Monocyte deactivation correlates with injury severity score, but not with heme oxygenase-1 levels in trauma patients

Traumatic injury induces a local and systemic release of pro-inflammatory cytokines, acute phase proteins, hormones, and other inflammatory mediators. The excessive release of these mediators plays an important role in the pathogenesis of shock. In parallel to this pro-inflammatory response, there is a regulatory response characterized by the release of anti-inflammatory mediators, which is thought to represent the host's attempt to restore immunological equilibrium. Studies in septic patients have suggested the compensatory anti-inflammatory response may result in an "immunodeficient state" that leaves the host susceptible to further infectious insults. A key feature of the anti-inflammatory state in septic patients is a change in the responsiveness of monocytes that has been termed "monocyte deactivation." This is supported by data that link monocyte deactivation to increased mortality in septic patients. Monocytes with reduced HLA-DR expression have been described in trauma patients. We collected blood from 25 severely injured patients and evaluated peripheral blood mononuclear cells (PBMC) for HLA-DR expression and TNF-α response to LPS stimulation as markers of monocyte deactivation. Levels of intracellular HO-1 were determined in each patient, as HO-1 has been implicated in monocyte deactivation in patients with severe systemic inflammatory response syndrome (SIRS). HLA-DR expression correlated inversely with Injury Severity Scores and TNF-α response to LPS stimulation, but failed to correlate with HO-1 levels in these patients. HLA-DR expression was decreased in normal monocytes stimulated with patient plasma, but this treatment had no effect on HO-1 levels. These results suggest monocyte deactivation in trauma patients is unlikely to be mediated by HO-1.     

Reduced expression of Toll-like receptor 4 contributes to impaired cytokine response of monocytes in uremic patients

Toll-like receptors (TLRs) play a pivotal role in pathogen recognition and subsequent cytokine synthesis by immune cells. Uremic patients have a high infectious morbidity, but it remains unclear if this arises from the defective innate immune responses related to TLRs. We studied TLR4 expression in monocytes and their intracellular cytokine synthesis in response to lipopolysaccharide (LPS) stimulation in 35 predialysis patients with chronic kidney disease (CKD) with or without predisposition to bacterial infections and 16 age-matched controls. Expression of TLR4 in unstimulated peripheral monocytes was determined by staining with anti-TLR4 antibody and analysis with flow cytometry. Monocytes were then stimulated by LPS, labeled with anti-CD14 antibody, and subjected to intracellular cytokine staining and flow cytometry. Tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, and IL-8 synthesis was examined in CD14(+) monocytes. TLR4 expression was constitutively diminished in CKD patients with reduced expression being more severe in those CKD patients who were predisposed to infections. Monocytes from these infection prone CKD patients exhibited significantly reduced synthesis of TNF-alpha, IL-1beta, IL-6, and IL-8 in response to LPS challenge compared with those from control subjects. The intensity of synthesis of each cytokine significantly correlated with TLR4 expression levels in monocytes (P<0.01). The capacity of monocytes to synthesize proinflammatory cytokines was significantly reduced in infection prone CKD patients, and this may possibly be due to the reduced monocyte expression of TLR4. Abnormal TLR4 expression by monocytes may play a role in the susceptibility of such patients to bacterial infections.     

核因子kB活化对烧伤血清诱导单核细胞细胞因子表达的影响

目的了解核因子(NF)κB活化对烧伤血清诱导单核细胞活化分泌细胞因子的作用,探讨烧伤血清激活单核细胞的机制。方法收集体外培养的人外周血单核细胞(PBMC),分别用正常人血清、烧伤患者血清、烧伤患者血清 吡咯烷二硫代氨基甲酸盐(PDTC)刺激后依次分为对照组、烧伤血清组、PDTC组。采用电泳迁移率分析法检测刺激前及刺激0.5、1.0、2.0、4.0h时PBMC的NF-κB活性;酶联免疫吸附测定法和原位杂交法检测刺激前及刺激1.0、2.0、4.0、6.0h时PBMC培养上清液中肿瘤坏死因子(TNF)α、白细胞介素(IL)8水平及TNF-αmRNA、IL-8mRNA的表达情况。结果血清刺激后,烧伤血清组PBMC NF-κB活性迅速升高,刺激1.0h时达峰值(30.2±3.5)×104积分灰度值,与对照组(4.4±0.8)×104积分灰度值比较差异有统计学意义(P<0.01).刺激2.0h后逐渐下降;而PDTC组NF-κB活性无明显升高,刺激1.0h时为(6.8±0.9)×104积分灰度值。烧伤血清组刺激PBMC1.0h时,TNF-αmRNA表达量和培养上清液中TNF-α水平即升高达峰值,并明显高于对照组(P<0·01);IL-8mRNA表达量和IL-8水平在刺激4.0h时达峰值,也明显高于对照组(P<0.01);而PDTC组PBMC培养上清液中TNF-α刺激1.0h时达峰值(0.52±0.06)μg/L;刺激4.0h时IL-8达峰值(239±20)ng/L,与对照组[(0.13±0.07)μg/L、<156ng/L]比较,差异有统计学意义(P<0·01).结论烧伤血清可通过活化NF-κB,启动PBMC对细胞因子的合成和释放,提示NF-κB活化在烧伤血清诱导PBMC分泌细胞因子的过程中起重要作用。     

Increased monocyte activation in elderly patients after surgical stress

OBJECTIVE: To investigate age-related changes in the host response to surgical stress. The clinical course, serum interleukin-6 (IL-6) levels, monocyte production of tumor necrosis factor-alpha (TNF-alpha), and monocyte expression of CD11b/CD18 were used as markers of the systemic response. METHODS: Patients with gastric cancer, undergoing distal gastrectomy were divided into 2 groups: >75 years of age (elderly group) and < or =75 years of age (young group). Serum IL-6 levels, TNF-alpha production and CD11b/CD18 expression by monocytes, and the postoperative clinical course were compared between the 2 groups. RESULTS: TNF-alpha production by lipopolysaccharide-stimulated monocytes and CD11b/CD18 expression on monocytes after surgical stress were significantly higher in the elderly than in the young group. Moreover, serum IL-6 levels on the first postoperative day in the elderly group were significantly higher than those in the young group. The incidence and duration of systemic inflammatory response syndrome (SIRS) were significantly greater in the elderly than in the young group. CONCLUSIONS: The activation of monocytes and hypercytokinemia occur readily after surgical stress in the elderly and may therefore contribute to SIRS and increased susceptibility to postoperative complications.     

Effects of a protease inhibitor on reduction of surgical stress in esophagectomy

OBJECTIVE: To evaluate the efficacy of gabexate mesilate (GM) in reducing surgical stress after esophagectomy. METHODS: In a prospective, randomized, clinical study, 11 patients with squamous cell carcinoma of the esophagus were randomly assigned to two groups: 5 patients were continuously administered gabexate mesilate 1.5 mg/kg per hour from the beginning of anesthesia until the third postoperative day (preop GM group); and 6 patients were administered gabexate mesilate 1.5 mg/kg per hour continuously from the end of surgery and for the same postoperative period (postop GM group). Blood samples were taken from all patients before surgery, immediately after it, and 3 days after surgery. Serum interleukin-6 (IL-6) level, tumor necrosis factor-alpha (TNF-alpha) production, and Mac-1 antigen expression of peripheral blood monocytes were measured. Clinical courses of patients in the two groups were compared. RESULTS: Time courses of serum IL-6 levels in the preop GM group were significantly lower than those in the postop GM group. Ex vivo TNF-alpha production by lipopolysaccharide (LPS) stimulated monocyte was much higher than that by monocyte without LPS stimulation. Gabexate mesilate showed a little inhibition of TNF-alpha production by monocyte without LPS stimulation. On the other hand, gabexate mesilate significantly inhibited TNF-alpha production by LPS stimulated monocyte. Mac-1 antigen expression by monocyte immediately after operation in the preop GM group was significantly lower than that in the postop GM group. Duration of systemic inflammatory response syndrome was significantly shorter in the preop GM group than in the postop GM group. CONCLUSIONS: Reduction of systemic inflammatory response syndrome duration after esophagectomy by the continuous administration of gabexate mesilate started before operation may be through the suppression of TNF-alpha production capacity and Mac-1 expression on monocytes immediately after operation, and to suppression of increase in serum IL-6 level.     

Drotrecogin alfa (activated) does not affect intracellular production of interleukin-6 and tumor necrosis factor-alpha in endotoxin-stimulated human monocytes

In this ex vivo laboratory study, we investigated the effects of drotrecogin alfa (activated) (DA(a)), a recombinant form of human activated protein C, on the intracellular expression of interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha on lipopolysaccharide (LPS)-stimulated human monocytes in a whole blood system. Whole blood samples from 10 healthy volunteers were stimulated with LPS (0.2 ng/mL) and incubated with 0.01, 0.1, 1, 10, and 100 nM of (final concentration) DA(a) for 3 h at 37 degrees C and 5% CO2. Intracellular expression of IL-6 and TNF-alpha was assessed by flow cytometry. Our investigation showed that DA(a), at any of the concentrations tested, did not affect intracellular IL-6 and TNF-alpha production in LPS-stimulated human monocytes after 3 h of incubation. The results of this investigation led us to conclude that any antiinflammatory activity of DA(a), if present, does not occur via detectible decreases in the production of proinflammatory cytokines IL-6 and TNF-alpha in human monocytes.     

Paradoxical cytoskeleton and microparticle formation changes in monocytes and polymorphonuclear leukocytes in severe systemic inflammatory response syndrome patients

BACKGROUND: Circulating monocytes and polymorphonuclear leukocytes (PMNLs) are considered as central regulators controlling systemic inflammatory response after severe insults. Recently, activated monocytes and PMNLs have been reported to produce microparticles (MPs) in vitro. The objective of this study was to evaluate production of MPs and changes of cytoskeleton in monocytes from severe systemic inflammatory response syndrome (SIRS) patients, and to compare them with those in PMNLs. METHODS: Twenty severe SIRS patients (SIRS criteria and serum C-reactive protein > 10 mg/dL) and 15 healthy volunteers were included. MP formation and F-actin content in monocytes and PMNLs were measured by flow cytometry in the presence or absence of lipopolysaccharide or formylmethionyl-leucyl-phenylalanine (FMLP). The membrane expression of human leukocyte antigen-DR and CD64 in monocytes and O2- production in PMNLs were also measured by flow cytometry. RESULTS: In severe SIRS patients, MP formation with and without lipopolysaccharide in monocytes significantly decreased in comparison with those in normal controls (p < 0.05), whereas those with and without FMLP in PMNLs increased (p < 0.05). F-actin content with and without FMLP in monocytes also significantly decreased in patients (p < 0.05), whereas those in PMNLs increased as compared with normal controls (p < 0.05). The expression of human leukocyte antigen-DR in monocytes significantly decreased in patients (p < 0.05), which indicated monocyte modulation. The O2- production in PMNLs increased in patients (p < 0.05), which showed PMNL activation. CONCLUSION: The changes of MP formation and cytoskeleton in circulating monocytes and PMNLs were paradoxically different in severe SIRS patients.     

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.     

Assessment of immunological status in the critically ill

The systemic inflammatory response (SIRS) results from various types of injuries such as severe infection, trauma, ischemia-reperfusion and major surgery including cardiac surgery with cardio-pulmonary bypass. This response involves immune cell activation and a complex network of proinflammatory cytokines, which may induce multiple organ failure when uncontrolled. The monocyte plsys a central role in the response to infection with the release of TNF-alpha, IL-1 beta, and IL-12. In addition, monocytes present antigens to T lymphocytes. An optimal antigen presentation requires the expression of MHC class II HLA-DR on monocytes surface and of costimulatory molecules such as CD54 on monocytes and LFA-1 on lymphocytes. It has become increasingly apparent that the proinflammatory response is balanced by concomitant anti-inflammatory mechanisms that results in monocyte deactivation, characterized by a decrease in HLA-DR expression and the release of anti-inflammatory cytokines such as IL-10. This counterregulatory response, if prolonged or predominant, may predispose the patient to a higher risk of infection. Further studies need to be conducted to precise: i) the intensity of depression of the surface molocule expression assessing monocyte function, such as HLA DR and CD54; ii) the level of IL-10 and IL-12 release in patients with severe sepsis; iii) the immuno-modulating effects of frequently used treatments in these patients with severe sepsis and in surgical patients; iv) the time course of recovery; v) if the monitoring of HLA-DR, CD54, IL-10 and IL-12 will better predict the clinical outcome than clinical parameters.     

Effect of hemoperfusion with polymyxin B-immobilized fiber on plasma endothelin-1 and endothelin-1 mRNA in monocytes from patients with sepsis

Hemoperfusion using polymyxin B-immobilized fiber (PMX-F) is reported to be an effective treatment for sepsis. The aim of the present study is to assess whether plasma endothelin-1 (ET-1) and ET-1 messenger RNA (mRNA) levels in peripheral-blood monocytes are altered in patients with sepsis and whether PMX-F treatment affects plasma ET-1 and monocyte ET-1 mRNA levels. Sixteen patients with sepsis and 20 healthy volunteers were included in this study. Plasma ET-1 concentration was measured by radioimmunoassay (RIA), and plasma levels of transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta) were measured by enzyme-linked immunosorbent assay (ELISA). Sixteen patients with sepsis were treated with direct hemoperfusion using PMX-F columns. Blood endotoxin levels decreased significantly from 35 to 10 pg/mL after two treatments of direct hemoperfusion, each for 2 hours. Patients with sepsis showed significantly increased levels of plasma ET-1 (P < 0.001) and monocyte ET-1 mRNA (P < 0.001) compared with healthy volunteers. However, no differences in plasma levels of TGF-beta, TNF-alpha, and IL-1beta existed between patients with sepsis and healthy volunteers. Increased plasma ET-1 levels and monocyte ET-1 mRNA levels in patients with sepsis decreased significantly after PMX-F treatment (P < 0.01). These data suggest that the secretion of ET-1 from peripheral-blood monocytes may be stimulated by endotoxin, and PMX-F treatment may be effective in reducing ET-1 secretion in patients with sepsis.     

肝移植术病人围术期中性粒细胞Toll样受体2表达与术后SIRS的关系

Objectlve investigate the role of Toll-like receptor 2 (TLR2) on polymorphonuclear neutrophil (PMN) during perioperative period in the development of postoperative systemic inflammatory response syndrome (SIRS) in patients undergoing orthotopic liver transplantation (OLT).Methods Twenty patients (18 male and 2 female, aged 33-58 yr and weighing 52-73 kg) with ASA Ⅲ or Ⅳ (NYHA Ⅱ or Ⅲ )undergoing OLT were studied. Blood samples were collected from the central vein for determination of TLR2 expression on PMN and plasma TNF-α, IL-1β and IL-8 concentrations before induction of anesthesia (T1, baseline), at 25 min of anhepatic phase (T2), 3 h (T3) and 24 h after beginning of reperfusion of the allograft (T4). The expression of TLR2 was measured by flow cytometry and the serum concentrations of TNF-α, IL-1β and IL-8 were measured by enzyme linked immunosorbant assay (ELISA). The patients were divided into SIRS and non-SIRS group depending on whether the patients developed SIRS or not within 7 days after operation. The diagnosis of SIRS was based on the criteria laid down by ACCP and SCCM in 1992.Results Ten patients developed SIRS within 7 days after operation. There was no significant difference in Child-Turcotte-Pugh (CTP) scores between the two groups. Compared with non-SIRS group, the TLR2 expression on PMN and the serum IL-1β concentration were significantly increased at T4 and the serum IL-8 concentration was significantly increased at T3 in SIRS group.There was positive correlation between serum TNF-α concentration and TLR2 expression on PMN in SIRS group ( r= 0.607, P ＜0.05).Conclusion The expression of TLR2 on PMN increases significantly at 24 h after beginning of reperfusion of allograft and may play an important role in the development of postoperative SIRS.     

Activation of Monocytes and Endothelial Cells Depends on the Severity of Surgical Stress

Surgical injury not only induces a systemic endocrine-metabolic response but also influences the function of the leukocytes and endothelial cells leading to various systemic responses. These responses appear to depend on the severity of surgical stress, which differs according to the surgical procedures. In this study, we investigated the response of monocytes and endothelial cells, and the development of systemic inflammatory response syndrome (SIRS) in relation to the severity of surgical stress. The postoperative clinical course was evaluated between patients undergoing an esophagectomy (ER group) and a distal gastrectomy (DG group). The tumor necrosis factor α (TNFα) production of monocytes, the serum interleukin 6 (IL-6) levels, the CD11b expression on either monocytes or granulocytes, and the intercellular adhesion molecule-1 (ICAM-1) expression on human umbilical vein endothelial cells (HUVECs) stimulated with culture supernatants of monocytes were compared between the 2 groups. The development of SIRS was observed in all patients in the ER group, whereas no patients demonstrated SIRS in the DG group. The serum IL-6 levels, TNFα production of monocytes, and CD11b intensity on monocytes or granulocytes in the ER group were higher than those in the DG group. In the ER group, the ICAM-1 intensity on HUVECs with monocytes immediately after operation significantly increased compared with before the operation. In conclusion, both the CD11b expression on monocytes and the TNFα production of monocytes are considered to reflect the degree of surgical stress, and the activation of endothelial cells stimulated with these activated leukocytes may therefore lead to both tissue and organ injury.     

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.     

肝移植术病人围术期中性粒细胞Toll样受体2表达与术后SIRS的关系

目的 研究肝移植术病人围术期中性粒细胞Toll样受体2(TLR2)表达与术后全身炎性反应综合征(SIRS)的关系.方法 择期行肝移植术的终末期肝病病人20例,年龄33～58岁,体重52～73 kg,心功能Ⅱ或Ⅲ级,ASAⅢ或Ⅳ级.于麻醉诱导前(T1)、无肝期25 min(T2)、新肝期3 h(T3)、新肝期24 h(T4)时,中心静脉采血样,采用流式细胞仪测定中性粒细胞TLR2表达;ELISA法测定血清肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)和白细胞介素-8(IL-8)浓度;按术后7 d内是否发生SIRS,分为SIRS组与非SIRS组.2组TNF-α、IL-1β和IL-8浓度和TLR2表达进行Spearman相关分析.结果 术后7 d内有10例病人发生SIRS.与T1时比较,SIRS组T4时中性粒细胞TLR2表达上调,T2-4时TNF-α浓度升高,T3,4时IL-1β浓度升高,T3时IL-8浓度升高(P＜0.05或0.01);与NSIRS组比较,SIRS组T4时中性粒细胞表达上调,血清IL-1β浓度升高,T3时血清IL-8浓度升高(P＜0.05或0.01).SIRS组TNF-α浓度与中性粒细胞TLR2表达呈正相关(r=0.607,P＜0.05).结论 肝移植术后病人发生SIRS可能与新肝期24 h中性粒细胞TLR2表达上调有关.     

Toll-Like Receptor and Heme Oxygenase-1 Signaling in Hepatic Ischemia/Reperfusion Injury

Ischemia/reperfusion injury (IRI) represents the major problem in clinical liver transplantation. We have shown that toll-like receptor 4 (TLR4) signaling is specifically required in initiating antigen-independent IRI leading to liver inflammation, whereas local induction of anti-oxidant heme oxygenase-1 (HO-1) is cytoprotective. This study analyzes in vivo interactions between HO-1 and sentinel TLR system in the pathophysiology of liver IRI. Using a 90-min lobar warm ischemia model, wild type (WT), TLR4 KO/mutant and TLR2 KO mice were first assessed for the severity of hepatocellular damage at 6 h postreperfusion. Unlike in WT or TLR2-deficient mice, disruption/absence of TLR4 pathway reduced IRI, as manifested by liver function (serum alanine aminotransferase levels), histology (Suzuki's scores), neutrophil infiltration (myeloperoxidase activity) and local/systemic TNF-alpha production (mRNA/protein levels). Moreover, defective TLR4 but not TLR2 signaling increased mRNA/protein HO-1 expression. In contrast, tin protoporphyrin-mediated HO-1 inhibition restored hepatic damage in otherwise IRI-resistant TLR4 mutant/KO mice. CoPP-induced HO-1 overexpression ameliorated hepatic damage in IRI-susceptible TLR2 KO mice, comparable with WT controls, and concomitantly diminished TLR4 levels. In conclusion, this study highlights the importance of cross talk between HO-1 and TLR system in the mechanism of hepatic IRI. Hepatic IRI represents a case for innate immunity in which HO-1 modulates proinflammatory responses that are triggered via TLR4 signaling, a putative HO-1 repressor.     

The effect of local anesthetics on monocyte mCD14 and human leukocyte antigen-DR expression

It has been demonstrated that local anesthetics have several effects on the immune system. Monocytes and macrophages are essential components of the host response to microbial infection; however, the effect of local anesthetics on monocyte surface receptor expression remains unclear. We designed this study to investigate the effects of local anesthetics on monocyte mCD14 and human leukocyte antigen (HLA)-DR expression and lipopolysaccharide (LPS)-induced or staphylococcal enterotoxin B (SEB)-induced tumor necrosis factor (TNF)-alpha production. Blood samples were obtained from 10 healthy volunteers. The effects of local anesthetics on LPS- or SEB-induced TNF-alpha production were determined by using an enzyme-linked immunosorbent assay. After different doses of local anesthetics were added, the blood was stimulated with LPS (10 ng/mL) or SEB (10 micro g/mL) for 4 h. The effects of local anesthetics on monocyte mCD14 and HLA-DR expression were measured by dual monoclonal antibody staining and flow cytometry. Local anesthetics showed no effect on LPS- or SEB-induced TNF-alpha production in human whole blood. Local anesthetics suppressed monocyte HLA-DR expression in a dose-dependent manner (P < 0.05) but had no effect on monocyte mCD14 expression. This study demonstrated that local anesthetics suppress HLA-DR expression on the surface of human monocytes. IMPLICATIONS: Monocyte surface receptors have a crucial role in the host response to microbial infection. We investigated the effects of local anesthetics on monocyte mCD14 and human leukocyte antigen (HLA)-DR expression. Our results show that local anesthetics suppress HLA-DR expression on the surface of human monocytes.     

Enhanced expression of intranuclear NF-kappa B in primed polymorphonuclear leukocytes in systemic inflammatory response syndrome patients

BACKGROUND: Nuclear factor-kappa B (NF-kappa B) plays a critical role in the cellular response to a variety of stimuli, and it regulates the production of various inflammatory cytokines, adhesion molecules, and enzymes. Polymorphonuclear leukocytes (PMNLs) play a central role in systemic inflammatory response after severe insult. The role of NF-kappa B in activation of PMNLs, however, has not been clear. We developed a simple flow cytometric method for quantifying expression of intranuclear NF-kappa B in PMNLs, and we used it to evaluate NF-kappa B activity in patients with systemic inflammatory response syndrome (SIRS). METHODS: Thirty patients who fulfilled the criteria for SIRS and 24 healthy volunteers were included as study subjects. Expression of intranuclear NF-kappa B with and without stimulation by lipopolysaccharide was quantified by our new method. Oxidative activity in PMNLs with and without formylmethionyl-leucyl-phenylalanine stimulation was measured by flow cytometry. Levels of interleukin-6, interleukin-8, PMNL elastase, and nitric oxide metabolites in blood were also measured. RESULTS: Expression of intranuclear NF-kappa B in PMNLs both with and without LPS stimulation was significantly elevated in SIRS patients in comparison with that of healthy volunteers. PMNL oxidative activity was significantly elevated in SIRS patients. Positive correlation was observed between intranuclear NF-kappa B expression and PMNL oxidative activity, whereas no relation was observed between intranuclear NF-kappa B expression and serum concentrations of chemical mediators. CONCLUSION: Our new flow cytometric method proved useful for quantifying intranuclear NF-kappa B expression in PMNLs. In PMNLs from SIRS patients, intranuclear NF-kappa B expression and oxidative activity were significantly elevated with positive correlation, and enhanced expression of NF-kappa B may play an important role in PMNL activation in SIRS.     

The isoflavone genistein inhibits LPS-stimulated TNFalpha, but not IL-6 expression in monocytes from hemodialysis patients and healthy subjects

BACKGROUND: Whole blood and peripheral blood mononuclear cells from hemodialysis (HD) patients show increased production and secretion of inflammatory cytokines. We determined the contribution of blood monocytes to the production of inflammatory cytokines in whole blood from HD patients. METHODS: Whole blood and isolated mononuclear cells from HD patients and healthy control subjects were preincubated with the isoflavone genistein and stimulated with LPS. TNFalpha, IL-6 and IL-10 formation in the whole blood was measured with ELISA and intracellular cytokine formation in CD 14-positive monocytes was determined by flow cytometry. RESULTS: Unstimulated blood levels of TNFalpha, IL-6 and IL-10 were significantly elevated in HD patients compared to controls, but intracellular monocyte content of these cytokines was identical between groups. LPS induced a robust TNFalpha response in both whole blood and monocytes, and TNFalpha formation was 2.3-fold higher in blood from HD patients compared to controls. A similar trend was observed in monocytes. Conversely, LPS stimulation increased IL-6 levels >1000-fold in whole blood, albeit without a noticeable difference between groups. Only minor increases in monocyte IL-6 content were observed. The isoflavone genistein did not inhibit IL-6 formation and did not alter basal TNFalpha levels, but genistein selectively blocked LPS-induced TNFalpha formation in whole blood and monocytes from both groups. CONCLUSION: Intracellular levels of TNFalpha, IL-6 and IL-10 in monocytes are indistinguishable between HD patients and healthy controls. However, monocytes from HD patients are selectively primed for enhanced TNFalpha secretion in response to LPS. The selective inhibition of monocyte TNFalpha production by genistein may explain the anti-inflammatory action of this phytochemical observed in vivo.     

Assessment of immunological status in the critically ill

The systemic inflammatory response (SIRS) results from various types of injuries such as severe infection, trauma, ischemia-reperfusion and major surgery including cardiac surgery with cardio-pulmonary bypass. This response involves immune cell activation and a complex network of proinflammatory cytokines, which may induce multiple organ failure when uncontrolled. The monocyte plays a central role in the response to infection with the release of TNF, IL-1, and IL-12. In addition, monocytes present antigens to T lymphocytes. An optimal antigen presentation requires the expression of MHC class II HLA-DR on monocytes surface and of co-stimulatory molecules such as CD54 on monocytes and LFA-1 on lymphocytes. It has become increasingly apparent that the pro-inflammatory response is balanced by concomitant anti-inflammatory mechanisms that results in monocyte deactivation, characterized by a decrease in HLA-DR expression and the release of anti-inflammatory cytokines such as IL-10. This counterregulatory response, if prolonged or predominant, may predispose the patient to a higher risk of infection. Further studies need to be conducted to precise: 1) the intensity of depression of the surface molecule expression assessing monocyte function, such as HLA DR and CD54; 2) the level of IL-10 and IL-12 release in patients with severe sepsis; 3) the immunomodulating effects of frequently used treatments in these patients with severe sepsis and in surgical patients; 4) the time course of recovery; 5) if the monitoring of HLA-DR, CD54, IL-10 and IL-12 will better predict the clinical outcome than clinical parameters.     

Bone morphogenic protein-7 inhibits monocyte-stimulated TGF-beta1 generation in renal proximal tubular epithelial cells

It has been demonstrated that bone morphogenic protein-7 (BMP-7) stimulates formation of hyaluronan (HA)-based cables on the cell surface of renal proximal tubular cells and that these cables mediate monocyte binding. Furthermore, interaction of monocytes with proximal tubule cell (PTC) surface intracellular adhesion molecule (ICAM) stimulates the synthesis of TGF-beta1. This study examined the effect of BMP-7 on monocyte-stimulated TGF-beta1 synthesis under conditions of basal and stimulated ICAM expression. Monocyte (U937 cells)-dependent stimulation of TGF-beta1 promoter activity and protein synthesis was reduced by addition of BMP-7 for 24 h before addition of U937 cells. Removal of cell surface HA or inhibition of monocyte interaction with HA using antibody to CD44 prevented this effect of BMP-7. These data suggest that BMP-7 enhances HA-dependent binding and reduces ICAM-dependent binding, which is known to stimulate TGF-beta1 synthesis. This hypothesis was examined further by stimulation of PTC ICAM expression by TNF-alpha. After TNF-alpha stimulation, monocyte-dependent TGF-beta1 synthesis increased. This was abrogated by inhibition of ICAM-CD18 interactions. TNF-alpha stimulation alone did not increase TGF-beta1 synthesis. TNF-alpha stimulation of PTC in the presence of BMP-7 failed to increase monocyte-dependent TGF-beta1 stimulation. Although stimulation of PTC by BMP-7 alone decreased cell surface ICAM expression, it did not affect TNF-alpha-induced ICAM expression. The effect of BMP-7 on TGF-beta1 synthesis in TNF-alpha-stimulated cells was abrogated by disruption of CD44-HA interactions, suggesting that it was due to increased monocyte binding to HA on the cell surface.