Modulation of eotaxin formation and eosinophil migration by selective inhibitors of phosphodiesterase type 4 isoenzyme

1. This study was undertaken to investigate the possible contribution of the blockade of eotaxin generation to the anti-eosinophilotactic effect of phosphodiesterase (PDE) type 4 inhibitors. In some experiments, the putative synergistic interaction between PDE type 4 inhibitors and the beta2-agonist salbutamol was also assessed. 2. Sensitized guinea-pigs aerosolized with antigen (5% ovalbumin, OVA) responded with a significant increase in eotaxin and eosinophil levels in the bronchoalveolar lavage fluid (BALF) at 6 h. Eosinophil recruitment was inhibited by both PDE type 4 inhibitors rolipram (5 mg kg(-1), i.p.) and RP 73401 (5 mg kg(-1), i.p.) treatments. In contrast, only rolipram inhibited eotaxin production. 3. Sensitized rats intrapleurally challenged (i.pl.) with antigen (OVA, 12 microg cavity(-1)) showed a marked eosinophil infiltration at 24 h, preceded by eotaxin generation at 6 h. Intravenous administration of a rabbit anti-mouse eotaxin antibody (0.5 mg kg(-1)) significantly reduced allergen-evoked eosinophilia in this model. 4. Local pretreatment with rolipram (40 microg cavity(-1)) or RP 73401 (40 microg cavity(-1)) 1 h before challenge reduced eosinophil accumulation evaluated in the rat pleural effluent, but only the former was active against eotaxin generation. The inhibitors of PDE type 3 (SK&F 94836) and type 5 (zaprinast) failed to alter allergen-evoked eosinophil recruitment in rats. 5. Local injection of beta2-agonist salbutamol (20 microg cavity(-1)) inhibited both eosinophil accumulation and eotaxin production following pleurisy. The former was better inhibited when salbutamol and rolipram were administered in combination. 6. Treatment with rolipram and RP 73401 dose-dependently inhibited eosinophil adhesion and migration in vitro. These effects were clearly potentiated by salbutamol at concentrations that had no effect alone. 7. Our findings indicate that although rolipram and RP 73401 are equally effective in inhibiting allergen-induced eosinophil infiltration only the former prevents eotaxin formation, indicating that PDE 4 inhibitors impair eosinophil accumulation by mechanisms independent of eotaxin production blockade.     

Inhibition of allergen-induced eosinophil recruitment by natural tetranortriterpenoids is mediated by the suppression of IL-5, CCL11/eotaxin and NFkappaB activation

The present study reports the anti-allergic activity of a group of six different tetranortriterpenoids (TNTP) isolated from the seeds of Carapa guianensis Aublet: 6a-acetoxygedunin, 7-deacetoxy-7-oxogedunin, andirobin, methyl angolensate, 6a-acetoxyepoxyazadiradione and gedunin. Oral pretreatment with TNTP significantly inhibited total leukocyte and eosinophil accumulation in C57BL/10 mice pleural cavities 24 h after the intrathoracic (i.t.) injection of ovalbumin (OVA), but had no effect on CD4, CD8 or gammadelta T lymphocyte accumulation. Pleural washes recovered from 6 h OVA-stimulated mice (OPW) pretreated with TNTP failed to induce shape change in eosinophil in vitro, indicating the inhibition of eosinophilotactic chemokines by TNTP. In accordance with such results, ELISA assays showed decreased levels of CCL11/eotaxin and IL-5 in OPW recovered from TNTP pretreated mice within 6 h. TNTP oral pretreatment inhibited nuclear factor-kappaB (NFkappaB) translocation into the nucleus in pleural leukocytes recovered from previously sensitized mice after antigenic challenge. In addition, the incubation of splenocytes recovered from previously sensitized mice with TNTP also inhibited NFkappaB activation after OVA stimulation. Taken together, these results indicate that the inhibition of allergic eosinophilia by TNTP is correlated with the inhibition of CCL11/eotaxin and IL-5 generation through NFkappaB signaling pathway impairment in mice.     

Regulation of antigen-specific CTL and Th1 cell activation through 5-Hydroxytryptamine 2A receptor

Serotonin (5-Hydroxytryptamine, 5-HT) is one of the most extensively studied neurotransmitters in the central nervous system. Also expressed in peripheral tissues, 5-HT participates in vasoconstriction and in aggregation of platelets through 5-HT(2A) receptor (5-HT2AR). However, there have been few studies regarding the interaction between 5-HT and 5-HT2AR in the immune system. In the current study, we analyzed the role of 5-HT and its 5-HT2AR in the activation of antigen-specific Th1 and cytotoxic T lymphocytes (CTL) in mice. RT-PCR and western blotting analyses confirmed the expression of 5-HT2AR in both CD4 and CD8 T cells. Both antigen-specific and anti-CD3 stimulation of IL-2 and IFN-γ production from these cells were inhibited by a selective 5-HT2AR inhibitor, sarpogrelate hydrochloride. Concanavalin A (ConA) activation of CD4(+) and CD8(+) T cells, which were purified from mouse spleen following depletion of endogenous 5-HT, was enhanced by a selective 5-HT2AR agonist, (R)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI). Such DOI-induced T cell activation was nullified by sarpogrelate. Moreover, an ELISPOT study showed that sarpogrelate also reduced antigen-specific induction of both CTL and Th1 cells in vivo following immunization of mice with cognate antigens. These data suggest that antigen-specific Th1 and CTL cells might be regulated by 5-HT signaling through 5-HT2AR on their surfaces and that 5-HT2AR inhibitor might have an immunosuppressive effect in vivo.     

Regulation of angiogenesis by Th1- and Th2-type cytokines

Angiogenesis is a complex process, where several cell types and mediators interact to establish a specific microenvironment suitable for the formation of new capillaries from pre-existing vessels. Such biological processes occur in several physiological conditions, such as embryo development and wound healing, as well as in pathological conditions, including tumours and diabetic retinopathy. T lymphocytes, neutrophils and monocytes fully participate in the angiogenic process by secreting cytokines that may control endothelial cell (EC) proliferation, their survival and apoptosis, as well as their migration and activation. Angiogenesis is the result of a net balance between the activities exerted by positive and negative regulators. This balance is conceptually very similar to that of the Th1/Th2 cells that modulate an appropriate and specific immune response. Th1 or Th2 cytokines may control angiogenesis directly, by acting on cell growth and differentiation, indirectly by inducing the release of other cytokines in the microenvironment, and by modulating the expression of specific receptors, involved in the control of angiogenic processes, such as EC proliferation and migration. In this review we will mainly discuss the role of Th1- and Th2-type cytokines in the angiogenic process, emphasizing the complexity of the cytokine and leukocyte/EC network, and highlighting the care that needs to be taken when designing new therapeutic interventions involving Th1 and Th2 cytokines.     

嗜酸粒细胞及白细胞介素5与小儿肺炎的关系

目的 探讨嗜酸粒细胞（EOS）及白细胞介素5（IL－5）与小儿支气管肺炎的关系。方法 对33例喘憋性肺炎、20例非喘憋性肺炎及18例正常儿童,以ELISA法检测血清IL－5和计数外周血EOS。结果 喘憋组肺炎患儿EOS数显著高于正常组（P＜0．01）,各组肺炎患儿IL－5水平显著高于正常组（P＜0．01和P＜0．05）,喘憋组肺炎患儿EOS数与IL－5水平呈显著正相关（r=0.598,P＜0．01）。结论 IL－5参与小儿肺炎的发病过程,而IL－5作用于EOS所导致气道炎症则可能是喘憋性肺炎的主要发病机制;EOS及IL－5应作为指导小儿肺炎诊断和治疗的一对重要检测指标。     

Regulation of neuronal cell function by glyco-signals

Gangliosides and proteoglycans with various sugar chains exist abundantly in the brain. They participate in intercellular recognition by revealing the sugar chains on the cell surface, and some of them show neurite-extension activity. Several recognition features that are mediated by the sugar chains are known such as saccharide-saccharide interaction and cell-surface sugar-chain receptor-mediated recognition. Experiments on animals lacking the sugar-chain synthetic system with the technique of gene targeting suggest that phylogenetically "old" sugar chains such as chondroitin sulfate appear necessary for early development of the organism while relatively "new" sugar chains such as gangliosides, which appear with further development of the brain, are necessary for differentiation maturity processes. On the other hand, research using primary cultured neurons showed similar effects of the gangliosides and chondroitin sulfate on cell differentiation. It is possible that these sugar chains share the glyco-receptor-mediated signal transduction system.     

Regulation of cell function by Rho GTPases

Small GTPases of the Rho family regulate a wide variety of cell functions. In this review, we briefly describe the biological activities of Rho GTPases. Using the Rac-regulated NADPH oxidase as an example, we discuss possible regulatory points that might be exploited for drug development. Finally, we explore strategies for specific targeting of Rho GTPase-regulated signaling pathways.     

清热止痒颗粒对特应性皮炎小鼠外周血Th1／Th2细胞的调控作用

目的探讨清热止痒颗粒对特应性皮炎小鼠外周血Th1／Th2细胞的调控作用。方法采用2,4-二硝基氟苯建立特应性皮炎小鼠模型,观察清热止痒颗粒对特应性皮炎小鼠的治疗效果,用流式细胞仪检测小鼠外周血Th1、Th2细胞浓度。结果特应性皮炎小鼠外周血Th1细胞水平、Th1/Th2比值低于正常小鼠,Th2细胞水平高于正常组小鼠（P〈0．01）。清热止痒颗粒治疗后,外周血Th1细胞水平、Th1／Th2比值上升,Th2细胞水平下降,且Th2细胞水平与正常小鼠比较差异无统计学意义（P〉0.05）。结论清热止痒颗粒能调节Th1、Th2细胞水平,纠Th1／Th2失衡,是其治疗特应性皮炎小鼠的可能机制之一。     

IL-5 accounts for the mouse pleural eosinophil accumulation triggered by antigen but not by LPS

The involvement of interleukin-5 (IL-5) in the pleural eosinophilia induced by LPS or allergen was investigated. The number of pleural eosinophils in actively sensitized mice increased 24 h after the intrathoracic (i.t) injection of ovalbumin (12 mg/cavity), peaked within 72 h, and persisted significantly increased for at least 120 h. Despite being less intense, the i.t. injection of LPS (250 ng/cavity) also increased the number of pleural eosinophils at 24 h, returning to basal levels within 72 h. Intraperitoneal pretreatment with monoclonal antibody to IL-5 (TRFK-4 and TRFK-5, 500 mg/kg) suppressed the eosinophil accumulation induced by IL-5 (200 units/cavity) or ovalbumin, but had no effect on the LPS-induced eosinophilia. Transfer of the cell-free pleural washing from LPS-treated donor mice to naive recipient animals led to a selective increase in the eosinophil counts. The co-incubation of the pleural washing from LPS-treated animals with monoclonal antibody to IL-5 failed to modify the pheonomenon. The results indicate that IL-5 plays an important role in the antigen-induced accumulation of eosinophils in vivo, but not in the eosinophilia triggered by LPS.     

IL-7和IL-2协同对肺结核患者Th1/Th2平衡调节作用的研究

目的探讨白细胞介素7(IL-7)、白细胞介素2(IL-2)协同对肺结核病患者外周血单个核细胞(PBMC)分泌干扰素γ(IFN-γ)、白细胞介素4(IL-4)的影响。方法随机将20例肺结核病患者和15例健康对照者PBMC按加入刺激物的不同分为RPMI-1640组、PPD组、PPD IL-7组、PPD IL-2组,PPD IL-7 IL-2组,培养72小时后收集上清液,采用酶联免疫吸附法(ELISA)检测各组培养上清液中IFN-γ、IL-4的水平。结果与PPD组相比,加入IL-7或IL-2均能有效促进肺结核患者及健康对照者PBMC分泌IFN-γ(P<0.05),IL-7可抑制IL-4的合成(P<0．05);同时加入IL-7和 IL-2可显著增加肺结核病患者及健康对照者PBMC分泌IFN-γ(P<0．01),抑制IL-4的合成(P< 0．05)。结论IL-7和IL-2协同可显著促进IFN-γ分泌,抑制IL-4合成,从而调节Th1／Th2平衡,对结核分枝杆菌感染患者发挥保护性的免疫反应。     

Regulation of microglial cell function by ATP]

Accumulated evidence suggests that extracellular ATP functions occur in neurons and glial cells in the nervous systems. Besides well-documented roles as a neurotransmitter or neuromodulator, ATP has also been demonstrated to have effects on glial cells. Reports have shown that ATP stimulates microglia to release various biologically active substances, such as interleukin-1 beta, tumor necrosis factor-alpha, and plasminogen. Microglial cell death was also demonstrated after stimulation with high-dose ATP. Although these responses were known to occur, via P2X7, we have recently found that ATP and ADP induced the formation of membrane ruffles and chemotaxis through Gi/o-coupled P2Y receptors. Taken together, it is suggested that two distinct P2X and P2Y receptor subtypes are involved in the diverse function of microglia in both physiological and pathological states.     

Long-term methylglyoxal intake aggravates murine Th2-mediated airway eosinophil infiltration

Asthma outcomes is aggravated in obese patients. Excess of methylglyoxal (MGO) in obese/diabetic patients has been associated with diverse detrimental effects on cell function. This study aimed to evaluate the effects of long-term oral intake of MGO on ovalbumin-induced eosinophil inflammation. Male C57/Bl6 mice received 0.5% MGO in the drinking water for 12 weeks. Mice were sensitized and challenged with ovalbumin (OVA), and at 48 h thereafter, bronchoalveolar lavage (BAL) fluid and lungs were collected for cell counting, morphological analysis, and ELISA, mRNA expressions and DHE assays. In MGO-treated mice, OVA challenge significantly increased the peribronchiolar infiltrations of inflammatory cells and eosinophils compared with control group. Higher levels of IL-4, IL-5, and eotaxin in BAL fluid were also detected in MGO compared with control group. In addition, lung tissue of MGO-treated mice displayed significant increases in mRNA expressions of NF-κB and iNOS whereas COX-2 expression remained unchanged. The high TNF-α mRNA expression observed in lungs of OVA-challenged control mice was not further increased by MGO treatment. In MGO group, OVA-challenge increased significantly the NOX-2 and NOX-4 mRNA expressions, without affecting the NOX-1 expression. Levels of reactive-oxygen species (ROS) were significantly higher in lungs of MGO-treated mice, and no further increase by OVA-challenge was observed. In conclusion, 12-week intake of MGO exacerbates Th2-mediated airway eosinophil infiltration by activation of NF-kB/iNOS-dependent signaling pathway and positive regulation of NOX-2 and NOX-4 in the lung tissues. Scavengers of MGO could be an option to prevent obesity-related asthma.     

Orally bioavailable allosteric CCR8 antagonists inhibit dendritic cell, T cell and eosinophil migration

The chemokine receptor CCR8 is associated with asthma. Herein, we describe that both mature and immature dendritic cells (DC) express CCR8, whereas only mature DC migrate towards CCL1. Moreover, transient LPS challenge significantly down-regulates CCR8 expression hence attenuating CCL1 chemotaxis. To inhibit CCR8 pathophysiology, we recently developed a novel series of small molecule CCR8 antagonists containing a diazaspiroundecane scaffold, which had micromolar potency. However, these first generation antagonists had high lipophilicity that endowed the compounds with poor physicochemical properties, and were thus not suitable for further development. By introducing polar bicyclic groups on the N-benzyl substituent and building in further polar interactions on the amide group we now show second generation diazospiroundecane antagonists with significantly improved overall properties. Potency is substantially improved from micromolar to nanomolar potency in CCR8 binding and inhibition of chemotaxis in human primary T cells, DC and in an eosinophil cell line. In addition to high potency, the most attractive antagonist, AZ084 showed excellent selectivity, high metabolic stability in vitro and an attractive in vivo PK profile with a long half-life in rat. Interestingly, in ligand saturation experiments and in wash-off experiments, CCL1 was shown to have two binding sites to CCR8 with K(d) at 1.2/68pM respectively, and on-off rates of 0.004 and 0.0035/0.02pMmin, respectively. The lead antagonist, AZ084, appears to act as an allosteric inhibitor with a K(i) at 0.9nM. Taken together, we herein report a novel oral allosteric CCR8 antagonist with predicted low once-daily dosing capable of potent inhibition of both human T cell and DC functions.     

Regulation of immune cell homeostasis and function by coronin 1

Coronin 1 is the most recent candidate in the list of genes causing severe combined immunodeficiency (SCID) in humans. A distinctive feature of the SCID induced by coronin 1 deficiency is selective naïve T cell lymphopenia in the presence of a normal thymus as well as normal B cell and natural killer cell numbers (T⁻B⁺NK⁺). Coronin 1 is a member of the evolutionarily conserved coronin protein family, members of which are widely expressed across the eukaryotic kingdom. Mammals express seven coronin molecules, numbered from coronin 1 to 7. The different coronin proteins have a distinct but overlapping tissue expression and have been reported to be involved in a wide array of cellular functions including calcium homeostasis, cytoskeletal dynamics, immune and inflammatory responses, neuromuscular transmission as well as cognition and behavior. In this minireview, we describe the role of coronin 1 in the maintenance of immune cell diversity and function.     

丹地化瘀汤对哮喘急性发作患儿肺功能及Th1/Th2细胞功能调节作用的影响

目的探讨丹地化瘀汤对哮喘急性发作患儿肺功能及Th1/Th2细胞功能调节作用的影响。方法 2013年1月至2014年1月选取在本院收治的120例哮喘急性发作期患儿为研究对象,根据随机数字表将患儿分为观察组及对照组各60例,对照组给予舒利迭雾化吸入治疗,50 mg/250 mg,2次·d-1,观察组在对照组基础上给予自拟地化瘀汤口服治疗,2组均持续治疗2周。对比分析2组患儿治疗效果及治疗前后肺功能及免疫功能的变化。结果观察组总有效率为96.67%,对照组总有效率为76.67%,差异有统计学意义(P<0.05)。观察组患儿治疗后第1 s呼吸气体的容积(FEV1)、用力呼吸的肺活量(FVC)及FVC占预计值百分比(FVC%)改善优于对照组(P<0.05)。观察组治疗后白细胞介素4(IL-4)、免疫球蛋白(Ig E)、干扰素(IFN-γ)和IL-4/INF-γ则显著低于对照组(P<0.05)。结论丹地化瘀汤能有效改善哮喘急性发作期患儿肺功能及免疫功能,提高哮喘患儿治疗效果。     

Regulation of human B cell function by sulfasalazine and its metabolites

Although sulfasalazine is a well-known disease-modifying antirheumatic drug (DMARD), the mechanisms of its action remain unclear. Indeed, it remains uncertain whether sulfasalazine itself or one of its metabolites is responsible for the antirheumatic effects of sulfasalazine. Since one of the characteristic features of rheumatoid arthritis (RA) is chronic stimulation of B cells, we compared the effects of sulfasalazine and its metabolites on the in vitro function of human B cells. Ig production was induced from highly purified B cells from healthy donors by stimulation with Staphylococcus aureus Cowan I (SA) plus IL-2. Sulfasalazine suppressed the production of IgM and IgG at its pharmacologically attainable concentrations (1-10 microg/ml). Of the metabolites of sulfasalazine, sulfapyridine (SP) and 5-aminosalicylic acid (5-ASA), but not 4-acethyl SP, also significantly suppressed the production of IgM and IgG at their pharmacologically relevant concentrations. By contrast, any of sulfasalazine, SP, 5-ASA and 4-acethyl SP did not suppress the IFN-gamma production of immobilized anti-CD3 stimulated CD4+ T cells. These results indicate that sulfasalazine and its metabolites preferentially suppress the function of B cells, but not that of T cells, at their pharmacologically attainable concentrations. The data therefore suggest that not only sulfasalazine, but its metabolites, might contribute to the beneficial effects of sulfasalazine.     

Th17细胞与白介素17在鼻息肉中的研究进展

白介素17是Th17细胞产生的炎性细胞因子,研究发现IL-17在鼻息肉组织中明显增多,且可能参与了鼻息肉形成过程中的黏膜重建并促进了血管生长因子的高表达,但其机制尚未明确。本文参阅近几年相关文献,就Th17细胞的分化及其产生的IL-17在鼻息肉中的作用做一综述。     

Murine myeloid progenitor responses to GM-CSF and eosinophil precursor responses to IL-5 represent distinct targets for downmodulation by prostaglandin E(2)

1. Because Prostaglandin E(2) (PGE(2)) and dibutiryl cyclic AMP (dbcAMP) modulate the production and effects of haemopoietic cytokines in allergy, we examined their ability to modulate responses of myeloid progenitors to GM-CSF, and of eosinophil precursors to IL-5. 2. The ability of PGE(2), dbcAMP, rolipram, forskolin, dbcGMP and PGD(2), to modulate the responses to GM-CSF and IL-5 in colony formation (progenitor) and eosinophil differentiation (precursor) assays using bone-marrow from nonsensitized or from intranasally-challenged, ovalbumin-sensitized mice of five strains was studied. 3. PGE(2) (10(-7) M) inhibited GM-CSF-stimulated colony formation in bone-marrow from BP-2 mice. This effect was duplicated by dbcAMP (0.3 - 1x10(-6) M), Rolipram (10(-5) M) and forskolin (3x10(-5) M), but not Prostaglandin D(2) (10(-6) M). Inhibition affected similarly all myeloid colony types. Progenitors from sensitized and challenged BP-2 mice were also inhibited by PGE(2) and cyclic AMP. PGE(2) inhibited progenitors from C57BL/10, CBA/J and A/J, but not BALB/c mice. However, BALB/c progenitors were sensitive to dbcAMP and Forskolin (10(-4) M). In contrast, in precursor assays, PGE(2) (10(-7) - 10(-9) M) blocked responses to IL-5 in bone-marrow from BP-2 and BALB/c mice, either na?ve or sensitized and challenged, to a similar extent. PGD(2) (10(-6) M) was ineffective, as was PGE(2) (10(-7) M), if added after 48 h of culture. 4. In conclusion, PGE(2) inhibits the responses of bone-marrow myeloid progenitors to GM-CSF and of eosinophil precursors to IL-5, in na?ve or ovalbumin sensitized and challenged mice. These effects are duplicated by cyclic AMP-elevating agents. In the BALB/c strain, the resistance of progenitors, but not precursors, to PGE(2) inhibition, indicates these developmental stages are separate targets for PGE(2) modulation.