John T. Curnutte

Staurosporine inhibits the soluble and membrane-bound protein tyrosine kinases of human neutrophils.

Superoxide production by neutrophils triggered with a chemotactic peptide or a phorbol ester is inhibited by the protein kinase antagonists staurosporine or 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7). We evaluated the effects of these antagonists on the protein tyrosine kinases and protein kinase C activities of neutrophils. Staurosporine completely inhibited all of these enzymes, whereas 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine was only substantially effective against protein kinase C. Thus, if a protein tyrosine kinase is involved in superoxide production, it is likely to function with a second kinase sensitive to 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine.

Failure to Detect Superoxide in Human Neutrophils Stimulated With Latex Particles (21)

Summary: Human neutrophils stimulated with either latex particles or opsonized zymosan exhibited equivalent rates of net oxygen consumption as well as hydrogen peroxide release. The quantity of superoxide (O2-) detected in latex-stimulated neutrophils was less than 2% of that seen with opsonized zymosan stimulation, and only several-fold greater than that of resting cells. The failure to detect O2- in the latex-stimulated neutrophils was due neither to latex acting as a O2- scavenger nor to its interference with the O2- - forming system of the neutrophil. An intracellular site of O2- generation could not be demonstrated. NADPH oxidase activity in cells exposed to latex particles was only 10% of that seen in cells comparably activated with opsonized zymosan. Latex particles have the unusual property of stimulating the respiratory burst of the human neutrophil without the extracellular release of O2-. The potential physiologic importance of this finding is discussed.

Activation of human neutrophil NADPH-oxidase in vitro by the catalytic fragment of protein kinase-C

Phorbol ester treatment of intact neutrophils both stimulates protein kinase C (PK-C) and causes the rapid proteolytic conversion to a cytosolic, co-factor independent fragment, protein kinase M (PK-M). In intact neutrophils, phorbol ester treatment activates the NADPH-oxidase, the enzyme responsible for the oxidative burst. Addition of purified PK-M to resting neutrophil light density membranes activated the NADPH-oxidase in the presence of PS, ATP and Mg2+. A 3.5-fold greater stimulation of oxidase (ca. 25 nmoles O2-/min/mg membrane protein) was obtained with comparable PK-M concentrations to that observed with the reconstituted PK-C system, and approximately 1/3 that obtained with arachidonic acid (AA) or SDS. In contrast to the reconstituted system using PK-C, PMA and Ca++ were neither required nor affected activity. The effect of PS was unexpected, since PK-M does not require phospholipids for enzymatic activity, and likely represents the action of PS on the oxidase itself or on another component in the plasma membrane fraction. Our studies demonstrate for the first time that purified PK-M permits reconstitution of a physiologic phorbol ester response.

Evidence that de novo protein synthesis participates in a time-dependent augmentation of the chemotactic peptide-induced respiratory burst in neutrophils: Effects of recombinant human colony stimulating factors and dihydrocytochalasin B☆

We examined the effects of the recombinant human colony stimulating factors GM-CSF and G-CSF, cycloheximide (a protein synthesis inhibitor) and dihydrocytochalasin B (a microfilament disrupting agent) upon FMLP (N-formyl-methionyl-leucylphenylalanine)-stimulated O2- production by neutrophils. We confirmed a time dependent augmentation of O2- production following preincubation of neutrophils either alone or with colony stimulating factors. Furthermore, we found that GM-CSF, but not G-CSF, increased O2- production at some concentrations of the stimulus. Preincubation of neutrophils with cycloheximide in the absence of CSF caused a marked fall in O2- -production that was first evident at 2 hours. The fall in O2- -forming capacity caused by cycloheximide was much less pronounced if dihydrocytochalasin B was also included in the preincubation buffer. These findings suggest a previously unrecognized role for de novo protein synthesis in maintaining the ability of neutrophils to manufacture O2-, and support earlier studies indicating that the cycling of FMLP receptors between the cell membrane and an intracellular compartment is important in determining the magnitude of the respiratory burst in FMLP-stimulated neutrophils.

Structure and Regulation of NADPH Oxidase of Phagocytic Leukocytes

When phagocytic cells of the immune system (neutrophils, monocytes, macrophages, and eosinophils) come into contact with opsonized microorganisms or a wide range of soluble stimuli, their consumption of oxygen increases dramatically (reviewed in refs. 1–3).