of L-selectin might impede adhesion.
Article
Evaluation of catecholamine modulation of PMNL extracellular and intracellular oxidant production may reflect beneficial and harmful effects of beta-adrenergic agonists in various disease states. We investigated the kinetics and potency of adrenaline-mediated inhibition of oxidant generation in FMLP- and zymosan-stimulated PMNLs. In FMLP-stimulated cells, the short-term burst of oxidant generation was inhibited by adrenaline in a dose-dependent fashion. Intra- and extracellular chemiluminescence and extracellular superoxide anion and hydrogen peroxide generation showed similar IC50 values for adrenaline (1.3-3.0 x 10(-8) M) indicating that both extracellular and intracellular events were inhibited with the same potency. In contrast, intracellular oxidant production evoked by the phagocytosis of zymosan was only minimally affected by 3 x 10(-5) -3 x 10(-12) M adrenaline. Extracellular inhibition of oxidant production was also apparent in zymosan-stimulated cells. In conclusion, adrenaline's ability to depress extracellular generation of oxygen metabolites while retaining prolonged intracellular oxidant production for phagocytosis supports its beneficial role as selectively targeted physiological protector.
Article
Studies performed on healthy volunteers have revealed that catecholamines down-regulate the lipopolysaccharide (LPS)-induced production of tumor necrosis factor (TNF)alpha, interleukin (IL)-6, and IL-1beta. We extended this observation and show that this effect is based on changes in the mRNA concentration of these cytokines. Catecholamines are increased in severe sepsis due to endogenous production and have to be administered exogenously when the disease has proceeded to the state of prolonged hypotension. We here investigated whether the immunomodulating effect of catecholamines could also be demonstrated in the blood of patients with prolonged severe sepsis and of those in prolonged septic shock. Blood was stimulated ex vivo with LPS in the presence and absence of epinephrine and the cytokine protein concentration was determined. In blood of healthy volunteers, epinephrine reduced the LPS-stimulated synthesis of TNFalpha by 62.5% (P< 0.0001), of IL-6 by 39% (P< 0.0001), and of IL-1beta by 40% (P= 0.015), and increased the LPS-stimulated IL-10 production by 77.8% (P < 0.0001). Correspondingly, in blood of patients with prolonged severe sepsis, TNFalpha was reduced by 67.2% (P < 0.0001) and IL-6 was reduced by 32.9% (P < 0.0001); IL-1beta and IL-10 were not modulated by catecholamines in these patients. In blood samples of patients in prolonged septic shock, epinephrine did not modulate cytokine levels of IL-6 and IL-10, and decreased TNFalpha only by 36.4% (P < 0.0001). Interestingly, epinephrine suppressed the IL-1beta production by 73% (P < 0.0001) in blood of patients in prolonged septic shock, which was twice as much as in blood samples of healthy volunteers. The altered response of septic blood to catecholamines might be due to an altered reactivity of leukocytes in the prolonged disease although an additional role of preexisting catecholamines cannot be completely excluded.
Article
This study was designed to evaluate the effects of epinephrine (0.01-1 microM) on superoxide production by, and release of elastase from human neutrophils activated with the chemotactic tripeptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) (1 microM) in vitro, and to relate alterations in these responses to changes in adenosine 3,5' cyclic monophosphate (cAMP) and cytosolic free Ca(2+). Cyclic AMP, superoxide production and elastase release were measured by radioimmunoassay, lucigenin-enhanced chemiluminescence, and a colorimetric procedure respectively. Cytosolic Ca(2+) fluxes were measured by fura-2 spectrofluorimetry in combination with radiometric procedures that enable distinction between net efflux and influx of the cation. Epinephrine treatment of neutrophils resulted in increased cAMP and dose-related inhibition of both superoxide production and elastase release, which was potentiated by the type 4 phosphodiesterase inhibitor, rolipram, and attenuated by propranolol, but not by selective beta(1)-, alpha(1)- or alpha(2)-adrenoreceptor antagonists. Although epinephrine did not affect the FMLP-activated abruptly-occurring increase in fura-2 fluorescence intensity, indicating no effects on the release of Ca(2+) from neutrophil intracellular stores, this agent accelerated the rate of decline in fluorescence in the setting of decreased efflux and a reduction in store-operated influx of Ca(2+). These effects of epinephrine on the clearance of Ca(2+) from the cytosol of FMLP-activated neutrophils were attenuated by propranolol, and are compatible with enhancement of the activity of the cAMP-dependent Ca(2+) sequestering/resequestering endo-membrane Ca(2+)-ATPase. We conclude that epinephrine down-regulates the pro-inflammatory activities of neutrophils by cAMP-mediated enhancement of the clearance of cytosolic Ca(2+).
Article
Delta(9)-Tetrahydrocannabinol (Delta(9)-THC) is the major psychoactive component of marijuana and elicits pharmacological actions via cannabinoid receptors. Anandamide (AEA) and 2-arachidonoyl-glycerol (2-AG) are endogenous ligands for cannabinoid receptors, which because of their structural similarities to arachidonic acid (AA), AEA, and 2-AG could serve as substrates for lipoxygenases and cyclooxygenases (COXs) that metabolize polyunsaturated fatty acids to potent bioactive molecules. In this study, we have compared the effects of Delta(9)-THC, AEA, 2-AG, and another cannabinoid agonist, indomethacin morpholinylamide (IMMA), on lipopolysaccharide (LPS)-induced NO, IL-6, and PGE(2) release from J774 macrophages. Delta(9)-THC, IMMA, and AEA diminish LPS-induced NO and IL-6 production in a concentration-dependent manner. 2-AG inhibits the production of IL-6 but slightly increases iNOS-dependent NO production. Delta(9)-THC and IMMA also inhibit LPS-induced PGE(2) production and COX-2 induction, while AEA and 2-AG have no effects. These discrepant results of 2-AG on iNOS and COX-2 induction might be due to its bioactive metabolites, AA and PGE(2), whose incubation cause the potentiation of both iNOS and COX-2 induction. On the contrary, the AEA metabolite, PGE(2)-ethanolamide, influences neither the LPS-induced NO nor IL-6 production. Taken together, direct cannabinoid receptor activation leads to anti-inflammatory action via inhibition of macrophage function. The endogenous cannabinoid, 2-AG, also serves as a substrate for COX-catalyzing PGE(2) production, which in turn modulates the action of CB2.
Article
The effects of cannabinoids (CB) that have been reported in various leukocyte populations were mainly immunosuppressive or immunomodulatory. Almost nothing is known, however, about direct interactions of cannabinoids with human polymorphonuclear cells (PMN), although m-RNA for the cannabinoid receptor-2 (CB(2)) was found in human PMN. In order to investigate a potential influence of cannabinoids on human PMN, the migration and phagocytosis of PMN were studied in the presence of Delta(9)-Tetrahydrocannabinol (Delta(9)-THC) at final concentrations between 10(-10) and 10(-5) M. No effect was detectable on these essential PMN functions; and besides, no CB(2)-receptor expression could be detected using the Western blotting technique. Thus, circulating human PMN from healthy individuals remain unaffected by Delta(9)-THC due to the absence of functional CB(2)-receptor expression.
Article
We have reported that injection of marijuana cannabinoids, such as Delta(9)-tetrahydrocannabinol (THC), into mice, followed by infection with Legionella pneumophila (Lp), suppresses the development of cell-mediated immunity T helper 1 (Th1) activity. These effects are accompanied by suppression of interleukin (IL)-12 and interferon (IFN) gamma production and enhancement of IL-4 production suggesting THC-induced T helper cell biasing. In the current report, other T helper cell biasing mechanisms were studied. Mice were injected with THC followed 18 h later by a challenge infection with Lp. Two-hour post-infection, spleens were removed and analyzed for mRNA to either IL-12Rbeta2 or GATA3 gene products. The results showed that THC suppressed IL-12Rbeta2 but increased GATA3. Receptor antagonists for CB1 (SR141716A, SR1) and CB2 (SR144528, SR2) were also injected to analyze the involvement of cannabinoid receptors. It was determined that SR1 attenuated the THC suppression of IL-12Rbeta2, while SR2 attenuated the increase in GATA3 mRNA. These results suggest that THC suppresses Th1 biasing activity such as IL-12Rbeta2 by a CB1 mediated mechanism and enhances the Th2 biasing activity, GATA3, by a CB2 mechanism. This dichotomy of receptor involvement might result from differential expression and/or signaling function of CB1 and CB2 on Th1 and Th2 cells.
Article
Cannabinoids have been shown to affect various immune functions. To date, almost no data exist on PMN, which provide the first line antimicrobial defense. The objective of the present study was to investigate the effects of the synthetic dibenzopyrane ligand CP55 940, the endogenous cannabinoid anandamide and methanandamide on the "respiratory burst" of isolated human PMN in vitro. After preincubation with high micromolar concentrations of CP55 940, fMLP-stimulated PMN showed a reduction in superoxide production, whereas the spontaneous burst activity of resting PMN remained unaffected. This inhibitory effect of CP55 940 was not CB-receptor-mediated. In contrast, anandamide and methanandamide did not alter the oxidative microbicidal PMN function.
Article
The use of flow cytometry with fluorescently labeled particles provides the means to examine quantitatively the phagocytotic capacity of an individual phagocyte. This report describes an improved flow cytometric method of analysis for kinetic measurement of phagocytosis of fluorescein isothiocyanate (FITC)-labeled zymosan particles by human leukocytes. FITC-labeled zymosan was incubated with leukocyte suspension, and at selected time intervals fluorescence positive neutrophils were divided by phagocytotic gates into three subpopulations: neutrophils that were neither binding nor ingesting particles, neutrophils that were only binding particles (binding cells), and neutrophils that were binding and ingesting particles (ingesting cells). For the distinction between internalized and surface-bound FITC-labeled zymosan, trypan blue (1.2 mg/ml) was used to quench surface-bound fluorescence. The technical challenges related to settings of phagocytotic gates and derivation of phagocytotic equations were presented. From 28 control samples, numerical values of mean fluorescence intensities and percentages of phagocytotic subpopulations inside phagocytotic gates before and after quenching were inserted into phagocytotic equations and corrected phagocytotic parameters were calculated. Calculated parameters were surprisingly constant across individuals. Essential elements of the present method appeared to be partial quenching of extracellular fluorescence with trypan blue and distinguishing between overlapping populations of binding and ingesting cells. Corrections using derived phagocytotic equations proved necessary for accurate kinetic phagocytotic measurements. Corrections were less necessary when the ingestion process was finished.
Article
Immunomodulatory effects of endogenous and exogenous cannabinoids have been investigated in numerous studies, mostly performed with isolated cells or transformed cell lines, but only sparse data exist on human polymorphonuclear neutrophils (PMNs). We therefore investigated the respiratory burst reaction of human whole-blood PMNs under the influence of cannabinoids using flow cytometry. In their natural whole-blood milieu, a CB(2) receptor-dependent stimulation of the PMN respiratory burst was found at nanomolar concentrations of CP55 940 [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol] and methanandamide after a 3-h incubation period, whereas the short-living and rapidly hydrolyzed endogenous ligand anandamide did not alter the burst reaction of whole-blood PMNs under the same experimental conditions. The stimulatory cannabinoid effect was totally absent in isolated PMNs but could be transferred onto isolated PMNs by adding the cell-free low-molecular mass plasma fraction (<5000 Da) of cannabinoid-incubated blood, indicating an indirect mechanism depending on humoral products or mediators. Results of our further experiments suggest that products of the arachidonic acid metabolism are mediators of the cannabinoid-induced enhancement of the respiratory burst reaction of whole-blood PMNs.