Inhibition Of Cytokine Release Research Articles

Selective α7-nicotinic acetylcholine receptor agonist GTS-21 improves survival in murine endotoxemia and severe sepsis*

Tumor necrosis factor and high mobility group box 1 are critical cytokine mediators of inflammation. The efferent vagus nerve inhibits cytokine release through alpha7-nicotinic acetylcholine receptor-mediated cholinergic signaling. Here we studied whether GTS-21, a selective alpha7-nicotinic acetylcholine receptor agonist, inhibits proinflammatory cytokines in vitro and in vivo and improves survival in murine endotoxemia and severe sepsis. Randomized and controlled in vitro and in vivo study. Research laboratory and animal facility rooms. RAW 264.7 cells and BALB/c mice treated with endotoxin or subjected to cecal ligation and puncture (CLP). RAW 264.7 cells were exposed to endotoxin (4 ng/mL or 10 ng/mL) in the presence or absence of GTS-21 (1-100 muM), and tumor necrosis factor and high mobility group box 1 release and nuclear factor-kappaB activation were analyzed. Mice were treated with GTS-21 (0.4 mg/kg or 4 mg/kg, intraperitoneally) or saline 30 mins before endotoxin (6 mg/kg, intraperitoneally), and serum tumor necrosis factor was analyzed 1.5 hrs after the onset of endotoxemia. In survival experiments, mice were treated with GTS-21 (0.4 or 4.0 mg/kg, intraperitoneally) or saline 30 mins before and 6 hrs after endotoxin and then twice daily for 3 days. Severe sepsis was induced by CLP. Mice were treated with GTS-21 (4 mg/kg) or saline immediately and 6 hrs and 24 hrs after CLP, and serum high mobility group box 1 was analyzed 30 hrs after CLP. In survival experiments, GTS-21 (0.4 or 4 mg/kg) treatment was initiated 24 hrs after CLP and continued twice daily for 3 days. GTS-21 dose-dependently inhibited tumor necrosis factor and high mobility group box 1 release and nuclear factor-kappaB activation in vitro. GTS-21 (4 mg/kg) significantly inhibited serum tumor necrosis factor during endotoxemia and improved survival (p < .0001). GTS-21 (4 mg/kg) significantly inhibited serum high mobility group box 1 levels in CLP mice and improved survival (p < .0006). These findings are of interest for the development of alpha7-nicotinic acetylcholine receptor agonists as a new class of anti-inflammatory therapeutics.

RR Interval Variability Is Inversely Related to Inflammatory Markers: The CARDIA Study

Recent evidence reveals that the immune system is under the direct control of the vagus nerve via the "cholinergic anti-inflammatory pathway." Stimulation of vagus nerve activity significantly inhibits cytokine levels in animal models, and cholinergic agents inhibit cytokine release by human macrophages. Moreover, when vagus nerve activity is decreased or absent, cytokines are overproduced. Atherosclerosis is an inflammatory disease characterized by elevated levels of CRP and IL-6, but the relationship between cardiac vagal activity and cytokine levels in healthy humans is not well understood. Here we measured RR interval variability, an index of cardiac vagal modulation, and CRP and IL-6 in 757 subjects participating in a subset of the year 15 data collection in the CARDIA study of the evolution of risk factors in young adults. Univariate analysis revealed that all indices of RRV were strongly and inversely related to IL-6 (log pg/mL b=-0.08 and -0.17 for HF and LF power, P<0.001 respectively) and CRP (log mg/L b=-0.14 and -0.26 for HF and LF power, P<0.001 respectively) levels. In the multivariate model including gender, race, age, smoking, physical activity, SBP, BMI, and disease, the inverse relationship between RRV and inflammatory markers, although slightly attenuated, remained significant. These findings are consistent with the hypothesis that diminished descending vagal anti-inflammatory signals can allow cytokine overproduction in humans.

Physiology and immunology of the cholinergic antiinflammatory pathway

Cytokine production by the immune system contributes importantly to both health and disease. The nervous system, via an inflammatory reflex of the vagus nerve, can inhibit cytokine release and thereby prevent tissue injury and death. The efferent neural signaling pathway is termed the cholinergic antiinflammatory pathway. Cholinergic agonists inhibit cytokine synthesis and protect against cytokine-mediated diseases. Stimulation of the vagus nerve prevents the damaging effects of cytokine release in experimental sepsis, endotoxemia, ischemia/reperfusion injury, hemorrhagic shock, arthritis, and other inflammatory syndromes. Herein is a review of this physiological, functional anatomical mechanism for neurological regulation of cytokine-dependent disease that begins to define an immunological homunculus.

Neuro-immune interactions via the cholinergic anti-inflammatory pathway

The overproduction of TNF and other cytokines is associated with the pathophysiology of numerous diseases. Controlling cytokine synthesis and release is critical for preventing unrestrained inflammation and maintaining health. Recent studies identified an efferent vagus nerve-based mechanism termed “the cholinergic anti-inflammatory pathway” that controls cytokine production and inflammation. Here we review current advances related to the role of this pathway in neuro-immune interactions that prevent excessive inflammation. Experimental evidence indicates that vagus nerve cholinergic anti-inflammatory signaling requires alpha7 nicotinic acetylcholine receptors expressed on non-neuronal cytokine-producing cells. Alpha7 nicotinic acetylcholine receptor agonists inhibit cytokine release and protect animals in a variety of experimental lethal inflammatory models. Knowledge related to the cholinergic anti-inflammatory pathway can be exploited in therapeutic approaches directed towards counteracting abnormal chronic and hyper-activated inflammatory responses.

The effect of pentoxifylline on the pulmonary response to high tidal volume ventilation in rats

Background Volume-induced lung injury is associated with lung inflammation. Pentoxifylline inhibits cytokine release and modulates neutrophil function. Objective To evaluate the efficacy of pentoxifylline in the attenuation of lung inflammation induced by high tidal volume ventilation. Design Adult rats were randomly assigned to receive saline as placebo or pentoxifylline (100 mg/kg over 30 min, followed by 50 mg/kg/h) before and during 4 h of high tidal volume ventilation (20 ml/kg). Bronchoalveolar fluid inflammatory mediators were measured at baseline and after 4 h of ventilation. Lung tissue myeloperoxidase activity and wet/dry lung weight were assessed upon completion of the study. Results Bronchoalveolar tumor necrosis factor- α (pentoxifylline vs. placebo; 192±61 vs. 543±99 pg/ml; p < 0.007 ) and thromboxane B 2 (262±26 vs. 418±49 pg/ml; p < 0.02 ) concentrations, lung myeloperoxidase activity (0.5±0.1 vs. 1.2±0.2 U/mg; p < 0.003 ) and wet/dry weight (6.1±0.2 vs. 7.1±0.3; p < 0.01 ) were all significantly lower in the pentoxifylline-treated group. Conclusion Pentoxifylline was effective in reducing inflammatory lung injury associated with high tidal volume ventilation.

The effects of tea extracts on proinflammatory signaling

BackgroundSkin toxicity is a common side effect of radiotherapy for solid tumors. Its management can cause treatment gaps and thus can impair cancer treatment. At present, in many countries no standard recommendation for treatment of skin during radiotherapy exists. In this study, we explored the effect of topically-applied tea extracts on the duration of radiation-induced skin toxicity. We investigated the underlying molecular mechanisms and compared effects of tea extracts with the effects of epigallocatechin-gallate, the proposed most-active moiety of green tea.MethodsData from 60 patients with cancer of the head and neck or pelvic region topically treated with green or black tea extracts were analyzed retrospectively. Tea extracts were compared for their ability to modulate IL-1β, IL-6, IL-8, TNFα and PGE2 release from human monocytes. Effects of tea extracts on 26S proteasome function were assessed. NF-κB activity was monitored by EMSAs. Viability and radiation response of macrophages after exposure to tea extracts was measured by MTT assays.ResultsTea extracts supported the restitution of skin integrity. Tea extracts inhibited proteasome function and suppressed cytokine release. NF-κB activity was altered by tea extracts in a complex, caspase-dependent manner, which differed from the effects of epigallocatechin-gallate. Additionally, both tea extracts, as well as epigallocatechin-gallate, slightly protected macrophages from ionizing radiationConclusionTea extracts are an efficient, broadly available treatment option for patients suffering from acute radiation-induced skin toxicity. The molecular mechanisms underlying the beneficial effects are complex, and most likely not exclusively dependent on effects of tea polyphenols such as epigallocatechin-gallate.

Leukemia Targeting Via Cytotoxic T Cells Expressing a CD33 Specific Chimeric Receptor.

CD33 is a surface protein expressed on the malignant blast cells of most acute myeloid leukemia (AML) and is a potential tumor-associated target antigen. Immunotoxin-based therapy against CD33+ myeloid leukemia has shown promising results with no long-term side effects on CD33+ progenitor cells, but is associated with liver toxicity. We describe a cell therapy approach targeting CD33 for AML, which should be more effective than the immunotoxin and have lower non-specific toxicity. The genetic modification of human T cells to express tumor specific chimeric immune receptors (TCR) can produce large numbers of tumor-specific T cells. Unfortunately, these modified T cells have only a limited response to tumor cells in vivo. By contrast, EBV-specific cytotoxic T cells (EBV-CTLs) expand and have long-term activity in vivo. We exploited this phenomenon and redirected EBV-CTLs against CD33. EBV-CTLs were generated from six EBV-seropositive healthy donors and transduced using a retroviral vector encoding the chimeric CD33 specific TCR. The cRCD33-EBV-CTL maintained their pre-transduction immunophenotype and specifically lyzed EBV-LCL and CD33+ targets (specific lysis of 30% and 35% at E:T ratio 25:1). They also produced Th1 and Th2-type cytokines on exposure to CD33+ targets. Addition of the CD28 intracellular domain did not significantly increase cytotoxicity to CD33+ targets, but resulted in a 5–7 and 2–3 fold increase in IFN-γand IL-5 production. We confirmed the specificity of the TCR interactions with CD33 by inhibiting them with MHC class I, class II and CD33 blocking monoclonal antibodies (MoAb). Preincubation of CD33+ target cells with the CD33-blocking MoAb resulted in 40% inhibition of lysis and 60% inhibition of cytokine release by cRCD33-EBV-CTLs, whereas preincubation of CD33+ targets with class I and II blocking MoAb had no significant effect. Incubation of autologous LCLs with MHC Class I and Class II MoAb showed MHC class I was the major restriction element for the native antigen receptor of both nontransduced and modified EBV-CTLS. Of note, the cRCD33-EBV-CTLs proliferated only in response to autologous LCLs and not to CD33+ targets. Failure to proliferate was not overcome by incorporating the CD28 endodomain into the CAR. By contrast, cRCD33-EBV-CTLs can be expanded and maintain their specific activities toward CD33+ targets by co-culture with LCLs, and these effects are sustained for over 30 days in culture. Despite their activity against CD33+ blast cells, long-term culture-initiating cell assay demonstrated that neither cRCD33-EBV-CTLs nor cRCD33-CD28-EBV-CTLs had measurable toxicity against CD33+ progenitor cells isolated from bone marrow samples. To assess activity in vivo, we injected NOD-SCID mice bearing a human CD33+ AML tumor with EBV-CTLs x4 weekly, starting 5 days after tumor inoculation. Whereas the administration of PBS or untransduced EBV-CTLs had no effect on tumor progression, cRCD33-EBV-CTLs showed a trend to reduced tumor growth, the statistical significance of which is being confirmed on a larger series of animals. Incorporation of the CD28 endodomain had no additive effect on anti-tumor activity. Importantly, no adverse effects wee seen on normal CD33+ progenitor cells in vivo. We conclude that EBV-CTL expressing the CD33 chimeric receptor may be appropriate as a therapeutic modality for the treatment of myeloid leukemia.

Acetylcholinesterase inhibitors for Alzheimer’s disease: anti-inflammatories in acetylcholine clothing!

The pathogenesis of Alzheimer's disease (AD) has been linked to a deficiency in the brain neurotransmitter acetylcholine. Subsequently, acetylcholinesterase inhibitors (AChEIs) were introduced for the symptomatic treatment of AD. The prevailing view has been that the efficacy of AChEIs is attained through their augmentation of acetylcholine-medicated neuron to neuron transmission. However, AChEIs also protect cells from free radical toxicity and beta-amyloid-induced injury, and increased production of antioxidants. In addition, it has been reported that AChEIs directly inhibit the release of cytokines from microglia and monocytes. These observations are supported by evidence showing a role for acetylcholine in suppression of cytokine release through a 'cholinergic anti-inflammatory pathway'. Based on the accumulating research data so far, it is no longer appropriate to consider that the sole action of AChEIs in AD is through direct acetylcholine-medicated enhancement of neuronal transmission. Evidence points to a possible anti-inflammatory role for these agents as well.

The effect of adenosine receptor agonists on cytokine release by human mononuclear cells depends on the specific Toll-like receptor subtype used for stimulation

In the present study, we determined whether the immunomodulatory effect of adenosine receptor stimulation depends on the Toll-like Receptor (TLR) used for stimulation of cytokine release. Therefore, human mononuclear cells were stimulated by different TLR agonists in the absence and presence of A 1 (CPA), A 2a (CGS21680), and A 3 (Cl-IB-MECA) adenosine receptor agonists. Effects of these agonists on Il-6, Il-10, IFN-γ, TNF-α, and Il-1β production were expressed as percentage inhibition/stimulation after TLR stimulation. CGS21680 inhibited TLR4-mediated TNF-α release and potentiated TLR3- and TLR5-mediated IL-6 release. Cl-IB-MECA inhibited TLR4-agonist-induced IFN-γ release. Interestingly, CPA en Cl-IB-MECA tended to inhibit cytokine release only after TLR4 stimulation. In more detail, CPA potentiated TLR5-mediated IL-6 production, TLR3-mediated IFN-γ production and TLR3-mediated Il-1β-production compared to TLR4-mediated stimulation. Cl-IB-MECA potentiated TLR5-mediated IL-6 and Il-1β formation as compared to TLR4-mediated stimulation. Finally, CGS21680 potentiated TLR5-mediated IL-6 production compared to TLR1-2 stimulation, and potentiated TLR3- and TLR5-mediated IL-10 production compared to TLR1-2-mediated stimulation. In conclusion, the effect of adenosine agonists on cytokine production depends on the specific TLR agonist used for stimulation. These findings suggest that well-known anti-inflammatory effects of adenosine agonists on LPS-induced inflammation cannot be extrapolated to situations in which stimulation of other TLR subtypes is involved.

Inhibition of rat RAW264.7 macrophage inflammatory cytokines release by small hairpin RNAi targeting Toll-like receptor

To construct a eukaryotic expression vector carrying the small hairpin RNA (shRNA) for Toll-like receptor 4 (TLR4) mRNA and a reporter gene of enhanced green fluorescence protein (EGFP) and study the inhibition of cytokine release by rat RAW264.7 macrophages induced by lipopolysaccharide (LPS) stimulation through transfection and expression of shRNA targeting TLR4 gene via the RNAi mechanism. The H1 promotor and double BbsIrestrict endoenzyme site from the plasmid psiRNA-hH1neo were cloned into the reporter gene plasmid pEGFP-C1 at the MluIrestrict endoenzymic site, thus forming the plasmid pEGFP-H1/siRNA containing Bbs site and reporter EGFP gene. Then an oligo nuclear hairpin sequence targeting TLR4 gene was designed by the internet tool siRNA Wizard and then inserted into the plasmid pEGFP-H1/siRNA so as to form the plasmid pEGFP-H1/TLR4-siRNA. Rat macrophages of the line RAW264.7 were cultured and transfected with pEGFP-H1/TLR4-siRNA mediated by lipofectamine 2000. Another RAW264.7 cells were transfected with pEGFP-H1/control sequence-siRNA or blank plasmid. Lipopolysaccharide was added into the 3 kinds of culture fluid for 2 and 68 hours respectively. ELSA was used to detect the levels of tumor necrosis factor-alpha (TNF-alpha) in the supernatants. Restriction endonuclease analysis showed that the construction pEGFP-H1/TLR4-siRNA carrying hairpin RNA for TLR4 gene and reporter EGFP gene was successful. The expression of EGFP gene was 50% +/- 8%. The TNF-alpha level of the TLR4-siRNA transfection group 2 hours and after transfection was 825 pg/ml +/- 136 pg/ml, significantly lower than those of the pEGFP-H1/control sequence-siRNA and blank plasmid groups (2190 pg/ml +/- 359 pg/ml and 1265 pg/ml +/- 283 pg/ml respectively, both P < 0.01). The TNF-alpha level of the TLR4-siRNA transfection group 8 hours and after transfection was 1179 pg/ml +/- 240 pg/ml, significantly lower than those of the pEGFP-H1/control sequence-siRNA and blank plasmid groups (4720 pg/ml +/- 227 pg/ml and 4689 pg/ml +/- 310 pg/ml respectively, both P < 0.01). shRNA targeting TLR4 gene can inhibit the TNF-alpha release by RAW264.7 cells evoked by LPS.

Effect of Ono-EI-600 elastase inhibitor on high-tidal-volume-induced lung injury in rats

We tested the effect of Ono-EI-600, an elastase inhibitor that suppresses cytokine release, on ventilator-induced lung injury in a rat model. After Wistar rats (aged 8-11 weeks) were anesthetized and tracheostomized, they were randomly assigned to four groups: high tidal volume (V(T)) group (H group: n = 10) receiving peak inspiratory pressure (PIP) 30 cmH(2)O for 240 min; high V(T) with drug group (HD group: n = 10) receiving the same ventilation settings as H group and also intravenous infusion 10 mg x kg(-1) x h(-1) of Ono-EI-600 during the protocol; the lower V(T) group (L group: n = 5) receiving PIP 10 cmH(2)O for 240 min; and control group (C group: n = 5) receiving the same ventilation as L group for 30 min. The cytokine levels (IL-6 and CINC-1) in the bronchoalveolar lavage fluid (BALF) of the H group were significantly higher than those of the C and L groups (P < 0.05). However, for the H and HD groups, no differences were found in arterial blood gas data, cytokine levels in BALF, and histological injury scores. Our experiment provided no evidence that elastase inhibitor Ono-EI-600 protects against lung injury induced by high V(T) ventilation.

Relative Corticosteroid Insensitivity of Peripheral Blood Mononuclear Cells in Severe Asthma

Patients with severe asthma have a poor therapeutic response to corticosteroid therapy, and corticosteroid responsiveness cannot be easily measured in these patients. We hypothesized that this poor response is associated with a reduced effect of corticosteroids to inhibit cytokine release from activated peripheral blood mononuclear cells (PBMCs). Patients with severe asthma were defined by American Thoracic Society criteria. We compared the suppression of LPS-induced cytokine release (monocyte chemotactic protein-1 [MCP-1], macrophage inflammatory protein [MIP] 1alpha, RANTES, tumor necrosis factor alpha, interleukin 1beta (IL-1beta), IL-8, IFN-gamma, IL-6, IL-10, and granulocyte-macrophage colony-stimulating factor [GM-CSF]) by dexamethasone from PBMCs of patients with severe asthma (n = 16), patients with nonsevere asthma (n = 19), and normal volunteers (n = 10). There was no difference in baseline spontaneous or stimulated release of these cytokines among groups. LPS-induced release of 10 cytokines was less suppressed by dexamethasone (10(-6) M) in patients with severe asthma compared with patients with nonsevere asthma, with statistical significance achieved for IL-1beta (p < 0.03), IL-8 (p < 0.03), and MIP-1alpha (p < 0.003), and borderline significance for IL-6 (p = 0.054). There was less difference between the two groups for dexamethasone at 10(-8) M. Nuclear histone deacetylase (HDAC) and histone acetyltransferase activities were reduced in patients with severe asthma compared with patients with nonsevere asthma (p < 0.01). HDAC activity reduction correlated directly to the degree of steroid insensitivity of GM-CSF (r = 0.57, p < 0.01) and IFN-gamma (r = 0.56, p < 0.05) release. Reduction in histone acetyltransferase activity related to corticosteroid use rather than asthma severity. Patients with severe asthma have diminished corticosteroid sensitivity of PBMCs when compared with patients with nonsevere asthma, associated with a reduction in HDAC activity that parallels the impaired corticosteroid sensitivity.

Triglyceride-Rich HDL3 from Patients with Familial Hypercholesterolemia Are Less Able to Inhibit Cytokine Release or to Promote Cholesterol Efflux

Familial hypercholesterolemia (FH) is associated with heterogeneity of the onset and severity of coronary heart disease (CHD). In this study, we investigated different low-grade proinflammatory markers and the atheroprotective function of the HDL3 subfraction in FH-patients (n = 13) with identical LDL-receptor mutations and in age- and sex-matched healthy controls (n = 11). Compared with healthy controls, FH-patients had greater gene expressions of the proatherogenic mediators TNF-alpha and IL-8 in circulating peripheral blood mononuclear cells. In addition, they had a higher serum concentration of intercellular adhesion molecule-1 (ICAM-1) and a lower net antioxidant capacity. FH-derived HDL3 with a high level of triglycerides had a reduced capacity to inhibit the release of IL-8 from TNF-alpha-stimulated human umbilical vein endothelial cells (HUVEC) [1.864 mg/L (1.461-2.208 mg/L) vs. 1.466 mg/L (1.225-1.643 mg/L); P < 0.05; median (range)], and a reduced capacity to promote cholesterol efflux from lipid-loaded macrophages [12% (12-14%) vs. 15% (14-18%); P < 0.05; median (range)] compared with HDL3 with a lower triglyceride content. Notably, the degree of inhibition of IL-8 release from HUVEC by HDL3 was correlated with the ability of HDL3 to promote cholesterol efflux (r = -0.80, P = 0.03). In conclusion, compared with healthy controls, FH-patients are characterized by higher levels of low-grade proinflammatory markers, and FH-derived HDL3 with high triglyceride content may be more proatherogenic. These triglyceride rich-HDL3 might be partly responsible for the phenotypic variation among FH-patients with identical LDL-receptor mutations.

Use of Autologous Serum in Corneal Epithelial Defects Post-Lamellar Surgery

To evaluate the efficacy of an autologous serum treatment of post-LASIK (laser in situ keratomileusis) corneal epithelial defects in a rabbit model. Five milliliters blood samples from 10 New Zealand rabbits were obtained by venepuncture. The serum was aseptically separated and diluted with saline solution to 20%. The final preparation was placed into 3-mL bottles with ultraviolet protection and maintained at 4 degrees C. Corneas were de-epithelialized using a 7-mm optical zone marker. A 160-microm thick flap was created in both eyes of all rabbits using an automatic corneal shaper microkeratome. Right eyes were treated with serum drops 6 times per day. Left eyes were treated with preservative-free artificial tears. Vital staining of the ocular surface and the area of corneal epithelial defect was measured daily for 1 week. Rabbits were humanely euthanized at postoperative day 7, and corneas were fixed and sectioned. Hematoxylin and eosin staining and immunohistochemical analysis were performed. Corneas treated with autologous serum had a statistically significant increase in the epithelial healing rate compared with those treated with artificial tears. Serum-treated corneas showed significantly less terminal transferase-mediated dUTP nick-end labeling (TUNEL) staining in the interface, minimal inflammatory cell infiltration, and less induced synthesis of stromal chondroitin sulfate than did corneas treated with preservative-free artificial tears. Treatment with autologous serum could be an efficient way to provide essential components to the ocular surface in the treatment of post-LASIK epithelial defects. Autologous serum induces faster epithelial healing than do artificial tears, leading to (1) a decrease in keratocyte apoptosis and migration of fibroblasts and myofibroblasts in the wound site, (2) a decrease in the migration of inflammatory cells, and (3) a consequent inhibition of cytokine release. This treatment could improve long-term refractive results post-LASIK.

Zinc-Mediated Inhibition of Cyclic Nucleotide Phosphodiesterase Activity and Expression Suppresses TNF-α and IL-1β Production in Monocytes by Elevation of Guanosine 3′,5′-Cyclic Monophosphate

The trace element zinc affects several aspects of immune function, such as the release of proinflammatory cytokines from monocytes. We investigated the role of cyclic nucleotide signaling in zinc inhibition of LPS-induced TNF-alpha and IL-1beta release from primary human monocytes and the monocytic cell line Mono Mac1. Zinc reversibly inhibited enzyme activity of phosphodiesterase-1 (PDE-1), PDE-3, and PDE-4 in cellular lysate. It additionally reduced mRNA expression of PDE-1C, PDE-4A, and PDE-4B in intact cells. Although these PDE can also hydrolyze cAMP, only the cellular level of cGMP was increased after incubation with zinc, whereas cAMP was found to be even slightly reduced due to inhibition of its synthesis. To investigate whether an increase in cGMP alone is sufficient to inhibit cytokine release, the cGMP analogues 8-bromo-cGMP and dibutyryl cGMP as well as the NO donor S-nitrosocysteine were used. All three treatments inhibited TNF-alpha and IL-1beta release after stimulation with LPS. Inhibition of soluble guanylate cyclase-mediated cGMP synthesis with LY83583 reversed the inhibitory effect of zinc on LPS-induced cytokine release. In conclusion, inhibition of PDE by zinc abrogates the LPS-induced release of TNF-alpha and IL-1beta by increasing intracellular cGMP levels.

Loss of shear stress induces leukocyte‐mediated cytokine release and blood–brain barrier failure in dynamic in vitro blood–brain barrier model

Brain ischemia is associated with an acute release of pro-inflammatory cytokines, notably TNF-alpha and IL-6 and failure of the blood-brain barrier. Shear stress, hypoxia-hypoglycemia, and blood leukocytes play a significant role in blood-brain barrier failure during transient or permanent ischemia. However, these mechanisms have not been studied as independent variables for in vitro ischemia. The present study, using a dynamic in vitro blood-brain barrier model, showed that flow cessation/reperfusion under normoxia-normoglycemia or hypoxia-hypoglycemia without blood leukocytes in the luminal perfusate had a modest, transient effect on cytokine release and blood-brain barrier permeability. By contrast, exposure to normoxic-normoglycemic flow cessation/reperfusion with blood leukocytes in the luminal perfusate led to a significant increase in TNF-alpha and IL-6, accompanied by biphasic blood-brain barrier opening. Enhanced permeability was partially prevented with an anti-TNF-alpha antibody. In leukocyte-free cartridges, the same levels of IL-6 had no effect, while TNF-alpha caused a moderate increase in blood-brain barrier permeability, suggesting that blood leukocytes are the prerequisite for cytokine release and blood-brain barrier failure during reduction or cessation of flow. These cells induce release of TNF-alpha early after ischemia/reperfusion; TNF-alpha triggers release of IL-6, since blockade of TNF-alpha prevents IL-6 release, whereas blockade of IL-6 induces TNF-alpha release. Pre-treatment of blood leukocytes with the cyclooxygenase (COX) inhibitor, ibuprofen, inhibited cytokine release and completely preserved blood-brain barrier permeability during the reperfusion period. In conclusion, loss of flow (flow cessation/reperfusion) independent of hypoxia-hypoglycemia plays a significant role in blood-brain barrier failure by stimulating leukocyte-mediated inflammatory mechanisms.

Modulation of Macrophage Phenotype by Soluble Product(s) Released from Neutrophils

The regulation of macrophage phenotype by neutrophils was studied in the s.c. polyvinyl alcohol sponge wound model in mice made neutropenic by anti-Gr-1 Ab, as well as in cell culture. Wounds in neutropenic mice contained 100-fold fewer neutrophils than those in nonneutropenic controls 1 day after sponge implantation. Wound fluids from neutropenic mice contained 68% more TNF-alpha, 168% more IL-6, and 61% less TGF-beta1 than those from controls. Wound fluid IL-10 was not different between the two groups, and IL-4 was not detected. Intracellular TNF-alpha staining was greater in cells isolated from neutropenic wounds than in those from control wounds. The hypothesis that wound neutrophil products modulate macrophage phenotype was tested in Transwell cocultures of LPS-stimulated J774A.1 macrophages and day 1 wound cells (84% neutrophils/15% macrophages). Overnight cocultures accumulated 60% less TNF-alpha and IL-6 than cultures of J774A.1 alone. The suppression of cytokine release was mediated by a soluble factor(s), because culture supernatants from wound cells inhibited TNF-alpha and IL-6 release from LPS-stimulated J774A.1 cells. Culture supernatants from purified wound neutrophils equally suppressed TNF-alpha release from LPS-stimulated J774A.1 cells. Wound cell supernatants also suppressed TNF-alpha and superoxide release from murine peritoneal macrophages. The TNF-alpha inhibitory factor has a molecular mass <3000 Da and is neither PGE2 nor adenosine. The present findings confirm a role for neutrophils in the regulation of innate immune responses through modulation of macrophage phenotype.

Autonomic neural regulation of immunity

The 'cytokine theory of disease' states that an overproduction of cytokines can cause the clinical manifestations of disease. Much effort has been expended to determine how cytokines are regulated in normal health. Transcriptional, translational and other molecular control mechanisms protect the host from excessive cytokine production. A recent discovery revealed an unexpected pathway that inhibits macrophage cytokine production. The inflammatory reflex is a physiological pathway in which the autonomic nervous system detects the presence of inflammatory stimuli and modulates cytokine production. Afferent signals to the brain are transmitted via the vagus nerve, which activates a reflex response that culminates in efferent vagus nerve signalling. Termed the 'cholinergic anti-inflammatory pathway', efferent activity in the vagus nerve releases acetylcholine (ACh) in the vicinity of macrophages within the reticuloendothelial system. ACh can interact specifically with macrophage alpha7 subunits of nicotinic ACh receptors, leading to cellular deactivation and inhibition of cytokine release. This 'hard-wired' connection between the nervous and immune systems can be harnessed therapeutically in animal models of inflammatory disease, via direct electrical stimulation of the vagus nerve, or through the use of cholinergic agonists that specifically activate the macrophage alpha7 subunit of the ACh receptor. Autonomic dysfunction has been associated with human inflammatory diseases including rheumatoid arthritis, diabetes and sepsis; whether this dysfunction results from the inflammatory component of these diseases, or is actually an underlying cause, is now less clear. The description of the cholinergic anti-inflammatory now brings to the fore several new therapeutic strategies for inflammatory disease, and suggests that many of these diseases may actually be diseases of autonomic dysfunction.

A potential role of YC-1 on the inhibition of cytokine release in peripheral blood mononuclear leukocytes and endotoxemic mouse models

To evaluate the anti-sepsis potential of YC-1, we have examined the effect of YC-1 on the regulation of cytokine production in human leukocytes and endotoxemic mice. The data demonstrated that YC-1 showed a preferential inhibition on proinflammatory cytokine production without inhibition of cell growth or induction of cytotoxicity in human leukocytes. On the other hand, in the septic mouse model, treatment with an intraperitoneal application of LPS caused a cumulative death within 27 hours. The post-treatment administration of YC-1 significantly increased the survival rate in endotoxemic mice. Furthermore, several mediators were detected and the data showed that YC-1 profoundly blocked LPS-induced NO as well as TNF-alpha production, and prevented lung damage by histological examination. Samples from the animal model showed that LPS-induced NF-kappaB/DNA binding activity and consequent up-regulation of iNOS expression in tissues were abolished by post-administration of YC-1. Furthermore, YC-1, by itself, did not modify cGMP content while significantly inhibit LPS-induced cGMP formation, suggesting that YC-1-mediated effect was not through a cGMP-elevating pathway. Taken together, it is evident that the post-treatment administration of YC-1 after LPS application significantly inhibits NF-kappaB activation, iNOS expression, NO over-production, and cytokine release reaction resulting in an improved survival rate in endotoxemic mice. It is suggested that YC-1 may be a potential agent for the therapeutic treatment of sepsis.

Immunosuppresive role of principal toxin (crotoxin) of Crotalus durissus terrificus venom

The composition of the crotalic venom and the immunochemistry and/or pathophysiological characterization and main components were well studied. However, few studies have been carried out to investigate the effect of toxins of this venom on the development of the immune response. The objective of this work was to find out if venom or crotoxin of Crotalus durissus terrificus was able to modulate the immune response through its ability to change the mediators involved in the immune response by an unrelated antigen. We observed in the murine model, that venom as well as crotoxin have inhibitory effect on splenic cells proliferation induced by Con-A. Moreover, CB did not inhibit the proliferative response, suggesting that the integrity of crotoxin complex is necessary for the development of this phenomenon. Moreover, we showed that the effect on cellular proliferation was unrelated to cytotoxicity activity. We also observed that venom or crotoxin inhibited cytokine release induced in HSA immunised mice, mainly IL-2, IL-4 and IL-10, however, crotoxin did not inhibit the release of IFN-γ. The involvement of T or B cells in the suppressive effect of venom was evaluated through the transference of purified splenic cells from venom-mice to normal mice that also produced low IgG1 anti-HSA levels, indicating the participation of these cells in this process. Mechanism of action of the crotalic venom on development of immune response to an unrelated antigen is much more complex, therefore it must not only involve the interaction of distinct cellular populations, but activation or inhibition of signalling proteins, need to be further investigated.