Lipopolysaccharide Stimulus Research Articles

Inflammatory Activation of Prostaglandin Production by Microglial Cells Antagonized by Amyloid Peptide

The murine cell line MMGT-16 is of microglial origin and capable of releasing immunoinflammatory cytokines. When stimulated by the proinflammatory stimulus lipopolysaccharide (LPS), MMGT-16 cells secrete large amounts of prostaglandin E2 (PGE2). This PGE2 production is nearly abolished if amyloid β-peptide (Aβ (1–40)) is present in the incubation medium. In addition, Aβ (1–40) inhibits cyclooxygenase-2 (COX-2) induction by LPS. Since these effects are not reproduced by the reverse control Aβ (40–1), these results suggest a novel, intriguing modulatory role for amyloid β peptide in the inflammatory response of microglial cells.

Apolipoprotein E Receptors Mediate the Effects of β-Amyloid on Astrocyte Cultures

We have previously shown that beta-amyloid (Abeta) induces astrocyte activation in vitro and that this reaction is attenuated by the addition of exogenous apolipoprotein E (apoE)-containing particles. However, the effects of Abeta on endogenous apoE and apoJ levels and the potential role of apoE receptors in astrocyte activation have not been addressed. Three activating stimuli (lipopolysaccharide, dibutyryl cAMP, and aged Abeta 1-42) were used to induce activation of rat astrocyte cultures, as assessed by changes in morphology and an increase in interleukin-1beta. However, only Abeta also induced approximately 50% reduction in the amount of released apoE and apoJ and an 8-fold increase in the levels of cell-associated apoE and apoJ. Experiments using two concentrations of receptor-associated protein, an inhibitor of apoE receptors with a differential affinity for the low density lipoprotein receptor (LDLR) and the LDLR-related protein (LRP), suggest that LRP mediates Abeta-induced astrocyte activation, whereas LDLR mediates the Abeta-induced changes in apoE levels. Receptor-associated protein had no effect on apoJ levels or on activation by either dibutyryl cAMP or lipopolysaccharide. These data suggest that apoE receptors translate the presence of extracellular Abeta into cellular responses, both initiating and modulating the inflammatory response induced by Abeta.

Impaired ex vivo lipopolysaccharide-stimulated whole blood tumour necrosis factor production may identify patients in the intensive care unit with ‘sepsis’ and multiple system organ failure

Abstract Background There is currently no reliable diagnostic test to identify patients with sepsis in the intensive care unit (ICU) or patients with multiple system organ failure (MSOF). It was previously found that in vitro pretreatment of monocytes with lipopolysaccharide (LPS) induced blunted tumour necrosis factor (TNF) release to LPS rechallenge. It was hypothesized that patients exposed to LPS or septic stimuli in vivo would produce less TNF when stimulated ex vivo with LPS. This preliminary study sought to determine whether impaired ex vivo TNF release was associated with measurable differences in clinical outcome. Methods Heparinized whole blood was obtained from 27 ICU patients and five healthy controls, and incubated immediately with or without LPS 10 ng ml−1 at 37°C for 3 h, then centrifuged to recover serum. Serum TNF levels were measured using an enzyme-linked immunosorbent assay and expressed as mean(s.e.m.). Clinical data, such as ICU length of stay (LOS), duration of mechanical ventilation, white blood cell count and positive cultures, were obtained retrospectively. Quartiles (I, 0–25 per cent; II, 26–50 per cent; III, 51–75 per cent; and IV, 76–100 per cent) were identified on the basis of the distribution of plotted LPS-stimulated whole blood TNF values. Statistical analysis was by χ2 and Student's t tests. Results A wide range of LPS-stimulated whole blood TNF production was observed in ICU patients (5·1(0·7) ng ml−1) and controls (6·6(1·0) ng ml−1). Patients identified in the lowest quartile (n = 6) of TNF producers (less than 2 ng ml−1) had significantly lower TNF production and a higher incidence of infection (83 versus 38 per cent), and longer LOS (21·8 versus 9·0 days) and duration of mechanical ventilation (18·3 versus 6·0 days) than patients in quartiles II–IV (n = 21). Patients in the lowest quartile had significantly lower TNF production (1·1(0·2) ng ml−1) than normal controls (6·6(1·0) ng ml−1) (P < 0·05) or ICU patients in any other quartile (quartile II: 2·9(0·2) ng ml−1, quartile III: 5·3(0·3) ng ml−1, quartile IV: 10·4(1·3) ng ml−1) (P < 0·05). Conclusion The lowest levels of ex vivo LPS-stimulated whole blood TNF production were associated with a prolonged ICU stay, a higher incidence of positive cultures and a prolonged need for mechanical ventilation. Impaired TNF release may be a manifestation of monocyte endotoxin tolerance and may be a marker of monocytic dysfunction. Determination of whole blood ex vivo LPS-stimulated TNF production could be useful in the diagnosis of severe sepsis and MSOF in patients in the ICU.

Lipopolysaccharide tolerance in relation to intrabronchial influx of neutrophils in the rat.

Lipopolysaccharide (LPS) is a potent chemotactic component for polymorphonuclear leukocytes (PMN, neutrophils). Since LPS tolerance was first described, many studies have been reported about the hyporesponsiveness in vitro corresponding to attenuating production of proinflammatory cytokines. We hypothesized that in vivo daily exposure to LPS stimuli impairs neutrophil accumulation in the rat airway. Interleukin 8 (IL-8) and/or CXC-chemokine, a neutrophil chemoattractant and activating cytokine, have been implicated as proinflammatory mediators in gram-negative respiratory tract infections. It is possible that the tolerance to LPS has occurred in relation to this chemoattractant cytokine production. To settle this issue, we examined whether the neutrophil count in bronchoalveolar lavage fluid (BALF) decreases after daily inhalation of Pseudomonas aeruginosa LPS into the rat airway. Repeated inhalation of LPS into the airway resulted in reduction in neutrophil recruitment. We measured rat CXC-chemokine (rat GRO/CINC1) levels in recovered BALF. There were noted reductions of rat GRO corresponding to the diminished neutrophil trafficking. We also confirmed that the HLA-DR positive lymphocyte number in BALF gradually increased after daily inhalation of LPS. These results suggest that continuous stimuli of LPS mitigate the accumulation of inflammatory cells in the airway by reducing chemokine production with a consequent change in the appearance of local inflammation to a chronic state.

Analysis of differentially regulated mRNAs in peripheral blood monocytes of berylliosis patients after in vitro stimulation.

In berylliosis and other granulomatous diseases the macrophage is regarded as effector cell in granuloma formation. However, little is known about granuloma-associated regulation of genes in these cells. Differential display reverse transcription polymerase chain reaction (DDRT-PCR) is an attractive method for detection of differentially expressed genes. Since DDRT-PCR requires a comparably low quantity of RNA, its application to rare and limited amounts of clinical samples is convenient. In the present study we applied DDRT-PCR in a multiple and complex comparison of expressed sequence tags induced in response to various granuloma-associated and control stimuli. Since we are interested in granuloma-restricted changes, we tested peripheral blood monocytes from berylliosis patients by DDRT-PCR stimulated with up to nine different stimuli, including BeSO4, the causal agent of berylliosis. Comparison of a total of 1663 sequence tags in four berylliosis patients revealed a mean of 32.5-37.4% differentially regulated sequence tags in peripheral blood monocytes of berylliosis patients, caused by stimuli including beryllium or Mycobacterium tuberculosis and control stimuli such as Latex or Zymosan. In 7.7-28.0% of the analyzed sequence tags we detected a differential regulation restricted to the presence of granuloma-associated stimuli BeSO4, HgS, LiCO3, NiSO4, lipopolysaccharide, and/or heat killed M. tuberculosis; 1.4-12.3% were induced by more than one granuloma-associated stimulus. Alterations associated with BeSO4 and one of the named stimuli were detected in 1.4-4.5%. An exclusive association with BeSO4 was found in 2.6-5.7% of the analyzed sequence tags.

In vitro growth inhibition of a broad spectrum of tumor cell lines by activated human dendritic cells

AbstractDendritic cells (DCs) are critical subsets of leukocytes providing antigen presentation for initiation of humoral and cellular immune responses. Their role as effector cells in tumor resistance, however, is less known. We report here that human DCs generated by culturing plastic-adherent peripheral blood monocytes in the presence of granulocyte-monocyte colony–stimulating factor (GM-CSF) and interleukin-4 have potent growth-inhibition activity in vitro on a wide spectrum of human tumor lines of different tissue origin. Proinflammatory stimuli lipopolysaccharide (LPS) and interferon-γ, but not tumor necrosis factor–α and CD40 signaling, can further enhance DC-mediated inhibition of tumor growth. The growth inhibition requires contact between DCs and tumor cells while LPS treatment enhances the antitumor activity in DC culture supernatants. Our results suggest that in addition to their predominant role as regulatory cells, activated DCs are also potential effector cells in tumor immunity.

In vitro growth inhibition of a broad spectrum of tumor cell lines by activated human dendritic cells

Dendritic cells (DCs) are critical subsets of leukocytes providing antigen presentation for initiation of humoral and cellular immune responses. Their role as effector cells in tumor resistance, however, is less known. We report here that human DCs generated by culturing plastic-adherent peripheral blood monocytes in the presence of granulocyte-monocyte colony–stimulating factor (GM-CSF) and interleukin-4 have potent growth-inhibition activity in vitro on a wide spectrum of human tumor lines of different tissue origin. Proinflammatory stimuli lipopolysaccharide (LPS) and interferon-γ, but not tumor necrosis factor– and CD40 signaling, can further enhance DC-mediated inhibition of tumor growth. The growth inhibition requires contact between DCs and tumor cells while LPS treatment enhances the antitumor activity in DC culture supernatants. Our results suggest that in addition to their predominant role as regulatory cells, activated DCs are also potential effector cells in tumor immunity.

Analysis of differentially regulated mRNAs in monocytic cells induced by in vitro stimulation.

Macrophages are known to be effector cells in several granulomatous disorders. However, little is known about granuloma-associated up- or downregulation of genes in these cells. Differential display reverse transcription polymerase chain reaction (DDRT-PCR) is an attractive method for the detection of differentially expressed genes. Although this method entails a number of drawbacks, its application to rare and limited amounts of clinical samples is still convenient. In this study, we introduce a screening procedure for detecting differentially regulated sequence tags in samples of patients suffering from granulomatous diseases. We applied DDRT-PCR in a multiple and complex comparison of expressed sequence tags in response to various granuloma-associated stimuli. The histiocytic cell line U937 was used as a model. The cells had been stimulated with granuloma-associated agents such as Mycobacterium tuberculosis, BeSO4, lipopolysaccharide, or HgS and unspecific stimuli such as phorbol myristate acetate, phytohemagglutinin, Zymosan, and Latex. Comparative analysis of 2237 sequence tags obtained from 55 primer combinations revealed 22.4% differentially amplified PCR products. Notably, only 8.0% of the differentially expressed sequence tags showed an association restricted to in vitro cultivation in the presence of M. tuberculosis, lipopolysaccharide, BeSO4, and/or HgS, while 1.0-1.9% of the tags were altered exclusively as a consequence of stimulation with one of the granuloma-associated agents. Our data provide evidence that this strategy may function as a preselection for appropriate primer combinations to discover sequence tags which could be specifically associated with granulomatous disorders. This approach could shorten laborious screening, save consumption of valuable and rare samples, and could reduce the number of false-positive results.

Lipopolysaccharide Inhibits the Expression of the Scavenger Receptor Cla-1 in Human Monocytes and Macrophages

Human Cla-1 is the likely homologue of the murine scavenger receptor class B type I (SR-BI). SR-BI mediates selective transfer of cholesterol to high-density lipoprotein (HDL) and the efflux of endogenously synthesized and plasma membrane sterols to HDL. HDL protects against atherosclerosis but also reduces endotoxic activity by complexation and neutralization of LPS. We found that Cla-1 is upregulated during phagocytic as well as dendritic differentiation of monocytes, indicating a function of this receptor for cholesterol homeostasis in phagocytes and antigen-presenting cells. Cla-1 expression is suppressed by the proinflammatory stimuli lipopolysaccharide, interferon-γ, and tumor necrosis factor α in monocytes and macrophages. Downregulation of Cla-1 mRNA by LPS is likely due to a modification and subsequent destabilization of the mRNA. We propose that suppression of Cla-1 expression may help to stabilize the lipoprotein status in the blood compartment important for host defense.

Hairy cell leukemia with translocation (11;20)(q13;q11) and overexpression of cyclin D1

We report on a male Japanese patient with hairy cell leukemia (HCL). A cytogenetic study with lipopolysaccharide stimuli showed a novel translocation (11;20)(q13;q11) in 10% of the analyzed cells. Northern blot analysis and RT-PCR analysis for cyclin D1 revealed the overexpression of cyclin D1, although the southern blot analysis of PRAD1 gene showed no rearrangement. In this particular case, the t(11;20)(q13: q11) might play some role in the oncogenesis of HCL and the overexpression of cyclin D1.

Dendritic cells and MPIF-1: chemotactic activity and inhibition of endogenous chemokine production by IFN-gamma and CD40 ligation.

We have examined the biological activity of the CC chemokine myeloid progenitor inhibitory factor 1 (MPIF-1) on human dendritic cells. MPIF-1 has chemotactic activity on dendritic cells derived from either peripheral blood monocytes or cord blood CD34+ progenitors. However, chemokine treatment did not induce further cell activation or maturation. In addition, MPIF-1 is constitutively released by monocyte-derived dendritic cells but not macrophages or monocytes (resting or stimulated). The proinflammatory stimuli lipopolysaccharide and tumor necrosis factor alpha, which induced the release of monocyte chemotactic protein-1, macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and interleukin-8, did not affect MPIF-1 release. In contrast, CD40 ligation and interferon-gamma treatment, while stimulating the production of the other chemokines, caused a pronounced reduction of MPIF-1 transcript and protein release. Thus, in dendritic cells the regulation of the production and release of MPIF-1 is distinct in comparison to other CC and CXC chemokines.

Assessment of the role of leucocyte function-associated antigen-1 in homotypic adhesion of activated B lymphocytes.

In this study we investigated how T-cell-dependent stimuli, via interleukin-4 (IL-4) or CD40 ligation, influence homotypic B-cell adhesion when compared with induction by the T-cell-independent stimulus lipopolysaccharide (LPS). Using primary murine B cells, we found that T-cell-dependent stimulation led to increased aggregation as compared to that induced by LPS. The adhesion was to a large extent dependent on the adhesion molecule, lymphocyte function-associated antigen-1 (LFA-1). We found that activation of B cells with the mitogenic stimuli induced an increased avidity of LFA-1 for its ligand, intercellular adhesion molecule-1 (ICAM-1). The increase was stable and different from that induced by phorbol esters. Although adhesion was reduced using B cells from LFA-1(-/-) mice, aggregation occurred in response to T-cell-dependent stimuli. Our data suggest that adhesion of B lymphocytes is regulated in different modes. One is induced by antigen and leads to a transient conformational change of the LFA-1 molecule. Another is induced by mitogenic stimuli and leads to stable avidity increase of LFA-1, possibly via activation of cytoskeletal anchorage. A third is LFA-1 independent, of low avidity and is induced by T-cell-dependent stimuli.

Calcitriol modulates in vivo and in vitro cytokine production: A role for intracellular calcium

Calcitriol modulates in vivoand in vitro cytokine production: A role for intracellular calcium. Background. Several immunomodulatory properties of calcitriol are currently known, however, only little information is available regarding the in vivo and in vitro effects of calcitriol on cytokine production in chronic renal failure. Methods. To study the in vitro effect of calcitriol on lipopolysaccharide (LPS)-induced cytokine production, peripheral blood mononuclear cells (PBMC, 2.5 ml/ml) from 12 chronic dialytic (HD), 15 undialyzed chronic renal failure (CRF) patients and 10 normal subjects (N) were incubated at 37 degrees for 12 hours with 100 ng of LPS (E. coli and P. maltofilia). Increasing doses of calcitriol from 10-10 to 10-9 M were added and cell associated TNF-alpha and IL-1beta were determined by immunoreactive tests after three freeze-thaw cycles. The intradialytic TNF-alpha and IL-1beta production were evaluated in vivo in 12 HD patients before and after three months of intravenous calcitriol treatment (6 microgram/week). Intracellular calcium [Ca++]i was determined on PBMC with a cytofluorimetric assay using FLUO-3 AM as the indicator. Results. In vitro, TNF-alpha increased from 3.6 +/- 1.9 pg/cell to 1797 +/- 337 in N, from 4.5 +/- 1.7 to 1724 +/- 232 in CRF and from 3.4 +/- 2.3 to 1244 +/- 553 in HD after the LPS stimulus. The production of TNF-alpha was inhibited by calcitriol in a dose-dependent manner [LPS + Vit.D3 100 ng, 2.9 +/- 2.1 in N, 3.7 +/- 1.9 in CRF and 3.4 +/- 1.7 in HD; LPS + Vit.D3 50 ng, 263 +/- 296 (N), 6.73 +/- 11 (CRF), 38 +/- 28 (HD); LPS + Vit.D3 25 ng = 873 +/- 583 (N), 325 +/- 483 (CRF), 588 +/- 507 (HD); LPS + Vit.D3 12.5 ng, 954 +/- 483 (N), 912 +/- 510 (CRF), 875 +/- 527 (HD)]. Comparable data were observed on IL-1beta production. In vivo, the intradialytic TNF-alpha increase (from 8.5 +/- 2.3 to 19 +/- 5.6 pg/2.5 x 106 cell) during hemodialysis was markedly reduced after calcitriol therapy (from 6.6 +/- 3.1 to 11 +/- 4.7). [Ca++]i decreased from 105 +/- 25 to 72 +/- 18 nM (P < 0.05) and a positive correlation between cytokine levels and [Ca++]i was found (r = 0.79; P < 0.001). Conclusions. The in vitro increase of cell-associated cytokine after LPS challenge was inhibited by calcitriol in a dose-dependent manner. These data suggest a possible in vivo modulatory effect of calcitriol therapy on cytokine production in hemodialysis.

Expression of cyclooxygenase-2 in foetal rat hepatocytes stimulated with lipopolysaccharide and pro-inflammatory cytokines.

Cyclooxygenase-2 (COX-2) is involved in the biosynthesis of prostanoids in the course of inflammatory reactions. This isoenzyme is regulated at the transcription level and many cells express COX-2 upon challenge with lipopolysaccharide (LPS) or pro-inflammatory cytokines. Since hepatocytes respond to LPS and pro-inflammatory stimuli, we investigated the expression of COX-2 in foetal and adult hepatocytes upon challenge with these substances. COX-2 was expressed in foetal hepatocytes incubated with LPS, tumour necrosis factor-alpha and interleukin-1beta. This response rapidly decreased after birth and was absent in hepatocytes from animals aged 2 days or more and treated under identical conditions. The expression of COX-2 was determined at the mRNA, protein and enzyme activity levels using Northern and Western blot, and following the synthesis of prostaglandin E2, respectively. The use of NS 398, a specific pharmacological inhibitor of COX-2, confirmed the expression of this isoenzyme in activated foetal hepatocytes. Synergism in COX-2 expression was observed between LPS, tumour necrosis factor-alpha and interleukin-1beta. Interleukin-6 and permeant analogues of cyclic AMP failed to induce COX-2 or to synergize with LPS. Also, transforming growth factor-beta inhibited the LPS- and pro-inflammatory cytokines-dependent expression of COX-2. These results indicate that foetal hepatocytes are competent to express COX-2 upon challenge with pro-inflammatory stimuli, a process lost completely in hepatocytes isolated from animals aged 2 days.

Nitric oxide inhibits stress-induced endothelial cell apoptosis.

To determine a mechanism by which nitric oxide alters induction of stress-induced endothelial cell apoptosis in vitro. Apoptosis is a form of cellular suicide that has been implicated in the pathogenesis of multiple organ dysfunction syndrome. Prospective, controlled trial. Research laboratory of a large, academic medical center. Cultured primary porcine aortic endothelial cells. Cells were treated with a range of doses of agents that either spontaneously generate nitric oxide (S-nitroso-N-acetyl-D,L-penicillamine [SNAP] or (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1,2-diolate [DETA-NO]) or block nitric oxide production (Nomega-methyl-L-arginine [L-NMA]). The ability of these agents to alter the rate of cell death by apoptosis (induced by the sequence stimuli lipopolysaccharide [LPS] followed by sodium arsenite) was measured. Mechanistic studies included examining the ability of: a) nitric oxide "donors" to alter nuclear factor kappa B (NF-kappaB) DNA binding activity and the level of IkappaBalpha accumulation; and b) a stable cyclic guanosine monophosphate (cGMP) analog (8-bromo-cGMP) to mimic the effect of nitric oxide donors. The sequence LPS/sodium arsenite increased the rate of endothelial cell apoptosis (47.4%, p< .05 vs. control), as measured by fluorescent-activated cell scanning using annexin V/propidium iodide staining. DETA-NO generated nitric oxide (as indicated by an increase in the concentration of the stable end-products of nitric oxide metabolism) and decreased the rate of endothelial cell apoptosis (20.6% at a dose of 2 mM, p=.0001 vs. control). DETA-NO also decreased NF-kappaB DNA binding activity and the apparent accumulation of its endogenous inhibitor, IkappaBalpha. The 8-bromo-cGMP did not mimic the effects of nitric oxide donors (DETA-NO) on apoptosis. These data suggest that exogenous nitric oxide can block stress-induced endothelial cell apoptosis in vitro. The mechanistic studies are consistent with our hypothesis that inhibitors of NF-kappaB DNA binding activity are associated with protection against apoptosis-inducing stimuli. The results do not support a role for cGMP in mediating the protective effect of DETA-NO in our model.

Cytokine Gene Expression in Human LPS- and IFNγ-stimulated Mononuclear Cells Is Inhibited by Heparin

In addition to its well-understood anticoagulant activity, heparin is known to modulate a variety of biological functions including immunologic responses. In order to investigate whether heparin influences the humoral immunity by interacting with cellular elements and affecting gene expression in blood circulating cells. we studied the effect of heparin on IL-1beta, IL-6 and TNFalpha mRNAs in human lipopolysaccharide-(LPS)- or interferon-gamma(IFN-gamma)-stimulated mononuclear cells. The study of mRNA was carried out by an initial PCR screening followed by a Northern blot quantitative analysis. Heparin (0.5 U/ml) turned out to inhibit all three cytokine gene expressions. The mRNA decrease was 37 +/- 6% for IL-1beta, 53 +/- 3% for IL-6 and 47 +/- 4% for TNFalpha with LPS stimulus. No differences could be observed in the inhibitory effect of heparin on IFNgamma-stimulated cells. This effect of heparin was confirmed in a subset of experiments performed on purified monocytes. These results suggest an important immunosuppressive effect of heparin on cell-mediated immune responses.

Effect of heat stress on LPS-induced fever and tumor necrosis factor.

Exposure to heat stress leads to both short-term and long-term effects on morbidity. Male rats were exposed to a high ambient temperature of 40 degrees C, which resulted in biotelemetered core body temperature rising to approximately 42 degrees C. This treatment led to a marked enhancement in lipopolysaccharide (LPS)-induced fever at 24 h after exposure to heat stress. The increase in fever was accompanied by a significant suppression in the circulating concentration of tumor necrosis factor. Heat-shock protein-70 measured in liver was elevated by the heat exposure (but not further elevated by the injection of LPS). An enhanced fever to LPS and other inflammatory stimuli found in heat-stressed human subjects could explain the apparent increase in susceptibility to disease.

Involvement of diacylglycerol production in activation of nuclear factor kappaB by a CD14-mediated lipopolysaccharide stimulus.

Exposure of Chinese hamster CHO-K1 transfectant cells expressing mouse CD14 (CHO/CD14 cells) to lipopolysaccharide (LPS) induced rapid elevation of the cellular diacylglycerol (DAG) and choline/phosphocholine levels and nuclear translocation of nuclear factor kappaB (NFkappaB). When cells were incubated with short-chain DAG analogues or bacterial phospholipase C, NFkappaB activation occurred even without the LPS stimulus. Treatment of CHO/CD14 cells with tricyclo[5.2.1.0(2.6)]decyl-(9[8])xanthogenate (D609), an inhibitor of phosphatidylcholine-specific phospholipase C and phospholipase D, almost completely inhibited not only the LPS-dependent production of DAG and choline/phosphocholine but also the LPS-dependent NFkappaB activation. In contrast, treatment of cells with 1-(6-{[3-methoxyoestra-1,3, 5(10)-trien-17beta-yl]-1H-pyrrole-2,5-dione (U73122), an inhibitor of phosphatidylinositol-specific phospholipase C in vitro, did not affect the LPS-dependent activation of NFkappaB. Production of DAG and activation of NFkappaB after the LPS stimulus were observed in mouse macrophage-like J774.1 cells, and this response to LPS by J774. 1 cells was also inhibited by D609. These results suggest that the production of DAG from phosphatidylcholine was upstream of NFkappaB activation in response to a CD14-mediated LPS stimulus.

Preexposure of mouse peritoneal macrophages to lipopolysaccharide and other stimuli enhances the nitric oxide response to secondary stimuli.

The aim of this study was to compare the regulation of the production of tumor necrosis factor-alpha (TNF-alpha) and secondary nitric oxide (NO) in macrophages submitted to a sequence of two stimulations. Pre-exposure for 18 h of mouse thioglycollate-elicited peritoneal macrophages to low doses (1-10 ng/ml) of lipopolysaccharide (LPS), in the presence or absence of serum, induces on one hand a desensitization (endotoxin tolerance) for secondary TNF-alpha responses to LPS and, on the other hand, a 4 fold increase (priming) of secondary NO responses. Preexposure to components from Gram-positive bacteria (lipoteichoic acid, peptidoglycan) and to a synthetic lipid structurally related to lipid A (compound M4), induced similar effects. In contrast to the desensitization for TNF-alpha secretion, the priming for NO production was not mimicked by sodium nitroprusside, a generator of NO. The results suggest that concomitant but distinct activation pathways induced by LPS and other agents can be dissociated by serum-independent modulation processes elicited by pre-exposure of the cells to LPS itself, or to other stimuli.

Late Acquisition of Hyporesponsiveness to Lipopolysaccharide by Mycobacterium avium-Infected Human Macrophages in Producing Tumor Necrosis Factor- but Not Interleukin-l and -6

To investigate whether infection with Mycobacterium avium modifies the cytokine response of human macrophages (Mphi) to lipopolysaccharide (LPS), the release of interleukin (IL)-1 beta, IL-6, and tumor necrosis factor (TNF)-alpha was determined in infected and uninfected Mphi, unstimulated or stimulated with LPS. In unstimulated Mphi, the release of IL-1 beta and IL-6 increased with the progress of infection while that of TNF-alpha progressively decreased. When Mphi were stimulated with LPS, IL-1 beta and IL-6 levels were always higher in infected than in uninfected cells, but levels of TNF-alpha significantly decreased in infected Mphi. A similar trend was obtained for TNF-alpha mRNA expression. Altogether, these results indicate that infected Mphi react to LPS stimulus with enhanced levels of IL-1 beta and IL-6 but are unable to restore the production of TNF-alpha impaired by the growth of the intracellular mycobacteria.