Effects of TNF inhibitor on innate inflammatory and Th17 cytokines in stimulated whole blood from rheumatoid arthritis patients

Background

Recent studies point to important roles for IL-17 and Th17 cells in sustaining chronic inflammation and articular destruction in rheumatoid arthritis (RA). We investigated the effects of TNF inhibitor on innate inflammatory and Th17 cytokines production by ex vivo lipopolysaccharide (LPS)-stimulated whole blood in patients with RA and the associations of cytokine levels in whole blood cultures with autoantibodies and markers of disease activity.

Materials and methods

Whole blood cultures from 18 healthy volunteers and 19 RA patients on etanercept therapy were stimulated with LPS and the production of IL-6, TNF-??, IL-23, IL-17A and IL-21 was measured by ELISA.

Results

After stimulation with LPS, the interleukin (IL)-17A (p?=?0.020) and IL-21 (p?=?0.0001) secretions were significantly higher in patients with RA than in controls, while the TNF-?? (p?=?0.002) was significantly lower at baseline. Etanercept significantly decreased IL-21 production (p?=?0.007), while IL-6 production (p?=?0.005) significantly increased after 6?months of therapy. IL-21 significantly correlated with RF (r?=?0.917, p?<?0.01) and antimutated citrullinated vimentin antibodies (r?=?0.770, p?<?0.01) at baseline. Logistic regression analysis revealed that baseline IL-21 levels (p?=?0.004) were significant predictors of DAS28-ESR at 6?months follow-up.

Discussion

Stimulation with LPS increased production of Th17 cytokines in whole blood cultures in patients with RA. Etanercept therapy decreased IL-21 secretion, while the capacity of whole blood cells to produce IL-6 increased. IL-21 production is strongly associated with the levels of autoantibodies. Our findings suggest that IL-21 production in LPS-stimulated whole blood cultures may be predictive of clinical response to etanercept treatment in patients with RA.     

Effect of antidepressants on melatonin metabolite in depressed patients

Antidepressants increase melatonin levels, but it is still unclear whether this effect is related to the improvement of depressive symptoms or to unrelated pharmacological action of antidepressants. To answer this question, the effect of antidepressants on 6-sulphatoxymelatonin (aMT6s), the main melatonin urinary metabolite, was examined in drug-free depressed patients - most of them antidepressant-naive. aMT6s was evaluated in 34 depressed patients, before and after 8 weeks of placebo (n = 12) or antidepressant (n = 22; fluoxetine, duloxetine or Hypericum perforatum). Both groups showed an improvement of depressive symptoms after treatment compared to baseline (Hamilton Depression scores): 17.0 +/- 1.4 vs. 9.0 +/- 2.8, P = 0.007 for placebo, and 18.6 +/- 1.1 vs. 11.8 +/- 1.6, P < 0.001 for antidepressants). After treatment, aMT6s levels increased after antidepressants (P < 0.01), but not after placebo (P > 0.05). As depressive symptoms improved both in patients taking antidepressant and in those taking placebo, but an effect of antidepressants could only be seen in those taking antidepressants, we suggest that melatonin changes after antidepressants are more likely due to a pharmacological action of these drugs on melatonin secretion.     

Effects of a selective cyclooxygenase-2 inhibitor, celecoxib, on bone resorption and osteoclastogenesis in vitro

The effects of an important new anti-inflammatory agent, the selective cyclooxygenase-2 inhibitor celecoxib, on bone resorption and osteoclastogenesis elicited by the inflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), the endotoxin lipopolysaccharide (LPS), and the systemic hormones 1alpha,25-dihydroxyvitamin D(3) and parathyroid hormone were examined in vitro. Bone resorption was evaluated by measuring calcium released into the culture medium in a neonatal mouse calvarial bone organ culture. Osteoclastogenesis was evaluated by measuring tartrate-resistant acid phosphatase activity in the cells in cocultures of bone marrow cells and osteoblastic cells and in macrophage-colony-stimulating factor-dependent bone marrow cell cultures. Celecoxib (0.1 microM) completely inhibited the calcium release induced by IL-1beta, TNF-alpha, and LPS. The resorptive effect of 1alpha,25-dihydroxyvitamin D(3) was inhibited partially by celecoxib. In contrast, celecoxib did not inhibit the calcium release elicited by parathyroid hormone or prostaglandin E(2). Celecoxib (0.1 microM) also markedly inhibited osteoclastogenesis induced by these stimulators of bone resorption except for PGE(2) in the coculture system, whereas it failed to inhibit osteoclastogenesis in macrophage-colony-stimulating factor-dependent bone marrow cell cultures. These results indicate that, under certain conditions, cyclooxygenase-2-dependent prostaglandin synthesis is critical for the bone resorption induced by IL-1beta, TNF-alpha, and LPS, and for the osteoclastogenesis induced by these pro-inflammatory molecules and calciotropic hormones. The prevention of prostaglandin synthesis by inflammatory cytokines in bone cells could contribute to the efficacy of celecoxib in preventing bone loss in rheumatoid arthritis.     

Effects of antidepressants on the production of cytokines

There is now evidence that major depression is associated with an up-regulation of the inflammatory response system (IRS). One of the major factors in this IRS activation is the hyperproduction of pro-inflammatory cytokines. Recently, a number of studies examined whether there is a causative role of these inflammatory mediators in the aetiology of major depression. Studies with animal models and cytokine immune therapy in humans suggest that pro-inflammatory cytokines induce depressive symptomatology. Moreover, these depressive symptoms can be effectively reversed by antidepressant treatment. Thus, it may be suggested that antidepressants suppress pro-inflammatory cytokine production and/or action, resulting in improvement of depressive symptoms. The influence of antidepressants on cytokine production has been examined in culture systems in vitro, and in animal models of depression - in which cytokine production is induced by endotoxin administration. Results suggest that antidepressants of several classes decrease the production of pro-inflammatory cytokines such as interferon-gamma and tumour necrosis factor-alpha, and increase that of interleukin-10, an anti-inflammatory cytokine. Further, the effect of antidepressive treatment on cytokine secretion and on plasma levels of cytokines in depressed patients has been studied. Unfortunately, different approaches to examine cytokine production and different techniques to measure cytokines in plasma are used in these studies. Despite this, current data indicate a normalization of cytokine plasma levels and cytokine production after antidepressant treatment. It is clear, however, that more research is warranted and we strongly argue the need for higher standardization in the methodology used to examine the cytokine network in depressed patients.     

Inhibitory effects of amantadine on the production of pro-inflammatory cytokines by stimulated in vitro human blood

Treatment with amantadine (AMA), an N-methyl-D-aspartate (NMDA) receptor antagonist and antidepressant drug, increased the antidepressant activity of subsequent drugs in experimental studies and in patients suffering from treatment-resistant depression (TRD). Recent evidence indicates that depression may be accompanied by activation of an inflammatory response. These data indicate that pro-inflammatory cytokines may play a role in the etiology of depression, particularly in TRD. The present in vitro study shows the ability of AMA, used at concentrations between 10^?7 to 10^?5 M, to reduce the production of the pro-inflammatory cytokines, specifically interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). In addition, AMA treatment increased the production of the negative immunoregulator, interleukin-10 (IL-10). Furthermore, the combined treatment of AMA with fluoxetine (FLU), but not imipramine (IMI), had a stronger immunomodulatory effect on cytokine production than AMA alone. The above data provide additional rationale for the treatment of patients suffering from depression with a combination of AMA and a selective serotonin reuptake inhibitor.     

Cognitive performance in depressed patients after chronic use of antidepressants

RATIONALE: Depressive disorders are conditions that often require continuous treatment, and it is therefore important to evaluate the consequences of prolonged administration. There are few studies assessing cognitive functions of depressed patients after long-term use of antidepressants. OBJECTIVES: This study evaluated the cognitive performance of depressed patients treated with antidepressants for at least 6 months. METHODS: Patients with major depression (DSM-IV) using imipramine for 2.4+/-0.6 years (mean+/-SE), clomipramine for 2.8+/-1.2 years, fluoxetine for 1.8+/-0.3 years and sertraline for 1.5+/-0.3 years were compared to matched controls (sex, age and educational level) without any psychiatric diagnosis. Memory evaluation consisted of episodic, implicit and working memory tests as well as metamemory assessment. RESULTS: (a) Psychomotor performance of patients taking imipramine was worse than that of controls in inserting pins and a visual reaction time task; on the performance of tapping the difference from controls varied according to dose/weight for patients taking clomipramine and fluoxetine. (b) For memory tests, differences between patients taking sertraline and controls were observed in the number of digits and words recalled; the difference between patients and controls varied according to dose/weight on the number of familiar words correctly completed for patients taking clomipramine and on digit span backward for those taking sertraline. (c) Metamemory was worse in all patient groups irrespective of patients' clinical state. CONCLUSIONS: The impairment in psychomotor and memory performances associated with these antidepressants seems to be of low intensity and of questionable clinical relevance.     

大黄素对体外诱导人血来源树突状细胞的影响

目的探讨大黄素对体外诱导培养的人血来源树突细胞(DC)的影响。方法分离健康人外周血单核细胞,经培养后获得未成熟DC(iDC),重组人粒-巨噬细胞集落刺激因子(rhGM-CSF,106IU.L-1)和重组人白细胞介素4(rhIL-4,8×105IU.L-1)诱导获得成熟DC(mDC)。实验分为iDC组、mDC组和大黄素组,mDC组于d 5加入脂多糖(LPS,1 mg.L-1)刺激,大黄素组采用mDC在d 5经LPS刺激后于d 7加入大黄素(100 mg.L-1)共培养2 d。用倒置显微镜和电镜观察DC细胞形态,用流式细胞仪检测DC表面共刺激分子的表达水平。结果经rhGM-CSF、rhIL-4诱导和LPS刺激,培养9 d后可获得表面有丰富分叉状胞浆突起的毛刺状mDC。大黄素组DC表面突起短而少,呈iDC形态。大黄素组CD80、CD83和CD86表达率分别为(13.4±6.6)%、(9.3±2.2)%和(84.2±6.3)%,低于mDC组[分别为(39.3±8.6)%、(30.7±5.6)%和(95.4±3.2)%,P<0.01];大黄素组CD14表达率为(8.4±2.8)%显著高于mDC组的(3.7±2.3)%(P<0.01)。大黄素组HLA-DR及CD11c表达与mDC组比较无显著差异(P>0.05)。结论大黄素能干扰DC表面突起的形成和表面共刺激分子的表达。     

Effects of two antidepressants on memory performance in depressed outpatients: a double-blind study

Forty outpatients with primary depression were randomly assigned on a double-blind basis to treatment with amitriptyline (a tricyclic antidepressant) or clovoxamine (a nontricyclic, experimental antidepressant). Memory and depression were assessed during a pretreatment baseline period and at the end of days 4, 7, and 28 of drug treatment. A signal detection recognition memory task and conventional memory measures (including the Benton Visual Retention, Wechsler Logical Memory, and verbal learning tests) were used to assess memory.Although both drugs led to comparable clinical improvement in depression, they affected memory performance differently. The signal detection recognition memory task detected an impairment in memory after chronic amitriptyline administration, as contrasted with an improvement in memory after chronic administration of clovoxamine. The memory impairment in the amitriptyline group and improvement in the clovoxamine group were the result of changes in sensitivity [P(A)]. No changes in response bias (BO were detected. Conventional memory tests failed to detect drug-related differences in memory between the two groups. On the Benton, errors decreased over time within both drug treatment groups, whereas correct reproductions increased within the amitriptyline group only. However, between-group differences on the Benton did not reach significance.Results from the signal detection task suggest an amitriptyline-associated memory impairment. However, this interpretation is tempered by the finding that conventional memory measures failed to detect differences in memory performance between the two groups. We discuss the limitations of traditional memory measures and the utility of a signal detection approach in studies of psychopharmacologic influences on memory.     

Effects of antidepressants on isolated,electrically stimulated rat epididymal fat pads

The isolated, electrically stimulated rat epididymal fat pad in vitro was tested under various conditions as a model for neuronally induced lipolysis. A 24-hr fast, or a preincubation without electrical stimulation, decreased spontaneous lipolysis as measured by glycerol release; however, only slight alterations were seen in the response of the pads during a 40-min period of electrical stimulation. Spontaneous (nonstimulated) lipolysis was reduced by pretreatment with alpha-methyl-paratyrosine-methylester and increased by pretreatment with reserpine; both of these treatments abolished the response to stimulation. The effects of addition of a variety of agents to the incubation medium confirm the role of a sympathetic nervous-cyclic 3′,5′-adenosine-monphosphate mediated response system.     

Effects of nimesulide,a cyclooxygenase-2 selective inhibitor,on colitis induced tumors

Cyclooxygenase-2 (COX-2) is known to suppress sporadic colorectal cancer, but effect of selective COX-2 inhibitor in UC-associated neoplasia is still unknown. This study investigated effect of a selective COX-2 inhibitor on colorectal carcinogenesis in experimental murine UC. Chronic colitis was induced in mice by four cycles of administration of dextran sulfate sodium (DSS) (i. e., 5 % DSS for 7 days and distilled water for the following 14 days), and the mice were sacrificed 120 days after the end of the fourth cycle. The mice were divided into the following five groups: Group A, served as a disease control; Group B, received a diet mixed with 400 ppm of nimesulide (NIM), a selective COX-2 inhibitor, during the whole period; Group C, received NIM during the four cycles of DSS administration; Group D, received NIM for 120 days from the end of the fourth cycle; Group E, served as a normal control. In Group D, NIM significantly suppressed the occurrence of dysplasia and/or cancer. The results show that NIM inhibited both dysplasia and cancer in DSS-treated mice, thus showing that NIM has preventive effects on the remission phase of carcinogenesis. Received 20 July 2006; revised 21 August 2006; accepted 4 September 2006     

Effects of trazodone treatment on alpha-2 adrenoceptor function in depressed patients

Tricyclic antidepressant drugs reduce the sensitivity of ₂ adrenoceptors during long-term treatment. In the present study, the ₂ adrenergic agonist clonidine was administered to 11 depressed patients before and during treatment with the triazolopyridine antidepressant trazodone (TRZ). Clonidine's ability to decrease blood pressure (BP) and plasma levels of the norepinephrine metabolite 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG), and to increase sedation and plasma growth hormone (GH), were measured. TRZ had little effect on these indices of pre- and postsynaptic ₂ receptor function, suggesting that the antidepressant properties of TRZ are not related to changes in ₂ adrenoceptor sensitivity.     

Development of a discriminating in vitro dissolution method for a poorly soluble NO-donating selective cyclooxygenase-2 inhibitor

A discriminating dissolution method using a USP apparatus 2 dissolution tester was developed for a nitric oxide donating selective COX-2 inhibitor to support phase I and II formulation development, clinical supplies release and stability testing of an immediate release oral tablet. The BCS class II compound showed very low aqueous solubility and required the use of surfactant-containing (sodium lauryl sulfate (SLS)) dissolution medium in order to achieve an appropriate release profile. The dissolution method utilized 900 mL of 2% SLS (w/v). Samples were withdrawn at five specified time-points over 60 min, at a paddle speed of 75 rpm. Analysis of samples was performed using a validated HPLC method. Despite the use of high levels of SLS, the ability to discriminate variations in physical properties such as drug particle size, granule particle size and tablet compression force was demonstrated. In order to confirm the relationship between these physical parameters and the tablet in vivo release profile, oral dosing of the formulations in fasted beagle dogs was performed to determine if the changes observed in the dissolution profiles were biorelevant. The results of the dissolution and corresponding in vivo experiments helped identify the critical processing parameters likely to influence product bioavailability.     

Serum levels and cardiovascular effects of tricyclic antidepressants and selective serotonin reuptake inhibitors in depressed patients

Tricyclic antidepressants (TCAs) and selective serotonin reuptake inhibitors (SSRIs) are used to treat depression. Whereas cardiovascular effects have occasionally been reported during controlled studies with SSRIs, TCA treatment poses a well-known problem in this respect. To investigate the putative correlation between antidepressant dose or serum levels and adverse effects, the authors devised a naturalistic study to evaluate the tricyclic antidepressants' and SSRIs' effect on the cardiovascular system. The authors also compared antidepressant serum levels to adverse effects. Inpatients treated with TCAs or SSRIs were included; an electrocardiogram (ECG) and a Schellong test were carried out on the day patients entered the hospital and during steady-state treatment with antidepressant drugs when blood was drawn for therapeutic drug monitoring. The patient population consisted of 114 acutely depressed patients; 81 patients were treated with TCAs and 33 with SSRIs. The TCAs comprised amitriptyline (n = 43), clomipramine (n = 11), doxepin (n = 19) and imipramine (n = 8); the SSRIs comprised fluvoxamine (n = 14) and paroxetine (n = 19). In TCA-treated patients, the authors observed the same type of abnormalities in conduction and orthostatic hypotension as had been observed earlier. The authors also observed cases of first-degree atrioventricular block, prolonged QTc interval, and orthostatic hypotension in SSRI-treated patients. Thus SSRIs also appear to affect the cardiovascular system, which might pose a problem for patients with preexisting conduction disease. The authors observed a strong correlation between the decrease in systolic pressure and antidepressant serum concentration (except for clomipramine and paroxetine), suggesting that antidepressant serum level is a better correlate than dose.     

脐血输注对再生障碍性贫血患者细胞因子的影响

目的探讨再生障碍性贫血(AA)患者脐血输注治疗前后细胞因子水平的变化。方法用ELISA法检测脐血输注前后20例AA患者骨髓及外周血中白细胞介素(IL)2、IL-3I、L-11、促血小板生成素(TPO)、干扰素γ(IFN-γ)水平变化。结果与输注前比较,脐血输注后AA患者外周血中IL-2、IFN-γ水平降低[(127.40±79.83)pg/ml vs.(90.77±4.86)pg/ml、(9.86±5.35)pg/ml vs.(4.48±3.35)pg/ml](P<0.05),IL-3水平升高[(99.13±55.31)pg/ml vs.(141.22±33.13)pg/ml)](P<0.05);脐血输注后,AA患者骨髓IL-2水平明显降低[(40.20±17.40)pg/ml vs.(5.87±0.96)pg/ml](P<0.05)。结论脐血输注可能通过改变造血环境中细胞因子的浓度或比例改善AA患者的造血功能。     

加味三棱丸含药血清对子宫内膜异位症内膜细胞芳香化酶及环氧合酶-2的影响

目的:观察子宫肌瘤与子宫内膜异位症(内异症)患者在位内膜芳香化酶(aromatase)和环氧合酶-2(cyclooxygenase-2,COX-2)表达上的差异,探讨加味三棱丸(SLW)含药血清对内异症在位内膜芳香化酶、COX-2表达的影响.方法:Westernblot和RT-PCR方法分别检测子宫肌瘤组、给予SLW含药血清前后内异症组芳香化酶、COX-2蛋白和mRNA的表达,发光免疫分析法及放射免疫分析法分别检测上述各组细胞上清中雌二醇及前列腺素E2的含量.结果:以未给药的内异症内膜组为对照,子宫肌瘤组芳香化酶、COX-2 mRNA蛋白的表达,分泌雌二醇及前列腺素E2水平均明显低于对照组,差异有显著性(P<0.05).SLW 5.0,2.5 g·kgll·d-1组芳香化酶、COX-2蛋白和mRNA的表达低于对照组,差异有显著性或极显著性(P<0.05或P<0.01).SLW 1.25 g·kg-1·d-1组芳香化酶mRNA和蛋白、COX-2蛋白的表达也明显低于对照组(P<0.01),但COX-2 mRNA的表达与对照组比较差异无显著性(P0.05).SLW 5.0,2.5,1.25 g·kg-1·d-1组分泌的雌二醇和前列腺素E2水平明显低于对照组(P<0.05).结论:SLW可抑制子宫内膜异位症在位内膜细胞芳香化酶和COX-2的表达,从而降低局部雌激素及前列腺素E2的水平.     

Pharmacological separation between peripheral and central functions of cyclooxygenase-2 with CIAA, a novel cyclooxygenase-2 inhibitor

There are many reports concerning the physiological and pathological involvement of cyclooxygenase (COX)-2 in the central nervous system and peripheral tissue cells. Selective COX-2 inhibitors that mainly distribute peripherally have not been reported thus far. Therefore central and peripheral roles of COX-2 remain classified pharmacologically. In this study, in vivo pharmacological profiles of CIAA ([6-chloro-2-(4-chlorobenzoyl)-1H-indol-3-yl]acetic acid), a novel selective COX-2 inhibitor which distributes at higher concentrations in plasma than in brain, were compared with those of well-known selective COX-2 inhibitors, celecoxib and rofecoxib. Additionally, the possibility of pharmacological separation between peripheral and central actions of COX-2 with the inhibitors was investigated. CIAA selectively inhibited COX-2 activity compared with COX-1 in in vitro assays with rat whole blood. The compound exhibited lower brain penetration and higher plasma concentration (the brain/plasma concentration ratio was approximately 0.02) than celecoxib and rofecoxib after oral administration. Therefore, CIAA is mainly expected to act peripherally. Edema and prostaglandin E2 (PGE2) production in Carrageenan-injected rat paws, and pyrexia and PGE2 production in the brain in lipopolysaccharide (LPS)-injected rats were measured in in vivo experiments. CIAA exhibited lower ratios of anti-pyretic/anti-edematous activities and of inhibitory activities of PGE2 production in brain/paw than those of celecoxib and rofecoxib, and these ratios well-reflected brain/plasma concentration ratios. In conclusion, we discovered a novel selective COX-2 inhibitor, CIAA, which distributes at higher concentrations in plasma than in brain, which would make possible the pharmacological separation of the peripheral and central functions of COX-2.     

Reduction of whole blood serotonin in depressed patients treated with a new,selective serotonin-uptake inhibitor,femoxetine

Femoxetine, a phenylpiperidine derivative with potent serotonin (5-HT)-uptake inhibitory properties, was investigated in 12 depressive patients for its clinical effect as well as its effect on 5-HT concentration in whole blood. The global evaluation after 6 weeks showed a good effect in six patients. The 5-HT concentration was found to be reduced from a mean value of 0.21 to about 0.05 g/ml, indicating a total depletion of 5-HT from the thrombocytes. The steady-state concentrations of femoxetine resulted in reduction of 5-HT to the same level in all patients. No correlation between degree of 5-HT reduction and therapeutic effect was found.     

Localization of cyclooxygenase-2 in mice vas deferens and its effects on fertility upon suppression using nimesulide: a preferential cyclooxygenase-2 inhibitor

Accumulating evidence on constitutive expression of cyclooxygenase-2 (COX-2), one of the isoforms of enzyme cyclooxygenase (COX) the other isoform being cyclooxygenase-1 (COX-1), questions the safety profile of non-steroidal anti-inflammatory drugs (NSAIDs). This COX-2 isoform which is induced not only during inflammation but also by factors such as cytokines, steroid hormones and mitogenic stimuli is constitutively expressed in brain, kidney and reproductive organs. Present NSAIDs, particularly COX-2 inhibitors is no longer considered safe since suppression of COX-2 in tissues which it is constitutively expressed may lead to adverse effects. Though intense expression of COX-2 in vas deferens is proved, lack of information with respect to its function has attracted a wide scope for research as to whether COX-2 in vas deferens contributes to male fertility. In the present study, the authors investigated the localization of COX-2 as well as COX-1 in mice vas deferens and also assessed the activity of COX-2 and total prostaglandin (PG) levels in vas deferens. Further they suppressed the expression of COX-2 using a preferential COX-2 inhibitor nimesulide and analyzed the sperm from vas deferens for any defects. COX-2 was intensely expressed in the epithelial cells of mice vas deferens and nimesulide was able to effectively suppress most of COX-2 expression. A decrease in PG levels was observed initially but interestingly, the levels tend to rise on sustained suppression of COX-2. The motility of sperm was affected severely after 6h of nimesulide administration that suggested a crucial role of COX-2 towards fertility of mice sperm.     

细胞因子IL-4对小鼠骨髓来源树突状细胞培养的影响

目的:建立从小鼠骨髓中分离、纯化、培养、扩增树突状细胞(DC)前体的方法,研究细胞因子IL-4对小鼠DC体外增殖、分化成熟的影响.方法:将健康小鼠股骨骨髓细胞分离,去除悬浮细胞,将贴壁细胞分为2组,第1组加入重组细胞/巨噬细胞集落刺激因子(GM-CSF)培养;第2组加入GM-CSF和白细胞介素-4(IL-4)联合培养.诱导的DC经相差显微镜观察.结果:分离的DC前体经8～9d的培养,镜下随机取8个视野进行细胞计数,第1组细胞数量是85.38±3.42;第2组细胞数量是119.25±3.96,两组间DC数量有显著差异(t=3.19,P<0.05).且细胞纯度达90%以上,具有典型的树枝状或裙褶状突起.结论:用GM-CSF和IL- 4联合作用更能促进DC的体外扩增及分化.     

Suppressive effect of selective cyclooxygenase-2 inhibitor on cytokine release in human neutrophils

To clarify whether a selective cyclooxygenase-2 (COX-2) inhibitor can affect various functions in human peripheral blood neutrophils. For this purpose, the effects of selective COX-2 inhibitors, NS-398 and nimesulide, on the expression of COX-2, PGE2 release and respiratory burst, degranulation and cytokine release in activated neutrophils were examined. Peripheral blood neutrophils were stimulated with formyl-methionyl-leucyl-phenylalanine (FMLP; 100 nM) or opsonized zymosan (OZ; 200 microg/ml). Then, the expression of COX-2 at protein and mRNA levels was detected by Western blot analysis and RT-PCR. The concentration of prostaglandin E2 (PGE2) and cytokines in the culture supernatant of neutrophils was determined using ELISA. Superoxide generation was measured by the cytochrome c reduction method. Elastase activity was measured using a chromogenic substrate assay specific for human neutrophil elastase. FMLP and OZ enhanced PGE2 release through induction of COX-2 protein and mRNA expression. FMLP- or OZ-induced PGE2 release was abolished by the addition of NS-398 or nimesulide; nevertheless, even a high concentration of COX-2 inhibitor did not change FMLP- or OZ-induced expression of COX-2 at message and protein levels. Although FMLP- or OZ-induced superoxide generation and elastase release were not affected by the addition of COX-2 inhibitor, cytokine release such as interleukin (IL)-1beta, IL-6 and IL-8 was significantly inhibited by high concentration of COX-2 inhibitor, but tumor necrosis factor-alpha (TNF-alpha) was partially attenuated. These studies showed that selective COX-2 inhibitors, NS-398 and nimesulide, suppressed PGE2 and proinflammatory cytokine release in activated neutrophils. These results suggest that selective COX-2 inhibitors may contribute to resolution of acute inflammation through the reduction of inflammatory cytokine release in activated neutrophils.