scholarly journals High-Affinity Interaction between Gram-Negative Flagellin and a Cell Surface Polypeptide Results in Human Monocyte Activation

2000 ◽  
Vol 68 (10) ◽  
pp. 5525-5529 ◽  
Author(s):  
Patrick F. McDermott ◽  
Federica Ciacci-Woolwine ◽  
James A. Snipes ◽  
Steven B. Mizel
Keyword(s):  
Cell Surface ◽  
Mutational Analysis ◽  
Hypervariable Region ◽  
Gram Negative Bacteria ◽  
Human Monocytes ◽  
Necrosis Factor Alpha ◽  
Potent Inducer ◽  
Gram Negative ◽  
High Affinity ◽  
Tnf Α

ABSTRACT Flagella from diverse gram-negative bacteria induce tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) synthesis by human monocytes (F. Ciacci-Woolwine, P. F. McDermott, and S. B. Mizel, Infect. Immun. 67:5176–5185, 1999). In this study, we establish that purified flagellin (FliC or FljB), the major filament protein from Salmonella enterica serovar Enteritidis,S. enterica serovar Typhimurium, and Pseudomonas aeruginosa, is an extremely potent inducer of TNF-α production by human monocytes and THP-1 myelomonocytic cells. Fifty percent of maximal TNF-α production (EC50) was obtained with 1.5 × 10−11 M flagellin (0.75 ng/ml). Mutagenesis studies revealed that the central hypervariable region of flagellin is essential for the TNF-α-inducing activity of the protein. Although less active than the wild-type protein, a Salmonellaflagellin mutant composed of only the central hypervariable region retained substantial TNF-α-inducing activity at nanomolar concentrations. In contrast, the conserved amino- and carboxy-terminal regions are inactive. Mutational analysis of the hypervariable region revealed that it contains two equally active TNF-α-inducing domains. The ability of THP-1 cells to respond to purified flagellins is dramatically reduced by mild trypsin treatment of the cells. Taken together, our results demonstrate that the cytokine-inducing activity of flagellins from gram-negative bacteria results from the interaction of these proteins with high-affinity cell surface polypeptide receptors on monocytes.

scholarly journals Interaction of Antimicrobial Peptide Temporin L with Lipopolysaccharide In Vitro and in Experimental Rat Models of Septic Shock Caused by Gram-Negative Bacteria

2006 ◽  
Vol 50 (7) ◽  
pp. 2478-2486 ◽  
Author(s):  
Andrea Giacometti ◽  
Oscar Cirioni ◽  
Roberto Ghiselli ◽  
Federico Mocchegiani ◽  
Fiorenza Orlando ◽  
...  
Keyword(s):  
Septic Shock ◽  
Antimicrobial Peptide ◽  
Gram Negative Bacteria ◽  
Amphibian Skin ◽  
Necrosis Factor Alpha ◽  
Gram Negative ◽  
Rat Models ◽  
E Coli ◽  
Tnf Α

ABSTRACT Sepsis remains a major cause of morbidity and mortality in hospitalized patients, despite intense efforts to improve survival. The primary lead for septic shock results from activation of host effector cells by endotoxin, the lipopolysaccharide (LPS) associated with cell membranes of gram-negative bacteria. For these reasons, the quest for compounds with antiendotoxin properties is actively pursued. We investigated the efficacy of the amphibian skin antimicrobial peptide temporin L in binding Escherichia coli LPS in vitro and counteracting its effects in vivo. Temporin L strongly bound to purified E. coli LPS and lipid A in vitro, as proven by fluorescent displacement assay, and readily penetrated into E. coli LPS monolayers. Furthermore, the killing activity of temporin L against E. coli was progressively inhibited by increasing concentrations of LPS added to the medium, further confirming the peptide's affinity for endotoxin. Antimicrobial assays showed that temporin L interacted synergistically with the clinically used β-lactam antibiotics piperacillin and imipenem. Therefore, we characterized the activity of temporin L when combined with imipenem and piperacillin in the prevention of lethality in two rat models of septic shock, measuring bacterial growth in blood and intra-abdominal fluid, endotoxin and tumor necrosis factor alpha (TNF-α) concentrations in plasma, and lethality. With respect to controls and single-drug treatments, the simultaneous administration of temporin L and β-lactams produced the highest antimicrobial activities and the strongest reduction in plasma endotoxin and TNF-α levels, resulting in the highest survival rates.

scholarly journals Moesin Functions as a Lipopolysaccharide Receptor on Human Monocytes

1999 ◽  
Vol 67 (7) ◽  
pp. 3215-3220 ◽  
Author(s):  
Ziad N. Tohme ◽  
Salomon Amar ◽  
Thomas E. Van Dyke
Keyword(s):  
Interleukin 1 ◽  
Biologically Active ◽  
Sequence Motif ◽  
Human Monocytes ◽  
Tissue Destruction ◽  
Necrosis Factor Alpha ◽  
Gram Negative ◽  
Tnf Α ◽  
Partial Blocking ◽  
Lps Binding

ABSTRACT Bacterial endotoxin (lipopolysaccharide [LPS]), a glycolipid found in the outer membranes of gram-negative bacteria, induces the secretion of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), and IL-6 by monocytes/macrophages. The secretion of these biologically active compounds leads to multiple pathological conditions, such as septic shock. There is substantial evidence that chronic exposure to LPS mediates, at least in part, the tissue destruction associated with gram-negative infection. CD14, a 55-kDa protein, has been identified as an LPS receptor. In conjunction with a serum protein, LPS binding protein (LBP), LPS-CD14 interactions mediate many LPS functions in the inflammatory response. However, CD14 lacks a cytoplasmic domain, or any known signal transduction sequence motif, suggesting the existence of another cell surface domain capable of transducing signals. In this paper, we report a second, CD14-independent LPS binding site, which, based on biological activity, appears to be a functional LPS receptor. Cross-linking experiments were performed to identify LPS binding sites. Two molecules were identified: a 55-kDa protein (CD14) and a second, 78-kDa band. Sequencing of the 78-kDa protein by mass spectroscopic analysis revealed 100% homology with moesin (membrane-organizing extension spike protein). Antibody to CD14 induced partial blocking of the LPS response. However, antimoesin monoclonal antibody completely blocked the LPS-induced TNF-α response in human monocytes, without blocking CD14 binding of LPS. Irrelevant isotype controls had no effect. Additional experiments were performed to evaluate the specificity of the antimoesin blocking. Separate experiments evaluated antimoesin effects on monocyte chemotaxis, IL-1 production in response to IL-1 stimulation, and TNF-α secretion in response toStaphylococcus aureus stimulation. Antimoesin blocked only LPS-mediated events. The data suggest that moesin functions as an independent LPS receptor on human monocytes. The role of moesin in transduction of CD14-mediated signals is discussed.

scholarly journals Activation of Interleukin-1 Receptor-Associated Kinase by Gram-Negative Flagellin

2001 ◽  
Vol 69 (7) ◽  
pp. 4424-4429 ◽  
Author(s):  
Marlena A. Moors ◽  
Liwu Li ◽  
Steven B. Mizel
Keyword(s):  
Cell Line ◽  
Salmonella Enteritidis ◽  
Mutant Cell ◽  
Interleukin 1 ◽  
Central Component ◽  
No Production ◽  
Gram Negative Bacteria ◽  
Necrosis Factor Alpha ◽  
Gram Negative ◽  
Tnf Α

ABSTRACT Flagellin from various species of gram-negative bacteria activates monocytes to produce proinflammatory cytokines. We have analyzed the pathway by which Salmonella enteritidis flagellin (FliC) activates murine and human monocyte/macrophage-like cell lines. Since lipopolysaccharide (LPS), the principal immune stimulatory component of gram-negative bacteria, is known to signal through Toll-like receptor 4 (TLR4), we tested the possibility that FliC also signals via TLR4. When murine HeNC2 cells were stimulated with LPS in the presence of a neutralizing anti-TLR4 monoclonal antibody, tumor necrosis factor alpha (TNF-α) and nitric oxide (NO) production were markedly reduced. In contrast, FliC-mediated TNF-α and NO production were minimally affected by the anti-TLR4 antibody. Furthermore, FliC, unlike LPS, stimulated TNF-α production in the TLR4 mutant cell line, GG2EE, indicating that TLR4 is not essential for FliC-mediated signaling. To test the possibility that FliC signals via another TLR, we measured FliC-mediated activation of interleukin-1 (IL-1) receptor-associated kinase (IRAK), a central component in IL-1R/TLR signaling. FliC induced IRAK activation in HeNC2 and GG2EE cells as well as in the human promonocytic cell line THP-1. IRAK activation was rapid in HeNC2 cells, with maximal activity observed after 5 min of treatment with FliC. In addition, FliC-mediated IRAK activation exhibited the same concentration dependence as was demonstrated for the induction of TNF-α. These results represent the first demonstration of IRAK activation by a purified bacterial protein and strongly suggest that a TLR distinct from TLR4 is involved in the macrophage inflammatory response to FliC.

scholarly journals Comparative Effects of Ciprofloxacin and Ceftazidime on Cytokine Production in Patients with Severe Sepsis Caused by Gram-Negative Bacteria

2004 ◽  
Vol 48 (8) ◽  
pp. 2793-2798 ◽  
Author(s):  
C. A. Gogos ◽  
A. Skoutelis ◽  
A. Lekkou ◽  
E. Drosou ◽  
I. Starakis ◽  
...  
Keyword(s):  
Severe Sepsis ◽  
Group Versus ◽  
Gram Negative Bacteria ◽  
Proinflammatory Response ◽  
Necrosis Factor Alpha ◽  
Gram Negative ◽  
Tnf Α ◽  
Factor Alpha ◽  
Anti Inflammatory ◽  
Necrosis Factor

ABSTRACT In the present study the effect of ciprofloxacin versus ceftazidime on concentrations of pro- and anti-inflammatory cytokines in the sera of patients with severe sepsis was evaluated. The study included 58 previously healthy patients suffering from severe sepsis caused by gram-negative bacteria, treated with either ciprofloxacin or ceftazidime after thorough clinical and microbiological evaluation and followed up for clinical outcome. Levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-1b (IL-1b), IL-6, and IL-8 and of the anti-inflammatory cytokine IL-10, as well as of IL-1 receptor antagonist and soluble TNF receptors I and II, in serum were measured at baseline and 24 and 48 h after the first antimicrobial dose. Mean SAPS-II scores, development of septic shock, and mortality rates were similar in the two groups (43.2 ± 9.2, 21.4%, and 14.3% in the ceftazidime group versus 49.8 ± 11.3, 20%, and 13.3% in the ciprofloxacin group). Serum TNF-α and IL-6 levels at 24 and 48 h were significantly lower in the ciprofloxacin group, while the IL-10/TNF-α ratio was significantly higher, than those for the ceftazidime group. Among patients with high baseline TNF-α levels, there were significant increases in the IL-10/TNF-α ratio at both 24 and 48 h over that at admission for the ciprofloxacin group, while no differences were noted in the ceftazidime group. These results indicate that ciprofloxacin may have an immunomodulatory effect on septic patients by attenuating the proinflammatory response, while there is no evidence that differences in the cytokines measured have any impact on the final outcome.

scholarly journals Gram-Positive and Gram-Negative Bacteria Do Not Trigger Monocytic Cytokine Production through Similar Intracellular Pathways

2001 ◽  
Vol 69 (7) ◽  
pp. 4590-4599 ◽  
Author(s):  
Lila Rabehi ◽  
Théano Irinopoulou ◽  
Béatrice Cholley ◽  
Nicole Haeffner-Cavaillon ◽  
Marie-Paule Carreno
Keyword(s):  
Kinase Inhibitors ◽  
Cytokine Production ◽  
Kinase Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Gram Negative Bacteria ◽  
Gram Positive ◽  
Necrosis Factor Alpha ◽  
Gram Negative ◽  
Tnf Α ◽  
P38 Pathway

ABSTRACT Toll-like receptors (TLRs) are involved in human monocyte activation by lipopolysaccharide (LPS) and Staphylococcus aureus Cowan (SAC), suggesting that gram-positive and gram-negative bacteria may trigger similar intracellular events. Treatment with specific kinase inhibitors prior to cell stimulation dramatically decreased LPS-induced cytokine production. Blocking of the p38 pathway prior to LPS stimulation decreased interleukin-1α (IL-1α), IL-1ra, and tumor necrosis factor alpha (TNF-α) production, whereas blocking of the ERK1/2 pathways inhibited IL-1α, IL-1β, and IL-1ra but not TNF-α production. When cells were stimulated by SAC, inhibition of the p38 pathway did not affect cytokine production, whereas only IL-1α production was decreased in the presence of ERK kinase inhibitor. We also demonstrated that although LPS and SAC have been shown to bind to CD14 before transmitting signals to TLR4 and TLR2, respectively, internalization of CD14 occurred only in monocytes triggered by LPS. Pretreatment of the cells with SB203580, U0126, or a mixture of both inhibitors did not affect internalization of CD14. Altogether, these results suggest that TLR2 signaling does not involve p38 mitogen-activated protein kinase signaling pathways, indicating that divergent pathways are triggered by gram-positive and gram-negative bacteria, thereby inducing cytokine production.

scholarly journals Annexin A5 Binds to Lipopolysaccharide and Reduces Its Endotoxin Activity

mBio ◽  
2012 ◽  
Vol 3 (2) ◽  
Author(s):  
Jacob H. Rand ◽  
Xiao-Xuan Wu ◽  
Elaine Y. Lin ◽  
Alexander Griffel ◽  
Philip Gialanella ◽  
...  
Keyword(s):  
Lipid A ◽  
Clinical Problem ◽  
Annexin A5 ◽  
Renal Tubules ◽  
Necrosis Factor Alpha ◽  
Gram Negative ◽  
Therapeutic Implications ◽  
High Affinity ◽  
Tnf Α

ABSTRACTAnnexin A5 (AnxA5) has a high affinity for phosphatidylserine. The protein is widely used to detect apoptotic cells because phosphatidylserine, a phospholipid that is normally present in the inner leaflets of cytoplasmic membranes, becomes translocated to the outer leaflets during programmed cell death. Here we report the novel observation that AnxA5 binds to Gram-negative bacteria via the lipid A domain of lipopolysaccharide (LPS). Binding of AnxA5 to bacteria was measured quantitatively, confirmed by fluorescence microscopy, and found to be inhibited by antibodies against lipid A. AnxA5 also bound to purified dot-blotted LPS and lipid A. Through ellipsometry, we found that the binding of AnxA5 to purified LPS was calcium dependent and rapid and showed a high affinity—characteristics similar to those of AnxA5 binding to phosphatidylserine. Initial functional studies indicated that AnxA5 can affect LPS activities. AnxA5 inhibited LPS-mediated gelation in theLimulusamebocyte lysate assay. Incubation of LPS with the protein reduced the quantity of tumor necrosis factor alpha (TNF-α) released by cultured monocytes compared to that released upon incubation with LPS alone. Initialin vivoexperiments indicated that injection of mice with LPS preincubated with AnxA5 produced serum TNF-α levels lower than those seen after injection of LPS alone. These data demonstrate that AnxA5 binds to LPS and open paths to investigation of the potential biological and therapeutic implications of this interaction.IMPORTANCEAnxA5 is highly expressed in cells that have a barrier function—including, among others, vascular endothelium, placental trophoblasts, and epithelial cells lining bile ducts, renal tubules, mammary ducts, and nasal epithelium. The protein has been well characterized for its binding to phospholipid bilayers that contain phosphatidylserine. This report of a previously unrecognized activity of AnxA5 opens the door to investigation of the possibility that this binding may have biological and therapeutic ramifications. In view of the tissue expression of the protein, the present results suggest the possibility that AnxA5 plays a role in modulating the host defense against lipopolysaccharide at these anatomic sites, where cells may interface with microorganisms. These results also raise the intriguing possibility that AnxA5 or analogous proteins or peptides could provide novel approaches to addressing the difficult clinical problem of Gram-negative sepsis.

scholarly journals Mutational Analysis of the TolA C-Terminal Domain of Escherichia coli and Genetic Evidence for an Interaction between TolA and TolB

2002 ◽  
Vol 184 (16) ◽  
pp. 4620-4625 ◽  
Author(s):  
Jean François Dubuisson ◽  
Anne Vianney ◽  
Jean Claude Lazzaroni
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Mutational Analysis ◽  
Membrane Stability ◽  
Genetic Evidence ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Terminal Domain ◽  
Suppressor Mutants

ABSTRACT The Tol proteins are involved in the outer membrane stability of gram-negative bacteria. The C-terminal domain of TolA was mutagenized to identify residues important for its functions. The isolation of suppressor mutants of tolA mutations in the tolB gene confirmed an interaction between TolAIII and the N-terminal domain of TolB.

T1808 TNF-α Inducing Activity in Gram Negative Bacteria From Patients With Crohn's Disease

2010 ◽  
Vol 138 (5) ◽  
pp. S-583
Author(s):  
Maiko Sasaki ◽  
Jan-Michael A. Klapproth
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Tnf Α
Sign in / Sign up

Export Citation Format

Share Document