Kenneth J. Balazovich

Human neutrophil annexin I promotes granule aggregation and modulates Ca(2+)-dependent membrane fusion.

The mechanism and cofactor requirements of exocytotic membrane fusion in neutrophils are unknown. Cytosolic proteins have been implicated in membrane fusion events. We assessed neutrophil cytosol for the presence of fusogenic proteins using a liposome fusion assay (lipid mixing). A fusogenic 36-kD protein containing amino acid sequence homology with human annexin I was purified from the cytosol of human neutrophils. This protein also shared functional characteristics with annexin I: it associated with and promoted lipid mixing of liposomes in a Ca(2+)-dependent manner at micromolar Ca2+ concentrations. The 36-kD protein required diacylglycerol to promote true fusion (contents mixing) at the same Ca2+ concentrations used for lipid mixing. The 36-kD protein exhibited a biphasic dose-response curve, by both promoting and inhibiting Ca(2+)-dependent lipid-mixing between liposomes and a plasma membrane fraction. The 36-kD protein also promoted Ca(2+)-dependent increases in aggregation of a specific granule fraction, as measured by a turbidity increase. Antiannexin I antibodies depleted the 36-kD protein from the cytosol by greater than 70% and diminished its ability to promote lipid mixing. Antiannexin I antibodies also decreased by greater than 75% the ability of neutrophil cytosol to promote Ca(2+)-dependent aggregation of the specific granules. These data suggest that annexin I may be involved in aggregation and fusion events in neutrophils.

Clinical and biologic effects of granulocyte colony stimulating factor in the treatment of myelokathexis

Successful treatment of a patient with myelokathexis, a rare form of chronic neutropenia associated with recurrent infections, is described. Rapid mobilization of bone marrow neutrophils and improved myeloid morphologic features were observed after treatment with human granulocyte colony stimulating factor. Transient thrombocytopenia and bone pain were observed during treatment. Although neutrophil chemotaxis, superoxide production, and FcRIII surface expression were reduced, the patient improved clinically after restoration of a normal neutrophil count.

Changes in protein kinase C activity are associated with the differentiation of Friend erythroleukemia cells

We investigated the activity and cellular distribution of protein kinase C during the dimethylsulfoxide (DMSO) and hypoxanthine-induced differentiation of Friend murine erythroleukemia cells. Most of the cellular protein kinase C activity was found in the soluble fraction of unstimulated Friend cells. Within 15 min of the addition of DMSO or hypoxanthine, protein kinase C underwent a dramatic and prolonged reversal of this distribution which was accompanied by a gradual decline in total cellular protein kinase C activity over the ensuing 5 days. The loss of total activity was found to be dose dependent although maximal translocation from soluble to insoluble components occurred at even lower concentrations of the inducers tested. Two clones of Friend cells, selected for their failure to differentiate in response to DMSO, showed alterations in protein kinase C activity and/or distribution following DMSO addition when compared to wild-type Friend cells. These data show that different inducers of Friend cell differentiation have similar effects on cellular protein kinase C, that the protein kinase C changes accompanying this process are immediate but prolonged, and that changes in protein kinase C activity and distribution are associated with Friend cell differentiation.

Transforming growth factor-β1 stimulates degranulation and oxidant release by adherent human neutrophils

The signal transduction pathways that are activated by cytokines and growth factors binding to their receptors on human neutrophils (PMN) are poorly understood. When PMN in suspension encounter many of these agonists they are not activated, but rather are primed for subsequent activation. We and others reported that when PMN are plated onto fibrinogen and stimulated with cytokines or with the chemotactic peptide N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP) they respond by releasing hydrogen peroxide (H2O2) and the specific granule component lactoferrin. Transforming growth factor‐β1 (TGF‐β1) is released by many cells including PMN. It has been reported that TGF‐β1 stimulates chemotaxis but not exocytosis or superoxide production by cells in suspension. We hypothesized that TGF‐β1 would activate PMN to release H2O2 when they were adherent to fibrinogen, a response mediated by β2 integrin receptors. In this study, we determined whether TGF‐β1 stimulated H2O2 and lactoferrin release by PMN adherent to fibrinogen. TGF‐β1 stimulated H2O2 and lactoferrin release from adherent PMN in a concentration‐dependent manner, with effects seen in the range of 0.1 to 100 pg/mL. Both H2O2 and lactoferrin release were detected by 60 min and continued for at least 180 min. Adhesion and spreading of PMN paralleled H2O2 and lactoferrin release. Ethanol (200 mM) blocked both H2O2 and lactoferrin release, suggesting the involvement of the phospholipase D pathway. In PMN labeled with lyso[3H]phoephatidylcholine, we observed that TGF‐β1 treatment caused an increase in [3H]phoephatidate. Propranolol (150 μM), an inhibitor of phosphatidate phosphohydrolase, blocked both H2O2 and lactoferrin release, suggesting that the conversion of phosphatidic acid to diradylglycerol is an important step in PMN activation by TGF‐β1. Overall, these results are similar to those reported for fMLP activation of adherent PMN and suggest that a common pathway is involved in both chemoattractant and cytokine activation. J. Leukoc. Biol. 60: 772–777; 1996.

Calcium-dependent fusion of the plasma membrane fraction from human neutrophils with liposomes

A cell-free assay monitoring lipid mixing was used to investigate the role of Ca2+ in neutrophil membrane-liposome fusion. Micromolar concentrations of Ca2+ were found to directly stimulate fusion of inside-out neutrophil plasma membrane enriched fractions (from neutrophils subjected to nitrogen cavitation) with liposomes (phosphatidylethanolamine:phosphatidic acid, 4:1 molar ratio). In contrast, right-side-out plasma membranes and granule membranes did not fuse with liposomes in the presence of Ca2+. Similar results were obtained with two different lipid mixing assays. Fusion of the neutrophil plasma membrane-enriched fraction with liposomes was dependent upon the concentration of Ca2+, with threshold and 50% maximal rate of fusion occurring at 2 microM and 50 microM, respectively. Furthermore, the fusion was highly specific for Ca2+; other divalent cations such as Ba2+, Mg2+ and Sr2+ promoted fusion only at millimolar concentrations. Red blood cell (RBC) membranes were used in control studies. Ca2(+)-dependent fusion did not occur between right-side-out or inside-out RBC-vesicles and liposomes. However, if the RBC-vesicles were exposed to conditions which depleted spectrin (i.e., low salt), then Ca2(+)-dependent fusion was detected. Other quantitative differences between neutrophil and RBC membranes were found; fusion of liposomes with RBC membranes was most readily achieved with La3+ while neutrophil membrane-liposome fusion was most readily obtained with Ca2+. Furthermore, GTP gamma S was found to enhance Ca2(+)-dependent fusion between liposomes and neutrophil plasma membranes, but not RBC membranes. These studies show that plasma membranes (enriched fractions) from neutrophils are readily capable of fusing with artificial lipid membranes in the presence of micromolar concentrations of Ca2+.

Human neutrophil protein kinase C: calcium-induced changes in the solubility of the enzyme do not always correlate with enzymatic activity

We hypothesized that calcium and 1,2-diacylglycerols stimulated human neutrophil (PMN) protein kinase C (EC 2.7.1.37) in a two-step mechanism. The proposed mechanism entails (1) increased insoluble protein kinase C activity and (2) endogenous protein phosphorylation, events which have not been biochemically dissociated. PMN which were treated with 100 nM ionomycin shifted protein kinase C activity from being mostly soluble to insoluble. Concentrations of ionomycin greater than 300 nM stimulated a doubling of total cellular (soluble + insoluble) protein kinase activity and stimulated increased endogenous phosphorylation of PMN proteins. Intracellular calcium (measured with fura-2) increased from 65 nM (basal) to 680 nM using 500 nM ionomycin; calcium increases were dose-dependent. The anti-inflammatory agents acetylsalicylic acid and sodium salicylate (but not ibuprophen, indomethacin or acetaminophen) inhibited ionomycin-induced protein kinase C activation and protein phosphorylation in a dose-dependent manner by inhibiting the production of diacylglycerols. 1-Oleoyl-2-acetylglycerol reversed the inhibitory effect of salicylates. In contrast to the effect of acetylsalicylates on protein kinase C functional activity the distribution of phorbol receptors was unaffected in acetylsalicylate-treated, ionomycin-stimulated PMN using a phorbol-binding assay. Our results show that ionomycin increased intracellular diacylglycerol levels 3.5-fold over those present in control PMN, while acetylsalicylate decreased diacylglycerol production in ionomycin-stimulated PMN below baseline values. These results support the hypothesis that increased intracellular calcium activated protein kinase C leading to protein phosphorylation in two distinct dissociable events: (1) increased intracellular calcium; and (2) increased 1,2-diacylglycerol levels.

'Depletion' of ATP by 2-Deoxyglucose: Secretion by Electroporated Human Neutrophils Is Not Restored by Readdition of ATP

Studies of intracellular signal transduction are facilitated by the use of permeabilized cell systems, which permit the ready manipulation of the cytosol. These model systems have helped to define the roles that small solutes, particularly Ca2+ and nucleotides, play in stimulus-response coupling. In circumstances where the full depletion of intracellular ATP contents is required, some investigators have resorted to prior treatment with metabolic toxins, with the expectation that the role of ATP in signal transduction could then be more unambiguously studied. However, in the work reported here, we found that treatment with 2-deoxyglucose (2-DOG) irreversibly altered the cells: when poisoned human neutrophils were then permeabilized, the cells failed to degranulate well in response to Ca2+, and their sensitivity to Ca2+ could not be recovered by the readdition of ATP. Inhibition of secretion by 2-DOG was most pronounced when low concentrations of Ca2+ were used as the stimulus. Preincubation of the cells with only 1 mM 2-DOG for 10 min at 37 degrees C (prior to washing and permeabilizing the cells) was sufficient for maximal inhibition. Even without preincubation, high concentrations of 2-DOG directly inhibited secretion. The refractory nature of poisoned cells was not restored by the presence of Mg2+ and/or ATP. The protein kinase C agonist phorbol myristate acetate also did not restore sensitivity of secretion to Ca2+. Addition of ATP and/or GTP to the permeabilization medium (to maximize penetration of the nucleotides) failed to restore sensitivity; tracer studies demonstrated that these conditions were adequate for repletion of the nucleotide pool. These data indicate that human neutrophils poisoned with 2-DOG were irreversibly altered, such that restoration of the putative deficiency (ATP) was without effect. Experiments in which such preincubation measures are employed should be viewed with caution.