The neutrophil-activating factor (NAF) purified from the conditioned medium of lipopolysaccharidestimulated human monocytes was sequenced and found to consist of 72 amino acids: SAKELRCQCIKTYSKPFHPKFI-KELRVIESGPHCANTEIIVKLSDGRELCLDP-KENWVQRVVEKFLKRAENS. Purified preparations of natural NAF contained, in addition to this main form, minor amounts of three amino-terminal variants with 77 (+AVLPR), 70, and 69 residues. A gene coding for the 72-amino acid NAF was synthesized, cloned, and expressed in Escherichia coli. Western (immunologic) blot analysis of crude bacterial extracts, with an antiserum raised against natural NAF, revealed a single band that comigrated with natural NAF. Recombinant NAF purified to homogeneity had identical amino-and carboxyl-terminal sequences to the 72-amino acid natural NAF. Recombinant NAF was tested on human neutrophils and had the same activity and potency as natural NAF in inducing chemotaxis, rapidly increasing cytosolic free Ca2+, activating the respiratory burst, and releasing specific and azurophilic granular contents.

The signal pathways of neutrophils following stimulation with the complement fragment C3a (C3a) were studied in neutrophils and compared to the pathways activated by complement fragment C5a (C5a). Analysis of polyphosphoinositol lipid turnover showed that C5a, but not C3a, activated phosphatidylinositol‐bisphosphate‐3‐kinase (PtdInsP2 3‐kinase) indicating that different signal pathways are activated by the two anaphylatoxins. To examine whether C3a stimulated Ca2+ transients, cytosolic free Ca2+ levels were analyzed in Fluo‐3‐labelled neutrophils by flow cytometry. C3a stimulated a fast and concentration‐dependent increase of cytosolic free Ca2+. Comparison of the C3a response with that of C5a revealed a more pronounced C5a‐triggered Ca2+ rise. Addition of EGTA to the extracellular buffer prior to stimulation did not significantly alter the initial Ca2+ rise at low C5a concentrations, but reduced the time course of the Ca2+ transients at high concentrations. In marked contrast, EGTA completely blocked the Ca2+ response stimulated by C3a in neutrophils labeled with either Indo‐1/AM or Fluo‐3. Preincubation of neutrophils with pertussis toxin inhibited both C3a‐ and C5a‐stimulated Ca2+ transients, indicating the involvement of guanine‐nucleotide‐binding proteins (G proteins) in these processes. In order to examine whether the C3a receptor is coupled to G proteins, binding of guanosine 5′‐O‐(3‐[35S]thiotriphosphate) ([35S]GTP[S]) to purified neutrophil plasma membranes was studied. Both C3a and C5a stimulated high‐affinity binding of [35S]GTP[S] up to 1.5‐fold and 3‐fold, respectively. These data suggest that the two anaphylatoxins activate pertussis‐toxin‐sensitive G proteins, which then trigger different signal transduction pathways. C3a specifically stimulated Ca2+ influx from the extracellular medium, whereas C5a additionally activated the PtdInsP2 3‐kinase and stimulated Ca2+ mobilization from intracellular stores.

No abstract

The binary botulinum C2 toxin ADP-ribosylated the actin of human neutrophils. Treatment of human neutrophils with botulinum C2 toxin for 45 min increased FMLP-stimulated superoxide anion (O°) production 1.5-5-fold, whereas only a minor fraction of the cellular actin pool (-20%) was ADP-ribosylated. Effects of botulinum C2 toxin depended on toxin concentrations, presence of both components of the toxin, and incubation time. Cytochalasin B similarly enhanced O2 production. The effects of botulinum C2 toxin and cytochalasin B were additive at submaximally, but not maximally effective concentrations and incubation time of either toxin. Botulinum C2 toxin also enhanced stimulation of O2 production by Con A and platelet-activating factor, but not by phorbol 12-myristate 13-acetate (PMA). Botulinum C2 toxin increased FMLP-induced release of N-acetyl-glucosaminidase by 100-250%; release of vitamin B12-binding protein induced by FMLP and PMA was enhanced by -150 and 50%, respectively. Botulinum C2 toxin blocked both random migration of neutrophils and migration induced by FMLP, complement C5a, leukotriene B4, and a novel monocyte-derived chemotactic agent. The data suggest that botulinum C2 toxin-catalyzed ADP-ribosylation of a minor actin pool has a pronounced effect on the activation of human neutrophils by various stimulants.

A non-detergent urea emulsion cleanser and a detergent cleanser with added moisturizers were compared for their effects on stratum corneum moisture, surface lipids and transepidermal water loss (TEWL) of atopic skin. Following a single wash with either cleanser, low corneometry and sebumetry values increased and elevated TEWL values decreased. Over the course of more than 6 h, all induced changes gradually returned to their starting points. In all instances, the changes induced by the urea emulsion lasted significantly longer than those caused by the detergent cleanser. The sebumetry increase after a wash with the lipid-free detergent cleanser indicated that this method recognized not only true lipids but also the lipid-derived and skin lipid-depleting detergents. The transient TEWL normalization with either cleanser could not be attributed to a passing barrier restoration nor to an occlusion. It is speculated that the TEWL changes were related to stratum corneum water binding capacity.

Interleukin-8 (IL-8) and GRO alpha are leukocyte-attracting peptides of the chemokine family. To study the priming potential of these chemokines, we measured superoxide anion production and up-regulation of N-formyl peptide receptors in human neutrophils. IL-8 and GRO alpha themselves did not stimulate production of significant amounts of superoxide anions but potentiated N-formyl peptide-induced superoxide anion production in a concentration-dependent manner. Binding measurements by flow cytometry at 37 degrees C with fluorescein-labeled N-formyl peptide revealed enhanced total N-formyl peptide binding after pretreatment of neutrophils with IL-8 and GRO alpha. Binding measurements performed at 4 degrees C indicated that the chemokines stimulated the up-regulation of N-formyl peptide receptors at the cell surface but did not alter their affinity for the ligand. This study indicates that IL-8 and GRO alpha, in addition to their known chemotactic activity, prime neutrophils for superoxide anion production, presumably by up-regulating the number of receptors for strong superoxide-anion-triggering stimuli.

No abstract

The sulfonated shale oil extract, Ichthyol, was studied for its effect on the migration of human neutrophilic granulocytes by the Boyden chamber technique. When presented to the cells in a concentration gradient, Ichthyol induced a directed migration. There was little or no chemokinetic effect of Ichthyol when added to the cell compartment of the Boyden chamber. The chemotactic migration towards the tripeptides, formyl-methionyl-leucyl-phenylalanine and formyl-norleucyl-leucyl-phenylalanine, towards the arachidonic acid-derived eosinophil chemotactic factor released from neutrophils by the ionophore A 23187, and towards complement-derived chemotactic activity of normal human serum was inhibited or abrogated by Ichthyol. The Ichthyol effect on f-MLP chemotaxis could be partly overcome by excessive f-MLP concentrations. It was reversible when Ichthyol-incubated cells were washed and resuspended in regular buffer. It is suggested that various substances contained in Ichthyol interacted with either the chemotactic factors or the cell membrane or both and thus blocked cell stimulation. The results could help to explain the cell accumulation and abscess formation observed with Ichthyol in inflammatory skin lesions and the anti-inflammatory properties of the drug.