(1/2131) Lipopolysaccharide (LPS) from Burkholderia cepacia is more active than LPS from Pseudomonas aeruginosa and Stenotrophomonas maltophilia in stimulating tumor necrosis factor alpha from human monocytes.

Whole cells and lipopolysaccharides (LPSs) extracted from Burkholderia cepacia, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Escherichia coli were compared in their ability to stimulate tumor necrosis factor alpha (TNF-alpha) from the human monocyte cell line MonoMac-6. B. cepacia LPS, on a weight-for-weight basis, was found to have TNF-alpha-inducing activity similar to that of LPS from E. coli, which was approximately four- and eightfold greater than the activity of LPSs from P. aeruginosa and S. maltophilia, respectively. The LPS-stimulated TNF-alpha production from monocytes was found to be CD14 dependent. These results suggest that B. cepacia LPS might play a role in the pathogenesis of inflammatory lung disease in cystic fibrosis, and in some patients it might be responsible, at least in part, for the sepsis-like cepacia syndrome.  (+info)

(2/2131) Signal transduction triggered by lipid A-like molecules in 70Z/3 pre-B lymphocyte tumor cells.

The lipid A (endotoxin) moiety of lipopolysaccharide (LPS) elicits rapid cellular responses from many cell types, including macrophages, lymphocytes, and monocytes. In CD14 transfected 70Z/3 pre-B lymphocyte tumor cells, these responses include activation of the MAP kinase homolog, p38, activation of NF-kappaB, and transcription of kappa light chains, leading to the assembly of surface IgM. In this work, we explored the specificity of the response with regard to lipid structure, and the requirement for p38 kinase activity prior to NF-kappaB activation in control and CD14 transfected 70Z/3 (CD14-70Z/3) cells. A p38-specific inhibitor, SB203580, was used to block p38 kinase activity in cells. CD14-70Z/3 cells were incubated with 1-50 microM SB203580, and then stimulated with LPS. Nuclear extracts were prepared, and NF-kappaB activation was measured using an electrophoretic mobility shift assay. SB203580 did not inhibit LPS induced NF-kappaB activation. In addition, LPS failed to activate p38 tyrosine phosphorylation in 70Z/3 cells lacking CD14, in spite of rapid NF-kappaB activation and robust surface IgM production with appropriate higher doses of LPS. LPS stimulation of p38 phosphorylation, NF-kappaB activation, and surface IgM expression were all blocked completely by lipid A-like endotoxin antagonists whether or not CD14 was present. Acidic glycerophospholipids and ceramides did not mimic lipid A-like molecules either as agonists or antagonists in this system. Our data support the hypothesis that lipid A-mediated activation of cells requires stimulation of a putative lipid A sensor that is downstream of CD14, but upstream of p38 and NF-kappaB.  (+info)

(3/2131) Enhanced production of tumor necrosis factor-alpha and interleukin-6 due to prolonged response to lipopolysaccharide in human macrophages infected in vitro with human immunodeficiency virus type 1.

Elevated levels of circulating tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 have been detected in human immunodeficiency virus (HIV) type 1 infection. The overproduction of these cytokines could contribute to AIDS pathogenesis. Thus, the expression of TNF-alpha and IL-6 in human macrophages infected with HIV-1 was investigated. HIV-1 infection, per se, did not induce any TNF-alpha or IL-6 production or cytokine-specific mRNA expression. In contrast, HIV-1 primed macrophages to a prolonged TNF-alpha and IL-6 response to lipopolysaccharide (LPS) stimulation with respect to uninfected cells. Time-course analysis and flow cytometry demonstrated that cytokine production stopped at 6 h in uninfected macrophages but continued up to 24 h in HIV-1-infected cells. RNA studies suggested that HIV-1 interfered with late steps of cytokine synthesis. No modulation of membrane CD14 was found to account for the enhanced response to LPS. Finally, the effect of HIV-1 on cytokine response could not be abolished by the antiviral compound U75875.  (+info)

(4/2131) Long-term fetal microchimerism in peripheral blood mononuclear cell subsets in healthy women and women with scleroderma.

Fetal CD34(+) CD38(+) cells have recently been found to persist in maternal peripheral blood for many years after pregnancy. CD34(+) CD38(+) cells are progenitor cells that can differentiate into mature immune-competent cells. We asked whether long-term fetal microchimerism occurs in T lymphocyte, B lymphocyte, monocyte, and natural-killer cell populations of previously pregnant women. We targeted women with sons and used polymerase chain reaction for a Y-chromosome-specific sequence to test DNA extracted from peripheral blood mononuclear cells (PBMC) and from CD3, CD19, CD14, and CD56/16 sorted subsets. We also asked whether persistent microchimerism might contribute to subsequent autoimmune disease in the mother and included women with the autoimmune disease scleroderma. Scleroderma has a peak incidence in women after childbearing years and has clinical similarities to chronic graft-versus-host disease that occurs after allogeneic hematopoietic stem-cell transplantation, known to involve chimerism. Sixty-eight parous women were studied for male DNA in PBMC and 20 for PBMC subsets. Microchimerism was found in PBMC from 33% (16 of 48) of healthy women and 60% (12 of 20) women with scleroderma, P =.046. Microchimerism was found in some women in CD3, CD19, CD14, and CD56/16 subsets including up to 38 years after pregnancy. Microchimerism in PBMC subsets was not appreciably more frequent in scleroderma patients than in healthy controls. Overall, microchimerism was found in CD3, CD19, and CD14 subsets in approximately one third of women and in CD56/16 in one half of women. HLA typing of mothers and sons indicated that HLA compatibility was not a requirement for persistent microchimerism in PBMC subsets. Fetal microchimerism in the face of HLA disparity implies that specific maternal immunoregulatory pathways exist that permit persistence but prevent effector function of these cells in normal women. Although microchimerism in PBMC was more frequent in women with scleroderma than healthy controls additional studies will be necessary to determine whether microchimerism plays a role in the pathogenesis of this or other autoimmune diseases.  (+info)

(5/2131) Monocytic cell necrosis is mediated by potassium depletion and caspase-like proteases.

Apoptosis is a physiological cell death that culminates in mitochondrial permeability transition and the activation of caspases, a family of cysteine proteases. Necrosis, in contrast, is a pathological cell death characterized by swelling of the cytoplasm and mitochondria and rapid plasma membrane disruption. Necrotic cell death has long been opposed to apoptosis, but it now appears that both pathways involve mitochondrial permeability transition, raising the question of what mediates necrotic cell death. In this study, we investigated mechanisms that promote necrosis induced by various stimuli (Clostridium difficile toxins, Staphylococcus aureus alpha toxin, ouabain, nigericin) in THP-1 cells, a human monocytic cell line, and in monocytes. All stimuli induced typical features of necrosis and triggered protease-mediated release of interleukin-1beta (IL-1beta) and CD14 in both cell types. K+ depletion was actively implicated in necrosis because substituting K+ for Na+ in the extracellular medium prevented morphological features of necrosis and IL-1beta release. N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a broad-spectrum caspase inhibitor, prevented morphological features of necrosis, plasma membrane destruction, loss of mitochondrial membrane potential, IL-1beta release, and CD14 shedding induced by all stimuli. Thus, in monocytic cells, necrosis is a cell death pathway mediated by passive K+ efflux and activation of caspase-like proteases.  (+info)

(6/2131) Pronase destroys the lipopolysaccharide receptor CD14 on Kupffer cells.

CD14 is a lipopolysaccharide (LPS) receptor distributed largely in macrophages, monocytes, and neutrophils; however, the role of CD14 in activation of Kupffer cells by LPS remains controversial. The purpose of this study was to determine if different methods used to isolate Kupffer cells affect CD14. Kupffer cells were isolated by collagenase (0.025%) or collagenase-Pronase (0.02%) perfusion and differential centrifugation using Percoll gradients and cultured for 24 h before experiments. CD14 mRNA was detected by RT-PCR from Kupffer cell total RNA as well as from peritoneal macrophages. Western blotting showed that Kupffer cells prepared with collagenase possess CD14; however, it was absent in cells obtained by collagenase-Pronase perfusion. Intracellular calcium in Kupffer cells prepared with collagenase was increased transiently to levels around 300 nM by addition of LPS with 5% rat serum, which contains LPS binding protein. This increase in intracellular calcium was totally serum dependent. Moreover, LPS-induced increases in intracellular calcium in Kupffer cells were blunted significantly (40% of controls) when cells were treated with phosphatidylinositol-specific phospholipase C, which cleaves CD14 from the plasma membrane. However, intracellular calcium did not increase when LPS was added to cells prepared by collagenase-Pronase perfusion even in the presence of serum. These cells were viable, however, because ATP increased intracellular calcium to the same levels as cells prepared with collagenase perfusion. Tumor necrosis factor-alpha (TNF-alpha) mRNA was increased in Kupffer cells prepared with collagenase perfusion 1 h after addition of LPS, an effect potentiated over twofold by serum; however, serum did not increase TNF-alpha mRNA in cells isolated via collagenase-Pronase perfusion. Moreover, treatment with Pronase rapidly decreased CD14 on mouse macrophages (RAW 264.7 cells) and Kupffer cells. These findings indicate that Pronase cleaves CD14 from Kupffer cells, whereas collagenase perfusion does not, providing an explanation for why Kupffer cells do not exhibit a CD14-mediated pathway when prepared with procedures using Pronase. It is concluded that Kupffer cells indeed contain a functional CD14 LPS receptor when prepared gently.  (+info)

(7/2131) Bacterial lipopolysaccharide inhibits dengue virus infection of primary human monocytes/macrophages by blockade of virus entry via a CD14-dependent mechanism.

Monocytes/macrophages (MO/Mphi) are the major target cells for both dengue virus (DV) and bacterial lipopolysaccharide (LPS), and the aim of this study was to define their interactions. We had found that LPS markedly suppressed DV infection of primary human MO/Mphi when it was added to cultures prior to or together with, but not after, viral adsorption. The inhibitory effect of LPS was direct and specific and was not mediated by LPS-induced secretion of cytokines and chemokines such as tumor necrosis factor alpha, interleukin-1beta (IL-1beta), IL-6, IL-8, IL-12, alpha interferon, MIP-1alpha, and RANTES. In fact, productive DV infection was not blocked but was just postponed by LPS, with a time lag equal to one viral replication cycle. Time course studies demonstrated that LPS was only effective in suppressing DV infection of MO/Mphi that had not been previously exposed to the virus. At various time points after viral adsorption, the level of unbound viruses that remained free in the culture supernatants of LPS-pretreated cultures was much higher than that of untreated controls. These observations suggest that the LPS-induced suppression of DV replication was at the level of virus attachment and/or entry. Blockade of the major LPS receptor, CD14, with monoclonal antibodies MY4 or MoS39 failed to inhibit DV infection but could totally abrogate the inhibitory effect of LPS. Moreover, human serum could significantly enhance the LPS-induced DV suppression in a CD14-dependent manner, indicating that the "binding" of LPS to CD14 was critical for the induction of virus inhibition. Taken together, our results suggest that LPS blocked DV entry into human MO/Mphi via its receptor CD14 and that a CD14-associated cell surface structure may be essential for the initiation of a DV infection.  (+info)

(8/2131) Selective activation and functional significance of p38alpha mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils.

Activation of leukocytes by proinflammatory stimuli selectively initiates intracellular signal transduction via sequential phosphorylation of kinases. Lipopolysaccharide (LPS) stimulation of human neutrophils is known to result in activation of p38 mitogen-activated protein kinase (MAPk); however, the upstream activator(s) of p38 MAPk is unknown, and consequences of p38 MAPk activation remain largely undefined. We investigated the MAPk kinase (MKK) that activates p38 MAPk in response to LPS, the p38 MAPk isoforms that are activated as part of this pathway, and the functional responses affected by p38 MAPk activation. Although MKK3, MKK4, and MKK6 all activated p38 MAPk in experimental models, only MKK3 was found to activate recombinant p38 MAPk in LPS-treated neutrophils. Of p38 MAPk isoforms studied, only p38alpha and p38delta were detected in neutrophils. LPS stimulation selectively activated p38alpha. Specific inhibitors of p38alpha MAPk blocked LPS-induced adhesion, nuclear factor-kappa B (NF-kappaB) activation, and synthesis of tumor necrosis factor-alpha (TNF-alpha). Inhibition of p38alpha MAPk resulted in a transient decrease in TNF-alpha mRNA accumulation but persistent loss of TNF-alpha synthesis. These findings support a pathway by which LPS stimulation of neutrophils results in activation of MKK3, which in turn activates p38alpha MAPk, ultimately regulating adhesion, NF-kappaB activation, enhanced gene expression of TNF-alpha, and regulation of TNF-alpha synthesis.  (+info)