Intracellular cytokine production by fetal and adult monocytes

BACKGROUND/PURPOSE: In light of the neonate's increased susceptibility to systemic infection, the authors hypothesized that adult and fetal monocytes have different cytokine expression profiles in response to lipopolysaccharide (LPS), and interleukin (IL)-11, a counter-inflammatory cytokine. METHODS: Samples of cord blood (n = 30) and adult blood (n = 30) were obtained and treated as follows: control (baseline expression), LPS exposure, and IL-11 or IL-11+LPS exposure. After incubation with a protein transport inhibitor, mononuclear cells were stained for intracellular tumor necrosis factor (TNF)-alpha, IL-1beta, IL-6, and IL-8. Each sample was then analyzed by flow cytometry for cytokine expression. Cytokine production was measured by the percent positive as well as the fluorescence index for each cytokine. Analysis of variance (ANOVA) and Students t tests were used for statistical analysis. RESULTS: Baseline levels of IL-8 were significantly higher for fetal monocytes (P <.0001). After LPS exposure, fetal monocytes produced less TNF-alpha (P =.0105) and more IL-8 (P <.0007) relative to adult cells. IL-11 treatment reduced baseline production of IL-8 in fetal and adult monocytes (P <.05). CONCLUSIONS: These results suggest that neonatal monocytes portray a different cytokine expression profile compared with adult monocytes. IL-11 treatment appears to alter the IL-8 expression of resting fetal and adult monocytes.     

Enhanced expression of intracellular heme oxygenase-1 in deactivated monocytes from patients with severe systemic inflammatory response syndrome

BACKGROUND: Monocyte deactivation is an important contributor to infectious susceptibility in critically ill patients. However, the mechanism of monocyte deactivation has not been fully elucidated. Recently, intracellular heme oxygenese-1 (HO-1), an anti-inflammatory heat-shock protein, was reported to be activated by Toll-like receptors (TLRs), and to inhibit inflammatory cytokine production such as that of TNF-alpha. In the present study, we evaluated the expression of intracellular HO-1 and TLRs in monocytes from patients with severe systemic inflammatory response syndrome (SIRS) and examined the role of HO-1 in monocyte deactivation. PATIENTS: Twenty-seven patients who fulfilled the criteria for severe SIRS and had a serum C-reactive protein (CRP) level >10 mg/dL were included in this study. The cause of SIRS was sepsis in 16 patients, trauma in 7, and other in 4. Expression of intracellular HO-1, surface TLR2 and TLR4, and intracellular cytokines (TNF-alpha, Interleukin-6) stimulated via TLR activation were measured in circulating monocytes by flow cytometry. Intracellular HO-1 expression was evaluated in normal monocytes stimulated with patient serum. Serum cytokine levels were also measured. Patient data were compared with data from healthy volunteers (n = 16). RESULTS: Cytoplasmic HO-1 was clearly detected by fluorescence microscopy. Expression of HO-1, TLR2, and TLR4 in monocytes was significantly enhanced in patients with severe SIRS compared with that in healthy volunteers, whereas intracellular TNF-alpha expression with peptidoglycan was significantly decreased (p < 0.05) in patients compared with that in healthy volunteers. HO-1 expression was significantly enhanced in normal monocytes stimulated with patient serum. Intracellular HO-1 levels were positively related to serum TNF-alpha levels in patients (r = 0.46). CONCLUSIONS: Expression of intracellular HO-1 and of TLRs was enhanced in deactivated monocytes from patients with SIRS. Increased production of intracellular HO-1 in response to serum factors may play a role in monocyte deactivation after systemic inflammation.     

右美托咪啶对脂多糖诱导大鼠外周血单核细胞Toll样受体4mRNA表达的影响

目的 探讨右美托咪啶对脂多糖(LPS)诱导大鼠外周血单核细胞Toll样受体4(TLR4)mRNA表达的影响.方法 健康雄性Wistar大鼠40只,取外周血分离培养单核细胞,采用随机数字表法,将其随机分为5组(n=8),A组:阴性对照;B组:单核细胞中加入LPS(终浓度为1μg/ml);C组:单核细胞中加入LPS(终浓度为1μg/ml)+右美托咪啶(终浓度为0.5 ng/ml);D组:单核细胞中加入LPS(终浓度为1μg/ml)+右美托咪啶(终浓度为5.0 ng/ml);E组:单核细胞中加入LPS(终浓度为1μg/ml)+右美托咪啶(终浓度为50.0 ng/ml).孵育24 h后,收集上清液,采用ELISA法测定TNF-α、IL-1β和IL-6的浓度,采用RT-PCR法测定TLR4 mRNA的表达.结果 与A组比较,B组TNF-α、IL-1p、IL-6的浓度升高,TLR4 mRNA表达上调(P＜0.01);与B组比较,C组、D组和E组TNF-α、IL-1β、IL-6的浓度降低,TLR4 mRNA表达下调(P＜0.05或0.01);与C组比较,D组和E组TNF-α、IL-1β、IL-6的浓度降低(P＜0.01),TLR4 mRNA表达差异无统计学意义(P＞0.05);D组和E组各指标比较差异无统计学意义(P＞0.05).结论 右美托咪啶可通过下调TLR4 mRNA表达,抑制TLR4的合成,从而抑制LPS诱导大鼠外周血单核细胞TNF-α、IL-1β和IL-6的生成与释放.

Abstract：

Objective To investigate the effects of different concentrations of dexmedetomidine on the expression of Toll-like receptor 4 (TLR4) mRNA in rat peripheral blood monocytes exposed to lipopolysaccharide ( LPS ). Methods Peripheral blood monocytes isolated from male Wistar rats were seeded in 24-well plate in RPMI 1640 liquid culture medium in CO2 incubator at 37 ℃ and 5% CO2 for 2 h, and were randomly divided into 5 groups ( n = 8 each): group A negative control; group B was exposed to LPS 1 μg/ml and C, D and E groups were exposed to LPS 1 μg/ml + dexmetomidine 0.5, 5.0 and 50.0 ng/ml respectively. The monocytes were then incubated for 24 h. The concentrations of TNF-α, IL-1β and IL-6 in the supernatant of the cultured monocytes were detected by ELISA. The expression of TLR4 mRNA in the monocytes was detected by RT-PCR.Results Exposure to LPS significantly increased the expression of TLR4 mRNA and the concentrations of TNF-α, IL-1β and IL -6 in group B as compared with group A ( P ＜ 0.01 ). Dexmedetomidine attenuated the LPS-induced increase in the expression of TLR 4 mRNA and the concentrations of TNF-α, IL-1β and IL-6 in a dose-dependent manner ( P ＜0.05or 0.01 ). Conclusion Dexmedetomidine can inhibit the synthesis of TLR4 and inhibit the secretion and dilivery of TNF-α, IL-1β and IL-6 by down-regulating the gene expression of TLR4 in rat peripheral blood monocytes exposed to LPS.     

Toll-like receptor 4 expression on circulating leucocytes in hemolytic uremic syndrome

Lipopolysaccharide stimulation of toll-like receptor 4 (TLR4) activates signal transduction pathways leading to proinflammatory cytokine secretion. We investigated TLR4 surface receptor expression on peripheral blood neutrophils and monocytes and their ability to modulate inflammatory cytokine release in 15 patients 1, 3, and 10 days after hemolytic uremic syndrome (HUS) onset. Seven patients with Escherichia coli (EHEC)-associated diarrhea and seven healthy controls were also studied. Isolated leucocytes from HUS-onset patients exhibited significantly higher messenger RNA (mRNA) TLR4 expression than controls. Moreover, TLR4 protein expression on neutrophils, determined by flow cytometry, was upregulated, driving dependent proinflammatory cytokine, tumor necrosis factor alpha (TNF-α), and interleukin 8 (IL-8) increase, and decreased anti-inflammatory IL-10 release at HUS onset compared with patients with EHEC diarrhea and controls. TLR4 expression on neutrophils was positively correlated with serum TNF-α levels. Conversely, significant reduction of neutrophil TLR4 receptor expression and lack of cytokine-responsive element activation was shown in patients 3 and 10 days after HUS onset. No differences were demonstrated in TLR4 receptor expression on monocytes among the studied groups. Our results suggest TLR4 expression may be differently regulated on neutrophils and monocytes. They could be dynamically modulated across the early development of HUS on neutrophils, resulting in negative regulation preceded by TLR4 overactivation.     

Innate immunity in hemodialysis and continuous ambulatory peritoneal dialysis patients: impaired cytokine synthetic response to ex-vivo stimuli in mononuclear cells

Dialysis patients are weak in immune host defense, which is associated with their high morbidity of infection. Polymorphonuclear leukocytes (PMNLs) and mononuclear cells play a key role in innate host defense. PMNLs and monocytes have bactericidal activity through the process of phagocytosis. Monocytes and lymphocytes contribute to the development of innate immunity by their cytokine actions. We studied the intracellular cytokine syntheses in response to ex-vivo stimuli, which may reflect the potential reactivity of immune cells in cytokine syntheses when pathogens invade humans. Furthermore, phagocytic activity was assessed in granulocytes and monocytes. Twenty HD, 15 CAPD, and 10 age-matched controls were enrolled in this study. One milliliter of whole blood from each subject was incubated with lipopolysaccharides (LPS) or mitogens for 4 hours at 37 degrees C. Monoclonal antibodies to CD14+ and CD4+ were used for identifying monocytes and helper T cells, respectively. Intracellular cytokines were stained using FASTIMMUNE staining kits. Interleukin-1beta and TNF-alpha syntheses were examined in monocytes, which are the most important early-response cytokines in innate immunity. IFN-gamma and IL-4 syntheses were examined in helper T cells to observe their polarization into Th1 and Th2 cells. IFN-gamma is a key factor in establishing innate immunity. The percentage of cells that stained positive for each cytokine was analyzed using a flow cytometer. The following results were obtained: 1) In CAPD patients, IL-1beta and TNF-alpha response to LPS in monocytes were significantly reduced, as compared to other subjects. Polarization of helper T cells was reduced, resulting in a significant decrease in Th1 cells. 2) In HD patients, monokine responses were not altered, but polarization of helper T cells was skewed toward a Th1 type. Phagocytic activities were not impaired in both dialysis groups. In conclusion, mononuclear cells from CAPD patients have the potential to exhibit failure of a cytokine response to ex-vivo stimuli in terms of innate immunity.     

Intracellular monocyte cytokine production and CD 14 expression are up-regulated in severe vs mild chronic heart failure.

BACKGROUND: The role of circulating monocytes in the process of low-grade inflammation, characteristic of chronic heart failure (CHF), has recently been questioned. Lipopolysaccharide (LPS) desensitization has been proposed to mediate reduced monocyte cytokine elaboration in patients with severe CHF. METHODS: Intracellular monocyte production of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumor necrosis factor (TNF)-alpha, and monocyte CD 14 expression were measured flow-cytometrically without and after 8-hour LPS stimulation in 46 patients with CHF and in a healthy control group. RESULTS: Basal cytokine concentrations were similar for the control and the mild CHF groups (New York Heart Association [NYHA] Class I or II). After LPS stimulation, IL-6 (p=0.002) and TNF-alpha levels (p=0.001) were lower in the latter group, whereas IL-1 beta production was comparable. For the moderate-severe CHF patients, unstimulated IL-1 beta (p=0.04) was higher, whereas IL-6 (p=0.2) and TNF-alpha (p=0.1) levels were not different from the controls. Measurement of LPS-stimulated cytokine production showed no differences between the control group and patients with moderate-severe CHF (all p= 0.5). Upon comparing mild vs moderate-severe CHF patients, higher levels of unstimulated cytokine production (IL-1 beta, p=0.002; IL-6, p=0.01; TNF-alpha, p=0.003), stimulated IL-1 beta (p=0.002) and IL-6 (p=0.008) were found in the latter patients. CD 14 expression in the moderate-severe CHF group was higher than in the mild-CHF group (p = 0.03) and was strongly related to stimulated IL-1 beta (r=0.62, p<0.0001), IL-6 (r=0.56, p=0.0002) and TNF-alpha (r=0.41, p=0.006) production. CONCLUSIONS: CD 14 expression and monocyte cytokine production, both unstimulated and after LPS stimulation, are increased in moderate-severe CHF when compared with mild CHF. These data suggest that circulating monocytes, possibly via increased CD 14 expression, may play a significant role in the immunologic dysbalance observed in advanced CHF.     

Impaired antiviral activity of monocytes from patients on hemodiafiltration

BACKGROUND: The aim of our study was to determine whether intermittent hemodiafiltration (HDF) leads to an alteration in monocyte antiviral activity as well as in the in vitro release of cytokines such as interleukin-12 (IL-12), tumor necrosis factor-alpha (TNF-alpha) and interferon-alpha (IFN-alpha) by the same cells. METHODS: We enrolled 25 patients undergoing HDF for 3.5-4 hours 3 times a week (12 men, 13 women; mean age 58 +/- 6.7 years) and 25 healthy donors (ND) (12 men, 13 women; mean age 57 +/- 8 years). Monocytes from peripheral blood mononuclear cells were isolated with a Monocyte Isolation Kit II. Monocytic cells were infected with herpes simplex virus type 2 (HSV-2). Cytokines were assayed in supernatants. RESULTS: The in vitro antiviral activity of monocytes from HDF patients was significantly impaired with respect to ND. Furthermore, monocytes from post-HDF patients were more prone to viral infection. Lipopolysaccharide (LPS) stimulation induced significant viral inhibition only in monocytes from NDs (p<0.05). The cytokine pattern (TNF-alpha, IFN-alpha and IL-12) in monocytes stimulated with LPS was markedly inhibited in HDF patients compared with ND (p<0.05). A basal production of TNF-alpha was found in monocytes from pre-HDF and post-HDF patients. No IFN-alpha production was found in LPS-stimulated and HSV-2-infected monocytes from pre-HDF and post-HDF patients. IL-12 production appeared significantly decreased after HDF in all experimental conditions (p<0.05). CONCLUSIONS: The significant increase of viral replication in monocytes from HDF patients compared with healthy donors could be related to a significant reduction of cytokine production. Moreover, the dialytic session influenced the intrinsic antiviral activity of monocytes, favoring viral replication.     

The effects of suplatast tosilate (IPD-1151T) on innate immunity and antigen-presenting cells

We previously demonstrated that the anti-allergic drug, suplatast tosilate (IPD-1151T), prolonged rat survival after heterotopic heart transplantation (HHT) and suppressed mixed lymphocyte reaction (MLR). In the present study, we investigated the effects of suplatast on T cells, lipopolysaccharides (LPS), or peptidoglycan (PGN)-stimulated cells and dendritic cells (DCs). The addition of suplatast to concanavalin A (ConA) blasts inhibited the proliferation of cells in which the gene expression of T-helper-1 (Th1) and T-helper-2 (Th2) cytokines including interferon (IFN)-gamma, interleukin (IL)-2, IL-4, and IL-10 were down-regulated with decreased concentration of the IFN-gamma and IL-10 in the supernatants of ConA blast cells. Suplatast also showed down-regulation of the toll-like receptor (TLR)2, TLR4, and CD14 gene expressions on splenocytes stimulated by LPS and PGN, TLR2 or TLR4 agonist, respectively. DCs treated with suplatast expressed lower levels of CD40, CD80, and CD86 and reduced IL-12 production. These results suggest that suplatast may modulate the TLRs on antigen-presenting cells (APCs) and thus block the pathway of Th1/Th2 cytokine production.     

The phenotypical and functional characteristics of cord blood monocytes and CD14(-/low)/CD16(+) dendritic cells can be relevant to the development of cellular immune responses after transplantation

Umbilical cord blood (UCB) has been used as an alternative source of haematopoietic progenitors for transplantation presenting advantages over bone marrow (BM) that are related with known shortages of newborns' immune system at adaptive and innate levels. Using flow cytometry, we studied the expression of Toll-like receptors (TLRs) and chemokine receptors (CKRs) and the production of pro-inflammatory cytokines by monocytes and CD14(-/low)/CD16(+)DCs from peripheral blood (PB; n=10), and umbilical cord blood (UCB; n=10). CKRs and cytokines were studied before and after stimulation of cells with LPS plus IFN-gamma. We also identified the two populations in normal bone marrow samples (BM; n=5). BM presented lower frequencies of both studied populations when compared to UCB and PB. CD14(-/low)/CD16(+)DCs presented a pattern of TLR expression different from mature monocytes reflecting distinct functions for these two populations. UCB cells presented reduced expression of TLR-4 and lower capability to produce cytokines prior stimulation. The populations studied presented different patterns of CKR expression reflecting distinct migratory pathways. Moreover, UCB cells presented higher expressions of CXCR4 and CCR7 that may be involved in immune system maturation and stem cell homing. Monocytes and CD14(-/low)/CD16(+)DCs present functional and phenotypical characteristics that may contribute to the lower incidence and severity of GVHD.     

Immunparalyse von T-Lymphocyten und Monocyten in der postoperativen abdominellen Sepsis

In vitro functions of stimulated peripheral T cells and monocytes were investigate in patients experiencing sepsis following major visceral surgery. Cell culture supernatants were analyzed by ELISA for IL-2, IFN-gamma, IL-4, IL-10, TNF-alpha, IL-1 beta, and IL-12p40. In addition, monocyte HLA class II expression was determined by flow cytometry. T cell secretion of IL-2, TNF-alpha, and in part IFN-gamma (but not IL-4) was significantly diminished in non-survivors throughout the entire course of sepsis, compared to controls and sepsis survivors. Production of IL-1 beta and IL-12 p40 by monocytes was strongly reduced in both survivors and non-survivors at the onset of sepsis. Persistence of depressed monocyte cytokine secretion correlated with lethality. Thus, overall suppression of cytokine production by T cells and monocytes was already observed at the beginning of postoperative sepsis. HLA class II expression by monocytes exhibited a strong and sustained down-regulation with no significant differences between sepsis survivors and non-survivors. In summary, suppression of both T cell and monocyte functions develops early during postoperative sepsis. Recovery of immune functions and severity of immune defects are associated with outcome.     

Toll样受体在强直性脊柱炎患者MSC中的作用研究

目的研究强直性脊柱炎(AS)患者间充质干细胞(MSC)中的Toll样受体表达及其对MSC免疫调节的调控作用。方法首先利用流式细胞分析的方法鉴定AS患者骨髓腔中分离培养MSC是否满足特定细胞表型;然后利用RT-PCR的方法检测AS患者MSC中的Toll样受体表达情况;接着分别应用q RT-PCR检测AS患者MSC中TLR3和TLR4的表达水平;最后分别用特定激动剂激活TLR3和TLR4后,利用ELISA检测AS患者MSC中IL-6的分泌情况。结果 AS患者MSC不表达CD34、CD45和HLA-DR,表达CD29、CD90和CD105;RT-PCR结果显示ASMSC表达TLR1、2、3、4、5、6、8,几乎不表达TLR7、9、10;q RT-PCR显示AS患者MSC中TLR3较正常人降低47.5%,TLR4较正常人降低了34.8%;TLR3和TLR4激活后,AS患者MSC中IL-6的分泌较前增加,分别增加了8.3倍13.8倍。结论 AS患者MSC中TLR3和TLR4表达和功能均异常,在AS患者MSC免疫调节中起重要的调控作用。     

Impaired monocyte cytokine production in critically ill patients with acute renal failure

BACKGROUND: Plasma levels of pro- and anti-inflammatory cytokines are predictive of mortality in patients with acute renal failure (ARF). Anti-inflammatory strategies are postulated to be beneficial in treatment. However, there are few studies simultaneously examining monocyte cytokine production and plasma cytokine levels in patients with ARF. METHODS: Study populations consisted of 20 critically ill patients with ARF, 19 critically ill patients without ARF (CRIT ILL), 28 healthy subjects (HS), 19 patients with chronic kidney disease (CKD), and 15 patients with end-stage renal disease (ESRD). Monocyte intracellular content of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-8, and tumor necrosis factor-alpha (TNF-alpha) was determined by flow cytometry in whole blood. Plasma interleukin 6 and TNF-alpha concentrations were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: At baseline, there were no differences in intracellular monocyte cytokine levels between groups. After lipopolysaccaride stimulation, monocyte production of IL-1beta, TNF-alpha, and IL-6 in ARF patients was reduced by 41%, 84%, and 45%, respectively, compared to healthy subjects (P < 0.01 in each case), and similarly reduced compared to CKD and ESRD patients, and were similar to CRIT ILL patients. Plasma IL-6 levels were significantly higher in ARF patients than healthy subjects, CKD, and ESRD patients (all P < 0.001). CONCLUSION: Critically ill patients with acute renal failure have impaired monocyte cytokine production and elevated plasma cytokine levels in a pattern that closely resembles critically ill patients without ARF, and that is dissimilar to CKD and ESRD patients.     

Reciprocal regulation of LTA(4) hydrolase expression in human monocytes by gamma-interferon and interleukins 4 and 13: potential relevance to leukotriene regulation in glomerular disease

BACKGROUND/AIMS: Leukotriene A(4) (LTA(4)) hydrolase catalyzes the final step in the synthesis of leukotriene B(4) (LTB(4)). TH-1- and TH-2-derived cytokines may regulate LTB(4) synthesis by monocytes through their actions on the expression of LTA(4) hydrolase. METHODS: Freshly isolated monocytes were incubated with pro- and anti-inflammatory cytokines for 36 h. mRNA expression was determined by Northern blot, protein expression was determined by Western blot and LTB(4) synthesis was determined by ELISA. RESULTS: Interferon-gamma (a TH-2-derived cytokine) increased significantly LTA(4) hydrolase mRNA expression, whereas interleukin (IL)-4 and IL-13 (both TH-2-derived cytokines) decreased LTA(4) hydrolase mRNA expression in these cells. The same effects were seen on the expression of immunoreactive LTA(4) hydrolase after incubating the monocytes with either TH-1- or TH-2-derived cytokines. The monocyte-derived cytokine IL-1 beta did not show any significant effect on LTA(4) hydrolase mRNA expression. When LTB(4) release was measured, both IL-1 beta and interferon-gamma significantly increased LTB(4) production by monocytes, while TH-2 cytokines (IL-4 and IL-13) decreased it. CONCLUSION: The opposing effects of TH-1- and TH-2-derived cytokines on the expression of LTA(4) hydrolase mRNA may regulate LTB(4) synthesis by monocytes during inflammation.     

Reduced secretion of proinflammatory cytokines of monosodium urate crystal-stimulated monocytes in chronic renal failure: an explanation for infrequent gout episodes in chronic renal failure patients?

BACKGROUND: In gouty arthritis, monosodium urate (MSU) crystals interact with monocytes and neutrophils to produce inflammatory reactions associated with acute synovitis. In patients with end-stage renal disease (ESRD), gouty arthritis is a rare condition despite often severe hyperuricaemia. We wondered whether differences in the secretion of proinflammatory cytokines by MSU crystal-stimulated monocytes might be one explanation for the low incidence of gouty arthritis in patients with ESRD compared with healthy controls. METHODS: Thirteen patients with ESRD on intermittent haemodialysis treatment, six patients with chronic renal failure not yet on dialysis, and 15 age- and sex-matched healthy controls were examined. Monocytes, purified from peripheral blood mononuclear cells (PBMC) by immunomagnetic bead separation, were incubated for 18 h in the presence of MSU crystals, Escherichia coli lipopolysaccharide (LPS) or medium alone. The supernatants were studied for the presence of interleukin (IL)-1beta, IL-6 and tumour necrosis factor-alpha (TNF-alpha) using cytokine-specific enzyme-linked immunosorbent assays. RESULTS: Monocytes from patients with ESRD produced significantly lower amounts of IL-1beta, IL-6 and TNF-alpha after stimulation with MSU crystals or LPS than did monocytes from healthy subjects. Cytokine production was not significantly different between ESRD patients on haemodialysis and chronic renal failure patients not yet on dialysis. Artificial MSU crystals were stronger stimuli than tophus-derived 'natural' MSU crystals. CONCLUSION: We demonstrate that monocyte-associated immunosuppression in ESRD leads to reduced secretion of proinflammatory cytokines in response to stimuli such as MSU crystals. This may be one of the factors preventing many ESRD patients from the manifestation of acute gout despite often severe hyperuricaemia.     

Toll样受体4信号对结肠癌细胞免疫抑制性细胞因子的调控

目的探讨Toll样受体（TLRs）对原位结肠癌细胞免疫抑制性细胞因子的调控作用及其机制。方法分别采用RT—PCR和蛋白印迹法对HT-29细胞中TLRs mRNA及蛋白质的表达进行检测。ELISA法检测经LPS刺激后及NF—κKB被抑制后,HT-29细胞所分泌的免疫抑制性细胞因子的改变。结果HT-29细胞可表达不同TLRs,以TLR4的表达为最高。经LPS刺激后,HT-29细胞中TLR4的mRNA和蛋白质水平,以及所分泌的转化生长因子（TGF）-β、VEGF、IL-8、CCL20和IL-6均显著升高（P〈0．01）。TGF—β、VEGF、IL-8和CCL20的上调表达不能被NF—κB抑制剂吡咯烷二硫代氨基甲酸盐（PDTC）所抑制,但IL-6的上调表达则依赖于NF—κB的活性。结论结肠癌细胞TLRs通过识别病原体相关模式分子．启动免疫抑制性细胞因子的表达．使肿瘤细胞逃避免疫监视。     

Whole blood production of monocytic cytokines (IL-1beta, IL-6, TNF-alpha, sIL-6R, IL-1Ra) in haemodialysed patients.

BACKGROUND: The production of monocytic cytokines by isolated mononuclear cells after stimulation by phytohaemagglutinin (PHA) and lipopolysaccharide (LPS) is generally increased in haemodialysed (HD) patients. We performed whole blood (WB) cultures to evaluate cytokine production by blood cells inside their complex cellular and humoral network. METHODS: Diluted whole blood from HD patients (collected before dialysis) and controls was cultured alone with PHA (2.5 microg/ml) or LPS (1 and 3 microg/ml). Supernatants were collected after 24 and 48 h of culture, and concentrations of IL-1 beta, IL-6, TNF-alpha, sIL-6R and IL-1Ra were determined by ELISA. RESULTS: The low spontaneous production of IL-1beta, IL-6 and TNF-alpha in both patients and controls was not significantly modified by PHA. The lower dose of LPS (1 microg/ml) induced a significant but lower increase in production of IL-1beta, IL-6 and TNF-alpha in patients than in controls. In contrast, while it did not further increase their production in controls, the higher concentration of LPS (3 microg/ml) still increased their production in patients to the same level than in controls. The plasma concentrations of sIL-6R were higher in patients than in controls. In both groups, the sIL-6R concentration did not vary during the culture period whether the cells were stimulated or not with LPS or PHA. This suggests that the increased plasma levels of sIL-6R were not produced by blood cells. Despite a similar significant LPS and PHA induced production of IL-1Ra, the IL-1Ra/IL-1beta ratio was always higher in patients than in controls. CONCLUSION: Monocytes from HD patients in WB cultures are hyporesponsive to PHA and LPS for their IL-1beta, TNFalpha and IL-6 production in contrast to isolated monocytes that demonstrate signs of activation. If it reflects the in vivo situation it could partly explain the immune defect in uraemic and haemodialysed patients. Higher sIL-6R/IL-6 and IL-1Ra/IL-1beta ratios could also participate to the complex immune disturbances of HD patients by reducing the biological activity of two cytokines playing a major role in the immune and inflammatory network.     

Impairment of innate cellular response to in vitro stimuli in patients on continuous ambulatory peritoneal dialysis.

BACKGROUND: Most crucial in the initial stages of host defence against invading micro-organisms is innate immunity, in which peripheral mononuclear cells, in particular cytokines, are pivotal. Mortality from infections is high in dialysis patients, but it remains unclear if this arises from the ineffectiveness of innate immune mechanisms. METHODS: In 20 haemodialysis (HD) patients, 20 patients on continuous ambulatory peritoneal dialysis (CAPD), and 15 age-matched controls, we studied cytokine production by monocytes and helper T-cells in response to in vitro stimuli. The most important early-response cytokines for innate immunity, tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta, were tested in monocytes, and interferon-gamma and IL-4 were studied as indicators of polarization of helper T-cells into type 1 and type 2 cells. Peripheral blood cells stimulated with lipopolysaccharide or mitogen were labelled with anti-CD14+ and -CD4+ antibodies and then subjected to intracellular cytokine staining and flow cytometry. RESULTS: CAPD patients showed significantly reduced synthesis of TNF-alpha and IL-1beta and inhibited T helper phenotype development compared with HD patients and control subjects. In contrast, HD patients showed an unaltered monokine response and a marked polarization of helper T-cells towards the type 1 phenotype. We also found that a single HD treatment potentiated monocytes to synthesize TNF-alpha. CONCLUSIONS: Circulating immune cells in CAPD patients may be hyporeactive against infections, indicating an unfavourable innate host defence.     

Monocyte activation in peripheral blood and dialyser eluates: phenotypic profile and cytokine release

BACKGROUND/AIMS: Monocyte activation and subsequent cytokine generation is presumed to be involved in haemodialysis (HD)-related morbidity. The present study was designed to investigate HD-induced changes in monocytes, with respect to their phenotypic profile and cytokine release, both in peripheral blood (PB) and dialyser eluates (DE). In addition, the effect of the type of dialyser on monocyte activation was assessed. METHODS: Dialyser elution was performed in 8 patients after 3 h of HD, using cuprammonium (CU) and polysulfon (PS) dialysers in a randomised cross-over design. PB samples and DE were analysed for both the expression of a variety of monocyte cell surface markers (CD62L, CD11b, CD25, HLA-DR, CD64 and CD14) by flow cytometry and IL-1beta levels. Monocytes were identified by dual labelling with antibodies against CD14. RESULTS: In PB, the expression of CD11b increased during HD with both devices, but was more pronounced with CU (CU versus PS: p < 0.05). CD62L decreased during HD, but only significantly for PS (p < 0.02). HLA-DR was downregulated during HD with CU (p = 0.056). The expression of CD64 was higher during HD with CU (p = 0.02). Finally, CD14 increased during HD with both dialysers (p < 0.03). DE yielded a mean cell count of 51 x 10(6) cells. The proportion of monocytes in DE was 3% for CU and 4% for PS. In eluted monocytes, a significant upregulation of CD11b, CD25, and HLA-DR was observed. CD62L was downregulated when compared to PB at t(180) (p < 0.001). In DE, no correlation was found between the type of dialyser and the phenotypic changes. In 10 of 16 DE supernatants, 6 CU and 4 PS, IL-1beta release could be demonstrated, CU yielding significantly more of this cytokine than PS (p = 0.03). CONCLUSIONS: According to both their phenotypic profile and cytokine release, monocytes sticking to the dialyser membrane after HD are considerably more activated than circulating monocytes. Activation of eluted monocytes appeared independent of the type of dialyser, suggesting an effect of mechanical stress rather than bioincompatibility. In contrast, phenotypic activation of peripheral blood monocytes and cytokine release in the DE supernatant were mainly dialyser-dependent.