Monocyte Surface Expression Research Articles

Hydroxy alkenal phospholipids regulate inflammatory functions of endothelial cells

Monocyte recruitment into the vessel wall plays an important role in atherogenesis. Polar lipid components of minimally modified/oxidized LDL were shown to activate endothelial cells to increase the synthesis of monocyte chemotactic factors and surface expression of adhesion molecules. We previously reported regulation of endothelial cell inflammatory functions by oxidized 1-palmitoyl-2-arachidonoyl- sn-glycero-3-phosphocholine (Ox-PAPC) and three component oxidized phospholipids, containing oxovaleroyl (POVPC), glutaroyl (PGPC) and epoxyisoprostane (PEIPC) groups at the sn-2 position of oxidized phospholipids. In the present study, we demonstrate the presence of γ-hydroxy-α,β-unsaturated aldehydic phospholipid, 1-palmitoyl-2-(5-hydroxy-8-oxooct-6-enoyl)- sn-glycero-3-phosphocholine (HOOA-PC; m/ z 650.4), in Ox-PAPC by liquid chromatography/mass spectrometry (LC/MS), LC/MS/MS, derivatization and tandem mass spectrometric analyses. This was further unambiguously confirmed by the identical chromatographic and mass spectrometric characteristics of Ox-PAPC-derived m/ z 650.4 with synthetic HOOA-PC. The time course of PAPC autoxidation showed that HOOA-PC accumulates with oxidation and represents about 2% of Ox-PAPC. We have also examined the effects of HOOA-PC on leukocyte–endothelial interactions. HOOA-PC dose-dependently activated human aortic endothelial cells (HAECs) to bind monocytes (twofold at 10 μg/ml) and caused a dose-dependent increase (two- to threefold) in levels of monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8)—chemokines that are important in monocyte entry into chronic lesions. HOOA-PC also inhibited LPS-induced expression of E-Selectin, a major adhesion molecule that mediates neutrophil endothelial interactions. The present study suggests that the HOOA-PC exerts its effects on endothelial cells as a free lipid. These studies demonstrate the importance of HOOA-PC as a new potential proinflammatory molecule that regulates leukocyte–endothelial interactions.

Proteolysis of Tissue Factor Pathway Inhibitor-1 by Thrombolysis in Acute Myocardial Infarction

In acute myocardial infarction (AMI), surface-bound tissue factor pathway inhibitor-1 (TFPI-1) inhibits an increased monocyte procoagulant activity. In addition, TFPI-1 is released from microvascular endothelial cells after treatment with heparin and thereby contributes to its antithrombotic properties. We examined 19 patients in a randomized study comparing intravenous fibrinolysis with alteplase (n=9) and revascularization by stent placement with additional abciximab treatment (n=10). We obtained blood samples for analysis of monocytic TFPI-1 surface expression by flow cytometry and plasma TFPI-1 concentrations by immunoassay before and after therapy. We found a significant decrease in surface TFPI-1 on circulating monocytes 24 hours after thrombolysis (P=0.006) that was not observed after stenting. Systemic plasma TFPI-1 concentrations increased immediately after stenting by 71+/-14% (P=0.008), whereas after thrombolysis, a decrease in TFPI-1 plasma concentrations of 21+/-11% was observed (P=0.075). In vitro experiments confirmed that plasmin decreased TFPI-1 surface expression dose-dependently. Activation of the fibrinolytic system by alteplase in AMI decreases surface-associated TFPI-1 on circulating monocytes and plasma TFPI-1. Reduced TFPI-1 may contribute to thrombotic complications after fibrinolysis in AMI.

Decreased monocyte surface expression of HLA-DQ is associated with prolonged duration of septic shock

There is increasing evidence for a protective role of Human-Leukocyte-Antigen (HLA)-DQ in response to infection. This study was performed: (1) to investigate the kinetics of monomorphic (m)HLA-II (HLA-DR,-DP,-DQ) and HLA-DQ expression on monocytes during septic shock; (2) to clarify whether maintaining mHLA-II and HLA-DQ expression within normal range (NR) could predict improved recovery and survival. In total, 16 patients (11 males, 5 females) in septic shock have been investigated. mHLA-II and HLA-DQ median fluorescence intensity (MFI) on monocytes have been monitored daily using flow cytometry. Normal range was determined in 19 healthy volunteers. MFI of mHLA-II and HLA-DQ expression throughout septic shock was significantly reduced when compared to control (P < 0.05). All patients showed median mHLA-II expression below normal range regardless of duration of septic shock and survival. In contrast, median HLA-DQ expression demonstrated negative correlation with the duration of septic shock (Phi-square = 0.73). Patients with HLA-DQ below normal range showed almost a three-fold prolonged term of septic shock as compared to patients with HLA-DQ expression within normal range (15.2 ± 6.3 days versus 5.5 ± 1.7 days; P < 0.015). Although, there was no correlation between median HLA-DQ and survival, all non-survivors showed HLA-DQ expression below normal range. In conclusion, HLA-DQ expression below normal range may predict prolonged duration of septic shock and increased mortality and could be an indicative marker of immune surveillance in critically ill patients.

Toll-like receptor 4 surface expression on human monocytes and B cells is modulated by IL-2 and IL-4

Human Toll-like receptor 4 (TLR4) has recently been identified, and it has been shown to be the main protein involved in recognizing gram-negative bacteria. We examined the regulation of TLR4 surface expression in human peripheral blood monocytes and B cells by interleukin-2 (IL-2) and IL-4. IL-2 up-regulated TLR4 surface expression on human peripheral blood monocytes, but did not change expression on human peripheral B cells. By contrast, IL-4 down-regulated TLR4 surface expression on human peripheral blood monocytes, but up-regulated TLR4 surface expression on human peripheral B cells. These results indicate that Th1 cytokine IL-2 enhances receptors involved in the response to gram-negative bacteria and that activation of cellular immunity may enhance defense against these pathogens through monocytes, but not B cells, whereas Th2 cytokine IL-4 modulates the receptor response to gram-negative bacteria and that activation of humoral immunity may enhance defense against these pathogens through B cells, but not monocytes.

Increased Expression of u-PA and u-PAR on Monocytes by LDL and Lp(a) Lipoproteins – Consequences for Plasmin Generation and Monocyte Adhesion

Monocyte-derived foam cells figure prominently in rupture-prone regions of atherosclerotic plaque. As urokinase/urokinase-receptor (u-PA/u-PAR) is the trigger of a proteolytic cascade responsible for ECM degradation, we have examined the effect of atherogenic lipoproteins on monocyte surface expression of u-PAR and u-PA. Peripheral blood monocytes, isolated from 10 healthy volunteers, were incubated with 10 to 200 microg/ml of native or oxidised (ox-) atherogenous lipoproteins for 18 h and cell surface expression of u-PA and u-PAR was analysed by flow cytometry. Both LDL and Lp(a) induced a dose-dependent increase in u-PA (1.6-fold increase with 200 microg/ml of ox-LDL) and u-PAR [1.7-fold increase with 200 microg/ml of ox-Lp(a)]. There is a great variability of the response among the donors, some of them remaining non-responders (absence of increase of u-PA or u-PAR) even at 200 microg/ml of lipoproteins. In positive responders, enhanced u-PA/u-PAR is associated with a significant increase of plasmin generation ( .9-fold increase with 200 microg/ml of ox-LDL), as determined by an amidolytic assay. Furthermore, monocyte adhesion to vitronectin and fibrinogen was significantly enhanced by the lipoproteins [respectively 2-fold and 1.7-fold increase with 200 microg/ml of ox-Lp(a)], due to the increase of micro-PAR and ICAM-1, which are receptors for vitronectin and fibrinogen. These data suggest that atherogenous lipoproteins could contribute to the development of atheromatous plaque by increasing monocyte adhesion and trigger plaque weakening by inducing ECM degradation.

Activated platelets induce monocyte chemotactic protein-1 secretion and surface expression of intercellular adhesion molecule-1 on endothelial cells.

Platelet/endothelium interaction plays an important role in the pathophysiology of inflammation and atherosclerosis. The role of platelets for monocyte chemotactic protein-1 (MCP-1) secretion and surface expression of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells has been assessed. Monolayers of human umbilical vein endothelial cells were incubated with nonstimulated or ADP-activated platelets for 6 hours, and secretion of MCP-1 and surface expression of ICAM-1 were determined by ELISA and flow cytometry, respectively. In the presence of ADP-activated platelets, both MCP-1 secretion and ICAM-1 surface expression were significantly increased compared with nonstimulated platelets (P<0.02). Activation of the transcription factor nuclear factor-kappaB (NF-kappaB) determined by electrophoretic mobility shift assay and kappaB-dependent transcriptional activity was enhanced in the presence of activated platelets. In addition, ADP-activated platelets induced MCP-1 and ICAM-1 promoter-dependent transcription. Liposomal transfection of a double-stranded kappaB phosphorothioate oligonucleotide, but not of the mutated form, inhibited MCP-1 secretion and surface expression of ICAM-1 on activated endothelium (P<0.05). The present study indicates that activated platelets modulate chemotactic (MCP-1) and adhesive (ICAM-1) properties of endothelial cells via an NF-kappaB-dependent mechanism. Platelet-induced activation of the NF-kappaB system might contribute to early inflammatory events in atherogenesis.

Antagonistic Action of IFN-β and IFN-γ on High Affinity Fcγ Receptor Expression in Healthy Controls and Multiple Sclerosis Patients

Abstract Monocyte-macrophage activation by IFN-γ is characterized by a pronounced increase of high affinity Fc receptors for IgG (FcγRI), capable of triggering respiratory burst, phagocytosis, Ab-dependent cytotoxicity, and release of proinflammatory cytokines. In view of the antagonism of IFN-β on IFN-γ action, of interest in the chronic inflammatory disorder multiple sclerosis, we examined the possible effect of IFN-β on IFN-γ induction of FcγRI gene expression. We found that IFN-β significantly down-regulated IFN-γ-induced FcγRI surface expression in peripheral blood monocytes from healthy donors, in a dose- and time-dependent manner. This down-regulation of FcγRI surface levels did not correspond to a decrease in FcγRI mRNA, suggesting a posttranscriptional effect of IFN-β. Down-regulation of FcγRI surface expression correlated with diminished cellular signaling through FcγRI, since the IFN-γ-induced increase in Fcγ receptor-triggered respiratory burst was nearly completely abrogated by simultaneous addition of IFN-β. Finally, the same antagonism between both IFNs on FcγRI surface expression was observed in peripheral blood monocytes derived from multiple sclerosis patients; inhibition by IFN-β was even increased (82 ± 11%), as compared with healthy controls (67 ± 4%). These results may partially help explain the beneficial effect of IFN-β in multiple sclerosis.

Inhibition of tissue factor surface expression in human peripheral blood monocytes exposed to cytokines.

Interleukin (IL)-4, IL-10, IL-13 and transforming growth factor beta (TGF-beta) are known to regulate several monocyte functions, including inhibition of the synthesis of different cytokines. Using quantitative RT-PCR and flow cytometry analysis we investigated the effects of these cytokines on bacterial lipopolysaccharide (LPS)-induced tissue factor (TF) expression in human monocytes. The effects of IL-4 and IL-10 on monocyte chemoattractant protein-1 (MCP-1)-and C-reactive protein (CRP)-induced TF expression were also studied. A direct comparison revealed that IL-4, IL-10 and IL-13 all down-regulated LPS-induced TF expression in a concentration-dependent manner without the need for priming. In contrast, TGF-beta required 4 h of priming to inhibit TF expression induced by LPS. IL-10 was the most powerful inhibitor, causing almost complete inhibition at 5 ng/ml. IL-4 and IL-13 exhibited a significantly lower inhibitory capacity even at concentrations of 100 ng/ml. IL-4 and IL-10 showed similar concentration-dependent inhibition of MCP-1- and CRP-induced TF expression. We also showed that the regulatory effect of the interleukins occurred at the mRNA level. In vivo, these inhibitory cytokines may play an important regulatory role in preventing thrombosis. IL-10, in particular, may be a possible candidate as a TF-preventing drug.

CD36 Induction on Human Monocytes upon Adhesion to Tumor Necrosis Factor-activated Endothelial Cells

Cell adhesion between circulating monocytes and the endothelium is a critical component of vascular thromboregulation and atherogenesis. The biochemical and genetic consequences of adhesion are poorly understood. We have found that monocyte surface expression of CD36, an integral membrane receptor for thrombospondin, collagen, and oxidized low density lipoprotein, increased dramatically upon adhesion to tumor necrosis factor-activated human umbilical vein endothelial cells (HUVEC). Expression was assessed by indirect immunofluorescence microscopy and immunoblotting using monoclonal antibodies to CD36. Steady-state CD36 mRNA levels, detected by RNase protection assay, also showed a similar pattern of up-regulation. To verify the adhesion dependence of the observed phenomenon, monocytes were co-cultured with tumor necrosis factor-activated HUVEC in a transwell apparatus that physically separated monocytes from the endothelial cells. Under these conditions, no increase in CD36 expression was detected, demonstrating that the enhanced monocyte CD36 expression observed is not due to soluble factors released by HUVEC. To characterize the specific adhesion molecules involved in the process, co-culture assays were performed on murine L cells transfected with either human E-selectin or intercellular adhesion molecule-1 cDNAs. A dramatic increase in CD36 mRNA was seen upon monocyte adhesion to E-selectin-transfected L cells compared with adhesion to intercellular adhesion molecule-1 or control transfectants. Furthermore, monoclonal antibodies to E-selectin inhibited the adhesion-dependent up-regulation of CD36 mRNA induced by transfected L cells or cytokine-activated endothelial cells. These findings demonstrate adhesion-dependent gene regulation of monocyte CD36 and suggest the possible involvement of E-selectin in initiating this process.

Human MCF Activates Monocytes to Produce IL-1 but not TNF or CSF-1

Monocyte cytotoxicity inducing factor (MCF) is a novel cytokine which was originally characterized by its ability to induce monocyte cytotoxicity against tumor targets. The present studies were undertaken to define the mechanisms of cytotoxicity. Since the cytotoxic state may be mediated, in part, by the secretion of monokines, MCF's effect on IL-1, TNF and CSF-1 were measured. MCF caused human peripheral blood monocytes to secrete large amounts of IL-1 but not TNF. In contrast monocytes produce CSF-1 in culture and this production was not enhanced by MCF. IL-1 neither appeared to directly lyse the target K562 nor was able to activate macrophages for cytotoxicity. However, IL-1 may be necessary for lysis of K562 by activated macrophages through its cytostatic effect on K562. MCF increased monocyte surface expression of DR-β (when studied by fluorescent microfluorometry) and messenger RNA for DR-β after 24 h in culture. These functional studies indicate that MCF is a cytokine with a distinct spectrum of biologic activities whose functions may be mediated by synthesis and secretion of IL-1.

Inhibition of Human Monocyte Adhesion by Interleukin-4

The adhesion of monocytes to vascular surfaces is central to inflammation and atherogenesis; however, very little is known about regulatory factors that can prevent these processes. Here we report the inhibition of human monocyte adhesion to human endothelial layers and plastic by interleukin-4 (IL-4), a T-cell-derived glycoprotein with pleiotropic activities. The inhibitory effects of IL-4 were seen with basal and cytokine-stimulated monocyte adhesion, were apparent at low concentration, and were abolished by inactivating IL-4. No direct toxic effect of IL-4 on monocytes was detected. Inhibition of adhesion was accompanied by small increases in monocyte surface expression of the leukocyte-functional antigen group of adhesion structures, suggesting that absolute levels of expression may be less important than the functional status of such molecules in the regulation of monocyte adhesion. In addition, inhibition by IL-4 of cytokine-stimulated monocyte adhesion was not associated with changes in the surface expression of cytokine receptors. These results suggest a role for IL-4 in the regulation of monocyte adhesion, and may provide for a common mechanism for the inhibitory effects of IL-4 on monocyte function.

Inhibition of human monocyte adhesion by interleukin-4

The adhesion of monocytes to vascular surfaces is central to inflammation and atherogenesis; however, very little is known about regulatory factors that can prevent these processes. Here we report the inhibition of human monocyte adhesion to human endothelial layers and plastic by interleukin-4 (IL-4), a T-cell-derived glycoprotein with pleiotropic activities. The inhibitory effects of IL-4 were seen with basal and cytokine-stimulated monocyte adhesion, were apparent at low concentration, and were abolished by inactivating IL-4. No direct toxic effect of IL-4 on monocytes was detected. Inhibition of adhesion was accompanied by small increases in monocyte surface expression of the leukocyte-functional antigen group of adhesion structures, suggesting that absolute levels of expression may be less important than the functional status of such molecules in the regulation of monocyte adhesion. In addition, inhibition by IL-4 of cytokine-stimulated monocyte adhesion was not associated with changes in the surface expression of cytokine receptors. These results suggest a role for IL-4 in the regulation of monocyte adhesion, and may provide for a common mechanism for the inhibitory effects of IL-4 on monocyte function.

Cytokines and endothelial cell biology

Ce travail resume les etudes qui ont permi la caracterisation de l'action specifique des cytokines sur les cellules endotheliales. Les cytokines agissent aussi sur l'endothelium dans des cas pathologiques cites dans cet expose

Molecular mechanisms of platelet activation.

Molecular mechanisms of platelet activation.

Human monocyte adherence to cultured vascular endothelium: monoclonal antibody-defined mechanisms.

We have evaluated the binding of human peripheral blood monocytes to cultured vascular endothelium as an in vitro model of monocyte interaction with the vessel wall. Monocytes were purified (91% +/- 4 SE esterase positive) by elutriation to avoid contact with surfaces before assay. Adherence of 51Cr-labeled monocytes after 45 min (36% +/- 11 SE) was significantly higher than that observed with autologous radiolabeled neutrophils (9% +/- 5 SE) and was greater on monolayers of human umbilical vein endothelium than on bovine aortic endothelium. Peripheral blood mononuclear cells treated with monoclonal antibody (MoAb) 60.3, a reagent that binds leukocyte membrane complex CDw18, implicated in multiple adherence-dependent functions, failed to adhere and flatten on artificial surfaces. Mononuclear cells treated with MoAb 60.3 simulated cells from a patient with recurrent infections whose phagocytes failed to react with MoAb 60.3 and failed to emigrate to extravascular sites in vivo. Incubation of monocytes with MoAb 60.3 inhibited (by 32 to 61%) monocyte adherence to endothelium in a dose-dependent manner for periods up to 24 hr, but had negligible effects on basal (unstimulated) neutrophil adherence. Basal monocyte adherence in the presence of MoAb 60.3 remained significantly greater than basal neutrophil adherence. Augmentation of phagocyte adherence to endothelial monolayers by autologous plasma or phorbol ester (PMA) was abrogated by incubation with MoAb 60.3. Studies with immunofluorescence flow cytometry indicated that PMA stimulation of monocytes resulted in a specific 40% increase in monocyte surface expression of the epitope recognized by MoAb 60.3. These in vitro findings, in conjunction with observations from two patients, support the hypothesis that monocyte adherence to endothelium and emigration to tissues is mediated by mechanisms both dependent upon and independent of the CDw18 complex and the epitope recognized by MoAb 60.3.