Characteristics of chondrocyte responses to a human interleukin 1-like factor

The objective of these studies was to characterize some aspects of collagenase production by rabbit articular chondrocytes cultured with stimulated monocyte supernatants. Supernatants from human monocytes stimulated with 20 ng/ml bacterial lipopolysaccharide (LPS) induced the synthesis and secretion of latent collagenase by the chondrocytes beginning at 6 hr. The time course and dose response of collagenase production by the chondrocytes were identical using crude monocyte supernatants or semipurified interleukin 1 (IL-1). Recombinant or purified human interleukin 2 failed to induce collagenase production in the cultured chondrocytes. The response of the chondrocytes was inhibited by actinomycin D or cycloheximide and not by corticosteroids. Phorbol myristate acetate (PMA) alone failed to directly stimulate the chondrocytes. However, PMA led to enhanced collagenase production by chondrocytes when incubated with submaximal amounts of LPS-stimulated monocyte supernatant or semipurified IL-1. LPS alone in amounts between 0.1 and 10.0 micrograms/ml directly stimulated collagenase production in chondrocytes between 4 and 11 days in culture. These data confirm those of other laboratories that IL-1 may be the active factor in monocyte supernatants responsible for inducing collagenase production in cultured chondrocytes. Further characterization of this response indicates that the collagenase is not preformed in the cells and stimulation of its production is not inhibited by corticosteroids. Cell supernatants or IL-1 preparations containing PMA as low as 1.0 ng/ml or LPS as low as 1.0 microgram/ml may give falsely high values for IL-1 activity when assayed by stimulation of collagenase production in cultured chondrocytes.     

Suppression of LPS-inducible cytotoxicity in cytomegalovirus-infected THP-1 monocytic cell cultures does not correlate with a decrease in TNF-alpha antigen.

We document suppression of tumor necrosis factor alpha (TNF-alpha)-associated cytotoxic activity in a human monocytic cell line (THP-1) infected with the mycoplasma free human cytomegalovirus (CMV) strain AD169. Addition of lipopolysaccharide (LPS) to cell cultures that had been infected with CMV for 24 h resulted in a significant reduction in released cytotoxic activity to mouse L929 cells at 3-22 h post-LPS compared with mock-infected cultures. However, using an ELISA to measure TNF-alpha antigen levels in these culture supernatants, we found infected cultures had significantly higher TNF-alpha antigen levels than in mock-infected cultures following LPS induction. CMV alone also induced TNF-alpha release and possibly TNF-alpha inhibitor(s) which may have blocked TNF-alpha associated cytotoxic activity in CMV-infected THP-1 cell culture supernatants.     

Serine protease inhibitors block priming of monocytes for enhanced release of superoxide.

Monocytes freshly isolated from human blood produced large amounts of superoxide when triggered by phorbol ester. After monocytes were cultured for 18-24 hr in endotoxin-free, non-adherent conditions, they produced low amounts of superoxide. Addition of lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), or platelet-activating factor (PAF) at the beginning of culture 'primed' the monocytes, causing them to maintain a high superoxide response for at least 96 hr. Also, in response to LPS, monocytes secreted TNF-alpha. The ability of LPS, IFN-gamma, TNF-alpha or PAF to maintain the high superoxide response was blocked by addition of inhibitors of serine proteases, either 4-(2-aminoethyl)-benzenesulphonyl fluoride (AEBSF) or 3,4-dichloroisocoumarin. AEBSF was most effective at 200 microns, and required 6 hr for maximum effect. AEBSF did not affect phorbol-triggered superoxide release by unprimed monocytes. AEBSF did not affect cell viability, nor did it interfere with the TNF-alpha secretion in response to LPS. An analogue of AEBSF that lacked ability to inhibit proteases did not affect monocyte responses. 3,4-Dichloroisocoumarin blocked priming at a low concentration, 1 microM. We conclude that activity of a monocyte serine protease is required to maintain the high superoxide response in monocytes primed with LPS, IFN-gamma, TNF-alpha, or PAF.     

C2-ceramide and C6-ceramide inhibited priming for enhanced release of superoxide in monocytes, but had no effect on the killing of leukaemic cells by monocytes.

Ceramide acts as an intracellular second messenger in cellular signal transduction. We examined the effects of two cell-permeable ceramides, C2-ceramide and C6-ceramide, on human monocyte functions. After monocytes were primed with lipopolysaccharide (LPS) or interferon-gamma (IFN-gamma) for 18 hr in suspension culture, they produced a high amount of superoxide (O2-) when triggered by phorbol myristate acetate. C2- or C6-ceramide inhibited O2- release from monocytes primed with LPS (1 ng/ml) or IFN-gamma (100 U/ml), but did not affect unprimed monocytes. An analogue, C2-dihydroceramide, was inactive. C2-ceramide was most effective at 6 microM, and C6-ceramide at 60 microM. C2- or C6-ceramide at these concentrations was not toxic for monocytes, as assessed by trypan blue exclusion and by the 3-[4, 5-dimethylthiazol-2-y1]-2,5 diphenyl tetrazolium bromide (MTT) assay which measures the ability of live cells to produce formazan. C2-ceramide (20 microM) had no effect on the killing of leukaemic cells (HL-60 and K562 cells) by monocytes treated with IFN-gamma, LPS, or both for 18 hr, with killing assessed by an 111 Indium-releasing assay. C2-ceramide (20 microM) induced secretion of low amounts of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) from the monocytes. But C2-ceramide did not alter the higher secretion of TNF-alpha or IL-1 beta from monocytes treated with IFN-gamma or LPS. Thus the cell-permeable ceramides acted like antagonists of LPS, rather than analogues of LPS, as has been proposed. The results here showed that the signal transduction pathway for O2- release by monocytes differed from that for the cytolysis of leukaemic cells, and confirmed that oxygen radicals are not involved in cytolysis.     

Paradoxical stimulation and inhibition by protein kinase C modulating agents of lipopolysaccharide evoked production of tumour necrosis factor in human monocytes.

Human blood monocytes were activated by bacterial lipopolysaccharide endotoxin (LPS) (10 ng/ml) for cytotoxicity of WEHI-164 mouse fibrosarcoma cells, determined by release of 51Cr from WEHI-164 tumour cells incubated with monocyte supernatants. The chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (FMLP) augmented LPS-induced cytotoxicity but had no effect alone. FMLP but not LPS stimulated phospholipase C (PLC), determined by the release of [3H]inositol phosphates. Addition of tumour promoter and protein kinase C stimulant, phorbol-12-myristate-13-acetate (PMA) at concentrations of 3 x 10(-10) M to 3 x 10(-9) M, resulted in an augmentation of 30-200% in LPS-evoked cytotoxicity. The effects of FMLP and PMA, like the effect of LPS alone, were completely blocked by antibody to recombinant human tumour necrosis factor-alpha (TNF-alpha), indicating that cytotoxicity induced by LPS, FMLP, and PMA were due solely to TNF release. Concentrations of PMA greater than 3 x 10(-9) M caused inhibition of TNF release. Okadaic acid (20 ng/ml), an inhibitor of phosphatases I and IIa, augmented the effects of LPS and the stimulatory effects of low levels of PMA, suggesting that phosphorylation was important in the actions of both LPS and PMA. The effects of LPS and of low levels of PMA were augmented by the protein kinase C (PKC) inhibitors H-7 (10-30 microM), staurosporine (2-10 nM) and calphostin C (0.1 microM). Higher concentrations of the inhibitors prevented LPS-evoked TNF release and its augmentation by low levels of PMA. However, they did not prevent the inhibition by high levels of PMA. One possible explanation for the results is that different isozymes of PKC may mediate the stimulatory as compared to the inhibitory effects of PKC on TNF production.     

Membrane molecules which trigger the production of interleukin-1 and tumor necrosis factor-alpha by lipopolysaccharide-stimulated human monocytes.

We have investigated the role of the membrane molecules CD11/CD18 and CD14 which may mediate the binding of lipopolysaccharide (LPS) to human monocytes, in the induction of the production and release of interleukin (IL)-1 and tumor necrosis factor-alpha (TNF-alpha) by LPS-stimulated cells. Blockade of CD11a, CD11b and CD18 with saturating concentrations of specific mAb did not inhibit the release of cytokines from LPS-stimulated monocytes. In contrast, inhibition of the release of IL-1 beta and TNF-alpha occurred in monocytes cultures that had been pretreated with either of two monoclonal antibodies (mAb) recognizing different epitopes on the CD14 molecule. The binding of LPS to CD14 has been previously shown to require serum factors. In the present study, we found that serum had an enhancing effect on the release of IL-1 and TNF-alpha from LPS-stimulated cultures of normal human monocytes. The inhibitory effect of anti-CD14 mAb was, however, observed in cultures performed in the presence or in the absence of serum, suggesting that triggering of IL-1/TNF-alpha release by CD14 is independent of LPS-binding proteins or other serum proteins. IL-1 beta and TNF-alpha were also released from LPS-stimulated cultures of monocytes from patients with paroxysmal nocturnal hemoglobinuria lacking expression of CD14. Thus, CD14 but not CD11/CD18 can trigger serum-dependent and independent cytokine release from endotoxin-stimulated normal human monocytes; CD14 is not, however, the only LPS receptor that is involved in the secretory response of endotoxin-stimulated cells.     

Tumor necrosis factor-alpha in gestation and puerperium of women with gestational hypertension and pre-eclampsia

PROBLEM: High plasma levels of tumor necrosis factor-alpha (TNF-alpha) in pregnant women have been associated with the pathogenesis of pre-eclampsia (PE). This study evaluated TNF-alpha plasma levels and monocyte production in gestational hypertension (GH) and PE during gestation and at puerperium. METHOD OF STUDY: This study included 128 women, of whom 20 were non-pregnant (NP) normotensive (NT), and 108 were pregnant: 36 NT, 27 with GH, and 45 with PE. Peripheral blood plasma was used for TNF-alpha and uric acid determination. TNF-alpha was determined in plasma and lipopolysaccharide (LPS)-stimulated and non-stimulated monocyte supernatants by L929 bioassay. RESULTS: Tumor necrosis factor-alpha and uric acid plasma levels were higher in PE than in GH pregnancies. In both hypertensive groups, these parameters positively correlated and were significantly more elevated than in NT and NP women. TNF-alpha plasma levels and monocyte production were higher in hypertensive than in NT women during gestation, and significantly decreased at puerperium. Although decreased, TNF-alpha release in LPS-stimulated PE monocytes, was still significantly higher than in the other pregnant groups. CONCLUSION: In vivo monocyte activation in GH and PE pregnant women was characterized by in vitro TNF-alpha production. The fact that higher circulating concentrations of TNF-alpha and uric acid were observed in PE than in GH suggests an association with disease severity.     

Human monocyte production of interleukin-1: parameters of the induction of interleukin-1 secretion by lipopolysaccharides

The role of lipopolysaccharide (LPS) as an activator of human monocyte interleukin-1 (IL-1) synthesis and secretion has been examined in this study. The results demonstrate that when blood monocytes are prepared under low endotoxin conditions, they do not spontaneously secrete IL-1 activity. When cells are exposed to LPS extracted from different bacterial species, there is variation seen in the potency, with LPS from Salmonella species being the most potent in inducing IL-1 activity from human monocytes. This material is tenfold more potent than LPS obtained from three different strains of Escherichia coli and 10,000-fold more potent than material obtained from two other bacterial species. Detoxified endotoxins are inefficient activators for IL-1 secretion. When monocytes are exposed to LPS, there is a rapid rise in the level of IL-1 activity detected. Activity can be detected in cell lysates after 1 hr with appreciable accumulation seen over the first 6 hr of culture. This is accompanied by IL-1 release into the surrounding medium after 2 hr of culture with subsequent accumulation. Monocyte synthesis of IL-1 activity appears to be sensitive to fg/ml levels of Salmonella minnesota LPS, while appreciable secretion of this activity by monocytes requires pg/ml levels.     

Administration of bacterial lipopolysaccharide elicits circulating tumor necrosis factor-alpha in neonatal calves.

The presence of tumor necrosis factor-alpha (TNF-alpha) during endotoxemia in ruminants has not been reported previously. In this study, we detected the in vivo release of bovine TNF-alpha by using WEHI-164 murine fibrosarcoma cells as targets in an 18-h cytotoxicity assay. Treatment of the WEHI-164 cells with 1 microgram of actinomycin D (dactinomycin) enhanced approximately twofold the susceptibility of the cells to TNF-alpha activity. TNF-alpha activity in sera from neonatal calves injected intravenously with 2.7 micrograms of Escherichia coli lipopolysaccharide (LPS) increased rapidly within the first 2 h postinjection and then declined until it was undetectable by 4 h postinjection. Sera taken before LPS administration had no TNF-alpha activity. LPS (10 micrograms/ml) and fetal, newborn, and pooled adult bovine sera alone and in combination had no direct cytotoxic effects on WEHI-164 cells. TNF-alpha cytotoxic activity is probably not due to the presence of interleukin-1 (IL-1), alpha interferon, or gamma interferon in the sera since recombinant human IL-1, natural bovine IL-1, and recombinant bovine alpha and gamma interferons had no direct cytotoxic effects on WEHI-164 cells. A monoclonal antibody that neutralizes recombinant human TNF-alpha significantly reduced the cytotoxic activity of sera from LPS-injected calves.     

Human recombinant IL-1 receptor antagonist (IL-1Ra) inhibits leukotriene B4 generation from human monocyte suspensions stimulated by lipopolysaccharide (LPS).

The effect of human recombinant IL-1 receptor antagonist (hrIL-1Ra) on leukotriene B4 (LTB4) release was investigated in activated human monocyte cultures. To stimulate LTB4 generation, LPS was used as an agonist. Detection was performed with the highly sensitive radioimmunoassay method. The cells were treated with scalar concentrations using LPS at 1-1000 ng/ml for different periods of time. The greater LTB4 stimulation was found at LPS 100 ng/ml for 18 h incubation time. Preincubation of monocytes with cytochalasin B (CB) (5 micrograms/ml) for 15 min augmented the release of LTB4 when LPS was used. A dose-dependent inhibition was found when human monocytes were pretreated for 10 min with hrIL-1Ra at different concentrations (0.25-250 ng/ml) and then treated with LPS 100 ng/ml for 18 h. Maximum inhibition was observed at the highest concentration of hrIL-1Ra (250 ng/ml). Macrophages treated with a non-selective 5-lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), used at 10 microM, added 15 min before LPS 100 ng/ml, produce a dose-dependent inhibition of LTB4. Cells pretreated with arachidonic acid, at various concentrations (10(-9)-10(-5) M) for 10 min and then treated with LPS 100 ng/ml for 18 h, were also inhibited in a dose-dependent manner by hrIL-1Ra in their production of LTB4. The inhibition of LTB4 release by hrIL-1Ra, in LPS-stimulated human monocytes, may suggest an important modulatory role for this new cytokine (monokine) in inflammation and immunity and may hold future therapeutic implications for diseases involving LTB4 as a mediator.     

Control by IFN-gamma and PGE2 of TNF alpha and IL-1 production by human monocytes.

There have been suggestions that the production of pro-inflammatory mediators by human monocytes in response to interferon-gamma (IFN-gamma) may be controlled by changes in prostaglandins. Therefore we investigated tumour necrosis factor alpha (TNF alpha) and interleukin-1 (IL-1) activities and prostaglandin E2 (PGE2) levels in the supernatants of highly purified human monocytes cultured for 18 hr with recombinant human IFN-gamma. IFN-gamma (100 U/ml) did not stimulate monocytes isolated by counter-current centrifugal elutriation for detectable TNF alpha or IL-1 activities, or PGE2 production. However, IFN-gamma synergistically enhanced lipopolysaccharide (LPS)-induced TNF alpha and IL-1 activities. In contrast, there was no consistent change in PGE2 levels upon addition of IFN-gamma to LPS-treated monocyte cultures. The TNF alpha and IL-1 activities induced by LPS and by LPS with IFN-gamma were reduced by PGE2, and stimulated by indomethacin. As reported previously for IL-1 activities, the regulation by cyclo-oxygenase products of TNF alpha activities reflected predominantly a control of the production of immunoreactive TNF alpha, rather than the measurement of TNF alpha bio-activity. However, the addition of indomethacin or PGE2 to monocyte cultures did not change the extent of IFN-gamma synergy with LPS for increased TNF alpha and IL-1 activities. The results of this study suggest that, despite control by cyclo-oxygenase products of TNF alpha and IL-1 production in human monocytes, IFN-gamma may enhance TNF alpha and IL-1 activities independently of this regulatory mechanism. These findings are contrary to those suggested for the regulation by prostanoids of IL-1 production by murine macrophages.     

Role of endotoxin in the expression of human monocyte cytotoxicity

The role of bacterial endotoxin in the expression of human monocyte cytotoxicity was studied. Endotoxin contamination of all reagents and steps of the experimental procedure were assessed by the limulus amebocyte lysate (LAL) assay. Peripheral blood monocytes were separated by adherence, and cytotoxicity was measured as [3H]-thymidine release from prelabeled mKSA-TU5 murine target cells in a 48-hr assay. Human monocytes expressed appreciable levels of spontaneous cytotoxicity under LAL- conditions irrespective of the serum source employed (LAL- or LAL+ fetal bovine serum, FBS, or LAL- human cord serum, HCS). HCS gave the highest cytotoxicity levels and LAL- FBS the lowest. Polymyxin B (10 micrograms/ml), which inhibited endotoxin-induced gelation of LAL and activation of mononuclear cells for procoagulant activity, did not affect the expression of spontaneous monocyte cytotoxicity with all three sera used. Three preparations of endotoxin used in the present study (Escherichia coli, S. typhosa, S. minnesota) caused small increases in monocyte killing in micrograms per milliliter concentrations only in 5 of 19 experiments performed. Human lymphoblastoid interferon (LAL-) and phytohemagglutinin-elicited lymphokine supernatants (LAL-) enhanced the tumoricidal activity of human monocytes under LAL- conditions and were not affected by Polymyxin B. It is concluded that exposure to endotoxin is not a prerequisite for the expression of spontaneous cytotoxicity by human blood monocytes.     

In vitro cultured human monocytes release fibroblast proliferation factor(s) different from interleukin 1

Human monocytes cultured in vitro released factors with growth stimulatory effect upon human dermal fibroblasts. Monocytes in RPMI 1640 released a growth enhancing activity in nearly constant amounts during in vitro differentiation to macrophage-like cells. The growth stimulatory effect mediated by supernatants was reduced when lipopolysaccharide (LPS) or opsonized zymosan particles (OpZ) were added to monocytes during the first 5 days of in vitro culture, thereafter reaching similar levels. The release of interleukin 1 (IL-1) from monocytes was restricted to the first 2 days of culture. IL-1 production by unstimulated monocytes was negligible, while LPS induced a high release of IL-1 from monocytes. A rabbit antibody against human IL-1 abolished the IL-1 activity of the monocyte supernatants as assessed in a mouse thymocyte proliferation assay, but caused only a small reduction of the fibroblast proliferation activity. Thus, the fibroblast growth stimulatory activity mediated by monocytes was caused by factor(s) in addition to IL-1.     

Cytokine regulation of the intracellular growth of Mycobacterium paratuberculosis in bovine monocytes.

In this study we examined the influence of various crude and recombinant cytokines on the ingestion and intracellular survival of Mycobacterium paratuberculosis within bovine monocytes and monocyte-derived macrophages. Cytokine pretreatment had little effect on the ingestion of M. paratuberculosis by bovine monocytes and macrophages. Monocytes that were continuously incubated with virus-induced crude bovine interferon (100 U) or recombinant bovine alpha interferon (100 U) significantly restricted the subsequent intracellular growth of M. paratuberculosis, as determined by microscopic counts of acid-fast bacilli and by recovery of CFU from lysed monocyte monolayers. In contrast to their effects on freshly adherent monocytes, these cytokines had little effect on the growth of M. paratuberculosis within monocyte-derived macrophages. In two separate experiments, we also observed inhibition of bacillary growth in monocytes treated with unpurified recombinant human granulocyte-macrophage colony-stimulating factor. Conversely, intracellular growth of M. paratuberculosis was enhanced in monocytes that were pretreated with culture supernatants from M. paratuberculosis-stimulated peripheral blood mononuclear cells obtained from an immunized calf. The growth-enhancing activity of these supernatants was labile at pH 2.0, suggesting a role for gamma interferon; however, subsequent experiments indicated that recombinant gamma interferon alone neither enhanced nor restricted intracellular bacillary growth. To determine the possible contributions of monocyte oxidative activity to cytokine-induced bacteriostasis, we compared the release of superoxide anion from cytokine-treated and control monocytes. No obvious relationship was observed between the release of superoxide anion and the subsequent intracellular fate of the bacilli.     

Differential production of tumor necrosis factor, macrophage colony stimulating factor, and interleukin 1 by human alveolar macrophages

Human alveolar macrophages (AMO) have been investigated for their ability to produce three monokines, tumor necrosis factor-alpha (TNF), macrophage colony stimulating factor (CSF-1), and interleukin 1-beta (IL-1). No TNF activity was found in supernatants of unstimulated AMO cultured for 20 h, although TNF mRNA was detected in the cells by Northern blot analysis. Stimulation of the cells with lipopolysaccharide (LPS) induced production and release of high levels of TNF into the culture supernatant. Increased levels of TNF mRNA were detectable at 90 min after LPS stimulation by dot blot analysis, reaching maximum expression between 4 and 8 h and declining thereafter. TNF activity peaked at approximately 8 h in the AMO supernatants. After 24 h TNF production had ended. Compared to autologous monocytes the AMO produced 5.7 times more TNF on a per cell basis (activity accumulated in 20 h supernatants). Uncultured AMO expressed CSF-1 mRNA which was translated into active protein recovered in supernatants upon culture in the absence of stimulus. The addition of LPS to AMO slightly reduced both mRNA levels and amount of factor in the supernatants. In contrast to the AMO, monocyte production of CSF-1 was enhanced by LPS. CSF-1 production by AMO continued for at least 48 h of culture. Spontaneous production of low amounts of IL-1 was found in one-third of the AMO samples, while low levels of IL-1 mRNA were present in all tested preparations. LPS stimulation induced increase in IL-1 mRNA within 90 min; mRNA levels peaked between 12 and 20 h and stayed high for at least 42 h. However, while all LPS-stimulated AMO expressed high levels of IL-1 mRNA biologically active IL-1 was again detected only in a fraction of the AMO supernatants. These results show that the production of monokines CSF-1, TNF, and IL-1 is differentially regulated by LPS in alveolar macrophages and has different responses as compared to monocytes. The longevity of the messages for each of the factors is possible indicators of the relative contribution of these factors to the response to endotoxin-induced injury and repair processes in the lung.     

Regulation by interleukin-3 of human monocyte pro-inflammatory mediators. Similarities with granulocyte-macrophage colony-stimulating factor.

Urokinase-type plasminogen activator (u-PA), tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) activities were measured for highly purified human monocytes cultured for 18 hr with recombinant human interleukin-3 (IL-3). IL-3 alone stimulated monocyte u-PA activity, but not TNF-alpha or IL-1 activity. However, IL-3, together with interferon-gamma (IFN-gamma), stimulated the TNF-alpha, but not IL-1, activities of monocytes from several donors. In parallel cultures, granulocyte-macrophage colony-stimulating factor (GM-CSF) behaved similarly. IL-3, like GM-CSF, synergized weakly and sometimes irregularly with lipopolysaccharide (LPS) for increased TNF-alpha and IL-1 activities. Thus, IL-3 can selectively stimulate monocyte mediator production depending on the costimulus present; however, the stimulatory properties of IL-3 vary from those of IFN-gamma and IL-4. The similarities in activity between IL-3 and GM-CSF may be explained by a common or associated IL-3/GM-CSF receptor(s), as suggested by biochemical studies.     

Lipopolysaccharide-Induced Tumor Necrosis Factor Alpha Production by Human Monocytes Involves the Raf-1/MEK1-MEK2/ERK1-ERK2 Pathway

During gram-negative sepsis, human monocytes are triggered to produce large quantities of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) in response to endotoxin (lipopolysaccharide [LPS]). Several studies have identified signal transduction pathways that are activated by LPS, including activation of nuclear factor-kappaB (NF-kappaB) and activation of mitogen-activated protein kinases (MAPKs), including ERK1 and ERK2, c-Jun N-terminal kinase, and p38. In this study, the relevance of ERK1 and ERK2 activation for LPS-induced TNF-alpha production by primary human monocytes has been addressed with PD-098059, which specifically blocks activation of MAPK kinase (MEK) by Raf-1. TNF-alpha levels in the monocyte culture supernatant, induced by 10 ng of LPS/ml, were reduced by PD-098059 (50 microM). In addition, PD-098059 also reduced TNF-alpha mRNA expression when cells were stimulated for 1 h with LPS. On the other hand, LPS-induced interleukin-10 (IL-10) levels in the monocyte supernatant were only slightly inhibited by PD-098059. Ro 09-2210, a recently identified MEK inhibitor, completely abrogated TNF-alpha levels at nanomolar concentrations. IL-10 levels also were strongly reduced. To show the efficacy of PD-098059 and Ro 09-2210, ERK1 and -2 activation was monitored by Western blotting with an antiserum that recognizes the phosphorylated (i.e., activated) forms of ERK1 and ERK2. Addition of LPS to human monocytes resulted in activation of both ERK1 and ERK2 in a time- and concentration (50% effective concentration between 1 and 10 ng of LPS/ml)-dependent manner. Activation of ERK2 was blocked by PD-098059 (50 microM), whereas ERK1 seemed to be less affected. Ro 09-2210 completely prevented LPS-induced ERK1 and ERK2 activation. LPS-induced p38 activation also was prevented by Ro 09-2210. These data further support the view that the ERK signal transduction pathway is causally involved in the synthesis of TNF-alpha by human monocytes stimulated with LPS.     

Role of endogenous prostaglandin E2 in interleukin 1 production by peripheral blood monocytes from patients with rheumatoid arthritis

We studied the effect of endogenous prostaglandin E2 (PGE2) on interleukin 1 (IL-1) production by peripheral blood monocytes from patients with rheumatoid arthritis (RA). IL-1 production by RA monocytes was not different from that of monocytes from normal controls, when the cells were either unstimulated or stimulated with lipopolysaccharide (LPS, 20 micrograms/ml), as measured by two different bioassays (thymocyte or fibroblast proliferation assay) and enzyme-linked immunosorbent assay. However, IL-1 production by LPS-stimulated monocytes from RA patients cultured in medium containing indomethacin, an inhibitor of PGE2 synthesis, was significantly greater than that of monocytes from normal controls. In addition, the levels of PGE2 in culture supernatants of unstimulated or LPS-stimulated monocytes from RA patients were higher than in culture supernatants of monocytes from normal controls. Moreover, the increase of in vitro IL-2 production by RA T cells stimulated by phytohemagglutinin (PHA) was observed when monocytes were removed from peripheral blood mononuclear cells. These results indicated that peripheral blood monocytes from RA patients could produce IL-1 in excess in vitro, but that in vivo IL-1 production by RA monocytes and IL-2 induction by RA T cells might be negatively regulated by endogenous PGE2.     

Exercise training-induced lowering of inflammatory (CD14+CD16+) monocytes: a role in the anti-inflammatory influence of exercise?

Exercise training or higher levels of physical activity are known to exert anti-inflammatory effects. CD14+CD16+ monocytes are potent producers of inflammatory proteins, and elevated levels of these "inflammatory" monocytes have been implicated in disease development. Little is known about the influence of exercise training on this cell population. On the basis of their physical activity pattern, male and female subjects, 65-80 years old, were assigned to a physically active (PA; n=15) or inactive (PI; n=15) group. The PI group performed 12 weeks (3 days/week) of endurance (20 min at 70-80% heart-rate reserve) and resistance exercise training (eight exercises, two sets at 70-80% of one repetition maximum). Subjects in the PA group maintained their habitual activity level. Flow cytometry was used to determine monocyte phenotype and monocyte TLR4 expression. ELISAs were used to measure whole blood, LPS-stimulated TNF-alpha production, and serum C-reactive protein (CRP). At baseline, the PA group had a lower percentage of CD14+CD16+ monocytes and lower unstimulated production of TNF-alpha than the PI group. CD14+CD16+ monocyte percentage and 1 ng/ml LPS-stimulated TNF-alpha production were reduced after the PI group underwent 12 weeks of exercise training. PI subjects also had higher TLR4 expression on classical monocytes, but there were no significant exercise training-induced changes in monocyte TLR4 expression. The PA group had significantly lower serum CRP than the PI group. Physical activity was associated with lower CD14+CD16+ monocyte percentage and LPS-stimulated TNF-alpha production. Exercise training-induced reductions in CD14+CD16+ monocytes may contribute to the anti-inflammatory effects of exercise training.     

Membrane expression and shedding of tumour necrosis factor receptors during activation of human blood monocytes: regulation by desferrioxamine.

Previous studies have shown that desferrioxamine (DFX), an iron chelator preventing the synthesis of hydroxyl radical (OH.), up-regulates the cell-surface expression of tumour necrosis factor-alpha (TNF-alpha) receptors on unactivated human blood monocytes. In the present study, we investigated the regulatory action of DFX on 125I-TNF-alpha binding to monocytes upon exposure to bacterial lipopolysaccharide (LPS). Exposure to LPS (1 microgram/ml) resulted in almost complete loss of 125I-TNF-alpha binding to the surface of monocytes. This down-regulation was reversible and the recovery observed after 18 hr was enhanced by addition of DFX (5 mM). However, binding studies on monocytes pre-exposed to low pH suggested that the DFX-induced increase of 125I-TNF-alpha binding was not due to differences in the number of receptors available but was probably due to a reduction of receptor occupancy by endogenously generated TNF-alpha. Time-course studies of TNF-alpha release from monocytes confirmed the ability of DFX to reduce the extracellular concentration of bioactive TNF-alpha through a decrease of its synthesis and an increase of its inactivation. The latter process was associated with an increased expression of the soluble form of TNF-alpha receptor type II. These results indicate that, in the presence of LPS, DFX increases the release of soluble TNF-alpha receptors from monocytes. Thus, conversely, OH. generated in situ could reduce the shedding of soluble TNF-alpha receptors and, hence, increase the widespread release of bioactive TNF-alpha.