Mechanism of extracellular release of human neutrophil calprotectin complex.

Calprotectin is an abundant cytosolic protein complex of human neutrophils with in vitro extracellular antimicrobial activity. Studies suggest that calprotectin may be actively secreted from intact HL-60 cells and that it can be translocated to polymorphonuclear neutrophil (PMN) cell membranes. To examine whether calprotectin is secreted extracellularly, we incubated soluble and particulate stimuli, including live and heat-inactivated Candida albicans, with whole blood and measured calprotectin levels in the plasma.

Immunomagnetic recovery of human neutrophil defensins from the human gingival crevice.

The human neutrophilic protein defensins are cationic, antimicrobial, cytotoxic, corticostatic chemotactic, opsonic peptides found in azurophilic granules and constitute about 5% of the total protein in human neutrophils. In the present study, defensins were recovered from the human gingival crevice using paramagnetic microspheres (beads), coated with anti-defensin antibodies. The bead-bound defensins were assayed using an enzyme-linked immunosorbent assay developed in this laboratory.

Porcine polymorphonuclear leukocytes generate extracellular microbicidal activity by elastase-mediated activation of secreted proprotegrins.

Antimicrobial peptides of several structural classes have been found in phagocytes and epithelial cells of many animals. The broadly microbicidal protegrins (PG1, -2, and -3) were originally isolated as 16 to 18-amino-acid peptides from pig neutrophil lysates, but the corresponding cDNA sequences encoded much larger precursors that belonged to the cathelicidin family of antimicrobial peptides. We explored the storage, secretion, and microbicidal activation of protegrins in porcine neutrophils and in a model system consisting of recombinant proprotegrin 3 (pPG3) and various serine proteases and their inhibitors.

Neutrophil activation in preeclampsia. Are defensins and lactoferrin elevated in preeclamptic patients?

Objective: To determine if human defensins and lactoferrin, both markers of neutrophil activation, are elevated in preeclamptic plasma.

Study Design: Blood samples were obtained from 18 preeclamptic and 29 normal pregnant women in the third trimester. Demographic and clinical data were obtained from the medical record.

Human neutrophil defensin and serpins form complexes and inactivate each other.

Defensins, antimicrobial and cytotoxic peptides of neutrophils, bind to and are inactivated by blood proteins. We identified defensin interactions with alpha 1-proteinase inhibitor (alpha 1-PI), alpha 1-antichymotrypsin (alpha 1-ACT), alpha 2-antiplasmin (alpha 2-AP), and antithrombin III (AT III) and examined defensin binding to alpha 1-PI and alpha 1-ACT in more detail. Defensin interactions with either alpha 1-PI or alpha 1-ACT were not affected by iodoacetamide or high salt concentration.

Identification of defensin binding to C1 complement.

In human serum we found strong defensin binding to the complexes of activated C1 complement (C1) and C1 inhibitor (C1i). Purified C1q, activated C1 tetramer (r2s2) and C1i did not bind defensin. When r2s2 was dissociated by EDTA, only the activated C1s (C1s) bound defensin.

Plasma defensin concentrations are elevated in patients with septicemia or bacterial meningitis.

We measured concentrations of defensins (human neutrophil peptides) in the plasma of healthy volunteers and patients with sepsis and meningitis. When a sensitive enzyme immunoassay was used, defensins were detected in plasma samples from 13 of 24 healthy blood donors, with a mean +/- SD of 42 +/- 53 ng/ml. Defensin levels in plasma samples from seven patients with sepsis at the onset of disease ranged from 900 ng/ml to 170,000 ng/ml.

The effect of biotinylation on the antigenic specificity of anti-defensin monoclonal antibodies.

We studied the effects of biotinylation on three monoclonal antibodies (Mabs) that were raised against carrier protein conjugates of human defensin HNP-1, and of rabbit defensins NP-2 and NP-5 respectively. Before biotinylation, each Mab specifically bound to its peptide hapten. Biotinylation of these Mabs by the N-hydroxysuccinimide-biotin (NHS-biotin) resulted in crossreactivity of each Mab with the two irrelevant defensin peptides.

Mononuclear cells from HIV-infected patients produce factors which enhance functional activity of polymorphonuclear neutrophils from healthy subjects.

The influence of mononuclear cell supernatants (MNCS) from nine healthy donors and 35 HIV-infected patients (17 with lymphoadenopathy syndrome (LAS), 15 with ARC and three with AIDS) on functional activity of polymorphonuclear neutrophils (PMN) from healthy donors was investigated. MNC after short-term cultivation (24 h) produced factors which enhanced chemiluminescence (CL) and chemotaxis of PMN. This augmentation did not depend on stimulation of MNC by mitogens (lipopolysaccharide Escherichia coli (LPS) and concanavalin A (Con A)) or on activation of PMN by FMLP.

A sensitive enzyme-linked immunosorbent assay for human interleukin-8.

In order to quantify human interleukin-8 (IL-8), which is chemotactic for T cells and basophils as well as neutrophils, we developed an enzyme-linked immunosorbent assay (ELISA). Since binding inhibition tests indicated that three monoclonal antibodies (mAbs; BS-1, WS-4, WS-6) blocked the binding of 125I-labelled IL-8 to neutrophils, we tested an ELISA using these mAbs as primary antibodies, rabbit anti-IL-8 Ab as the secondary antibody, and alkaline phosphatase-labelled goat anti-rabbit Ab as the conjugate. Among the three mAbs tested, WS-4 was the most sensitive with a detection limit of 16 pg/ml.

An enzyme immunoassay for human defensins.

We developed and optimized an enzyme immunoassay for human neutrophil defensins, cationic cysteine-rich peptides that participate in host defense and inflammation. The assay utilizes a sandwich design with a monoclonal capture antibody and a biotinylated monoclonal detecting antibody. Cetrimonium bromide is employed to obviate non-specific binding of defensins to surfaces.

Activated alpha 2-macroglobulin is a principal defensin-binding protein.

Defensins are highly abundant and variably cationic peptides that possess antimicrobial, cytotoxic, and chemoattractant properties and equip mammalian phagocytes for participation in host defense and inflammatory processes. We studied the binding of the human defensin HNP-1 by proteins in plasma and serum and identified activated (F-form) alpha 2-macroglobulin (alpha 2M) as a principal binding protein for HNP-1. In contrast, native (S-form) alpha 2M bound little HNP-1.

Tumor necrosis factor alpha induction in human monocytes.

The present study was undertaken to assess the presence of tumor necrosis factor (TNF)-alpha mRNA and protein in circulating human blood monocytes and to study the TNF-alpha gene expression in human monocytes isolated by continuous Percoll gradient fractionation. The technique of RNA isolation directly from the blood samples was used to study TNF-alpha mRNA expression in circulating human blood leukocytes. It was shown that human blood leukocytes of healthy donors contained no presynthesized pool of TNF-alpha mRNA as well as no TNF-alpha protein.

The use of syngeneic cells producing human somatotropic hormone (hSTH) for immunization of mice and development of hybridomas.

We studied the possibility of syngeneic cells expressing heterologous protein being used for sensitization of mice and production of hybridomas. Recombinant retroviral vector containing cloned human somatotropic hormone (hSTH) gene was used to express hSTH in BALB/3T3 cells. BALB/c mice were injected intrasplenically (i/s) or combination of intraperitoneally (i/p) and intrasplenically with hSTH-producing cells.

Induction of tumor necrosis factor synthesis in human monocytes treated by transcriptional inhibitors.

Human blood monocytes and lymphocytes were separated by Percoll gradient fractionation. The synthesis of RNA was inhibited by actinomycin D (AcD) or alpha-amanitin (Amn). Monocytes were stimulated with LPS, lymphocytes were stimulated with phytohaemagglutinin (PHA).