Enhancement of the surface expression of tumor necrosis factor alpha (TNFalpha) but not the p55 TNFalpha receptor in the THP-1 monocytic cell line by matrix metalloprotease inhibitors

The monocytic cell line THP-1 can be induced to express and release tumor necrosis factor alpha (TNFalpha) and both TNFalpha receptors (p55 and p75) upon exposure to bacterial lipopolysaccharide (LPS). The broad-spectrum matrix metalloprotease (MMP) inhibitors [4-(N-hydroxyamino)-2R-isobutyl-3S-(phenylthiomethyl)succinyl]-L-p henylalanine-N-methylamide (GI-129471) and marimastat [2S-[N4(R*),2R*,3S*]]-N4[2,2-dimethyl-1-[(methylamino)carbonyl]propyl]-N 1,2-dihydroxy-3-(2-methylpropyl)butanediamide (BB-2516) were effective inhibitors of LPS-induced TNFalpha (soluble) release with IC50 values of 0.2 and 4.0 microM, respectively. Upon LPS stimulation, the expression of pro-TNFalpha (membrane associated) on the cell surface (FACS analysis) could not be observed. However, in the presence of GI-129471, a concentration-dependent increase in TNFalpha surface expression was observed. Peak expression (percentage of cells expressing pro-TNFalpha and mean fluorescence units) in the presence of GI-129471 was at 2 hr, and steadily declined to return to near control levels by 8 hr. This time course was similar to TNFalpha release, which also peaked at 2-4 hr after LPS exposure and then declined. Stimulation of THP-1 cells with LPS + phorbol myristate acetate increased the percentage of cells expressing pro-TNFalpha by 10-fold. In the presence of GI-129471, these increases were augmented further and peaked between 2 and 4 hr, but also returned to near control levels of expression by 24 hr. This was in contrast to the release of soluble TNFalpha, which continued to accumulate over a 24-hr time course. TNFalpha receptor I (p55, TNFRI) and II (p75, TNFRII) shedding was also inhibited by GI-129471 (IC50 = 1.5 and 3.1 microM, respectively) and BB-2516 (IC50 = 14 and 15 microM, respectively). Unlike pro-TNFalpha surface expression, surface expression of both TNFalpha receptors steadily increased over 72 hr. In contrast to pro-TNFalpha surface expression, TNFRI surface expression was not augmented by these MMP inhibitors in THP-1 cells after LPS stimulation. Surface expression of TNFRII was augmented by these MMP inhibitors. These results suggest that even in the continued presence of LPS stimulation and an inhibitor of TNFalpha processing, the augmented surface expression of TNFalpha is transient. The potential "deleterious" implications of high levels of surface pro-TNFalpha expression in the presence of these inhibitors may be lessened by its transient nature.     

Activated isoforms of MMP-2 are induced in U87 human glioma cells in response to beta-amyloid peptide

The effect of stress- or lipopolysaccharide (LPS) endotoxin-induced release of ACTH, beta-endorphin (beta-END) and prolactin (PRL) was investigated in two groups of conscious male rats: (1) Rats pretreated with different H3 receptor agonists, which inhibit neuronal histamine (HA) synthesis and release, and (2) rats with bilateral posterior hypothalamic lesion, which destroys the histaminergic perikarya exclusively localized in the mammillary nuclei. The H3 receptor agonists R(alpha)methyl-HA, BP 2-94 or imetit injected intraperitoneally (ip) had no effect on basal secretion of ACTH or PRL but inhibited the ACTH and PRL responses to restraint stress and the ACTH response to LPS endotoxin. LPS had no effect on PRL secretion. The inhibitory effect of the agonists was prevented by prior ip administration of the H3 receptor antagonist thioperamide. Bilateral lesion of the posterior hypothalamus inhibited the ACTH, beta-END and PRL responses to restraint stress, ether stress and LPS endotoxin, whereas sham operation had no effect compared to nonoperated control rats. In addition, posterior hypothalamic lesion inhibited the PRL response but not the ACTH and beta-END responses to activation of serotonergic neurons induced by ip administration of the 5-HT precusor 5-hydroxytryptophan in combination with the 5-HT re-uptake inhibitor fluoxetine. Thus, serotonergic pathways were not damaged by the lesions. The present results support our previous findings that inhibition of neuronal HA synthesis by alpha-fluoromethylhistidine as well as blockade of H1 or H2 receptors inhibit the ACTH, beta-END and PRL responses to stress and LPS endotoxin and further substantiate an important role of histaminergic neurons in the mediation of the stress-induced release of pituitary stress hormones. Furthermore, in accordance with our previous findings, the lesion experiments indicated the existence of an interaction between the histaminergic and serotonergic system in regulation of the stress- and LPS-induced PRL release.     

Long-term results of pediatric liver transplantation: an analysis of 569 transplants

The present study has investigated the therapeutic potential of a type 4 phosphodiesterase (PDE) inhibitor, rolipram, in experimental lung injury. Acute lung injury was induced in the mouse by combined treatment with lipopolysaccharide (LPS; 10 mg/kg, i.v.) and zymosan (3 mg/kg, i.v.), and assessed using extravascular albumin accumulation; neutrophil sequestration in pulmonary capillaries was also measured. The results show that pretreatment with rolipram (5 mg/kg, i.p.) was protective against the induction of lung injury by combined LPS and zymosan; extravascular albumin accumulation was reduced by 89% and neutrophil sequestration in lung tissue, as assessed by lung myeloperoxidase (MPO) activity was reduced by 75%. Pretreatment with rolipram also attenuated increases in serum tumor necrosis factor alpha (TNFalpha) levels induced by LPS and zymosan treatment, measured after 2.5 h. The role of endogenous TNFalpha in the induction of lung injury was therefore assessed. Blockade of endogenous TNFalpha by treatment with the soluble receptor p55-IgG fusion protein or an anti-murine TNFalpha monoclonal antibody, TN3. 19.12, had no protective effect against LPS and zymosan-induced lung injury. This suggests that there is a disassociation between TNFalpha production and the induction of injury in this model. Administration of rolipram after LPS and before zymosan treatment obliterated the increase in pulmonary vascular permeability, but its effect on sequestration of neutrophils in pulmonary microvessels, as measured by MPO, was less marked. The results of the present study suggest that use of agents such as rolipram that inhibit PDE4 may have a therapeutic role in treatment of acute lung injury, since we have shown that it is effective in attenuation of neutrophil activation even after sequestration. However, its effect appears to be independent of TNFalpha inhibition.     

An autopsied case of multiple system atrophy with remarkable cerebral atrophy

OBJECTIVES: We tested the effects of NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthesis, on plasma levels of interleukin (IL) IL-6, IL-8, tumor necrosis factor-alpha (TNFalpha) and nitrite/nitrate (NO2-/ NO3-) in patients with severe septic shock. DESIGN: Prospective clinical study. SETTING: Surgical intensive care unit at a university hospital. PATIENTS: 11 consecutive patients with severe septic shock. INTERVENTIONS: Standard hemodynamic measurements were made and blood samples taken at intervals before, during, and after a 12-h infusion of L-NAME 1 mg x kg(-1) x h(-1) for determination of plasma IL-6, IL-8, TNFalpha and NO2-/NO3- concentration. MEASUREMENTS AND RESULTS: Patients with sepsis had increased plasma levels of IL-6, IL-8, TNFalpha and NO2-/NO3- (p < 0.05). Plasma levels of IL-6. IL-8, and NO2-/NO- were negatively correlated with systemic vascular resistance (r = -0.62, r = -0.65, and r = -0.78, respectively, all p < 0.05). Continuous infusion of L-NAME increased mean arterial pressure and systemic vascular resistance, with a concomitant reduction in cardiac output (all p < 0.01). No significant changes were seen in levels of plasma IL-6, IL-8, and NO-/NO3- during the 24-h observation period. Plasma levels of TNFalpha were significantly reduced during L-NAME infusion compared to baseline (p < 0.05). CONCLUSIONS: NO plays a role in the cardiovascular derangements of human septic shock. Inhibition of NO synthesis with L-NAME does not promote excessive cytokine release in patients with severe sepsis.     

SB 207499 (Ariflo), a second generation phosphodiesterase 4 inhibitor, reduces tumor necrosis factor alpha and interleukin-4 production in vivo

The ability of the second generation phosphodiesterase 4 inhibitor SB 207499 (Ariflo), [c-4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)-r-l-cyclohexane carboxylic acid], to inhibit inflammatory cytokine production in vivo was evaluated and compared to that of rolipram, a first generation phosphodiesterase 4 inhibitor. To examine human tumor necrosis factor alpha (TNFalpha) production, human monocytes were adoptively transferred into Balb/c mice and challenged with lipopolysaccharide (LPS). In this model, SB 207499 inhibited human TNFalpha production with oral ED50 of 4.9 mg/kg. Similarly, R-rolipram inhibited human TNFalpha production with an ED50 of 5.1 mg/kg, p.o. In contrast to their equipotent activity against TNFalpha production, SB 207499 (ED50 = 2.3 mg/kg, p.o.) was 10-fold less potent than R-rolipram (ED50 = 0.23 mg/kg, p.o.) in reversing reserpine-induced hypothermia, a model of antidepressant activity. In time course studies, SB 207499 (30 mg/kg, p.o.) inhibited TNFalpha production for at least 10 hr; substantial plasma concentrations of SB 207499 were detected over the same interval. The ability of SB 207499 to modulate interleukin-4 production in vivo was assessed in a chronic oxazolone-induced contact sensitivity model in Balb/c mice. In this model, topical administration of SB 207499 (1000 microgram) inhibited intralesional concentrations of interleukin-4 (55%; P <.01). The results demonstrate that SB 207499 is a potent inhibitor of inflammatory cytokine production in a variety of settings in vivo. Moreover, although it is as potent as R-rolipram in inhibiting TNFalpha production, it has substantially less central nervous system activity. Thus SB 207499 represents an excellent candidate with which to evaluate the antiinflammatory potential of PDE4 inhibitors.     

Lipopolysaccharide, central in vivo biogenic amine variations, and anhedonia

Systemic administration of lipopolysaccharide (LPS), a non-specific activator of proinflammatory cytokine release from macrophages, provokes sickness characterized by anorexia, soporific effects, and disturbances of locomotor activity and exploration. In addition, endotoxin treatment may provoke an anhedonic response. Assessment of anhedonia in appetitive paradigms, however, is compromised by the anorexia provoked by the treatment. The present investigation assessed the anhedonic effects of LPS on rewarding lateral hypothalamic brain stimulation. Using a simultaneous discrimination, current titration procedure in the assessment of intracranial self-stimulation (ICSS), it was found that acute systemic administration of LPS (50 microg, 100 microg or 200 microg) reduced ICSS during the ascending sequence of current presentations, but had little effect on responding to a series of descending currents. In a parallel experiment, peripheral administration of LPS (100 microg) increased in vivo dopamine (DA) efflux from the nucleus accumbens, a region thought to be involved in goal-directed responding to positively reinforcing stimuli. It is suggested that LPS alters ICSS in a manner different than that observed following stressor exposure or peripheral IL-2 treatment. Furthermore, LPS may engender an anhedonic effect (possibly secondary to sickness), and the decline of responding reflects the relation between the cost of responding given in the face of sickness and the reward received for responding.     

Interleukin-10 inhibits activation of coagulation and fibrinolysis during human endotoxemia

Interleukin-10 (IL-10) has been found to inhibit lipopolysaccharide (LPS)-induced tissue factor expression by monocytes in vitro. To determine the effects of IL-10 on LPS-induced activation of the hemostatic mechanisms in vivo, we performed a placebo-controlled, cross-over study of human endotoxemia. Two groups of eight volunteers were challenged with LPS (4 ng/kg) on two occasions: once in conjunction with placebo, and once with recombinant human IL-10 (rhIL-10; 25 microg/kg). In group 1, placebo or rhIL-10 was given 2 minutes before LPS challenge, group 2 received placebo or rhIL-10 1 hour after LPS administration. Pretreatment with rhIL-10 reduced both LPS-induced activation of the fibrinolytic system (plasma concentrations of tissue type plasminogen activator, plasmin-alpha2-antiplasmin complexes, and D-dimer), and inhibition of fibrinolysis (plasma levels of plasminogen activator inhibitor 1), whereas posttreatment only inhibited the latter response. Both IL-10 pre- and posttreatment attenuated activation of the coagulation system (plasma levels of prothrombin fragment F1 + 2 and thrombin-antithrombin complexes). These results indicate that rhIL-10, besides its well-described inhibitory effects on cytokine release, potently modulates the fibrinolytic system and inhibits the coagulant responses during endotoxemia.     

Resistance to endotoxin shock in spontaneously hypertensive rats

Septic shock involves systemic vasodilation mediated by proinflammatory cytokines. In essential hypertension, vascular and immune dysfunctions are closely associated. The response of hypertensive animals compared with normotensive controls to endotoxin (lipopolysaccharide; LPS) challenge is not known. Age-matched (12 weeks) normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were exposed to intravenous injection of 10 mg/kg LPS. Survival rate at 24 hours was markedly higher in SHR than in WKY (12 of 15 and 3 of 15, respectively; P<0.01). Survival of LPS-injected SHR was not related to their hypertension because hydralazine-treated SHR with normalized pressure had similar survival rates, and WKY made hypertensive by clipping of one renal artery showed fatality similar to that of normotensive WKY. Continuous arterial pressure and sequential plasma levels of interleukin-6 (IL-6) and tumor necrosis factor (TNF) were measured in LPS-treated SHR and WKY. Both the duration of the delayed hypotensive phase and the systemic release of IL-6 were much lower in SHR than WKY, whereas both acute hypotension and plasma TNF peak were equivalent. We further explored in vitro the inflammatory response and showed that LPS-activated whole blood from SHR produced less TNF and IL-6 than WKY LPS-activated whole blood. Our results indicate that SHR have a greater ability to resist endotoxic shock than WKY. This is not related to their hypertension but is associated with an attenuated inflammatory response to LPS.     

Prostaglandin E2 and bacterial lipopolysaccharide stimulate bioactive interleukin-1 release from rat hypothalamic explants

While interleukin-1 (IL-1) is intimately involved in locally modulating the acute inflammatory response, it is also able to influence processes at remote sites, i.e., in an endocrine manner. While there is as yet little evidence that IL-1 can cross the blood-brain barrier, many effects such as fever, increased slow-wave sleep, anorexia and the modulation of neuroendocrine function suggest an action of circulating IL-1 at regulatory sites within the hypothalamus. However, there is accumulating evidence for IL-1 originating within the central nervous system (CNS), and it is currently unclear as to whether the neurally mediated manifestations of the acute inflammatory response are due to activation of central or peripheral (circulating) IL-1. In this study we have characterized the release of IL-1 from rat hypothalamic explants, and we have investigated the effects of putative modulators of IL-1 release, lipopolysaccharide (LPS) and the prostaglandins E2 (PGE2) and F2 alpha (PGF2 alpha). After 1 h of incubation, IL-1-like activity in hypothalamic supernatants ranged between 175 and 2,304 munits/mg of protein; this was substantially inhibited by the addition to the bioassay system of antibodies (1:200) against IL-1 alpha, but not against IL-1 beta. LPS and PGE2 significantly stimulated IL-1 release at 100 and 1 ng/ml respectively, whereas PGF2 alpha had no effect in the range of doses tested. It is therefore concluded that the control of hypothalamic IL-1 release may be investigated by means of acute rat hypothalamic explants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alveolar macrophage release of tumor necrosis factor-alpha in chronic alcoholics without liver disease

Alcohol is an immunosuppressive drug, and chronic abuse has been associated with increased susceptibility to a variety of infections, including bacterial pneumonia and tuberculosis. Alveolar macrophages are the resident phagocytes of the lung and play a central role in lung host defenses against infection ranging from direct antibacterial activity to the release of proinflammatory cytokines such as tumor necrosis factor-alpha (TNFalpha). TNFalpha, in particular, plays a key role in the development of the early inflammatory response. In this study, we investigated the effects of chronic alcohol consumption on alveolar macrophage release of TNFalpha in vitro. We prospectively studied lipopolysaccharide (LPS)-stimulated release of TNFalpha from alveolar macrophages obtained from bronchoalveolar lavage fluid (BALF) in 22 alcoholic (18 smokers, 4 nonsmokers) and 7 nondrinking healthy volunteers (3 smokers, 4 nonsmokers). The total number of cells recovered by bronchoalveolar lavage (BAL) and their differential distribution were not significantly different in alcoholics versus controls (43 +/- 8 x 10(6) and 39 +/- 13 x 10(6), respectively). However, the total number of cells recovered from BALF was significantly higher in smokers (51 +/- 8 x 10(6)) than in nonsmokers (19 +/- 5 x 10(6)). Spontaneous (basal) release of TNFalpha by alveolar macrophages was the same in alcoholics and controls. In contrast, LPS-stimulated release of TNFalpha was significantly suppressed in alcoholics compared with that of controls (1343 +/- 271 vs. 3806 +/- 926 U TNF/ml/10(6) cells, respectively, p < 0.015). When controlled for smoking, LPS-stimulated TNFalpha production was suppressed in alcoholic nonsmokers (563 +/- 413 U TNF/ml/10(6)) compared with control nonsmokers (5113 +/- 1264 U TNF/ml/10(6)). LPS-stimulated TNFalpha production was also less in control smokers (2063 +/- 386 U TNF/ml/10(6) cells) than in control nonsmokers (5113 +/- 1264 U TNF/ml/10(6) cells). There was no difference in TNFalpha production between smoking alcoholics and smoking control subjects. We conclude that chronic alcohol consumption significantly suppresses LPS-stimulated alveolar macrophage production of TNFalpha. This effect is obscured if the subject also smokes. Because TNFalpha production is an important element in host defense, this may explain, in part, the susceptibility of chronic alcohol abusers to a variety of infections.     

The mitochondrial permeability transition is required for tumor necrosis factor alpha-mediated apoptosis and cytochrome c release

This study assesses the controversial role of the mitochondrial permeability transition (MPT) in apoptosis. In primary rat hepatocytes expressing an IkappaB superrepressor, tumor necrosis factor alpha (TNFalpha) induced apoptosis as shown by nuclear morphology, DNA ladder formation, and caspase 3 activation. Confocal microscopy showed that TNFalpha induced onset of the MPT and mitochondrial depolarization beginning 9 h after TNFalpha treatment. Initially, depolarization and the MPT occurred in only a subset of mitochondria; however, by 12 h after TNFalpha treatment, virtually all mitochondria were affected. Cyclosporin A (CsA), an inhibitor of the MPT, blocked TNFalpha-mediated apoptosis and cytochrome c release. Caspase 3 activation, cytochrome c release, and apoptotic nuclear morphological changes were induced after onset of the MPT and were prevented by CsA. Depolarization and onset of the MPT were blocked in hepatocytes expressing DeltaFADD, a dominant negative mutant of Fas-associated protein with death domain (FADD), or crmA, a natural serpin inhibitor of caspases. In contrast, Asp-Glu-Val-Asp-cho, an inhibitor of caspase 3, did not block depolarization or onset of the MPT induced by TNFalpha, although it inhibited cell death completely. In conclusion, the MPT is an essential component in the signaling pathway for TNFalpha-induced apoptosis in hepatocytes which is required for both cytochrome c release and cell death and functions downstream of FADD and crmA but upstream of caspase 3.     

Bacterial lipopolysaccharide induces uncoupling protein-2 expression in hepatocytes by a tumor necrosis factor-alpha-dependent mechanism

The liver is a target for bacterial lipopolysaccharide (LPS) and participates in the metabolic response to endotoxemia. Recently published evidence indicates that LPS increases the expression of mitochondrial uncoupling protein-2 (UCP-2) mRNAs in several tissues, including the liver. Because hepatocytes in the healthy liver do not express UCP-2, LPS was thought to induce UCP-2 in liver macrophages, which express UCP-2 constitutively. However, the present studies of cultured peritoneal macrophages indicate that LPS reduces steady state levels of UCP-2 mRNAs in these cells. In contrast, UCP-2 mRNAs are induced in hepatocytes isolated from LPS treated rats and transfection of these hepatocytes with UCP-2 promoter-reporter constructs demonstrates substantial increases in UCP-2 promoter activity. LPS induction of hepatocyte UCP-2 expression is virtually abolished by prior treatment of rats with neutralizing antibodies to tumor necrosis factor alpha (TNF). Futhermore, TNFalpha treatment induces UCP-2 mRNA accumulation in primary cultures of hepatocytes from healthy rats. Thus, hepatocytes are likely to be important contributors to endotoxin-related increases in liver UCP-2 via a mechanism that involves the LPS-inducible cytokine, TNFalpha.     

Effect of a recombinant dimeric tumor necrosis factor receptor on inflammatory responses to intravenous endotoxin in normal humans

To determine the role of tumor necrosis factor (TNF) in lipopolysaccharide (LPS)-induced inflammation, 12 healthy subjects received an intravenous injection with LPS (2 ng/kg) preceded by infusion of either a recombinant human dimeric TNF receptor type II-IgG fusion protein (TNFR:Fc; 6 mg/m2; n = 6) or vehicle (n = 6) from -30 minutes to directly before LPS injection. LPS elicited a transient increase in plasma TNF activity, peaking after 1.5 hours (219 +/- 42 pg/mL; P < .05). Infusion of TNFR:Fc completely neutralized endogenous TNF activity. LPS administration was associated with an early activation of fibrinolysis (plasma concentrations of tissue-type plasminogen activator, plasminogen activator activity, and plasmin-alpha2-antiplasmin complexes), followed by inhibition (plasma plasminogen activator inhibitor type I), changes that were completely prevented by TNFR:Fc. By contrast, TNFR:Fc did not influence LPS-induced activation of coagulation (plasma levels of prothrombin fragment F1 + 2 and thrombin-antithrombin III complexes). TNFR:Fc strongly inhibited endothelial cell activation (plasma levels of soluble E-selectin), modestly reduced neutrophil responses (neutrophilia and plasma concentrations of elastase-alpha1-antitrypsin complexes and lactoferrin), but did not affect the release of secretory phospholipase A2 or lipopolysaccharide-binding protein (P > .05). Infusion of TNFR:Fc only (without LPS) in another 6 normal subjects did not induce any inflammatory response. These data indicate that TNF is involved in only some inflammatory responses to intravenous LPS in humans.     

Mechanisms of the priming effect of lipopolysaccharides on the biosynthesis of leukotriene B4 in chemotactic peptide-stimulated human neutrophils

The goal of this study was to explain the priming effect of lipopolysaccharides (LPS) in human polymorphonuclear leukocytes on leukotriene B4 (LTB4) biosynthesis after stimulation with the receptor-mediated agonist formyl-methionyl-leucyl-phenylalanine (fMLP). This priming effect for LTB4 biosynthesis was maximal after a 30 min preincubation with LPS but was lost when incubations were extended to 90 min or longer. Priming with LPS resulted in an enhanced maximal activation of 5-lipoxygenase (5- to15-fold above unprimed cells) as well as a prolonged activation of the enzyme after stimulation with fMLP compared to that measured in unprimed cells. The activation of 5-lipoxygenase was associated with its translocation to the nuclear fraction of the cell after stimulation of LPS-primed cells but not of unprimed cells. Priming of cells with LPS also resulted in an enhanced capacity (fivefold increase) for arachidonic acid (AA) release after stimulation with fMLP compared to unprimed cells as measured by mass spectrometry. This release of AA was very efficiently blocked in a dose-dependent manner by the 85 kDa cytosolic phospholipase A2 (PLA2) inhibitor MAFP (IC50=10nM) but not by the 14 kDa secretory PLA2 inhibitor SB 203347 (up to 5 microM), indicating that the 85 kDa cPLA2 is the PLA2 responsible for AA release in response to receptor-mediated agonists. In accord with inhibitor studies, the LPS-mediated phosphorylation of cPLA2 followed the same kinetics as the priming for AA release, and a measurable fMLP-induced translocation of cPLA2 was observed only in primed cells. As with AA release and LTB4 biosynthesis, both the phosphorylation and capacity to translocate cPLA2 were reversed when the preincubation period with LPS was extended to 120 min. These results explain some of the cellular events responsible for the potentiation and subsequent decline of functional responses of human polymorphonuclear leukocytes recruited to inflammatory foci.     

Potential role of membrane internalization and vesicle fusion in adhesion of neutrophils in response to lipopolysaccharide and TNF

Human polymorphonuclear leukocytes (PMN) respond to LPS with strongly increased integrin-mediated adhesion. While the first step of this process has been identified as the interaction of LPS with CD14 on the cell surface, subsequent steps remain to be elucidated. The experiments presented here suggest that monomeric LPS is internalized in vesicles, and uptake may be required for signaling. Fluorescently labeled LPS presented as monomeric complexes with soluble CD14 appeared in the plasma membrane of PMN by 5 min and was concentrated in cytoplasmic vesicles by 20 min. Adhesion in response to LPS/soluble CD14 occurred only after a 15- to 20-min lag period, consistent with endocytosis occurring before signal generation. In contrast, there was no time lag for adhesion in response to the formyl peptide formyl-norleucyl-leucyl-phenylalanine (fNLLP). Adhesion in response to LPS, but not fNLLP, was completely blocked by lowering the temperature to 19 degrees C, a procedure that prevents vesicle fusion. These studies indicated that an event with the time and temperature dependence of endocytosis precedes signaling by LPS. Cytochalasin D, an inhibitor of phagocytosis, and wortmannin, an inhibitor of phosphatidylinositol 3-kinase that blocks vesicle fusion and phagocytosis, both completely blocked adhesion in response to LPS but not in response to fNLLP. These results support the idea that LPS internalization and early endosomal fusion may be required for signal transduction. Parallel studies showed that the adhesion response to TNF had time, temperature, and inhibitor sensitivities nearly identical with those of LPS, suggesting that responses to TNF may also include an obligate vesicle fusion step.     

Mature macrophage cell lines exhibit variable responses to LPS

Bacterial lipopolysaccharide (LPS) is a potent activator of cells of the macrophage/monocyte lineage. Two mature macrophage cell lines, P388D1 and RAW264.7, exhibit very different biological responses to LPS. Although RAW264.7 cells release arachidonic acid from phospholipid in response to LPS stimulation, P388D1 cells do not respond in this manner. However, LPS primes P388D1 cells to release arachidonic acid in response to other stimuli. The goal of this work is to contrast the biochemical events that occur in LPS-treated P388D1 and RAW264.7 macrophages. Enzyme assays indicate that LPS treatment induces the activation of cytosolic PLA2 in RAW264.7, but not in P388D1 cells. Phorbol ester (PMA), a receptor-independent stimulus, also fails to induce arachidonic acid release from P388D1 cells, suggesting that these cells may have a defect in the signal transduction machinery that is common to LPS and PMA. This hypothesis is supported by the observation that the expression of the LPS receptors CD14 and CD11b/CD18 is similar on P388D1 and RAW264.7 cells. Western blot analyses indicate that the erk kinases are activated upon LPS treatment of RAW264.7 but not P388D1 cells. LPS-induced arachidonic acid release is reduced in cells treated with the MEK inhibitor PD98059, suggesting that activated erk kinases mediate the phosphorylation and activation of cPLA2 in this system. Interestingly, the p42 isoform of erk (erk2) appears to be activated in resting P388D1 cells. This observation indicates that the MAP kinase cascade may be constitutively activated in P388D1 cells which may in turn limit their ability to respond to LPS. Together, these data provide evidence that mature macrophages from different sources can exhibit variable responses to LPS and highlight the danger of making generalizations regarding the effects of LPS on macrophages.     

Protein kinase C eta mediates lipopolysaccharide-induced nitric-oxide synthase expression in primary astrocytes

The signaling pathway involved in protein kinase C (PKC) activation and role of PKC isoforms in lipopolysaccharide (LPS)-induced nitric oxide (NO) release were studied in primary cerebellar astrocytes. LPS caused a dose- and time-dependent increase in NO release and inducible NO synthase (iNOS) expression. The tyrosine kinase inhibitor, genestein, the phosphatidylcholine-phospholipase C inhibitor, D609, and the phosphatidate phosphodrolase inhibitor, propranolol, attenuated the LPS effects, whereas the PI-PLC inhibitor, U73122, had no effect. The PKC inhibitors (staurosporine, Ro 31-8220, Go 6976, and calphostin C) also inhibited LPS-induced NO release and iNOS expression. However, long term (24 h) pretreatment of cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA) did not affect the LPS response. Previous results have shown that TPA-induced translocation, but not down-regulation, of PKCeta occurs in astrocytes (Chen, C. C., and Chen, W. C. (1996) Glia 17, 63-71), suggesting possible involvement of PKCeta in LPS-mediated effects. Treatment with antisense oligonucleotides for PKCeta or delta, another isoform abundantly expressed in astrocytes, demonstrated the involvement of PKCeta, but not delta, in LPS-mediated effects. Stimulation of cells for 1 h with LPS caused activation of nuclear factor (NF)-kB in the nuclei as detected by the formation of a NF-kB-specific DNA-protein complex; this effect was inhibited by genestein, D609, propranolol, or Ro 31-8220 or by PKCeta antisense oligonucleotides, but not by long term TPA treatment. These data suggest that in astrocytes, LPS might activate phosphatidylcholine-phospholipase C and phosphatidylcholine-phospholipase D through an upstream protein tyrosine kinase to induce PKC activation. Of the PKC isoforms present in these cells, only activation of PKCeta by LPS resulted in the stimulation of NF-kB-specific DNA-protein binding and then initiated the iNOS expression and NO release. This is further evidence demonstrating that different members of the PKC family within a single cell are involved in specific physiological responses.     

Peroxynitrite mediates IL-8 gene expression and production in lipopolysaccharide-stimulated human whole blood

Recent evidence indicates that free oxygen radicals, in particular hydroxyl radicals, may act as intracellular second messengers for the induction of IL-8, a potent chemoattractant and activator of neutrophil granulocytes. Here we report that peroxynitrite (ONOO-), formed by a reaction of nitric oxide (NO) with superoxide, mediates IL-8 gene expression and IL-8 production in LPS-stimulated human whole blood. The NO synthase inhibitors aminoguanidine and NG-nitro-L-arginine methyl ester (L-NAME) blocked IL-8 release by approximately 90% in response to LPS (1 microg/ml), but did not affect the production of IL-1beta or TNF-alpha. Both aminoguanidine and L-NAME blocked the induction of IL-8 mRNA by LPS. Authentic ONOO- (2.5-80 microM) augmented IL-8 mRNA expression and stimulated IL-8 release in a concentration-dependent manner, whereas the NO-releasing compounds, S-nitroso-N-acetyl-DL-penicillamine and sodium nitroprusside failed to induce cytokine production. Combination of the NO-generating chemicals with a superoxide-generating system (xanthine/xanthine oxidase) markedly increased IL-8 release. Enhanced ONOO- formation was detected in granulocytes, monocytes, lymphocytes, and plasma after challenge with LPS. Furthermore, pyrrolidine dithiocarbamate, an inhibitor of activation of nuclear factor-gammaB, markedly attenuated the induction of IL-8 mRNA expression and IL-8 release by either LPS or ONOO-. Our study identifies ONOO- as a novel signaling mechanism for IL-8 gene expression and suggests that inhibition of ONOO- formation or scavenging ONOO- may represent a novel therapeutic approach to inhibit IL-8 production that could lead to reduction of neutrophil accumulation and activation.