Tumor necrosis factor-alpha causes release of cytosolic interleukin-18 from human neutrophils.

Neutrophils (PMNs) are a vital part of host defense and are the principal leukocyte in innate immunity. Interleukin (IL)-18 is a proinflammatory cytokine with roles in both innate and adaptive immunity. We hypothesize that PMNs contain preformed IL-18, which is released in response to specific inflammatory stimuli. Isolated PMNs were stimulated with a battery of chemoattractants (5 min to 24 h), and IL-18 release was measured. PMNs were also separated into subcellular fractions and immunoblotted with antibodies against IL-18 or were fixed and probed with antibodies to IL-18 as well as to the contents of granules, intracellular organelles, and filamentous actin (F-actin), incubated with fluorescent secondary antibodies, and examined by digital microscopy. Quiescent PMNs contained IL-18 in the cytoplasm, associated with F-actin, as determined by positive fluorescence resonance energy transfer (FRET+). In turn, TNF-alpha stimulation disrupted the association of IL-18 with F-actin, induced a FRET+ interaction of IL-18 with lipid rafts, and elicited IL-18 release. Manipulation of F-actin status confirmed the relationship between IL-18 and F-actin in resting PMNs. Consequently, incubation with monomeric IL-18 binding protein inhibited TNF-alpha-mediated priming of the PMN oxidase. We conclude that human PMNs contain IL-18 associated with F-actin in the cytoplasm and TNF-alpha stimulation causes dissociation of IL-18 from F-actin, association with lipid rafts, and extracellular release. Extracellular IL-18 participates in TNF-alpha priming of the PMN oxidase as demonstrated by inhibition with the IL-18 binding protein.

Physiological levels of interleukin-18 stimulate multiple neutrophil functions through p38 MAP kinase activation.

Patients with sepsis and acute lung injury have increased interleukin (IL)-18 levels systemically. We hypothesize that IL-18 stimulates neutrophils (PMNs) at physiologic concentrations. IL-18 primed the oxidase at 15 min (10-100 ng/ml), 30 min (0.1-100 ng/ml), and 60 min (100 ng/ml; P<0.05) and caused translocation of p47(phox) to the membrane similar to lipopolysaccharides. CD11b surface expression was increased by IL-18 in a time- and concentration-dependent manner. IL-18 caused up-regulation of the formyl-Met-Leu-Phe receptor, changes in PMN size, and elastase release. Investigation of signaling demonstrated IL-18-mediated activation of p38 mitogen-activated protein (MAP) kinase in a concentration (0.1-100 ng/ml)-, time (5-15 min)-, and Ca2+-dependent manner. IL-18 directly increased cytosolic Ca2+ concentration. IL-18 activation of PMNs was blocked by inhibition of p38 MAP kinase activity (SB203580) or by inhibition of p38 MAP kinase activation by chelation of cytosolic Ca2+. We conclude that IL-18, at physiologic concentrations, is an effective PMN priming agent that requires p38 MAP kinase activity.

Lysophosphatidylcholines prime the NADPH oxidase and stimulate multiple neutrophil functions through changes in cytosolic calcium.

A mixture of lysophosphatidylcholines (lyso-PCs) are generated during blood storage and are etiologic in models of acute lung injury. We hypothesize that lyso-PCs stimulate polymorphonuclear neutrophils (PMNs) through Ca(2)(+)-dependent signaling. The lyso-PC mix (0.45-14.5 micro M) and the individual lyso-PCs primed formyl-Met-Leu-Phe (fMLP) activation of the oxidase (1.8- to 15.7-fold and 1.7- to 14.8-fold; P<0.05). Labeled lyso-PCs demonstrated a membrane association with PMNs and caused rapid increases in cytosolic Ca(2)(+). Receptor desensitization studies implicated a common receptor or a family of receptors for the observed lyso-PC-mediated changes in PMN priming, and cytosolic Ca(2)(+) functions were pertussis toxin-sensitive. Lyso-PCs caused rapid serine phosphorylation of a 68-kD protein but did not activate mitogen-activated protein kinases or cause changes in tyrosine phosphorylation. With respect to alterations in PMN function, lyso-PCs caused PMN adherence, increased expression of CD11b and the fMLP receptor, reduced chemotaxis, provoked changes in morphology, elicited degranulation, and augmented fMLP-induced azurophilic degranulation (P<0.05). Cytosolic Ca(2)(+) chelation inhibited lyso-PC-mediated priming of the oxidase, CD11b surface expression, changes in PMN morphology, and serine phosphorylation of the 68-kD protein. In conclusion, lyso-PCs affect multiple PMN functions in a Ca(2)(+)-dependent manner that involves the activation of a pertussis toxin-sensitive G-protein.

Psoralen Inactivation of Viruses: A Process for the Safe Manipulation of Viral Antigen and Nucleic Acid.

High consequence human pathogenic viruses must be handled at biosafety level 2, 3 or 4 and must be rendered non-infectious before they can be utilized for molecular or immunological applications at lower biosafety levels. Here we evaluate psoralen-inactivated Arena-, Bunya-, Corona-, Filo-, Flavi- and Orthomyxoviruses for their suitability as antigen in immunological processes and as template for reverse transcription PCR and sequencing. The method of virus inactivation using a psoralen molecule appears to have broad applicability to RNA viruses and to leave both the particle and RNA of the treated virus intact, while rendering the virus non-infectious.

Spo0A-dependent activation of an extended -10 region promoter in Bacillus subtilis.

At the onset of endospore formation in Bacillus subtilis the DNA-binding protein Spo0A directly activates transcription from promoters of about 40 genes. One of these promoters, Pskf, controls expression of an operon encoding a killing factor that acts on sibling cells. AbrB-mediated repression of Pskf provides one level of security ensuring that this promoter is not activated prematurely. However, Spo0A also appears to activate the promoter directly, since Spo0A is required for Pskf activity in a DeltaabrB strain. Here we investigate the mechanism of Pskf activation. DNase I footprinting was used to determine the locations at which Spo0A bound to the promoter, and mutations in these sites were found to significantly reduce promoter activity. The sequence near the -10 region of the promoter was found to be similar to those of extended -10 region promoters, which contain a TRTGn motif. Mutational analysis showed that this extended -10 region, as well as other base pairs in the -10 region, is required for Spo0A-dependent activation of the promoter. We found that a substitution of the consensus base pair for the nonconsensus base pair at position -9 of Pskf produced a promoter that was active constitutively in both deltaabrB and deltaspo0A deltaabrB strains. Therefore, the base pair at position -9 of Pskf makes its activity dependent on Spo0A binding, and the extended -10 region motif of the promoter contributes to its high level of activity.

Plasma and lipids from stored platelets cause acute lung injury in an animal model.

BACKGROUND: Transfusion of PLT concentrates may cause TRALI, a life-threatening reaction that has been linked to the infusion of anti-WBC immunoglobulins or older, stored PLTs that contain bioactive lipids. We hypothesize that lipids generated during storage of PLTs cause TRALI in a two-event animal model. STUDY DESIGN AND METHODS: Plasma from both whole-blood PLTs (WB-PLTs) and apheresis PLTs (A-PLTs) was isolated on Day 0 (D.0) and Day 5 (D.5) of storage and heat-treated before use. Rats were pretreated with saline or 2 mg per kg endotoxin (LPS), anesthetized, and the lungs were ventilated, isolated, and perfused with saline or 5-percent PLT plasma. Pulmonary artery pressure, pulmonary edema, and leukotriene B4 levels (perfusate) were measured. RESULTS: Plasma from D.5, but not D.0, of the identical WB-PLT and A-PLT units caused injury in lungs from LPS-pretreated rats (LPS/D.5) evidenced by increases in pulmonary edema and leukotriene B4 (p < 0.05). Lipid extracts and purified lipids from D.5 PLT plasma also elicited injury in lungs from LPS-pretreated rats (p < 0.05). Saline/D.5 plasma or lipids or LPS/D.0 did not cause pulmonary edema. Prestorage WBC reduction was ineffective in inhibiting TRALI. CONCLUSION: PLT-induced TRALI may be the result of two events: 1) the clinical condition of the patient and 2) the infusion of lipids in stored PLTs.

A two-insult in vitro model of PMN-mediated pulmonary endothelial damage: requirements for adherence and chemokine release.

Lysophosphatidylcholines (lyso-PCs), generated during blood storage, are etiologic in a two-insult, sepsis-based model of transfusion-related acute lung injury (TRALI). Individually, endotoxin (LPS) and lyso-PCs prime but do not activate neutrophils (PMNs). We hypothesized that priming of PMNs alters their reactivity such that a second priming agent causes PMN activation and endothelial cell damage. PMNs were primed or not with LPS and then treated with lyso-PCs, and oxidase activation and elastase release were measured. For coculture experiments, activation of human pulmonary microvascular endothelial cells (HMVECs) was assessed by ICAM-1 expression and chemokine release. HMVECs were stimulated or not with LPS, PMNs were added, cells were incubated with lyso-PCs, and the number of viable HMVECs was counted. Lyso-PCs activated LPS-primed PMNs. HMVEC activation resulted in increased ICAM-1 and release of ENA-78, GRO alpha, and IL-8. PMN-mediated HMVEC damage was dependent on LPS activation of HMVECs, chemokine release, PMN adhesion, and lyso-PC activation of the oxidase. In conclusion, sequential exposure of PMNs to priming agents activates the microbicidal arsenal, and PMN-mediated HMVEC damage was the result of two insults: HMVEC activation and PMN oxidase assembly.