Differences in LPS and PepG Induced Release of Inflammatory Cytokines in Orthopedic Trauma

Trauma is associated with immune paresis which may predispose to postoperative sepsis. We characterized the ex vivo cytokine responses to bacterial cell wall components in whole blood from 8 patients undergoing a major musculoskeletal trauma in the form of total hip replacement. Preoperatively, at the end of operation, and at days 1 and 6 postoperatively, patient blood was obtained, anticoagulated, and incubated at 37°C in the presence of peptidoglycan (PepG) or lipopolysaccharide (LPS). Plasma cytokines were measured by enzyme immunoassay. The numbers of leucocytes, monocytes, and neutrophils were unchanged at the end of surgery, while there were significant increases at postoperative days 1 and 6. We observed significant reductions in tumor necrosis factor–α (TNF-α) and interleukin 10 (IL-10) responses to PepG at the end of surgery, which was disproportional to the nonsignificant reductions in circulating monocytes, suggesting a functional suppression. However, at postoperative day 1 the responses were recovered. There were no significant changes in responses of TNF-α to LPS stimulation at the end of surgery, while there were significant depressions at postoperative days 1 and 6. The expression of IL-10 was significantly depressed at the end of surgery and at day 6. There were modest changes in PepG- and LPS-induced expression of interleukin 8 (IL-8) during the experiments. On the basis of these observations, we conclude that a musculoskeletal trauma is associated with reduced expression of TNF-α and IL-10 by whole blood leucocytes when exposed to endotoxin, but there is a difference between gram-positive endotoxin (PepG) and gram-negative endotoxin (LPS).     

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.     

beta(2)-adrenoceptor agonist suppresses renal tumour necrosis factor and enhances interleukin-6 gene expression induced by endotoxin.

BACKGROUND: beta(2)-Adrenoceptor activation regulates tumour necrosis factor (TNF)-alpha and interleukin-6 (IL-6) production in cultured renal cells. However, it remains uncertain whether, in vivo, the administration of beta(2)-adrenoceptor agonists regulate renal TNF-alpha and IL-6 mRNA following lipopolysaccharide (LPS) stimulation to cause endotoxaemia. This study was performed in order to evaluate the effect of beta(2)-adrenoceptor agonist on renal TNF-alpha and IL-6 production. METHODS: Four-week-old Wistar rats pre-treated with the beta(2)-adrenoceptor agonist terbutaline or formoterol, and/or the beta- and beta(2)-adrenoceptor antagonists (propanolol, ICI118,551), were injected with LPS (1 mg i.p.), and then 2, 4 or 6 h later, kidneys (cortex, medulla), spleen, thymus and plasma were collected to assay TNF-alpha and IL-6 mRNA levels and their respective protein release. RESULTS: Administration of beta(2)-adrenoceptor agonists suppressed TNF-alpha mRNA expression in the whole kidney, by 61% (P<0.05), as well as plasma, spleen and thymus TNF-alpha protein and mRNA expression 2 hours after injection of LPS. On the other hand, although IL-6 levels in plasma, spleen and thymus mRNA expression were suppressed significantly by administration of beta(2)-adrenoceptor agonists, the basal- and LPS-induced IL-6 mRNA levels in the whole kidney were increased 1.6- and 1.2-fold (P<0.05), respectively, by treatment with beta(2)-adrenoceptor agonists. beta(2)-Adrenoceptor agonist suppressed LPS-induced TNF-alpha mRNA expression by 35% (P<0.05) and stimulated LPS-induced IL-6 mRNA expression by 1.5-fold (P<0.05) in the medullary region of kidney. CONCLUSIONS: beta(2)-Adrenoceptor agonists down-regulate renal TNF-alpha mRNA expression following LPS-induced endotoxaemia. This effect was particularly apparent in the renal medulla. IL-6 mRNA expression in the renal medulla was up-regulated by the agonists whereas plasma, spleen and thymus IL-6 levels were completely inhibited by the agonist, which suggests the existence of tissue specific regulation of IL-6 production in the kidney by beta(2)-adrenoceptor activation.     

Cytokine modulation in experimental endotoxemia: characterization of an ex vivo whole blood model

A new model was developed to study cytokine regulation and modulation in whole blood ex vivo. The model is characterized by stable leukocyte counts and high leukocyte viability throughout the experimental period. Oxygen consumption per time decreased slowly, whereas carbon dioxide partial pressure increased accordingly throughout the experiment. In this model, the anti-inflammatory effects of recombinant human (rh) interleukin (IL)-4, rhIL-10 and rhIL-13 on lipopolysaccharide (LPS) stimulated (10 ng/ml) leukocytes were examined and compared by measuring their ability to inhibit the release and mRNA levels of tumor necrosis factor (TNF)alpha, IL-6 and IL-1beta. rhIL-10 potently inhibited the release of TNF-alpha, IL-6 and IL-1beta in a potent and dose-dependent manner, but did not influence the mRNA levels of these cytokines in CD14-positive cells. Also, rhIL-4 and rhIL-13 inhibited the release of IL-6 and IL-1beta in a potent and dose-dependent manner, however, stronger maximal inhibition of IL-1beta (85%) than of IL-6 (60%) was obtained. In contrast, rhIL-4 and rhIL-13 seemed to have both stimulatory and inhibitory effects on plasma values of TNF-alpha. The effects of 10 ng/ml LPS showed to be signalling through the CD14 receptor, since blood treated with a monoclonal anti-CD14 antibody did not produce any TNF-alpha. The whole blood model described in this study is in our opinion a useful tool for investigating immunomodulating effects on a mixed white blood cell population.     

The effect of 17beta-estradiol on production of cytokines in cultures of peripheral blood

Estrogen's action on bone may be mediated by cytokines produced by monocytes. We have reported a decreased ratio of interleukin-1beta (IL-1beta) to interleukin-1 receptor antagonist (IL-1ra) produced by whole blood cultures in vivo in women taking hormone replacement therapy (HRT). Also, one study has shown an effect of estradiol on tumor necrosis factor-alpha (TNF-alpha) secretion by separated monocytes in vitro. The aim of this study was to evaluate the effect of estrogen in vitro on the secretion of cytokines using whole blood cultures. Subjects consisted of 12 healthy postmenopausal women, ages 57-69 years, 4-20 years since menopause. Cytokines IL-1beta, interleukin-1alpha (IL-1alpha), IL-1ra, interleukin-6 (IL-6), TNF-alpha, and granulocyte macrophage-colony stimulating factor (GM-CSF) were measured in unstimulated and in stimulated (500 ng/mL lipopolysaccharide [LPS]) whole blood cultures treated with 17beta-estradiol (E(2)) at concentrations of 10(-12)--10(-6) mol/L. We found significant decreases in the spontaneous secretion of IL-6, TNF-alpha, IL-1ra, IL-1beta, and ratio of IL-1beta/IL-1ra compared with control, at physiological concentrations of E(2). The action of E(2) was blocked by the use of the antiestrogen ICI 182780 in coculture. A decrease in cytokine secretion was not observed when the inactive form of estrogen, 17alpha-estradiol, was used in place of 17beta-estradiol. GM-CSF and IL-1alpha were not detectable in unstimulated cultures. Cytokine levels measured in stimulated cultures were not attenuated by treatment with E(2). We conclude that E(2) inhibits the spontaneous secretion of cytokines measured in whole blood cultures at physiological concentrations, and that the powerful stimulatory effect of LPS prevents any significant inhibition by E(2) in stimulated cultures.     

Systemic administration of enamel matrix derivative to lipopolysaccharide-challenged pigs: effects on the inflammatory response

BACKGROUND: Periodontitis is the primary clinical indication for enamel matrix derivative (EMD). Recent investigations, showing that EMD inhibits the production of tumor necrosis factor-alpha (TNF-alpha) when added to human whole blood, indicate a novel role for EMD as a modulator of systemic inflammation. In the present study, we investigated the systemic effects of EMD in lipopolysaccharide (LPS)-challenged pigs. METHODS: In a preparatory study, seven pigs received a prophylactic EMD bolus injection (5 mg/kg), followed by a continuous infusion (50 mg/kg/min). Thirty minutes later, a continuous infusion of LPS (1.7 mcg/kg/h) was started. An additional 12 pigs were randomized into two groups. Six of these animals were given the same treatment, except that EMD was administered 30 min after LPS. The remainder served as controls. The groups were compared according to organ injury and function, hemodynamics, and systemic markers of inflammation. RESULTS: Prophylactic administration of EMD triggered transient hemodynamic instability in two of seven pigs. In the randomized pigs, no or only nonspecific changes were observed in biopsies from vital organs, independent of treatment. Enamel matrix derivative did not modify systemic TNF-alpha, interleukin (IL)-1 beta, or IL-6 concentrations. CONCLUSIONS: In the formulation and dosages used, EMD did not modulate the inflammatory response. No true allergic or immunotoxic reactions were seen. To be usable for systemic application, a new formulation should be developed, or the active part of the protein(s) should be identified and produced in a soluble form designed for infusion. The potential of EMD as a systemic immune modulator is still unsettled.     

Ketamine suppresses proinflammatory cytokine production in human whole blood in vitro.

The production of proinflammatory cytokines, such as tumor necrosis factor (TNF) a, interleukin (IL)-6, and IL-8, increases in patients with sepsis; marked production causes organ failure and septic shock. We previously reported that ketamine suppressed lipopolysaccharide (LPS)-induced TNF-alpha production in mice. However, there are no reports on the effect of ketamine on cytokine production in human whole blood. Therefore, in this study, we investigated the efficacy of ketamine on LPS-induced TNF-alpha, IL-6, and IL-8 production and recombinant human (rh) TNF-a-induced IL-6 and IL-8 production in human whole blood. After adding different doses of ketamine to whole blood, the blood was stimulated with LPS or rhTNF. After incubation, the plasma TNF-alpha activity and IL-6 and IL-8 concentrations were measured using the L929 cell cytotoxic assay or an enzyme-linked immunoassay. Ketamine significantly suppressed LPS-induced TNF-alpha production at concentrations >20 microg/mL. At concentrations >100 microg/mL, ketamine also significantly suppressed both LPS-induced and rhTNF-induced IL-6 and IL-8 production. In this study, we demonstrated that ketamine directly inhibits the production of proinflammatory cytokines such as TNF-alpha, IL-6, and IL-8 in human whole blood. IMPLICATIONS: We found that ketamine suppressed lipopolysaccharide-induced tumor necrosis factor alpha, interleukin (IL)-6, and IL-8 production and recombinant human tumor necrosis factor-induced IL-6 and IL-8 production in human whole blood. Ketamine directly suppresses proinflammatory cytokine production.     

Is circulating endotoxin the trigger for the systemic inflammatory response syndrome seen after injury?

OBJECTIVE: Patients with severe traumatic or burn injury and a mouse model of burn injury were studied early after injury to determine the relation of plasma endotoxin (lipopolysaccharide [LPS]) to the production of proinflammatory cytokines and subsequent resistance to infection. SUMMARY BACKGROUND DATA: Elevated levels of plasma LPS have been reported in patients after serious injury. It has been suggested that circulating LPS may be a trigger for increased proinflammatory cytokine production and may play a role in the septic syndromes seen in a substantial portion of such patients. Yet, despite multiple reports of leakage of LPS from the gut and bacterial translocation after injury in animal models, there is little direct evidence linking circulating LPS with production of inflammatory mediators. METHODS: The authors studied serial samples of peripheral blood from 10 patients with 25% to 50% surface area burns and 8 trauma patients (injury Severity Score, 25-57). Patients were compared with 18 healthy volunteers. The study was focused on the first 10 days after injury before the onset of sepsis or the systemic inflammatory response syndrome. Plasma samples were assayed for LPS, and adherent cells from the blood were studied for basal and LPS-stimulated production of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and interleukin-6 (IL-6). The correlation of increased plasma LPS with TNF-alpha production was studied as was the association of increased plasma LPS and increased TNF-alpha production with subsequent septic complications. We also studied a mouse model of 25% burn injury. Burn mice were compared with sham burn control subjects. Plasma samples were assayed at serial intervals for LPS, and adherent cells from the spleens were studied for basal- and LPS-stimulated production of TNF-alpha, IL-1 beta, and IL-6. Expression of the messenger RNAs for IL-1 beta and TNF-alpha also was measured. The relation of increased TNF-alpha production with mortality from a septic challenge, cecal ligation and puncture (CLP), was determined. Finally, the effect of administration of LPS to normal mice on subsequent mortality after CLP and on TNF-alpha production was studied. RESULTS: Elevated plasma LPS (> 1 pg/mL) was seen in 11 of the 18 patients within 10 days of injury and in no normal control subjects. In this period, patients as compared with control subjects showed increased stimulated production of TNF-alpha, IL-1 beta, and IL-6. Increased TNF-alpha production was not correlated with elevated plasma LPS in the same patients. Neither increased plasma LPS nor increased TNF-alpha production early after injury was correlated with subsequent development of systemic inflammatory response syndrome or sepsis in the patients. Burn mice, as compared with sham burn control subjects, showed elevated plasma LPS levels chiefly in the first 3 days after injury. Increased stimulated production of proinflammatory cytokines by adherent splenocytes from the burn mice also was seen at multiple intervals after injury and did not correlate with mortality from CLP. Increased production of TNF-alpha and IL-1 beta was associated with increased expression of messenger RNAs for these cytokines. Finally, two doses of 1 ng LPS administered 24 hours apart to normal mice had no effect on mortality from CLP performed 7 days later nor on the production of TNF-alpha at the time of CLP. CONCLUSIONS: These findings call into question the idea that circulating LPS is the trigger for increased proinflammatory cytokine production, systemic inflammatory response syndrome, and septic complications in injured patients.     

Modulation of interleukin-6 by beta2-adrenoceptor in endotoxin-stimulated renal macrophage cells.

BACKGROUND: Activation of the cAMP signaling pathway by means of beta2-adrenoceptor agonists has been shown to up-regulate interleukin-6 (IL-6) gene expression and to stimulate IL-6 production in macrophage cells. However, whether beta2-adrenoceptor activation can also modify the rate of IL-6 production in macrophage cells activated by the bacterial endotoxins has not yet been determined. Using renal resident macrophage cells treated with endotoxin, lipopolysaccharide (LPS), and beta2-adrenoceptor agonist, terbutaline, we investigated the role of cAMP pathway, tumor necrosis factor (TNF)-alpha and mitogen-activated protein kinase (MAPK) pathway (p42/p44) in regulating IL-6 production. METHODS: IL-6 protein, mRNA, and promoter activity were measured in these cells exposed to LPS (1 microg/ml) and/or terbutaline (10(-9) to 10(-6) M). Furthermore, the time course effects of terbutaline on cAMP, MAPK (p42/p44), and TNF-alpha release were evaluated in the cells. RESULTS: Terbutaline at high concentrations (10(-6) M) significantly up-regulated IL-6 by approximately 25% (P<0.05), whereas at a lower concentration (10(-8) M), it down-regulated IL-6 production by 42% (P<0.05). Terbutaline (10(-8) and 10(-6) M) caused a concentration- and time-dependent stimulation of cAMP (P<0.05) and TNF production (P<0.05) and a time-dependent decrease in MAPK activity (P<0.05). Following the addition of a cAMP inhibitor, IL-6 promoter activity was correlated with TNF-alpha levels and MAPK activity. CONCLUSIONS: A biphasic effect of beta2-adrenoceptor agonist on IL-6 production in renal resident macrophage cells became apparent when LPS was exposed to the cells. The terbutaline-induced down-regulation of IL-6 gene production was mediated by an inhibitory effect of terbutaline on TNF-alpha, which was exerted through the MAPK and cAMP pathways, whereas the up-regulation appeared to be due to a direct action of intracellular cAMP.     

Inosine exerts a broad range of antiinflammatory effects in a murine model of acute lung injury

OBJECTIVE: To investigate the effects of inosine on the acute lung inflammation induced by lipopolysaccharide (LPS) in vivo and on the activation and cytotoxicity elicited by proinflammatory cytokines on human lung epithelial (A549) cells in vitro. SUMMARY BACKGROUND DATA: Inosine is an endogenous purine recently shown to exert immunomodulatory and antiinflammatory effects. METHODS: Mice challenged with intratracheal LPS (50 microg) were treated after 1, 6, and 12 hours with inosine (200 mg/kg intraperitoneal) or vehicle. After 24 hours, bronchoalveolar lavage fluid was obtained to measure proinflammatory (tumor necrosis factor-alpha [TNF-alpha], interleukin [IL]-1beta, IL-6), and antiinflammatory (IL-10, IL-4) cytokines, chemokines (MIP-1alpha and MIP-2), myeloperoxidase activity and total cell counts, nitric oxide production, and proteins. Lung histology and immunohistochemical detection of 3-nitrotyrosine, a marker of nitrosative stress, were performed in inflated-fixed lungs. In vitro, cell viability and production of the chemokine IL-8 were evaluated in A549 cells stimulated with a mixture of cytokines in the presence or absence of inosine. RESULTS: Inosine downregulated the LPS-induced expression of TNF-alpha, IL-1beta, IL-6 and MIP-2 and tended to reduce MIP-1alpha, whereas it enhanced the production of IL-4. Total leukocyte counts, myeloperoxidase, nitric oxide production, and proteins were all significantly decreased by inosine. The purine also improved lung morphology and suppressed 3-nitrotyrosine staining in the lungs after LPS. Inosine attenuated the cytotoxicity and the expression of IL-8 induced by proinflammatory cytokines in A549 cells. CONCLUSIONS: Inosine largely suppressed LPS-induced lung inflammation in vivo and reduced the toxicity of cytokines in lung cells in vitro. These data support the proposal that inosine might represent a useful adjunct in the therapy of acute respiratory distress syndrome.     

Anti-inflammatory effects of sevoflurane and mild hypothermia in endotoxemic rats

BACKGROUND: Volatile anesthetics and hypothermia attenuate the inflammatory response. We aimed to compare the anti-inflammatory effects of sevoflurane and mild hypothermia during experimental endotoxemia in the rat. METHODS: Anesthetized, ventilated Sprague-Dawley (SD) rats were randomly treated as follows (n = 6 per group): lipopolysaccharide (LPS) only, animals received LPS [LPS 5 mg/kg, intravenously (i.v.)] with no further treatment. In the LPS-hypothermia group, rats were cooled down to a temperature of 33 degrees C 15 min after LPS-injection (LPS 5 mg/kg i.v.). In animals of the LPS-sevoflurane group, sevoflurane inhalation (1 MAC) was initiated 15 min after induction of endotoxemia. The LPS-sevoflurane-hypothermia group received combined sevoflurane and hypothermia 15 min after induction of endotoxemia. A Sham group served as control without endotoxemia or treatment. After 4 h of endotoxemia, plasma levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and IL-10 were measured. Alveolar macrophages (AM) were ex vivo cultured for nitrite assay. RESULTS: Inhalation of sevoflurane significantly attenuated plasma levels of TNF-alpha (-60%, P < 0.05) and IL-1beta (-68%, P < 0.05) as compared with the LPS-only group. Hypothermia and its combination with sevoflurane significantly reduced TNF-alpha levels (-46% and -58%, each P < 0.05), but not IL-1beta. Application of mild hypothermia and also its combination with sevoflurane resulted in a significant increase in plasma IL-10 as compared with endotoxemic controls. Nitrite release from AM was found to be significantly suppressed by sevoflurane (-83%), hypothermia (-73%) and by the combination of both (-67%) (P < 0.05, each). CONCLUSION: Our data suggest that sevoflurane and mild hypothermia attenuate the inflammatory response during endotoxemia in vivo thus contributing to their beneficial role in clinical organ protection.     

p38丝裂原活化蛋白激酶信号转导通路在严重烧伤大鼠枯否细胞肿瘤坏死因子α和白细胞介素1β产生中的作用

目的 探讨p38丝裂原活化蛋白激酶(MAPK)信号转导通路在严重烧伤大鼠枯否细胞(KCs)促炎性细胞因子肿瘤坏死因子α(TNF-α)和白细胞介素1β(IL-1β)产生中的作用。方法 健康成年的雄性SD大鼠32只,随机分为：假烫组;假烫 SB203580组;烧伤对照组;烧伤 SB203580组,每组8只。假烫或烧伤24h后分离出肝脏KCs,培养18h后加入50ng／ml的LPS进行刺激,18h后取上清液,用酶联免疫吸附法(ELISA)测定TNF-α和IL-1β的含量,并收集KCs,实时逆转录聚合酶链反应检测KCs内TNF-α和IL-1β mRNA表达的改变,蛋白印迹(Western blot)法检测KCs中p38MAPK和JNK活性的变化。结果 烧伤大鼠分离出的KCs培养上清液中TNF-α和IL-1β含量、KCs中TNF-α和IL-1β mRNA的表达均较假烫组的明显增强,同时KCs中p38 MAPK活性和JNK活性升高,SB203580能显著抑制大鼠KCs上清液中TNF-α和IL-1β含量、KCs中TNF-α和IL-1β mRNA的表达和p38MAPK活性的升高,对JNK活性无影响。结论p38MAPK信号转导通路介导了严重烧伤大鼠KCs促炎性细胞因子TNF-α和IL-1β的产生。     

The immunomodulatory effects of prolonged intravenous infusion of propofol versus midazolam in critically ill surgical patients

Both propofol and midazolam are known to inhibit immune function. The aim of this study was to investigate cytokine production in critically ill surgical patients as early markers of immune response to prolonged infusion of propofol and midazolam. The study enrolled 40 elective patients who were to receive long-term sedation for more than 2 days. Patients were randomly allocated to one of two equally sized groups. Central venous blood samples for measurement of interleukin-1beta (IL-1beta), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-8 (IL-8), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) were drawn prior to the start and after 48 h of infusion. After 48 h, propofol caused significant increases in IL-1beta (24%), IL-6 (23%) and TNF-alpha (4.8 times) levels, while midazolam caused significant decreases in IL-1beta (21%), IL-6 (21%) and TNF-alpha (19%). Both agents caused significant decreases in IL-8 levels (propofol: 30%, midazolam: 48%, p < 0.05). Propofol caused significant decreases in IL-2 levels (68%, p < 0.001) but increases in IFN-gamma (30%, p < 0.05), whereas there was no significant change with midazolam compared with the pre-infusion level. In conclusion, during 48 h of continuous infusion, propofol stimulated, while midazolam suppressed, the production of the pro-inflammatory cytokines IL-1beta, IL-6 and TNF-alpha, and both caused suppression of IL-8 production. Propofol inhibited IL-2 production and stimulated IFN-gamma production, whereas midazolam failed to do so. Therefore, sedative agents may have clinical implications in high-risk and immunocompromised patients.     

Reduced capacity of whole blood leucocytes to express tumour necrosis factor-alpha and interleukin-10 following major orthopaedic surgery

BACKGROUND: Severe trauma is a challenge to the immune response and may cause reduced immune capacity. As a marker of decreased cellular activity, studies with ex vivo lipopolysaccharide (LPS) stimulation of whole blood or isolated mononuclear cells from injured patients have revealed reduced production of inflammatory cytokines. To gain further insight into immune alterations in orthopaedic surgery, we studied LPS-induced tumour necrosis factor (TNF)-alpha and interleukin (IL)-10 in whole blood of patients during peri- and postoperative phases of total hip replacement. METHODS: Four females and 3 males undergoing elective total hip replacement were included in the study. Ex vivo LPS-induced TNF-alpha and IL-10 were measured in a whole blood assay before, during and at 1 and 6 days after operation. In addition, the counts of white blood cells were determined. RESULTS: During the operation, there were significant reductions in the number of monocytes, but at day 1 and 6 after surgery, there were significant increases as compared to the levels before surgery. The capacity of whole blood to express TNF-alpha and IL-10 did not change significantly during the operation and the following postoperative day. At day 6, however, there were significant reductions in expression of both TNF-alpha and IL-10 as compared to the levels before the operation. In relation to the values of monocytes, there was a significant reduction in the expression of TNF-alpha also at day 1 after operation. CONCLUSION: Our data indicate that in the course of at least 6 days after a major orthopaedic trauma, there is suppression of the whole blood capacity to express the inflammatory cytokine TNF-alpha and the anti-inflammatory cytokine IL-10 when exposed to LPS. During this time, then, the patient is particular susceptible to septic complications.     

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.     

Inflammatory response of interleukin-1beta and interleukin-6 in septic rats undergoing laparotomy and laparoscopy

It is generally accepted that greater inflammatory response is observed after laparotomy than laparoscopy in animal models. However, in a previous study, we reported there are no significant differences in the systemic response of tumor necrosis factor (TNF)-alpha between the laparotomy and laparoscopy groups in a rat model of endotoxic shock. The present study extends this investigation to the inflammatory response of 2 additional proinflammatory mediators, interleukin (IL)-1beta and IL-6, in septic rats after laparotomy and laparoscopy in the same animal model. Rats received lipopolysaccharide (LPS) intraperitoneally and underwent laparotomy (n = 5), laparoscopy (n = 5), or no surgical intervention (n = 5). A control group received anesthesia only (n = 5). Serum IL-1beta and IL-6 levels were significantly higher at 2, 4, and 8 hours after LPS injection and were equally suppressed in the laparotomy and laparoscopic groups (P < 0.05). Liver IL-1beta mRNA and protein levels were significantly inhibited at 2, 4, and 8 hours in the laparotomy and laparoscopic groups. Liver IL-6 mRNA (2 and 4 hours) and protein (4 hours) levels were also suppressed significantly in both the laparotomy and laparoscopic groups (P < 0.05). There were no significant differences in hepatic levels of mRNA and protein of IL-beta and IL-6 in both the laparotomy and laparoscopic groups. These results extend our previous finding demonstrating the suppression of TNF-alpha in both the laparotomy and laparoscopic groups. The behavior of the markers used in our study demonstrated that the inflammatory response does not differ between laparotomy and laparoscopic surgery in our rat model of endotoxic shock.     

Perioperative GLY-GLN infusion diminishes the surgery-induced period of immunosuppression: accelerated restoration of the lipopolysaccharide-stimulated tumor necrosis factor-alpha response

OBJECTIVE: To investigate whether the administration of different glutamine-containing dipeptides, glycyl-l-glutamine (GLY-GLN) and l-alanyl-l-glutamine, has a differing impact on perioperative immunomodulation. SUMMARY BACKGROUND DATA: Surgery leads to transitory immunosuppression, which is associated with decreased plasma glutamine (GLN) levels and increased susceptibility to infection and sepsis. A useful tool to detect immunocompetence is the ex vivo lipopolysaccharide (LPS)-stimulated tumor necrosis factor alpha (TNF-alpha) secretion in whole blood. METHODS: Forty-five patients undergoing major abdominal surgery were randomized prospectively to receive 0.5 g/kg/24 h GLN dipeptides administered as GLY-GLN or as ALA-GLN or isonitrogenous Vamin (a GLN-free amino acid solution; control group) as a continuous infusion over 72 hours, starting 24 hours before surgery. Blood samples were collected before infusion, at the end of surgery, and 48 hours postoperatively to determine the TNF-alpha release into whole blood stimulated with LPS. Groups were compared by analysis of variance. RESULTS: The groups were comparable in age, gender distribution, and length of operative time. At the end of surgery a significant reduction in ex vivo LPS-stimulated TNF-alpha production was observed in all groups. In patients who received GLY-GLN, the induced TNF-alpha production was restored after 48 hours. CONCLUSIONS: In this study perioperative infusion of GLY-GLN reduced immunosuppression. The effect of GLN-containing dipeptides seems to be different when administered in glycine or alanine form.