The first seconds of neutrophil activation: phosphoinositides, protein kinase C, and calcium movements.

Activation of the neutrophil by interaction of a ligand such as f-Met-Leu-Phe with its specific receptor elicits a prompt breakdown of PIP2 and the generation of PA via diacylglycerol. There is a rapid elevation of cytosolic calcium and activation of protein kinase C. Calcium and protein kinase C act synergistically to elicit the physiological responses. Although PIP2 breakdown and PA generation are prompt responses of neutrophils to receptor occupancy by chemoattractants, these steps can be bypassed by stimuli which directly activate protein kinase C or increase cytosolic calcium. Elevation of cytosolic Ca and activation of protein kinase C did not elicit breakdown of PIP2, indicating that phosphoinositide remodeling is not caused by activation of protein kinase C or by elevation of cytosolic calcium, nor is such a breakdown or the generation of phosphatidic acid required for the subsequent responses. The evidence indicates that PIP2 breakdown is an early event in stimulus-response coupling and is correlated with receptor-initiated generation of the signal.

Neutrophil stimulation: receptor, membrane, and metabolic events.

In the neutrophil, binding of ligands to their appropriate receptors initiates a sequence of events culminating in the physiological responses of aggregation, degranulation, and superoxide anion generation. Calcium has been proposed as a second messenger in the activation sequence of the neutrophil. Increments in cytosolic free calcium are one of the first measurable events subsequent to receptor occupancy, followed by enhanced plasmalemmal permeability to calcium, a process that may serve to enhance the physiological responses. In contrast to calcium, cyclic AMP (cAMP) does not act as a signal in the activation sequence of the neutrophil. Increments in cAMP that are triggered by complete secretagogues may act as an inhibitory feedback mechanism. Protein kinases, both cAMP- and calcium/phospholipid-sensitive enzymes, may play a role in the activation sequence. Phosphorylation of proteins occurs during neutrophil activation. A role for phosphatidylinositol/phosphatidic acid turnover in calcium gating has been proposed. In addition, modulation of phospholipids could serve to activate a protein kinase C. Finally, phospholipids can serve as a source for arachidonic acid, which is metabolized by a 5-lipoxygenase pathway in the neutrophil. Products of this pathway, such as leukotriene B4, may serve to mediate or modulate the activation sequence.

Stimulus response coupling in the human neutrophil. Differential requirements for receptor occupancy in neutrophil responses to a chemoattractant.

Activated neutrophils aggregate, generate superoxide (O-2), and degranulate. The role of Ca as "second messenger" in neutrophil activation was examined using as agonist the chemotactic peptide fMet-Leu-Phe and its antagonist t-butoxycarbonyl-Phe-Leu-Phe-Leu-Phe to systematically vary the time of receptor occupancy. Release of enzymes from specific and azurophil granules showed a finite requirement for receptor occupancy; the cells were committed to full degranulation after 10 s of receptor-agonist interaction. In contrast, continuous occupation of the receptor by agonist was required to initiate and maintain O-2 generation and aggregation. Cytosolic Ca ( Quin2 fluorescence) increased immediately in response to fMet-Leu-Phe, requiring less than 2 s of agonist-receptor interaction to initiate an optimal response. Mobilization of membrane-associated Ca (chlorotetracycline fluorescence) also demonstrated a finite time requirement; the cells were fully committed after 10 s of agonist-receptor interaction. Increased Ca permeability (45Ca uptake) was fully launched after 15 s of agonist-receptor interaction. The data indicate that Ca movements ( quin2 , chlorotetracycline fluorescence, 45Ca uptake) are both necessary and sufficient to account for degranulation by neutrophils activated by fMet-Leu-Phe. However, neutrophil aggregation and the generation and release of O-2 in response to the same stimulus require a further unknown factor(s) associated with receptor occupancy to maintain these responses.

Stimulus response coupling in the human neutrophil. II. Temporal analysis of changes in cytosolic calcium and calcium efflux.

The role of changes in cytosolic free calcium (Ca) in the activation sequence of the human neutrophil has been monitored by means of the fluorescent probe Quin2. "Complete" secretagogues such as the chemotactic peptide f-Met-Leu-Phe and aggregated IgG, as well as the "incomplete" secretagogue concanavalin A, elicited prompt rises in cytosolic Ca. The rise in cytosolic Ca was in all cases one of the earliest measurable events, consistent with the hypothesis that increments in cytosolic free Ca serve as a signal to activate subsequent physiological responses. The source of cytosolic Ca is principally intracellular, since removal of extracellular Ca had little effect on the rise in cytosolic Ca. Although increments in cytosolic Ca may be essential for neutrophil activation, they are evidently not sufficient; chemotactic levels of f-Met-Leu-Phe elicited optimal increments in cytosolic Ca without triggering degranulation, O-2 generation, and aggregation. Some other factor, associated with high levels of receptor occupancy, must be required for secretion. The tumor promoter phorbol myristate acetate was an exceptional stimulus, since it elicited degranulation, aggregation, and O-2 generation without triggering a rise in cytosolic Ca. Finally, an efflux of Ca is initiated which serves to maintain low intracellular levels of Ca.

Granulocytes without degranulation: neutrophil function in granule-depleted cytoplasts.

Neutrophils respond to a variety of stimuli by generating superoxide anion, degranulating, and aggregating. Because it has been suggested that fusion of granules with the plasmalemma (degranulation) is necessary for aggregation and superoxide anion generation, we have tested whether these responses can be demonstrated in "neutrophilic cytoplasts" (granule-free vesicles of cytoplasm enclosed by plasmalemma). When examined by electron microscopy, cytoplasts were found to be approximately 4 microns in diameter and essentially granule free. Cytoplasts exposed to fMet-Leu-Phe (0.1 microM) generated superoxide anion after a lag of 16 sec but released no detectable beta-glucuronidase, lysozyme, or elastase. Aggregation of cytoplasts, as measured by changes in light transmission, was also activated by fMet-Leu-Phe; no lag period was observed. Electron microscopy of the aggregates demonstrated clusters of cytoplasts with a scalloped appearance. Superoxide anion generation and aggregation of cytoplasts were also activated by phorbol 12-myristate 13-acetate, concanavalin A, and leukotriene B4. Exposure of cytoplasts to the dye 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)] led to dye uptake and enhancement of fluorescence, implying that the vesicles were sealed and maintained a membrane potential across the plasmalemma. Exposure of DiOC6(3)-loaded cytoplasts to fMet-Leu-Phe and PMA caused a rapid loss of dye fluorescence that was not inhibited by CN-, compatible with their lack of mitochondria. Exposure of dye-loaded cytoplasts to concanavalin A or leukotriene B4 caused an increase in fluorescence--i.e., a hyperpolarization. These results demonstrate that degranulation is not a prerequisite for aggregation or superoxide anion generation. The retention of ionic gradients and changes in membrane potential, as measured by DiOC6(3) fluorescence changes, suggest a fundamental role for ionic movements in activating superoxide anion generation and aggregation.

Granulocytes without degranulation: neutrophil function in granule-depleted neutroplasts.

Neutroplasts, which are vesicles consisting of cytoplasm enclosed by plasmalemma, have been prepared and found to be incapable of degranulation in response to f-Met-Leu-Phe. However, neutroplasts generate superoxide anion in response to f-Met-Leu-Phe and PMA, and therefore, degranulation is not essential for superoxide anion generation. In addition, neutroplasts aggregate and show shape changes in response to f-Met-Leu-Phe and PMA, and thus degranulation is not essential for aggregation. Neutroplasts take up the carbocyanine dye DiOC6(3), thereby providing evidence for ionic gradients. Dye-loaded neutroplasts show fluorescence changes in response to a variety of stimuli. This response is not CN--inhibitable; therefore, activation of neutroplasts is associated with changes in membrane potential at the plasmalemma, suggesting a role for ion fluxes in the activation sequence for aggregation and superoxide anion generation.

Murine complement component 3: genetic variation and linkage to H-2.

Two electrophoretic variants of murine complement component 3 (C3) were detected by using high-voltage electrophoresis of fresh mouse serum in agarose gels. Most of the inbred strains tested were homozygous for the S allele (for the slow-migrating variant); only four out of 46 strains had the alternative F allele (fast variant). Pen-bred Swiss-Webster animals belonged to one of three phenotypes--S, F, or SF--and the genes responsible for this variation segregated in a strictly Mendelian manner. In three such crosses, with 5* offspring, C3 segregated with H-2 in 46 instances, corresponding to a recombination frequency of approximately equal to 0.12.