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PubMed1990

Elevated tumor necrosis factor alpha production concomitant to elevated prostaglandin E2 production by trauma patients' monocytes.

The level of tumor necrosis factor alpha (TNF alpha), a monokine implicated in mediating septic shock, is elevated in the blood of some patients with sepsis. Monocytes from 11 trauma patients and 11 burn patients were suboptimally stimulated with interferon gamma and muramyl dipeptide, an analogue of bacterial wall products. The patients with sepsis showed significantly greater total TNF alpha levels (secreted in combination with cell-associated) 3 days before septic episodes, as compared with normal controls (32.38 to 2231.76 ng/10(6) monocytes per milliliter, median = 121.03 ng/10(6) monocytes per milliliter; normal control: 0.00 to 18.20 ng/10(6) monocytes per milliliter, median = 5.93 ng/10(6) monocytes per milliliter). Increases in patients' total monocyte TNF alpha levels greater than 30 ng/10(6) monocytes per milliliter correlated with septic episodes. In patients with sepsis, the total monocyte TNF alpha levels were increased despite a concomitant increase in their prostaglandin E2 levels in both stimulated (interferon gamma plus muramyl dipeptide) and unstimulated in vitro assays (9 patients: stimulated prostaglandin E2 range, 30.1 to 123.6 ng/10(6) monocytes per milliliter). Massively elevated monocyte TNF alpha and prostaglandin E2 production occurred simultaneously in patients with sepsis.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1998

Cytokine repertoire during maturation of monocytes to macrophages within spheroids of malignant and non-malignant urothelial cell lines.

Terminal maturation of human blood monocytes to macrophages (MAC) in vivo is believed to be important for the morphology, antigen expression and functional activity of the resulting MAC population. This process is modulated by the specific tissue micro-environment to which blood monocytes migrate upon leaving the vasculature. Tumor-associated macrophages (TAM) are a special type of MAC, and little is known about the modulating capacity of the tumor environment on monocyte-to-MAC differentiation. By co-culturing 3-dimensional multicellular spheroids (MCS) of the urothelial-bladder-carcinoma cell lines J82 and RT4 with human monocytes/MAC we generated TAM in vitro. For comparison, monocytes/MAC were co-cultured with the non-tumorigenic urothelial cell line HCV29. The effects on monocyte differentiation were analyzed, particularly with respect to cytokine release. Monocyte maturation was modulated within the tumor spheroid dependent upon the tumor cell type. Monocytes co-cultured with MCS of the poorly differentiated J82 carcinoma spontaneously produced high amounts of IL-1beta and IL-6, but only low amounts of TNF-alpha, which could be further increased by the addition of LPS. This cytokine pattern is characteristic for monocytes and remained constant for up to 8 days in J82-MCS co-cultures. However, in RT4-MCS and HCV29-MCS co-cultures, the initial cytokine pattern changed and after 8 days corresponded well to that of MAC differentiated in vitro without tumor contact. In addition to functional parameters, we analyzed the morphology of J82-MCS-TAM and found that they displayed a monocyte-like morphology. Our data indicate that (1) tumor cells can influence monocyte-to-MAC differentiation, giving rise to TAM with monocyte-specific phenotypic properties; and (2) this capacity is dependent on the type of tumor cell.

Cell Differentiation↗
PubMed1999

Ability of Ni-containing biomedical alloys to activate monocytes and endothelial cells in vitro.

Nickel-containing alloys commonly are used in medical and dental applications that place them into long-term contact with soft tissues. The release of Ni ions from these alloys is disturbing because of the toxic, immunologic, and carcinogenic effects that have been documented for some Ni compounds. In particular, Ni ions in solution recently have been shown to cause expression of inflammatory mediators, such as interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and intercellular adhesion molecules (ICAMs) from keratinocytes, monocytes, and endothelial cells. However, the ability of the solid alloys themselves to induce these inflammatory effects has not been demonstrated. An in vitro system was used to determine if Ni-containing biomedical alloys could cause secretion of either IL-1beta or TNF-alpha from monocytes or expression of ICAMs on endothelial cells. Pure nickel, titanium, and three biomedical alloys-18-8 stainless steel, NiTi, and Rexillium III-were evaluated. First, it was determined whether or not the alloys or pure metals could cause cytotoxicity to THP-1 human monocytes or human microvascular endothelial cells (HMVECs) by measuring the succinic dehydrogenase (SDH) activity of the cells. Then, using identical conditions of exposure, the secretion of IL-1beta or TNF-alpha from monocytes or ICAM-1 expression on the HMVECs was determined. Only pure nickel suppressed (by 48% compared to Teflon controls) the SDH activity of the HMVECs or THP-1 monocytes. No alloy or metal caused the HMVECs to express ICAM-1, but the NiTi alloy caused a significant (ANOVA/Tukey) secretion of IL-1beta from the THP-1 monocytes. Secretion of TNF-alpha induced by NiTi was detectable but not statistically significant. The levels of IL-1beta secretion from monocytes were sufficient to induce ICAM-1 expression on HMVECs. The release of Ni from the NiTi was a logical suspect in causing the IL-1beta secretion by monocytes, but its role was not confirmed since other alloys, such as Rexillium III, released the same quantities of Ni yet did not activate the THP-1 monocytes. Within the limitations of in vitro conditions, our results indicate that NiTi alloys pose a risk of promoting an inflammatory response in soft tissues by activating monocytes. Further study is needed to substantiate this finding in vivo.

Alloys↗
PubMed2000

Effect of anti-inflammatory medication on monocyte response to titanium particles.

Cytokines produced by macrophages in the periprosthetic membranes surrounding joint replacements have been implicated as causal agents in osteolysis and prosthetic loosening. The present study characterizes the response of human peripheral blood monocytes to titanium particles. Monocytes were obtained from donated blood and were cultured in the presence of different-sized titanium particles. Exposure to titanium-aluminum-vanadium particles significantly changed the release of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interleukin-1 (IL-1), whereas there was no significant effect on the release of prostaglandin E(2) (PGE(2)). When monocytes were cultured with particles, the titanium alloy particles induced significantly more release of TNF-alpha and less IL-1 secretion. Ciprofloxacin inhibited production of TNF-alpha, IL-6, IL-1, and PGE(2) in human monocytes exposed to titanium particles. In contrast to ciprofloxacin, indomethacin was not a potent inhibitor of TNF-alpha production but potentiated IL-6 production in titanium-stimulated monocytes. Indomethacin had no effect on the production of IL-1 and was a potent inhibitor of PGE(2) production in titanium-stimulated monocytes. Pentoxifylline had an inhibitor effect on TNF-alpha production in titanium-stimulated monocytes. Pentoxifylline potentiated IL-6 and IL-1 production in monocytes exposed to titanium particles and had a biphasic effect on the PGE(2) production. The results of this study support our hypothesis that human monocytes release bone resorption mediators after in vitro exposure to TiAlV alloy particles. The results also demonstrate the differences of bone-resorbing mediators in response to different wear particle size. The pharmacologic agents (ciprofloxacin, pentoxifylline, and indomethacin) that can modulate the release of bone resorbing mediators such as PGE(2), TNF-alpha, IL-1, and IL-6 release from human monocytes. The results help to elucidate the differences in cellular response to wear particles but may not be directly transposed to the human situation.

Anti-Infective Agents↗
PubMed1984

Human monocyte interaction with antibody-coated platelets. I. General characteristics.

The interaction of human monocytes with antibody-coated and normal platelets was studied using an assay system that employed rate zonal centrifugation to separate monocyte-associated (bound) platelets from free platelets. At a monocyte:platelet ratio of 1:10, monocytes bound 21.2 +/- 6.6% of antibody-coated platelets and less than 2.2 +/- 1.3% of control platelets. Monocyte binding of antibody-coated platelets was rapid and inhibited by monomeric IgG and staphylococcal protein A. Specific binding was positively related to the number of monocytes present and the amount of IgG displayed on the platelet surface. Transmission and scanning electron micrographs illustrate the membrane binding of antibody-coated platelets to monocytes and suggest that phagocytosis of platelet targets occurs as well. Thus, these observations indicate that monocytes can participate in Fc-receptor binding of anti-PlA1-sensitized platelets. This novel assay may be useful in the analysis of monocyte-macrophage Fc-receptor interaction with platelets sensitized with IgG from both iso- and autoimmune disorders as well as in the characterization of treatment modalities which may alter monocyte Fc-receptor recognition and binding.

Antibodies↗
PubMed2001

Evaluation of monensin and brefeldin A for flow cytometric determination of interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha in monocytes.

Flow cytometry has become a powerful technique to measure intracellular cytokine production in lymphocytes and monocytes. Appropriate inhibition of the secretion of the produced cytokines is required for studying intracellular cytokine expression. The aim of this study was to compare the capacity of cytokine secretion inhibitors, monensin and brefeldin A, in order to trap cytokine production (interleukin-1 beta [IL-1beta], IL-6, tumor necrosis factor-alpha [TNF-alpha]) within peripheral blood monocytes. A two-color flow cytometric technique was used to measure intracellular spontaneous and lipopolysaccharide (LPS)-stimulated IL-1beta, IL-6, and TNF-alpha production in monocytes (CD14+) of whole blood cultures. The viability of monensin-treated monocytes was slightly lower than that of brefeldin A-inhibited monocytes, as measured with propidium iodide (PI). The percentage of IL-6 and TNF-alpha-producing monocytes after 8 h of culture without stimulation revealed significant lower values for monensin-treated than for brefeldin A-treated monocytes. The percentages for stimulated cells did not differ. The spontaneous intracellular production in molecules of equivalent soluble fluorochrome units (MESF) of IL-1beta, IL-6, and TNF-alpha after 8 h of culture was higher in brefeldin A than in monensin-inhibited monocytes. The LPS-stimulated intracellular production of IL-1beta, IL-6, and TNF-alpha was increased in brefeldin A-inhibited monocytes. In conclusion, for flow cytometric determination of intracellular monocytic cytokines (IL-1beta, IL-6, and TNF-alpha), brefeldin A is a more potent, effective, and less toxic inhibitor of cytokine secretion than monensin.

Adult↗
PubMed1989

Up-regulation by granulocyte-macrophage colony-stimulating factor (GM-CSF) of induction of lymphokine (IL-2)-activated killer (LAK) cells by human blood monocytes.

The role of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) in monocyte-mediated up-regulation of lymphokine-activated killer (LAK) cell induction by IL-2 was examined. Treatment of blood mononuclear cells (MNC) of healthy donors with GM-CSF for 4 days in the presence of IL-2 resulted in a significant increase in LAK activity against natural killer (NK)-resistant Daudi cells, as assessed by the 4 hr 51Cr-release assay. For determination of the role of GM-CSF in LAK induction, highly purified lymphocytes (greater than 99%) and monocytes (greater than 90%) were isolated from MNC by counter-flow centrifugal elutriation (CCE). Pre-treatment of monocytes for 4 days with GM-CSF before addition of lymphocytes plus IL-2 resulted in a significant dose-dependent increase in monocyte-mediated up-regulation of LAK induction, but in the absence of monocytes GM-CSF had no effect on LAK cell induction. Similarly, GM-CSF augmented the proliferative response of lymphocytes to IL-2 in the presence of monocytes as assessed by 3H-TdR uptake. Treatment with anti-GM-CSF antibody completely abolished up-regulation of LAK induction by GM-CSF-treated monocytes. When blood monocytes were separated into 5 fractions by CCE, GM-CSF-responding monocytes were found to be responsible for up-regulation of LAK induction. These results suggest that GM-CSF may be important in monocyte-mediated up-regulation of LAK cell induction in vivo.

Colony-Stimulating Factors↗
PubMed1984

Characterization of purified cryopreserved human monocyte function in assays of superoxide production, accessory cell function, chemotaxis, and in fluorescent cell sorter analysis.

The ability to use highly purified cryopreserved human monocytes in various in vitro assays has a number of practical and theoretical advantages, including convenience and the potential for enhanced reproducibility. Large numbers (up to 1 X 10(9)) human monocytes can be isolated from a single donor in a purified suspension state, by a combination of leukapheresis and counter-current centrifugal elutriation (CCE) technologies. Following short- and long-term periods of cryopreservation, the viability and phagocytic function of these CCE-purified monocytes was unimpaired. Cryopreserved monocytes were similar to fresh cells in their ability to release superoxide anion (O2-), although unstimulated and stimulated O2- release values tended to increase slightly following weeks to months of cryopreservation. In contrast, even short-term cryopreservation diminished both the random migration and chemotactic responses of human monocytes; however, cryopreserved monocytes could be employed in this assay provided the calculated chemotactic ratio (chemotactic migration/spontaneous migration) was used. Cryopreserved monocytes demonstrate 70% of fresh monocyte accessory cell function in pokeweed mitogen-induced lymphocyte proliferation assays. When the binding of OKT3, OKM1, anti-DR, and fluoresceinated pokeweed mitogen to monocytes was analyzed in the fluorescence activator cell sorter (FACS), cryopreserved and fresh monocytes displayed a similar pattern of membrane reactivity.

Chemotaxis, Leukocyte↗
PubMed1986

Enhancement of human peripheral blood monocyte respiratory burst activity by aggregated C-reactive protein.

We had previously demonstrated that C-reactive protein (CRP), an acute phase reactant, when aggregated or coupled to a ligand, interacts with monocytes and certain human peripheral blood lymphocytes. The purpose of the present study, after further characterizing the binding interaction of CRP with human monocytes, was to focus on the biological response of monocytes to CRP binding. Flow cytometric analysis of human mononuclear leukocytes, following incubation with fluoresceinated heat-aggregated CRP (Agg-CRP), revealed that while greater than 70% of monocytes bound Agg-CRP, only 8% of lymphocytes demonstrated positive fluorescence. Furthermore, mean channel fluorescence values indicated that monocytes bound greater amounts of Agg-CRP per cell than did lymphocytes. Luminol-enhanced chemiluminescence (CL) was used as a measure of monocyte respiratory burst activity. Monocytes were stimulated only minimally by Agg-CRP alone; however, Agg-CRP substantially enhanced the CL response to heat-aggregated IgG. This Agg-CRP enhancing effect was selective for IgG-initiated monocyte activation, as no augmentation in CL was observed following cell stimulation with phorbol myristate acetate or serum-opsonized zymosan. These results demonstrate that aggregated CRP binds to the major proportion of human monocytes and selectively augments Fc receptor-mediated stimulation of monocyte oxidative metabolism.

C-Reactive Protein↗
PubMed1990

Multiple receptors on human monocytes are involved in adhesion to cultured human endothelial cells.

Monocytes exhibit significant basal (unstimulated) adherence to human umbilical vein endothelium (HUVE), which is only partially inhibited by an anti-CD18 monoclonal antibody (mAb) (60.3). We examined factors modulating the residual, CD18-independent monocyte binding to HUVE by pretreating monocytes with mAb 60.3 to eliminate CD18-dependent binding. Basal adherence was reduced from 32% +/- 2% to 14% +/- 2% with mAb 60.3 (means +/- SE of eight experiments; P less than 0.01). mAb 60.3-treated monocytes were incubated with tumor necrosis factor-gamma (TNF-alpha), interleukin-1 (IL-1), lipopolysaccharide (LPS), N-formylmethionyl-leucyl-phenylalamine (FMLP), or phorbol myristate acetate (PMA). Only PMA affected CD18-independent binding. Pretreatment with PMA alone reduced adherence to 21% +/- 2% (mean +/- SE of eight experiments; P less than 0.01). In conjunction with mAb 60.3, PMA virtually eliminated monocyte adherence to HUVE (7% +/- 1%, mean +/- SE of eight experiments; P less than 0.01). We also examined CD18-independent monocyte binding to endothelial-leukocyte adhesion molecules (E-LAMs) induced by pretreatment of HUVE with LPS. Monoclonal antibody 60.3-treated monocytes increased adherence from 14% +/- 2% with unstimulated HUVE to 37% +/- 2% with LPS-stimulated HUVE (mean +/- SE of four experiments; P less than 0.01). Monocytes pretreated with both mAb 60.3 and PMA increased adherence from 5% +/- 1% with the unstimulated HUVE to 18% +/- 1% with the LPS-stimulated HUVE (mean +/- SE of four experiments; P less than 0.01). This result implies the presence of a CD18-independent and PMA-insensitive receptor on human monocytes for an E-LAM induced by LPS. In summary, we have identified two CD18-independent mechanisms of monocyte adherence to HUVE; a PMA-sensitive mechanism mediating basal adherence and a PMA-insensitive mechanism involved in binding to E-LAMs.

Antibodies, Monoclonal↗
PubMed1992

Monocyte diapedesis through an in vitro vessel wall construct: inhibition with monoclonal antibodies to thrombospondin.

Human peripheral blood monocytes were examined for migration across an endothelial cell monolayer in an in vitro vessel wall construct. Few monocytes invaded in the absence of a chemotactic gradient, despite significant adhesion to the endothelial monolayer. However, the addition of zymosan-activated human plasma to the lower compartment, to create a chemotactic gradient across the vessel wall, resulted in significantly enhanced monocyte migration. Pretreatment of the monocytes with monoclonal antibodies to thrombospondin (TSP) dramatically inhibited monocyte diapedesis into the vessel wall. The same treatment inhibited monocyte adhesion to endothelial cells in two-dimensional monolayer cultures as well as in vessel wall constructs (no chemotactic gradient). Of interest, however, the monoclonal antibodies had no inhibitory effect on monocyte migration into collagen gels devoid of endothelial cells in response to the same chemotactic gradient, suggesting the importance of TSP in monocyte-endothelial cell interactions. Monoclonal antibodies to fibronectin and normal mouse immunoglobulin G did not inhibit migration in this model of a vessel wall. Furthermore, monoclonal antibodies to TSP showed no inhibition of human peripheral blood neutrophil migration. Previous studies have shown that monocytes synthesize TSP and express this moiety on their surface. The present data suggest that monocytes may utilize TSP to interact with endothelial cells lining the vessel wall during diapedesis.

Antibodies, Monoclonal↗
PubMed1996

T cell rescue of monocytes from apoptosis: role of the CD40-CD40L interaction and requirement for CD40-mediated induction of protein tyrosine kinase activity.

Circulating monocytes have a limited life span and will undergo apoptosis in the absence of specific stimuli. Recent studies have demonstrated that monocytes can be rescued from apoptosis via lipopolysaccharide (LPS) activation or stimulation with interleukin-1 or tumor necrosis factor-alpha. Based on previous studies from our laboratory, we hypothesized that, in nonseptic (e.g., autoimmune) inflammation, the presence of activated T cells may enhance monocyte longevity through T cell contact-dependent signaling. Plasma membranes prepared from 6 h activated (TmA) and resting (TmR) purified CD4+ T cells were added to resting elutriation-purified monocytes cultured in serum-free medium. Cells were assayed for degree of apoptosis occurring over a 72-h incubation using both agarose gel electrophoresis and flow cytometry. The addition of TmA (but not TmR) was capable of blocking monocyte apoptosis and the ability of TmA to rescue monocytes was abrogated by the addition of anti-CD40L antibodies. Rescue of monocytes from apoptosis could also be mediated by direct cross-linking of monocyte CD40. Inhibitors of tyrosine kinase activity blocked both TmA and anti-CD40-mediated rescue of monocytes from apoptosis, suggesting a primary role of a tyrosine kinase signaling pathway in the events controlling monocyte longevity.

Apoptosis↗
PubMed1997

Monocytes incubated with surfactant: a model for human alveolar macrophages?

Monocytes migrate to the lungs and enter the alveoli where they come into contact with surfactant and differentiate into alveolar macrophages. This study focused on the question of the extent to which monocytes and monocyte-derived macrophages (MDM) incubated with surfactant resemble alveolar macrophages. Surfactant-incubated monocytes shared with alveolar macrophages the intracellular presence of surfactant, efficient phagocytosis of opsonized Staphylococcus aureus, and poor intracellular killing of ingested bacteria. The suppressive effect of surfactant on bactericidal activities of monocytes could not be attributed to either the surfactant lipid fraction or surfactant protein A. Monocytes incubated with surfactant differed from alveolar macrophages with respect to expression of various Fc and complement receptors involved in intracellular killing of bacteria. Surfactant-incubated monocytes produced significantly more H2O2 upon stimulation with phorbol ester than alveolar macrophages, but significantly less than control monocytes. Together, monocytes and MDM incubated with surfactant, although similar to alveolar macrophages in some aspects, are not an adequate model for alveolar macrophages. Most likely, factors other than surfactant in the microenvironment of the alveoli, such as oxygen tension, play a role in the differentiation of monocytes to alveolar macrophages as well.

Animals↗
PubMed1993

Regulation of human B cell responsiveness by interferon-alpha: interferon-alpha-mediated suppression of B cell function is reversed through direct interactions between monocytes and B cells.

Previous studies have revealed that interferon-alpha (IFN-alpha) suppresses the B cell responses stimulated with Staphylococcus aureus (SA) + IL-2 in the complete absence of monocytes but enhances the responses of B cells contaminated with monocytes. The current studies therefore examined in detail the combined effects of IFN-alpha and monocytes on the B cell responses induced by SA + IL-2. Monocytes overcome the suppressive effects of IFN-alpha on the IgM production induced by SA + IL-2. Thus, in the presence of monocytes, IFN-alpha enhanced the IgM production by B cells stimulated with SA + IL-2. IFN-alpha still enhanced the IgM production induced by SA + IL-2 in the presence of monocytes and indomethacin. The IFN-alpha-mediated suppression of B cell responsiveness was not overcome by addition of factors generated from SA-activated monocytes, or any of IL-1 beta, IL-6, and TNF-alpha. Of note, the IFN-alpha-mediated suppression of B cell responsiveness was overcome only when B cells and monocytes were allowed to contact with each other. This reversal of the IFN-alpha-mediated suppression of B cell function was not blocked by any of the mAb to CD11a, CD18, CD54, or monomorphic determinants of HLA-DR. These results indicate that IFN-alpha enhances the B cell responses induced by SA + IL-2 through direct interactions between monocytes and B cells that do not involve lymphocyte-function-associated-1 molecules, intercellular adhesion molecule-1, or HLA-DR antigens. Thus, the data demonstrate the presence of unique direct interactions between B cells and monocytes that regulate human B cell responsiveness.

Adult↗
PubMed1995

Regulation of monocyte-derived interleukin 1 receptor antagonist by cisplatinum.

IL-1 biology has taken on a new dimension with the discovery of the IL-1 receptor antagonist (IL-1ra). The balance in the production of monocyte-derived IL-1 and IL-1ra may impact on subsequent IL-1-dependent inflammation. Cancer patients are known to have impaired monocyte biological function. Interestingly, cancer patients receiving chemotherapeutic regiments containing cisplatinum appear to regain enhanced monocyte cytolytic activity both in vitro and in vivo. We postulated that cisplatinum may enhance the normal biological function of monocytes via its effect on IL-1 biology. Monocytes isolated from normal healthy subjects cultured in the presence of graded concentrations of cisplatinum with or without lipopolysaccharide (LPS) demonstrated significantly attenuated production of monocyte-derived IL-1ra in a dose-dependent manner. Moreover, the delayed addition of cisplatinum to monocyte cultures (up to 4 h), in relation to LPS stimulation, significantly suppressed IL-1ra protein by 49%. The level of this regulation was inhibition of IL-1ra mRNA. In contrast, cisplatinum failed to significantly inhibit the production of monocyte-derived IL-1 beta or other pro-inflammatory cytokines. These findings support the notion that cisplatinum has disparate effects on monocyte-derived cytokines, and provide a potential mechanism for cisplatinum's effects on monocyte function in cancer patients.

Base Sequence↗
PubMed2000

Thrombin inhibits apoptosis of monocytes and plasminogen activator inhibitor 2 (PAI-2) is not responsible for this inhibition.

Plasminogen activator inhibitor 2 (PAI-2) has been shown to inhibit apoptosis in transfected cells. We have investigated this phenomenon in activated human monocytes, which are a physiological source of intracellular PAI-2. Apoptosis of monocytes was rapidly induced by removal of serum, addition of hydrogen peroxide, or binding of a monoclonal antibody to Fas. Treatment of monocytes with thrombin or lipopolysaccharide (LPS) inhibited apoptosis of monocytes and also up-regulated intracellular PAI-2. Increased apoptosis was accompanied with increased activity of caspases 3 and 8. Thrombin or LPS treatment of monocytes decreased the activity of both caspases, which correlated with protection from apoptosis. The role for PAI-2 in protection of monocytes from apoptosis was studied. Monocytes were transfected with antisense oligonucleotides that blocked PAI-2 antigen, and antisense for PAI-2 had no effect on apoptosis of monocytes. No interaction was evident between PAI-2 and recombinant caspases 3 and 8 in vitro. PAI-2 was not a substrate for caspases during apoptosis of monocytes, although some cleavage of recombinant PAI-2 by caspase 3 was evident in vitro. This study shows that thrombin or LPS protected monocytes from apoptosis and that PAI-2 did not mediate this inhibitory effect.

Apoptosis↗
PubMed1995

Tissue factor expression during coculture of endothelial cells and monocytes.

The role of monocytes as initiators of coagulation through the expression of tissue factor has been well documented in vitro, and the relationship of monocyte tissue factor to the thrombotic complications of atherosclerosis has been suggested. Tissue factor antigen has been identified in the plasma membranes of monocytes adherent to the vascular endothelium overlying atherosclerotic plaques and the presence of tissue factor in adherent mononuclear cells correlates with the polymerization of fibrin at these same sites. To further understand the relationship of cellular adhesion to tissue factor expression, human monocytes were cocultured for periods ranging from 30 min to 24 hr with endothelial cells isolated from human umbilical veins (HUVEC). Tissue factor antigen, as assayed by both ELISA and immunogold electron microscopy, was minimal on either monocytes or HUVEC maintained in homogeneous cultures or on the cells when cocultured for 1 hr or less. This was true whether the HUVEC were in a native state or if they had been stimulated with interleukin-1 (IL-1 beta) or lipopolysaccharide (LPS) prior to monocyte adhesion. Typically, less than 13% of the cells in short-term cocultures were positively labeled through anti-tissue factor immunogold microscopy. The level of tissue factor, however, was increased 3-fold above baseline when monocytes were cocultured with unstimulated HUVEC for 4 hr, and it was more than double this if the HUVEC had been exposed to IL-1 beta or LPS (7-fold increase). By 24 hr, the expression of tissue factor antigen was nearly 50-fold higher in cocultures involving stimulated HUVEC, and at later times greater than 70% of the cells were labeled with immunogold. Through the use of quantitative immunogold electron microscopy, the increase in tissue factor was most pronounced on monocytes which had three times greater increase in tissue factor than HUVEC in the same cultures. These studies document the stimulation of tissue factor expression by monocytes upon coculture with endothelial cells, and the data document an enhancement of this coculture effect upon HUVEC stimulation with cytokines. These observations have relevance to atherosclerotic disease by suggesting that interaction of monocytes with dysfunctional endothelial cells overlying atherosclerotic plaques would be sufficient to induce tissue factor and by so doing predispose to localized thrombotic events.

Cell Adhesion↗
PubMed1997

Immunodepressive effects of LPS on monocyte CD14 in vivo.

Having previously reported that septic patients displayed lower levels of monocyte CD14 (endotoxin receptor) as compared to normal individuals, we were interested in the hypothesis that lipopolysaccharide (LPS) modulates levels of monocyte CD14 in vivo. We examined CD14 expression in 13 human volunteers who were given a non-lethal injection of Escherichia coli LPS (4.0 ng/kg). Monocyte CD14 was assayed by direct immunofluorescent determination with appropriate anti-CD14 monoclonal antibodies using flow cytometry. To test for cell responsiveness, monocytes were additionally examined following in vitro stimulation by phorbol myristate acetate (PMA) and N-formyl-methionyl-leucyl-phenylalanine (FMLP). Following LPS infusion, all patients displayed significant monocytopenia and responded with fever and tachycardia. Plasma samples demonstrated elevated levels of TNF alpha. CD14 expression was down-regulated by 52% on monocytes obtained 3 hr following LPS infusion (P < 0.05, vs. pre-LPS levels). Monocytes obtained pre-LPS infusion were down-regulated following in vitro stimulation by PMA to levels 72 +/- 8% and by FMLP to levels 75 +/- 5% of unstimulated control cells. In contrast, monocytes obtained 3 hr post-LPS infusion failed to respond to PMA or FMLP with significant down-regulation. LPS down-regulated CD14 expression on monocytes in vivo and LPS also blunted the ability of monocytes to respond to other stimuli. We conclude that LPS desensitizes monocytes to itself and thereby renders an immunodepressive effect on these cells.

Escherichia coli↗