Characterization of cyclic-nucleotide phosphodiesterase activities in resting and N-formylmethionylleucylphenylalanine-stimulated human neutrophils.

Cyclic nucleotide phosphodiesterase activities in human neutrophils were characterized. Neutrophil sonicates exhibited high-affinity and low-affinity cAMP phosphodiesterase activities, with apparent Km values of 1.9 microM and 112 microM, respectively. No cGMP phosphodiesterase activity was detected. Approx. 70% of cAMP phosphodiesterase activity measured at 1 microM cAMP was present in the soluble subcellular fraction, and the remainder was localized in the particulate fraction. Chromatography of the soluble subcellular fraction on DE-52 ion-exchange resin yielded a low-affinity cAMP phosphodiesterase activity and a high-affinity cAMP phosphodiesterase activity. The soluble high-affinity cAMP phosphodiesterase activity exhibited moderate calmodulin sensitivity. After incubation of intact neutrophils with N-formylmethionylleucylphenylalanine (fMet-Leu-Phe), a 25-30% increase in the activity of the high-affinity cAMP phosphodiesterase activity was observed in the sonicate and in the soluble fraction. Maximal increases were achieved after 2 min of incubation and the increases persisted for at least 10 min. The increase in activity was independent of calmodulin and guanine nucleotide regulatory proteins. These results indicate that a soluble high-affinity cAMP phosphodiesterase comprises the majority of phosphodiesterase activity in neutrophils and that increases in this activity may contribute to the regulation of cAMP levels in neutrophils during activation.

The effect of some protease substrates and inhibitors on chemotaxis and protease activity of human polymorphonuclear leukocytes.

Both low- and high-molecular-weight inhibitors of serine proteases were found to inhibit chemotaxis by human polymorphonuclear leukocytes totally at widely varying concentrations. Synthetic low-molecular-weight substrates with trypsin-like or chymotrypsin-like specificity were also shown to be potent inhibitors of chemotaxis. Chemotactic inhibition was reversible except with a titrant for the active site of a serine protease. N-acetyl-L-tyrosine ethyl ester was found to be a suitable substrate for measuring protease activity of polymorphonuclear leukocyte. Concentrations of the various protease inhibitors that caused 100% chemotactic inhibition caused 80%-100% inhibition of protease activity of polymorphonuclear leukocytes.

PMN chemotactic inhibition associated with a cryoglobulin.

A child with recurrent pyogenic infections, eczema, markedly elevated serum concentrations IgA, and totally absent PMN chemotactic acticity is described. The chemotactic defect was characterized as a humoral inhibitor present in serumand plasma, directed toward autologous as well as homologous PMN's. Immunochemical studies revealed the purified inhibitor to be a cryoglobulin composed of an IgM-IgA complex. Incubation of the cryoglobulin complex with PMN's resulted in a quantitative dose-response curve.

Cell-directed inhibition of polymorphonuclear leukocyte chemotaxis in a patient with mucocutaneous candidiasis.

A patient with mucocutaneous candidiasis and impaired polymorphonuclear leukocyte (PMN) chemotaxis is described. The patient's PMN chemotaxis was markedly decreased in the presence of autologous plasma but normal in control plasma. Cell-directed inhibitory activity was found in whole patient plasma as well as the 40% ammonium sulfate precipitate fraction of both the patient's and the control plasma. The inhibitor was heat stable, reversible, nondialyzable, eluted from DEAE cellulose with 0.005 M sodium phosphate buffer, and migrated with IgG on immunoelectrophoresis. The supernate from 40% ammonium sulfate-fractionated patient and control plasma contained a cell-directed enhancer of PMN chemotaxis that antagonized the cell-directed inhibitor activity. It is possible that the patient's chemotactic defect may be caused by imbalance between plasma factors that regulate chemotaxis.