Endotoxin-stimulated Monocytes Research Articles

Extracellular vesicle glucose transporter-1 and glycan features in monocyte-endothelial inflammatory interactions

Monocyte-induced endothelial cell inflammation is associated with multiple pathological conditions, and extracellular vesicles (EVs) are essential nanosized components of intercellular communication. EVs derived from endotoxin-stimulated monocytes were previously shown to carry pro-inflammatory proteins and RNAs. The role of glucose transporter-1 (GLUT-1) and glycan features in monocyte-derived EV-induced endothelial cell inflammation remains largely unexplored. This study demonstrates that EVs derived from endotoxin-stimulated monocytes activate inflammatory pathways in endothelial cells, which are partially attributed to GLUT-1. Alterations in glycan features and increased levels of GLUT-1 were observed in EVs derived from endotoxin-stimulated monocytes. Notably, inhibition of EV-associated GLUT-1, through the use of fasentin, suppressed EV-induced inflammatory cytokines in recipient endothelial cells.

Microvesicular Caspase-1 Mediates Lymphocyte Apoptosis in Sepsis

ObjectiveImmune dysregulation during sepsis is poorly understood, however, lymphocyte apoptosis has been shown to correlate with poor outcomes in septic patients. The inflammasome, a molecular complex which includes caspase-1, is essential to the innate immune response to infection and also important in sepsis induced apoptosis. Our group has recently demonstrated that endotoxin-stimulated monocytes release microvesicles (MVs) containing caspase-1 that are capable of inducing apoptosis. We sought to determine if MVs containing caspase-1 are being released into the blood during human sepsis and induce apoptosis..DesignSingle-center cohort studyMeasurements50 critically ill patients were screened within 24 hours of admission to the intensive care unit and classified as either a septic or a critically ill control. Circulatory MVs were isolated and analyzed for the presence of caspase-1 and the ability to induce lymphocyte apoptosis. Patients remaining in the ICU for 48 hours had repeated measurement of caspase-1 activity on ICU day 3.Main ResultsSeptic patients had higher microvesicular caspase-1 activity 0.05 (0.04, 0.07) AFU versus 0.0 AFU (0, 0.02) (p<0.001) on day 1 and this persisted on day 3, 0.12 (0.1, 0.2) versus 0.02 (0, 0.1) (p<0.001). MVs isolated from septic patients on day 1 were able to induce apoptosis in healthy donor lymphocytes compared with critically ill control patients (17.8±9.2% versus 4.3±2.6% apoptotic cells, p<0.001) and depletion of MVs greatly diminished this apoptotic signal. Inhibition of caspase-1 or the disruption of MV integrity abolished the ability to induce apoptosis.ConclusionThese findings suggest that microvesicular caspase-1 is important in the host response to sepsis, at least in part, via its ability to induce lymphocyte apoptosis. The ability of microvesicles to induce apoptosis requires active caspase-1 and intact microvesicles.

ROS-induced ATF3 causes susceptibility to secondary infections during sepsis-associated immunosuppression.

Sepsis, sepsis-induced hyperinflammation and subsequent sepsis-associated immunosuppression (SAIS) are important causes of death. Here we show in humans that the loss of the major reactive oxygen species (ROS) scavenger, glutathione (GSH), during SAIS directly correlates with an increase in the expression of activating transcription factor 3 (ATF3). In endotoxin-stimulated monocytes, ROS stress strongly superinduced NF-E2-related factor 2 (NRF2)-dependent ATF3. In vivo, this ROS-mediated superinduction of ATF3 protected against endotoxic shock by inhibiting innate cytokines, as Atf3(-/-) mice remained susceptible to endotoxic shock even under conditions of ROS stress. Although it protected against endotoxic shock, this ROS-mediated superinduction of ATF3 caused high susceptibility to bacterial and fungal infections through the suppression of interleukin 6 (IL-6). As a result, Atf3(-/-) mice were protected against bacterial and fungal infections, even under conditions of ROS stress, whereas Atf3(-/-)Il6(-/-) mice were highly susceptible to these infections. Moreover, in a model of SAIS, secondary infections caused considerably less mortality in Atf3(-/-) mice than in wild-type mice, indicating that ROS-induced ATF3 crucially determines susceptibility to secondary infections during SAIS.

Mg2+ Is Required for Optimal Folding of the γ-Carboxyglutamic Acid (Gla) Domains of Vitamin K-Dependent Clotting Factors At Physiological Ca2+

Abstract 1172Equilibrium dialysis experiments indicate that the Gla domain of coagulant protein factor (F) VIIa and of anticoagulant protein C (PC) each bind seven Ca2+ in the absence and four in the presence of physiologic Mg2+ (0.6 mM). The previous x-ray structure of FVIIa/soluble (s) tissue factor (TF) in the presence of 5 mM Ca2+/45 mM Mg2+ (5Ca/45Mg) (Bajaj et al., J Biol Chem, 281, 24873–24888, 2006) revealed that sites 2,3,5 and 6 were occupied by Ca2+ while sites 1, 4 and 7 contained Mg2+ (Tulinsky numbering, Biochemistry 31, 2554–2566, 1992). Since these concentrations of metal ions were supraphysiologic, we sought to obtain crystals under concentrations of Ca2+ and Mg2+ approaching those found in blood. We have solved the structures of FVIIa/sTF under three new conditions: 2.5 mM Ca2+/1.25 mM Mg2+ at 2.8 Å (2.5Ca/1.25Mg); 5 mM Ca2+/2.5 mM Mg2+ at 1.8 Å (5Ca/2.5Mg); and 45 mM Ca2+/5 mM Mg2+ at 1.7 Å (45Ca/5Mg). For 2.5Ca/1.25Mg, the Gla domain of FVIIa was disordered indicating insufficient concentrations of Ca2+/Mg2+ to fold the Gla domain. Interestingly, folding of the Ω-loop for 5Ca/2.5Mg was similar to that reported under 5Ca/45Mg, with identical positions for four Ca2+ and three Mg2+. In contrast, while the folding of the Ω-loop at 45Ca/5Mg was similar to that reported only in the presence of Ca2+ (Banner et al., Nature, 380, 41–46,1996); however, positions 1 and 7 contained Mg2+ (Fig. 1A). Thus, it would appear that four Ca2+ and three Mg2+ ions are bound to the circulating FVII/FVIIa. Moreover, circulating FVII/FVIIa will need high Ca2+ concentrations to have its position four switched from Mg2+ to Ca2+. [Display omitted] All seven metal sites in the structure of PC-Gla complexed to endothelial protein C receptor (EPCR) had been refined with Ca2+ despite the presence of 5 mM Ca2+ and 5 mM Mg2+ in the crystallization buffer (Oganesyan et al., J Biol Chem, 277, 24851–24854, 2002). Based on our findings with VIIa/sTF, we revisited the PC-Gla/EPCR structure. We determined that positions 1 and 7 are Mg2+ sites while 2, 3, 4, 5 and 6 are Ca2+ sites in the PC-Gla/EPCR structure (Fig. 1B). This contrasts to four Ca2+ and three Mg2+ bound to VIIa/TF at 5Ca/2.5Mg. Since five Ca2+ and two Mg2+ were seen in VIIa/sTF at 45Ca/5Mg, it would appear that position 4 can accommodate either Ca2+ or Mg2+ depending upon conditions. We propose that at near physiologic Ca2+ and Mg2+, ligation of the Gla domain with EPCR and possibly phospholipid (PL) membranes facilitates substitution of the Mg2+ bound at the position four with Ca2+. This metal switch may be essential to achieve a favorable conformation of the Gla domain Ω-loop for optimal ligand of PL binding.Mg2+ also enhanced the Ca2+-dependent interaction of FVIIa or activated protein C (APC) to PL assessed by surface plasmon resonance. Binding of FVIIa and APC each was saturated by the physiological concentration of Ca2+ (1.1 mM) in the presence of physiological Mg2+ (0.6 mM). In contrast, only half-saturable binding was observed at the physiological concentration of Ca2+ in the absence of Mg2+. Further, 0.6 mM Mg2+ potentiated (∼2.5-fold) the PL-dependent activation of FX by FVIIa/TF at 1.1 mM Ca2+. Similarly, Mg2+ potentiated (∼3-fold) the activation of FX by FVIIa/TF assembled on the endotoxin-stimulated monocyte surface. PL-dependent inactivation of FVa by APC was also enhanced ∼3-fold by 0.6 mM Mg2+ at 1.1 mM Ca2+. At saturating Ca2+ (5 mM), the activation of FX by FVIIa/TF or the inactivation of FVa by APC was similar to that with 1.1 mM Ca2+/0.6 mM Mg2+. Thus Mg2+ at physiologic concentrations augments PL- or natural membrane-dependent coagulation and anticoagulation at the plasma concentration of Ca2+.We propose that vitamin K-dependent clotting and anti-clotting proteins circulate in blood with four Ca2+ ions bound. The remaining three (or more in FIX and FX) divalent metal binding sites in each Gla domain are occupied by Mg2+. The conformation of the Ω-loop in circulating vitamin K- dependent proteins is not favorable for binding to PL but it is achieved by switching Mg2+ at position four to Ca2+. Thus, the metal ion at position four regulates PL-dependent coagulation and anticoagulation reactions. Disclosures:No relevant conflicts of interest to declare.

Loss of Cystic Fibrosis Transmembrane Conductance Regulator Function Enhances Activation of p38 and ERK MAPKs, Increasing Interleukin-6 Synthesis in Airway Epithelial Cells Exposed to Pseudomonas aeruginosa

In cystic fibrosis (CF), the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) translates into chronic bacterial infection, excessive inflammation, tissue damage, impaired lung function and eventual death. Understanding the mechanisms underlying this vicious circle of inflammation is important to design better therapies for CF. We found in CF lung biopsies increased immunoreactivity for p38 MAPK activity markers. Moreover, when compared with their non-CF counterpart, airway epithelial cells expressing the most common mutation in CF (CFTRDeltaF508) were more potent at inducing neutrophil chemotaxis through increased interleukin (IL)-6 synthesis when challenged with Pseudomonas aeruginosa diffusible material. We then discovered that in CFTRDeltaF508 cells, the p38 and ERK MAPKs are hyperactivated in response to P. aeruginosa diffusible material, leading to increased IL-6 mRNA expression and stability. Moreover, although TLR5 contributes to p38 MAPK activation upon P. aeruginosa challenge, it only played a weak role in IL-6 synthesis. Instead, we found that the production of reactive oxygen species is essential for IL-6 synthesis in response to P. aeruginosa diffusible material. Finally, we uncovered that in CFTRDeltaF508 cells, the extracellular glutathione levels are decreased, leading to a greater sensitivity to reactive oxygen species, providing an explanation for the hyperactivation of the p38 and ERK MAPKs and increased IL-6 synthesis. Taken together, our study has characterized a mechanism whereby the CFTRDeltaF508 mutation in airway epithelial cells contributes to increase inflammation of the airways.

Fibronectin-adherent monocytes express tissue factor and tissue factor pathway inhibitor whereas endotoxin-stimulated monocytes primarily express tissue factor: physiologic and pathologic implications.

Monocytes are critical cells in initiating physiologic and/or pathologic tissue factor (TF)-induced intravascular and extravascular coagulation. Monocytes constitutively express small amounts of TF and tissue factor pathway inhibitor (TFPI). Non-adherent lipopolysaccharide (LPS)-stimulated monocytes express significant amounts of TF; however, increased expression of TFPI by these cells is controversial. Further, whether fibronectin-adherent monocytes (mimicking conditions in the extravascular space) express sufficient TFPI to inhibit TF-procoagulant activity (PCA) is unknown. To compare TF and TFPI expression by fibronectin-adherent and LPS-stimulated non-adherent monocytes. Monocytes were isolated from normal peripheral blood, adhered to fibronectin or stimulated with lipopolysaccharide (LPS) under non-adherent conditions and examined for expression of TF and TFPI using quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), ELISA and factor X (FX) activation. Under LPS-free conditions, the fibronectin-adherent monocyte TF mRNA, antigen and activity were markedly upregulated. Notably, cell and microparticle (MP)-associated TF and alternatively spliced TF (asTF) were all upregulated. TFPI mRNA and antigen were also upregulated in the fibronectin-adherent monocytes, which significantly inhibited TF-PCA. TFPI mRNAs for both alpha and beta forms were detected. The peak in TFPI activity occurred in tandem with the peak in TF-PCA. In contrast, LPS-stimulated monocytes, which expressed cell and MP-associated TF and asTF, demonstrated only minimal expression of TFPI as determined by mRNA, antigen or inhibition of TF activity. Both LPS-stimulated and fibronectin-adherent monocytes demonstrate a procoagulant phenotype by expressing TF but only fibronectin-adherent monocytes express significant amounts of TFPI to control thrombin generation and fibrin formation in the context of extravascular space.

Cyclooxygenase inhibition in human monocytes increases endotoxin-induced TNFα without affecting cyclooxygenase-2 expression

Human endotoxin-stimulated adherent monocytes were used in order to determine whether or not NSAIDs influence cyclooxygenase-2 and/or tumor necrosis factor (TNF)α expression within the range of inhibitor concentrations that are required to suppress prostaglandin biosynthesis. Exogenous prostaglandin E 2 (IC 50<5 nM) inhibited endotoxin-induced TNFα mRNA and protein while, up to 1 μM, it did not significantly affect cyclooxygenase-2 mRNA expression. Similar results were obtained using the membrane-permeable cAMP analogue db-cAMP, which caused preferential inhibition of TNFα expression. Indomethacin or lysine-acetylsalicylic acid concentration-dependently inhibited prostaglandin E 2 biosynthesis and, at concentrations causing near-complete inhibition, enhanced TNFα mRNA and protein expression without significantly influencing cyclooxygenase-2 mRNA. In addition, by facilitating endotoxin-induced TNFα expression, indomethacin or lysine-acetylsalicylic acid counteracted dexamethasone-induced inhibition of TNFα biosynthesis, thereby exhibiting an effect opposite to that of exogenous prostaglandin E 2. The results suggest that in human endotoxin-stimulated monocytes, NSAIDs can enhance TNFα expression through inhibition of cyclooxygenase and the resulting decrease in prostanoid biosynthesis.

Platelets enhance endotoxin-induced monocyte tissue factor (TF) activity in the horse

Endotoxaemia is the leading cause of death in horses. Disseminated intravascular coagulation (DIG), stimulated by induced monocyte proteins, is a prominent feature. Monocyte–platelet cellular interactions are central to the vascular dysfunction produced by circulating endotoxin and are implicated in many thrombotic diseases in the horse. This study reports that endotoxin (0.01–10 μg ml −1) and blood platelets (2.5 × 10 7 – 1× 10 8 ml −1) are potent inducers of expression and activity of monocyte tissue factor (TF), the primary activator of the blood coagulation protease cascade.The co-incubation of endotoxin-stimulated monocytes with platelets resulted in greater production of this protein. Cycloheximide (1 mM) inhibited part of the stimulatory effect of endotoxin and/or platelets, the uninhibited part indicating de-encryption of cell-surface TF. Hence, platelets are considered to be an important component of the endotoxin-stimulated response of equine monocytes. The role of platelets as potent stimulators of endotoxin-stimulated monocyte proteins and mediators in vitro is likely to be of significance in vivo in the clinical manifestations and management of endotoxaemia in the horse.

Inhibition of Prostaglandin Biosynthesis in Human Endotoxin-Stimulated Peripheral Blood Monocytes: Effects of Caffeine

There is an ongoing debate about possible advantages of the coadministration of caffeine with cyclooxygenase (COX) inhibitors in the treatment of pain. There are results suggesting interference by caffeine with COX expression and activity in rat immune cells. In the present study, we have used, therefore, human endotoxin-stimulated monocytes to investigate a possible influence of caffeine on indometacin-induced inhibition of prostaglandin E₂ (PGE₂) formation. Endotoxin caused a concentration- and time-dependent increase in immunoreactive PGE₂ that was dependent on CD14-mediated mechanisms. In order to investigate pharmacological inhibition of the COX activity, a submaximal concentration of 1 ng/ml endotoxin (4 h exposure time) was used. Indometacin caused a concentration-dependent inhibition of PGE₂ with an apparent IC₅₀ of 8.9 ± 1.4 × 10^–9 mol/l and an I_max of 1 x 10^–7 mol/l. Caffeine (5 × 10^–6 to 1.5 × 10^–4 mol/l) on its own produced no statistically significant effect on endotoxin-induced PGE₂ formation. In the presence of caffeine (5 × 10^–6 to 1.5 × 10^–4 mol/l), inhibition of PGE₂ biosynthesis by indometacin (1 × 10^–8 mol/l) was not significantly altered. These results show that, in human monocytes, caffeine, up to concentrations severalfold higher than those reached in patients, has no significant effect on endotoxin-induced PGE₂ formation nor on its inhibition by indometacin.

Adenosine Inhibits Tissue Factor Expression by LPS-stimulated Human Monocytes: Involvement of the A3 Adenosine Receptor

Tissue Factor (TF), an integral membrane glycoprotein that initiates the extrinsic pathway of blood coagulation, is thought to play a major part in coronary acute events. Adenosine, an endogenous nucleoside produced by the degradation of intracellular adenosine triphosphate, has been shown to exert many cardioprotective effects via an inhibition of platelets and neutrophils. This study was conducted to determine whether adenosine (ADO) could modulate the expression of TF by human monocytes. We found that ADO inhibited TF antigen and activity on endotoxin-stimulated monocytes in a dose-dependent manner. The mechanism was at least pre-translational since ADO caused a change in the TF mRNA level. Using ADO receptor-specific analogs, we showed that highly selective A3 agonist N6-(3-iodobenzyl)-adenosine-5'-N'-methyluronamide (IB-MECA) inhibited LPS-induced TF activity expression more potently than A1 agonist R-phenylisopropyladenosine (R-PIA) and A2 agonist CGS 2180. Furthermore, A1/A3 antagonist, xanthine amine congener (XAC) blocked the effect of ADO whereas A2a, A2b and A1 antagonists were ineffective. In addition, we observed that ADO agonists inhibited monocyte TF expression in LPS-stimulated whole blood. The rank order of agonist potency suggested that A2 and A3 receptors might be involved (2-Cado > CGS = IB-MECA > R-PIA). This was supported by the fact that A2 and A3 antagonists reversed the action of 2-Cado. We conclude that TF inhibition by ADO on human purified monocytes involved A3 receptors.

A tissue specific IL-1 receptor antagonist homolog from the IL-1 cluster lacks IL-1, IL-1ra, IL-18 and IL-18 antagonist activities.

Interleukin (IL)-1-like protein 1 (IL-1L1) is a 155-amino acid protein that shares 27% identity with IL-1beta and 47% with IL-1 receptor antagonist (IL-1ra). A 2.7-kb IL-1L1 mRNA was cloned from human placenta and is detectable in the trophoblastic cell line JEG-3, in macrophages and in endotoxin-stimulated monocytes. Expression of IL-1L1 is much less abundant and less widespread than IL-1ra. We have determined the human and mouse IL-1L1 cDNA sequences and the complete sequence of the human gene, IL1L1. IL1L1 consists of four coding exons, has two alternative non-coding first exons, lies between IL1B and IL1RN, is orientated in the same direction as IL1RN and is separated from it by approximately 53 kb. The predicted IL-1L1 protein lacks both signal sequence and glycosylation signals. A 17-kDa protein was recovered by immunoprecipitation with IL-1L1-specific antibodies from JEG-3. IL-1L1 did not stimulate IL-6 production from primary human fibroblasts or human umbilical vein endothelial cells nor did it block the IL-1alpha or IL-1beta-dependent activation of IL-6 expression. We conclude, contrary to a recent suggestion made by others, that IL-1L1 is not a functional IL-1ra. IL-1L1 also had no detectable agonistic or antagonistic effect on IFN-gamma production in response to IL-18 in KG-1 cells.

Sepsis after major visceral surgery is associated with sustained and interferon-γ–resistant defects of monocyte cytokine production

Background: Recent clinical trials failed to demonstrate beneficial effects of anti-inflammatory sepsis therapy. The present study therefore asked the following questions: Is there evidence for immunosuppression during postoperative sepsis? When, during the septic course, may immunosuppression develop? Can defective cellular functions be restored by in vitro treatment with interferon-γ (IFN-γ)? Methods: The study included 35 patients with sepsis after major visceral surgery and 85 control patients. Monocyte secretion of interleukin (IL)-1β, IL-12 p40 and p70, IL-18, tumor necrosing factor, and IL-10 with or without IFN-γ treatment and its correlation with the course and outcome of postoperative sepsis were determined. Results: Postoperative sepsis was associated with an immediate defect of endotoxin-stimulated monocyte production of IL-12 p40, IL-1β, and IL-10 in both surviving and nonsurviving patients. During the final phase of postoperative sepsis, a significant recovery of IL-12 p40 and IL-1β secretion, but not of IL-10 production, correlated with survival. Despite the exposure of monocytes in vitro to IFN-γ for 16 hours, the production of the biologically active IL-12 p70 heterodimer was severely suppressed both in survivors and nonsurvivors, although the secretion of the p40 subunit was restored. In contrast, IFN-γ treatment resulted in a significant suppression of monocyte IL-1β production in all patient subgroups. Alterations of monocyte tumor necrosing factor secretion were not observed. The production of IL-18 was below the limits of detection in all samples. Conclusions: Postoperative sepsis was associated with immediate monocyte defects that affected both pro- and anti-inflammatory cytokine secretion, which suggests that immunosuppression is a primary rather than a compensatory response to a septic challenge. Sepsis survival correlated with the recovery of the proinflammatory, but not the anti-inflammatory, response. The treatment of monocytes with IFN-γ did not reconstitute defective proinflammatory cytokine production. (Surgery 2000;127:309-15.)

Ex vivo interleukin (IL)-1 beta, IL-6, IL-12 and tumor necrosis factor-alpha responsiveness with monocytes from patients with head and neck carcinoma.

Seventy newly diagnosed Caucasian male patients with head and neck squamous cell carcinomas (HNSCC) were included in the study. All patients were less than 80 years of age, with no cachexia or auto-immune disease, and they were not taking immuno-active medications. Monocytes from these patients were cultured in vitro and supplemented with autologous serum under ex vivo conditions or cultured with serum-free medium. Comparison was made to monocytes from 59 patients with benign HN diseases. Similar physical activity levels prior to testing as well as a minimum stress load were ensured in both groups. Increased monocyte supernatant levels were determined under ex vivo conditions of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumor necrosis factor (TNF)-alpha, but not of interleukin-12 (IL-12) with endotoxin stimulated monocytes of HNSCC patients compared to control conditions. Increased monokine levels were not present with mononuclear cell cultures stimulated with a T-cell general stimulatory agent or with purified monocytes not specifically stimulated. With endotoxin-stimulated monocytes under in vitro conditions, an increased supernatant was shown for TNF-alpha, but not IL-6. With serum from the different patients cultured with monocytes employed from a healthy control, no difference between the groups was shown in the IL-6 and TNF-alpha response to endotoxin stimulation. The differences in IL-1 beta and TNF-alpha, but not IL-6 levels were differentiated statistically from the smoking and alcohol histories of the patients. These findings suggest that the function of monocytes in general, and thus possibly all mononuclear phagocyte system cells in HNSCC patients, are altered.

Endotoxin-Stimulated Monocytes Release Multiple Forms of IL-1β, Including a ProIL-1β Form Whose Detection Is Affected by Export

Abstract The processing and release of 31-kDa proIL-1β to the mature 17-kDa form of IL-1β are still poorly understood. To help elucidate the mechanisms involved in IL-1β processing and release, we measured IL-1β forms released from endotoxin-stimulated monocytes by immunoprecipitation of [35S]methionine-labeled protein, by Western blots, and by our recently developed ELISA specific for proIL-1β. Our studies demonstrate that in addition to the 17-kDa mature IL-1β, IL-1β is also released as 31-, 28-, and 3-kDa molecules. The 31-kDa-released form of proIL-1β represented 20–40% of the total released IL-1β, as measured by SDS-PAGE with densitometry. This released proIL-1β was susceptible to ICE processing; however, this proIL-1β was not detectable by either a mature or proIL-1β-specific ELISA, suggesting that release induces a conformational change. The ELISA inability to detect proIL-1β was not due to inadequate sensitivity or subsequent degradation in the ELISA. Furthermore, while immunoaffinity-purified cytosolic proIL-1β could complex the type II IL-1R, released proIL-1β did not. Finally, the absence of a band shift in nondenaturing gel electrophoresis excluded proIL-1β binding to another protein. These findings imply that IL-1β is exported from monocytes as 3-, 17-, 28-, and 31-kDa forms and that the released 31-kDa form differs from cytosolic proIL-1β.

Tumor necrosis factor measurement and use of different anticoagulants: possible interference in plasma samples and supernatants from endotoxin-stimulated monocytes.

Unfractionated heparin is frequently used as an anticoagulant during blood sampling and in cell culture experiments. In the present study we investigated whether heparin and other anticoagulants (citrate and EDTA) interfered with measurements of plasma tumor necrosis factor alpha (TNF alpha) concentrations or with TNF alpha release from endotoxin-stimulated monocytes. TNF alpha was measured by a WEHI 164 bioassay in the plasma of 16 septic patients anticoagulated with heparin, citrate, or EDTA. Anticoagulants were incubated with the bioassay cell line and cell lysis was monitored. To exclude falsely low TNF alpha concentrations, anticoagulants were incubated in increasing amounts with human recombinant TNF alpha/saline solution, and rTNF alpha recovery was measured either with the WEHI 164 bioassay or an ELISA test. Further, anticoagulants were incubated with monocytes isolated from healthy volunteers and stimulated with endotoxin. Supernatants were analyzed for TNF alpha with both test systems. No biologically active TNF alpha was detected in the plasma with heparin anticoagulation, whereas with citrate, reproducible, TNF alpha-induced cytotoxicity was detectable in blood samples of 13 of the 16 patients. Anticoagulation with EDTA resulted in fairly high, variable and poorly reproducible TNF alpha values. Only EDTA produced falsely high values by unspecific lysis of WEHI cells. Only heparin at a concentration of 20 I.U./ml or more was found to produce falsely low values by interaction with the TNF alpha bioassay, but also with the ELISA test. In monocyte culture experiments, heparin significantly attenuated the stimulatory effect of endotoxin on TNF alpha release already at the lowest concentration tested (25 I.U./ml). Heparin and EDTA may have significant adverse effects on TNF alpha measurement when used for blood sampling. Citrate does not interfere with the TNF alpha bioassay or ELISA, and seems, therefore, to be the anticoagulant of choice. Due to intrinsic interactions with various cell systems (including the WEHI cell and monocytes), one should be careful in using heparin in cell culture studies in which effects of TNF alpha or of endotoxin are being studied.

Gabexate mesilate, a synthetic protease inhibitor, attenuates endotoxin-induced pulmonary vascular injury by inhibiting tumor necrosis factor production by monocytes

In order to determine whether gabexate mesilate, a synthetic protease inhibitor with anticoagulant properties, is useful for the treatment of adult respiratory distress syndrome, we examined its effect on endotoxin-induced pulmonary vascular injury in rats. Prospective, randomized, controlled study. Research laboratory at a university medical center. Male Wistar rats (180 to 220 g.) Animals received intravenous infusions of endotoxin (5 mg/kg iv) or saline (control). Pulmonary vascular injury was assessed 6 hrs after administration of endotoxin in terms of the increase in vascular permeability. Rats received gabexate mesilate (10 mg/kg ip), heparin, antithrombin III, an inactive derivative of activated factor X (a selective inhibitor of thrombin generation), or N-[2-[4-(2,2-dimethyl-propionyloxy) phenylsulfonylamino] benzoyl] aminoacetic acid (ONO-5046) (a potent granulocyte elastase inhibitor) 30 mins before endotoxin administration. Leukocytopenia was induced by administration of methotrexate. The effects of the gabexate mesilate on the function of activated neutrophils and the production of tumor necrosis factor -alpha (TNF-alpha) by endotoxin-stimulated monocytes were examined in vitro using neutrophils and monocytes prepared from healthy human volunteers. Pulmonary vascular permeability was determined by measuring the vascular leakage of intravenously administered 125I-labeled bovine serum albumin. Intravenous administration of endotoxin significantly increased pulmonary vascular permeability. Gabexate mesilate significantly inhibited pulmonary vascular injury observed 6 hrs after the administration of endotoxin. Pulmonary vascular injury was not attenuated by the administration of heparin, heparin plus antithrombin III, or the inactive derivative of activated factor X, but pulmonary vascular injury was significantly attenuated in animals with methotrexate-induced leukocytopenia and in those animals treated with N-[2-[4-(2,2-dimethyl-propionyloxy) phenylsulfonylamino] benzoyl] aminoacetic acid. Gabexate mesilate in concentrations of 10(-4) to 10(-3) M inhibited the release of granulocyte elastase and leukocyte aggregation stimulated by N-formyl-methionyl-leucyl-phenylalanine and the opsonized zymosan-activated production of superoxide radical by neutrophils in vitro. Gabexate mesilate significantly inhibited the endotoxin-induced increase in the serum concentration of TNF-alpha in vivo and, at a concentration of 10(-8) M, the production of TNF-alpha by endotoxin-stimulated monocytes in vitro. Our findings suggest that gabexate mesilate attenuated endotoxin-induced pulmonary vascular injury mainly by inhibiting TNF-alpha production by monocytes, which may play a central role in sepsis-related lung injury.

Specific Regulation of Procoagulant Activity on Monocytes: INTRINSIC PATHWAY INHIBITION BY CHONDROITIN 4,6-DISULFATE

Hypercoagulability of blood, monocytic infiltration, and changes in pericellular and extracellular matrix glycosaminoglycans (GAGs) are observed in atherosclerosis, inflammation, and neoplasia. In the present studies, monocyte procoagulants and different GAGs including chondroitin sulfate (CS) A, CSB, CSC, CSD, CSE, and heparan sulfate, were tested either in clotting assays with whole plasma or in chromogenic assays with purified coagulation proteases. Procoagulant activity in plasma was inhibited by three of the seven GAGs, including heparan sulfate, CSE, and CSB. In contrast, activity of purified coagulation protease was inhibited only by CSE, and the inhibition was observed with intrinsic (factor VIIIa/IXa) but not extrinsic (tissue factor/factor VII) components. Reciprocal titration experiments with enzyme and substrate and Scatchard type analyses were consistent with concentration-dependent inhibitory interactions between CSE and sites on both factor VIIIa and IXa. On purified phospholipids, CSE concentration resulting in half-maximal inhibition (Ki) was 5 ng/ml for interaction with factor IXa and > 500 ng/ml for interaction with factor VIIIa. The Ki values were lower for reactions on purified lipid than for reactions on monocyte surfaces and for reactions on resting than on endotoxin-stimulated monocytes. Experiments with CSE oligosaccharides of defined size indicated that the smallest CSE fragment capable of inhibitory activity was composed of 12-18 monosaccharide units. Collectively, these results indicate that factor X-activating reactions are inhibited by GAGs expressed on monocyte membranes. Inhibition is specific with respect to the structure of both the GAG and the activating protease. Lack of inhibition by added CSA, CSB, and CSC in contrast to CSE strongly suggests a direct role of 4,6-di-O-sulfated N-acetylgalactosamine GAG structures in the inhibition of intrinsic pathway protease. These findings also suggest potential pharmacologic use of CSE as specific anticoagulant in the management of prothrombotic states mediated by intrinsic pathway coagulation reactions.

Cytokines affect pseudomonas binding to tracheal cells via a neutrophil-mediated process.

Critical illness is often associated with gram-negative bacterial colonization of the airways, increasing the risk of nosocomial pneumonia. Cytokines, released in response to endotoxin, might contribute to this phenomenon by causing changes in epithelial cell binding of bacteria. To investigate this possibility, human monocytes and hamster pulmonary macrophages were cultured without or with Escherichia coli endotoxin (10 micrograms/ml) for 4 and 24 h. Hamster and human tracheal epithelial cells were treated with supernates from monocyte cultures for 24 h, and subsequent binding of 14C-labeled Pseudomonas aeruginosa to the epithelial cells was measured (percent adherence). In separate experiments, recombinant human (rh) tumor necrosis factor-alpha (TNF-alpha) (25 to 100 ng/ml) and interleukin-1 beta (IL-1 beta) (2,000 to 8,000 pg/ml) were added to hamster monolayers. Neither monocyte supernates nor purified cytokines were toxic to the epithelial cells for up to 48 h. There was no significant change in P. aeruginosa adherence to either hamster or human tracheal epithelial cells after 24 h of exposure to culture supernates from either endotoxin-stimulated human monocytes or hamster macrophages. Similarly, purified rhTNF and rhIL-1 exposure did not increase bacterial adherence. However, when polymorphonuclear leukocytes were coincubated with the monocyte supernates and epithelial cells, P. aeruginosa adherence was significantly increased. Moreover, this effect was enhanced by an epithelial cell-derived substance. Thus, while inflammatory cytokines may participate in enhancing bacterial colonization of the lung in vivo, they do not do so by a direct action on tracheal epithelial cells but can act via a neutrophil-dependent mechanism.

Modulation of TNF alpha and IL-1 beta from endotoxin-stimulated monocytes by selective PDE isozyme inhibitors.

The effect of selective PDE isozyme inhibitors including vinpocetine (PDE-I), CI-930 and milrinone (PDE-III), rolipram and nitraquazone (PDE-IV) and zaprinast (PDE-V) on monocyte viability and production of tumor necrosis factor (TNF alpha) and interleukin-1 beta (IL-1 beta) elicited from endotoxin-stimulated human monocytes was investigated. None of the inhibitors affected monocyte viability at 10 microM or lower concentrations. PDE-IV inhibitors and to a lesser extent, PDE-III inhibitors suppressed TNF alpha production. Only high concentrations of PDE-IV inhibitors modestly suppressed IL-1 beta. Zaprinast stimulated IL-1 beta and to a lesser extent TNF alpha production. These data show that TNF alpha and IL-1 beta production are differentially regulated, and that PDE III, PDE-IV and PDE-V isozymes are functional in endotoxin-stimulated monocytes. Clinical trials will be needed to ascertain if PDE-IV inhibitors are able to suppress TNF alpha levels in man.

Regulation of tumor necrosis factor production in healthy humans and in patients with cancer.

To study the factors that influence cytokine release, the effect of endotoxin on in vitro tumor necrosis factor production by monocytes from debilitated patients with cancer (n = 6) was compared with that from healthy controls (n = 5). Spontaneous and endotoxin-stimulated monocyte tumor necrosis factor production was similar in patients with cancer and controls. However, with total peripheral blood mononuclear cells, enhancement of tumor necrosis factor production by endotoxin in patients with cancer (46 +/- 12, mean +/- SEM) was greater than in controls (0% +/- 7%). This enhanced response correlated with reduced peripheral blood mononuclear cell blastogenesis in response to phytohemagglutinin (r = .66) and could be partially reversed in vitro by addition of exogenous interleukin 2. Thus, a component of total peripheral blood mononuclear cells (probably T cells) seems to influence monocyte cytokine production in response to endotoxin. Moreover, this regulatory component is decreased in patients with cancer, correlates with decreased peripheral blood mononuclear cell blastogenesis, and can stimulated with interleukin 2.