Stimulation Of Leukocytes Research Articles

FCRL6 receptor: Expression and associated proteins

FCRL6 receptor is a more recently identified representative of the FCRL family. We generated a panel of mouse mAbs to baculovirus-derived recombinant FCRL6 protein. The clone 7B2 was found to specifically recognize a 63 kDa protein expressed preferentially on the surface of CD8 T and CD56 NK cells in human peripheral blood and spleen. The clone 7B2 reacts with FCRL6 in Western blotting, FACS, and immunohistochemistry. In the T cell lineage, FCRL6 functions in antigen-experienced cells. Mitogenic stimulation of PB leukocytes in vitro resulted in an abrogation of the FCRL6 gene expression. We found a significant decrease in the FCRL6 gene expression in peripheral T cells of patients with certain autoimmune and blood diseases, and its upregulation at the late stages of HIV infection. Study of the FCRL6 association with signaling molecules showed its ability to recruit SHP-1, SHP-2, SHIP-1, and SHIP-2 phosphatases, and also adaptor protein Grb2 through phosphorylated cytoplasmic tyrosines. The current results demonstrate inhibitory potential of FCRL6 and suggest its possible involvement in modulation of CTL effector functions in various immune disorders.

High cytokine levels in perforated acute otitis media exudates containing live bacteria

Acute otitis media (AOM) is an inflammatory response to microbes in the middle ear, sometimes associated with rupture of the tympanic membrane. Human leukocytes produce different patterns of inflammatory mediators in vitro when stimulated with Gram-positive and Gram-negative bacteria, respectively. Here, we investigated the cytokine and prostaglandin E2 (PGE2) responses in middle ear fluids (MEFs) from children with spontaneously perforated AOM, and related the mediator levels to the presence of pathogens detected by culture (live) or PCR (live or dead). Furthermore, the in vivo cytokine pattern was compared with that induced in leukocytes stimulated by dead bacteria in vitro. MEFs with culturable pathogenic bacteria contained more interleukin (IL)-1β (median: 110 μg/L vs. <7.5 μg/L), tumour necrosis factor (TNF) (6.3 μg/L vs. <2.5 μg/L), IL-8 (410 μg/L vs. 38 μg/L) and IL-10 (0.48 μg/L vs. <0.30 μg/L) than culture-negative fluids, irrespective of PCR findings. IL-6 and PGE2 were equally abundant (69–110 μg/L) in effusions with live, dead or undetectable bacteria. Cytokine levels were unrelated to bacterial species and to the presence or absence of virus. Similar levels of TNF and IL-6 as found in the MEFs were obtained by in vitro stimulation of leukocytes, whereas 11 times more IL-1β and 3.5 times more IL-8 were produced in vivo, and 22 times more IL-10 was produced in vitro. Vigorous production of proinflammatory cytokines accompanies AOM with membrane rupture, regardless of the causative agent, but the production seems to cease rapidly once the bacteria are killed and fragmented. IL-6 and PGE2, however, remain after bacterial disintegration, and may play a role in the resolution phase.

Searching for immunomodulatory sequences in sea bass ( Dicentrarchus labrax L.): Transcripts analysis from thymus

The thymus is a key organ of the immune system in most vertebrates and, for this reason, it has been used in this paper for the generation of a normalized cDNA library from sea bass ( Dicentrarchus labrax), one of the most extensively cultured species in South Mediterranean aquaculture. A total of 1632 ESTs from this library were initially analysed for sequence quality and vector sequences and, after this control, 1264 (77% of total clones sequenced) high-quality ESTs were further processed. The total collection of D. labrax thymus ESTs has been deposited in the EBI-GenBank-DBJ database (GenBank accession numbers from FN565576 to FN566839). The functional classification of ESTs was performed by Gene Ontology and KEGG annotation and, successively, the sequences were analysed using the ImmunomeBase software to identify potentially immuno-related genes. Using this approach, we found about 100 putative genes involved in immune system responses, most new in sea bass, that were analysed more in detail. Some of the potentially interesting genes identified by these in silico analyses were studied by real-time PCR to verify their expression both at basal level and after in vitro stimulation of sea bass head kidney leukocytes. The used strategy has been confirmed as a good approach to discover new immuno-related genes and to improve the knowledge of specific markers that could help the discrimination of T-cell subpopulations in sea bass and, in general, in Teleosts.

Chronic Cigarette Smoke Exposure Primes NK Cell Activation in a Mouse Model of Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is a debilitating, progressive lung disease punctuated by exacerbations of symptoms. COPD exacerbations are most often associated with viral infections, and exposure to cigarette smoke (CS) followed by viral infection has been shown experimentally to enhance lung inflammation, tissue destruction, and airway fibrosis. Despite this, however, the cellular mechanisms responsible for this effect are unknown. In this study, we examined NK cell function in a mouse model of COPD given the vital role of NK cells following viral infection. Ex vivo stimulation of lung leukocytes with poly(I:C), ssRNA40, or ODN1826 enhanced production of NK cell-derived IFN-gamma in CS-exposed mice. NK cells from CS-exposed mice exhibited a novel form of priming; highly purified NK cells from CS-exposed mice, relative to NK cells from filtered air-exposed mice, produced more IFN-gamma following stimulation with IL-12, IL-18, or both. Further, NK cell priming was lost following smoking cessation. NKG2D stimulation through overexpression of Raet1 on the lung epithelium primed NK cell responsiveness to poly(I:C), ssRNA40, or ODN1826 stimulation, but not cytokine stimulation. In addition, NK cells from CS-exposed mice expressed more cell surface CD107a upon stimulation, demonstrating that the NK cell degranulation response was also primed. Together, these results reveal a novel mechanism of activation of the innate immune system and highlight NK cells as important cellular targets in controlling COPD exacerbations.

Innate lymphoid cells drive interleukin-23-dependent innate intestinal pathology

The key role of IL-23 in the pathogenesis of autoimmune and chronic inflammatory disorders is supported by the identification of IL-23R susceptibility alleles associated with IBD, psoriasis and ankylosing spondylitis. IL-23 driven inflammation has primarily been linked to the actions of Th17 cells1. Somewhat overlooked, IL-23 also has inflammatory effects on innate immune cells2 and can drive T cell- independent colitis. However the downstream cellular and molecular pathways involved in this innate intestinal inflammatory response are poorly characterized. Here we show that bacteria-driven innate colitis is associated with increased IL-17 and IFN-γ production in the colon. Stimulation of colonic leukocytes with IL-23 induced IL-17 and IFN-γ production exclusively by innate lymphoid cells expressing Thy1, SCA-1, RORγt and IL-23R and these cells markedly accumulated in the inflamed colon. Importantly, IL-23 responsive innate intestinal cells are also a feature of T-cell dependent models of colitis. The transcription factor RORγt, which controls IL-23R expression, plays a functional role as Ror−/−Rag−/− mice failed to develop innate colitis. Lastly, depletion of Thy1+ innate lymphoid cells completely abrogated acute and chronic innate colitis. These results identify a novel IL-23 responsive innate lymphoid population that mediates intestinal immune pathology and may therefore represent a target in IBD.

Molecular and structural characterisation of a macrophage migration inhibitory factor from sea bass ( Dicentrarchus labrax L.)

The macrophage migration inhibitory factor (MIF) is a cytokine produced in numerous cell types, mainly T lymphocytes and macrophages, in response to inflammatory stimuli. In this paper we report the identification of a cDNA encoding a MIF molecule from sea bass ( Dicentrarchus labrax L.), its expression analysis and its 3D structure obtained by template-based modelling. The sea bass MIF cDNA consists of 609 bp that translates in one reading frame to give the entire molecule containing 115 amino acids. The sequence contains three cysteine residues in conserved positions compared to human MIF and most Teleost fishes, with the exception of zebrafish and carp. The Cys 57-Ala 58-Leu 59-Cys 60 motif, present inside the stretch important for JAB1-interaction and mediator of the thiol-protein oxidoreductase activity of MIF, is conserved in sea bass, together with the Pro 2 residue that is crucial for the tautomerase catalytic activity. Real-time PCR analyses revealed that MIF is constitutively expressed in all selected tissues and organs, with the highest mRNA level observed in thymus. MIF expression was induced after 4 h in vitro stimulation of head kidney leukocytes with LPS and decreased after 24 h. The predicted 3D model of sea bass MIF has been used to verify the presence of structural requirements for its known biological activities.

Assessment of Pharmacological Activity of Baminercept (LTBR-Ig) in Patients with Rheumatoid Arthritis

of Th2 lymphocytes and promotion of eosinophil transmigration across endothelium. IL-4 signals through type I receptor, which consists of IL4Rα and the common γ chain, and through the type II receptor composed of IL4Rα and IL13Rα1 subunits. While IL-4 serves as a sole ligand for type I receptor, both IL-4 and IL-13 are capable of signaling through the type II receptor. Therefore, targeting the shared subunit IL4Rα is expected to inhibit both IL-4and IL-13-induced signaling. To test this hypothesis, we developed a fully human antibody specific for IL-4Rα (REGN668/SAR231893) and tested its potential to block IL-4 mediated Type I and Type II signaling and IL-13 mediated Type II signaling using in vitro cell-based assays. Utilizing microarray technology, IL-4and IL-13induced expression profiles were generated for human leukocytes after in vitro stimulation. REGN668/SAR231893 was shown to antagonize both IL-4 and IL-13 signaling, evidenced by its ability to attenuate IL-4 and IL-13-mediated changes in gene expression. Significantly, IL-4 or IL-13 were shown to up-regulate expression of six chemokine mRNAs following stimulation of whole blood leukocytes: TARC (CCL17), eotaxin-3 (CCL26), MIP-4 (CCL13), PARC (CCL18), MDC (CCL22) and MIP-3 (CCL23). Significantly, REGN668/ SAR231893 was shown to reverse upregulation of TARC and eotaxin-3 levels measured by TaqMan arrays and ELISA. The chemokines TARC and eotaxin-3 have been reported as biomarkers in atopy. We conclude that REGN668/SAR231893 has potential clinical application in the treatment of the atopic disease.

Designed Surface with Tunable IgG Density as an in Vitro Model for Immune Complex Mediated Stimulation of Leukocytes

We present the design of an in vitro model for immune-complex-mediated stimulation of leukocytes and its functional characteristics with respect to monocyte adhesion. The model was based on the orientation-controlled immobilization of a humanized IgG1 monoclonal antibody (rituximab) via its interaction with a biotinylated peptide epitope derived from the CD20 marker. The peptide was linked to neutravidin covalently attached to a mixed self-assembled monolayer of carboxyl- and methoxy-terminated oligo(ethylene glycol) alkane thiolates on gold. The surface adhesion propensity of human monocytes (cell line U937) was highly dependent on the lateral IgG density and indicated that there exists a distance between IgG-Fc on the surface where interactions with Fc gamma receptors are optimal. This well-defined platform allows for a careful control of the size and orientation of artificial IgG immune complexes, it is easily made compatible with, for example, cellular imaging, and it will become useful for in vitro studies on the importance of Fc gamma receptor interactions in chronic immune-mediated diseases.

High cytokine levels in perforated acuteotitis mediaexsudates containing live bacteria

Acute otitis media (AOM) is an inflammatory response to microbes in the middle ear, sometimes associated with rupture of the tympanic membrane. Human leukocytes produce different patterns of inflammatory mediators in vitro when stimulated with Gram-positive and Gram-negative bacteria, respectively. Here, we investigated the cytokine and prostaglandin E2 (PGE2) responses in middle ear fluids (MEFs) from children with spontaneously perforated AOM, and related the mediator levels to the presence of pathogens detected by culture (live) or PCR (live or dead). Furthermore, the in vivo cytokine pattern was compared with that induced in leukocytes stimulated by dead bacteria in vitro. MEFs with culturable pathogenic bacteria contained more interleukin (IL)-1β (median: 110 μg/L vs. <7.5 μg/L), tumour necrosis factor (TNF) (6.3 μg/L vs. <2.5 μg/L), IL-8 (410 μg/L vs. 38 μg/L) and IL-10 (0.48 μg/L vs. <0.30 μg/L) than culture-negative fluids, irrespective of PCR findings. IL-6 and PGE2 were equally abundant (69-110 μg/L) in effusions with live, dead or undetectable bacteria. Cytokine levels were unrelated to bacterial species and to the presence or absence of virus. Similar levels of TNF and IL-6 as found in the MEFs were obtained by in vitro stimulation of leukocytes, whereas 11 times more IL-1β and 3.5 times more IL-8 were produced in vivo, and 22 times more IL-10 was produced in vitro. Vigorous production of proinflammatory cytokines accompanies AOM with membrane rupture, regardless of the causative agent, but the production seems to cease rapidly once the bacteria are killed and fragmented. IL-6 and PGE2, however, remain after bacterial disintegration, and may play a role in the resolution phase.

Mechanisms of Action and Chemical-Biological Interactions Between Ozone and Body Compartments: A Critical Appraisal of the Different Administration Routes

After a long initial stage obscured by empirism and misconceptions, oxygen-ozonetherapy has now become a scientific discipline where the reactions between ozone and human blood are within the realm of orthodox biochemistry, physiology and pharmacology. Most of the basic mechanisms of action have been clarified and ozone can be considered a pro-drug, which almost instantaneously reacts with antioxidants and unsaturated fatty acids. These reactions generate the actual ozone messengers represented by either hydrogen peroxide as a fast acting compound or a variety of lipid oxidation products as late effectors. While ozone is totally consumed, micromolar amounts of these messengers are able to enhance the delivery of oxygen via erythrocyte activation, the immune system by a bland leukocyte stimulation and most of the remaining body cells by up-regulating the antioxidant system. The hazard of ozone toxicity has been dispelled by using the gas only within a dose range perfectly calibrated against the potent blood antioxidant capability. Ozonetherapy can be very useful in patients with chronic vascular disorders and ischemic problems and should be extensively used by official medicine. An extraordinary facet of ozone is its medical application versatility, as represented by several administration routes, and the minimal cost of this drug.

The influence of harvesting intervals on chemiluminescence and enzyme activities of rat peritoneal leukocytes.

Rat leukocytes were obtained from the peritoneal cavity by stimulation with potassium caseinate. The chemiluminescence response of the polymorphonuclear leukocytes exposed to opsonized zymosan was influenced by the interval between instillation of caseinate and harvesting of the cells. With intervals increasing from four to 72 hours, the maximum activity was reached after 24 hours. The myeloperoxidase activity of all the leukocytes together increased gradually up to 72 hours after instillation of caseinate. After exposure to zymosan particles, only a negligible fraction of this enzyme was released from the cells. On the other hand, the lysozyme activity was highest in cells harvested early, and a large fraction of this enzyme was also released from these cells. The findings emphasize the importance of standardized conditions for stimulation and harvesting of rat peritoneal leukocytes.

Identification and characterization of TLR8 and MyD88 homologs in Atlantic salmon (Salmo salar)

Toll-like receptor 8 (TLR 8) belongs to a subgroup of the TLR family that recognizes nucleic acids and that is involved in the protection against viruses. In mammals, TLR7 and 8 have been characterized as receptors for viral and synthetic single-stranded RNA. Here we describe the cloning of a TLR8 homolog in Atlantic salmon (Salmo salar) and its proximal adaptor protein MyD88. The mRNA expression of SsTLR8 was tissue-restricted and its highest level was detected in the spleen while SsMyD88 was expressed in all of the tested organs. SsTLR8 and SsMyD88 mRNAs were up-regulated in TO cells treated with recombinant IFN α1 and IFN γ. In vivo, the expression of SsTLR8 was not significantly affected following challenge with salmon alphavirus subtype 3 (SAV3). By contrast, infection with SAV3 up-regulated SsMyD88 transcripts on day 7 post-challenge and the expression remained elevated at day 28. The SsMyD88 expression in vivo paralleled type I IFN expression. In vitro stimulation of salmon head kidney leukocytes with CpG ODNs and IFN γ also up-regulated SsMyD88 mRNA. Furthermore, ectopic expression of SsMyD88 in HEK cells was able to activate a NF-κB reporter construct indicating that the cloned salmon molecule was a functional MyD88 homologue.

5-Lipoxygenase: mechanisms of regulation

5-lipoxygenase (5-LO) catalyzes two steps in biosynthesis of leukotrienes (LTs), a group of lipid mediators of inflammation derived from arachidonic acid (AA). LT antagonists are used in treatment of asthma; more recently a potential role also in atherosclerosis has raised considerable interest. Furthermore, possible effects of 5-LO metabolites in relation to tumorigenesis have emerged. Thus, an understanding of the biochemistry of this lipoxygenase has potential implications for treatment of various diseases.

Transepithelial activation of human leukocytes by probiotics and commensal bacteria: Role of Enterobacteriaceae‐type endotoxin

The goal of the current study was to clarify whether commercially available probiotics induce greater trans-epithelial activation of human leukocytes than do commensal, food-derived and pathogenic bacteria and to identify the compounds responsible for this activation. Eleven different bacterial strains, and some of their pathogen-associated molecular patterns, were incubated apically on a confluent layer of intestinal epithelial cells (Caco-2), which were basolaterally co-cultured with human mononuclear leukocytes. Only Gram-negative bacteria having Enterobacteriaceae-type endotoxin (commensal Escherichia coli K12, probiotic E. coli Nissle, EPEC) induced basolateral production of TNF-alpha, IFN-gamma, IL 6, 8, and 10. Gram-positive probiotics (Lactobacillus spp. and Bifidobacterium spp.) had virtually no effect. In addition, commensals (Enterococcus faecalis, Bacteroides vulgatus) and food fermenters (Lactobacillus spp.) did not stimulate leukocytes transepithelially. Endotoxin from E. coli and Salmonella enterica Typhimurium induced comparable transepithelial stimulation of leukocytes, but not endotoxin from B. vulgatus or lipoteichoic acid from E. faecalis. Endotoxin-binding agents (polymyxin, colistin) completely abrogated transepithelial activation of leukocytes. Enterobacteriaceae-type endotoxin is a crucial factor in transepithelial stimulation of leukocytes, regardless of whether it is produced by probiotics or other bacteria. Hence, transepithelial stimulation ofleukocytes' innate immune response seems to not be linked to the health-promoting effects ofprobiotics.

In vitro stimulation of equine leukocytes with CpG-oligodeoxyribonucleotides—Cytokine production and proliferation

In vitro stimulation of equine leukocytes with CpG-oligodeoxyribonucleotides—Cytokine production and proliferation

Phosphatidylcholine Reverses Ethanol‐Induced Increase in Transepithelial Endotoxin Permeability and Abolishes Transepithelial Leukocyte Activation

Chronic alcohol abuse increases both intestinal bacterial overgrowth and intestinal permeability to macromolecules. Intestinal permeability of endotoxin, a component of the outer cell membrane of Gram-negative bacteria, plays a crucial role in the development of alcohol-induced liver disease (ALD). As impaired bile flow leads to endotoxemia and the bile component phosphatidylcholine (PC) is therapeutically active in ALD, we tested the hypothesis that conjugated primary bile salts (CPBS) and PC inhibit ethanol-enhanced transepithelial permeability of endotoxin and the subsequent transepithelial activation of human leukocytes. For this purpose, we used a model in which intestinal epithelial cells (Caco-2) were basolaterally cocultivated with mononuclear leukocytes. Cells were challenged apically with endotoxin from Escherichia coli K12 and were incubated with or without the addition of CPBS (1.5 mM), PC (0.38 mM), pooled human bile (2%) in combination with ethanol (0 to 66 mM). Ethanol decreased integrity of intestinal epithelial cell monolayer and enhanced transepithelial permeability of endotoxin. Both the transepithelial permeability of endotoxin and the transepithelial stimulation of leukocytes were nearly completely abolished after the apical supplementation of PC with CPBS, but not by CPBS alone. Ethanol up to 66 mM was not able to reverse this effect. A considerable part of the therapeutic and preventive effect of PC supplementation in ALD might result from a reduction of ethanol-enhanced permeability of endotoxin through the intestinal barrier.

Expression levels of immune markers in Actinobacillus pleuropneumoniae infected pigs and their relation to breed and clinical symptoms

BackgroundIn pigs little is known about the role of innate immune defence in bacterial infections of the respiratory tract, despite their major role in pig production. In the present study we characterized and compared in vitro and in vivo activation of immune markers of different pig breeds 7 days before, and 4 and 21 days after an experimental aerosol infection with Actinobacillus (A.) pleuropneumoniae.ResultsIn vitro stimulation of bronchoalveolar lavage fluid (BALF) and blood leukocytes with A. pleuropneumoniae, Streptococcus suis, PMA and LPS led to production of different amounts of H2O2, NO and TNF-α, depending on the stimulus, individual, breed and time of infection. Generally, significant responses to in vitro stimulation were observed only in blood leukocytes, whereas the alveolar macrophages showed a high basal activation. In addition, the production of haptoglobin and cytokines (TNF-α, IFN-γ and IL-10) in vivo was measured in plasma and BALF. Plasma haptoglobin levels mirrored the clinical manifestations at 4 days post-infection. In plasma and BALF TNF-α could not be detected, whereas variable levels of IFN-γ were found at pre- and post-infection times. IL-10 was found in some plasma but in none of the BALF samples. The different expression levels in individuals within the breeds correlated for some markers with the severity of clinical manifestations, e.g. H2O2, plasma haptoglobin and BALF IFN-γ for German Landrace pigs.ConclusionOur findings revealed differences in the activation of the immune markers with respect to infection time, individuals and breeds. Moreover, results showed different correlation grades between the immune markers produced in vitro or in vivo and the clinical manifestations. Further analyses will have to show whether these markers may serve as correlates of protection against porcine respiratory infections.

Infectious Bronchitis Virus induces acute interferon-gamma production through polyclonal stimulation of chicken leukocytes

Infectious Bronchitis Virus, a member of the Coronaviridae, is a respiratory pathogen in poultry. We found that in vitro stimulation with IBV resulted in ChIFN-γ production in splenocytes of both infected birds and uninfected birds. The non-specific stimulation did not occur when other avian viruses or other coronaviruses were used or when mammalian cells were stimulated with IBV. Inactivation of IBV reduced ChIFN-γ production, but ChIFN-γ remained elevated compared to unstimulated cells. An increase in ChIFN-γ mRNA was detected in splenocytes from IBV-infected and uninfected chickens as early as 1 h after stimulation with IBV. These results indicate that IBV acts as a polyclonal stimulus, inducing a rapid production of IFN-γ even without previous exposure to the virus.

Role of p38 MAPK in LPS induced pro-inflammatory cytokine and chemokine gene expression in equine leukocytes

Endotoxemia occurs when bacterial lipopolysaccharide (LPS) in the blood induces a dysregulated inflammatory response, resulting in circulatory shock and multi-organ failure. Laminitis is a common complication in endotoxemic horses and is frequently the reason for humane euthanasia of these cases. Blood leukocytes are a principal target of LPS in endotoxemia leading to activation of multiple signal transduction pathways involved in the induction of a number of pro-inflammatory genes. In other animal models, the p38 mitogen activated protein kinase (MAPK) pathway has been associated with induced expression of tumor necrosis factor-α (TNFα), interleukin (IL)-1β, IL-6 and IL-8. The goal of this study was to determine the role of the p38 MAPK pathway in the induction of these pro-inflammatory cytokine and chemokine genes in LPS-stimulated equine leukocytes. Stimulation of equine peripheral blood leukocytes resulted in an increase in TNFα, IL-1β, IL-6 and IL-8 mRNA levels. Pharmacological inhibition of p38 MAPK activity with SB203580 or SB202190 reduced the ability of LPS stimulation to increase mRNA concentrations for all four genes. However, only SB203580 pretreatment significantly reduced LPS-stimulated IL-1β and IL-8 mRNA expression and only pretreatment with SB202190 significantly reduced LPS-stimulated TNFα and IL-6 mRNA expression. From this study we conclude TNFα, IL-1β, IL-6 and IL-8 are induced upon LPS stimulation of equine leukocytes and that this induction of gene expression is dependent on the p38 MAPK pathway. However, there are differences in the efficacy of the p38 inhibitors tested here that may be explained by differences in specificity or potency. This study provides evidence for the use of selective p38 MAPK inhibitors as potential therapeutics for the treatment of equine endotoxemia.

Zinc Signals Are Essential for Lipopolysaccharide-Induced Signal Transduction in Monocytes

Cytosolic alterations of calcium ion concentrations are an integral part of signal transduction. Similar functions have been hypothesized for other metal ions, in particular zinc (Zn(2+)), but this still awaits experimental verification. Zn(2+) is important for multiple cellular functions, especially in the immune system. Among other effects, it influences formation and secretion of pro-inflammatory cytokines, including TNF-alpha. Here we demonstrate that these effects are due to a physiological signaling system involving intracellular Zn(2+) signals. An increase of the intracellular zinc ion concentration occurs upon stimulation of human leukocytes with Escherichia coli, LPS, Pam(3)CSK(4), TNF-alpha, or insulin, predominantly in monocytes. Chelating this zinc signal with the membrane permeable zinc-specific chelator TPEN (N,N,N',N'-tetrakis-(2-pyridyl-methyl)ethylenediamine) completely blocks activation of LPS-induced signaling pathways involving p38 MAPK, ERK1/2, and NF-kappaB, and abrogates the release of proinflammatory cytokines, including TNF-alpha. This function of Zn(2+) is not limited to monocytes or even the immune system, but seems to be another generalized signaling system based on intracellular fluctuations of metal ion concentrations, acting parallel to Ca(2+).