Kurt F. Seetoo

Neutrophil degranulation and phospholipase D activation are enhanced if the Na+/H+ antiport is blocked.

Neutrophils phagocytize high‐valency immune complexes (HIC) by an Fc receptor‐mediated mechanism. Engaging Fc receptors in this manner induces PMN to generate superoxide and release the contents of both their specific and azurophilic granules. Signaling events that precede and accompany PMN secretion include activation of phospholipase D (PLD), as well as changes in cytoplasmic [Ca2+] (δ[Ca2+]in) and pH (δpHin). Although the role of PLD and δ[Ca2+]in in mediating Fc receptor‐mediated PMN secretion has been studied, whether pHin plays a regulatory role has not yet been defined. HIC‐stimulated PMN undergo an intracellular acidification followed by a prolonged Na+/H+ antiport‐mediated alkalinization. To investigate the role of the pH transient in controlling degranulation, the Na+/H+ antiport was inhibited either with 100 μM dimethylamiloride (DMA) or by substituting N‐methyl‐glucamine for extracellular sodium. Blocking the antiport with DMA led to hyper‐acidified PMN, which exhibited an increase in degranulation, but did not affect generation of superoxide. DMA did not alter the ability of neutrophils to phagocytose and oxidize dichlorodihydrofluoresceinated HIC, suggesting the increase in degranulation was not the result of failed phagocytosis. Investigation into whether the observed increase in degranulation when the antiport was blocked was mediated by PLD or δ[Ca2+]in revealed that blocking the antiport increased HIC‐induced PLD activity but had no effect on HIC‐induced δ[Ca2+]in. Blocking the Na+/H+ antiport by ion substitution caused similar effects on PMN signaling and secretion as was seen with DMA. These results indicate that Na+/H+ antiport activity is not necessary for degranulation or superoxide release in HIC‐stimulated PMN and that hyperacidification of the cytoplasm can modulate degranulation. Therefore, pHin, via its effect on PLD, may be a control point of degranulation and may represent one way that neutrophils achieve differential control of their antibacterial products. J. Leukoc. Biol. 64: 98–103; 1998.

A cytosolic calcium transient is not necessary for degranulation or oxidative burst in immune complex‐stimulated neutrophils

Receptor‐mediated activation of neutrophils (PMN) initiates possibly interdependent events, including a rapid transient increase in [Ca2+]i, implicated as a second messenger. To investigate whether this transient is required for eventual degranulation, PMN were incubated with an intracellular Ca2+ chelator (BAPTA), then exposed to chemotactic peptide [N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP)] with or without cytochalasin B (CB) or to high‐valency immune complexes (HIC); δ[Ca2+]i, δpHi, oxidative burst, and elastase release were then evaluated (plus or minus EGTA 15 s before stimulation) after 2 and 15 min incubation in 0.9 mM Ca2+. With either fMLP plus CB or HIC stimulation, BAPTA‐treated cells were unable to achieve a Ca2+ transient with a 2‐min incubation, whereas a 15‐min incubation allowed the BAPTA‐treated cells to recover a portion of the δ[Ca2+]i. Even though BAPTA‐treated cells were unable to mount a δ[Ca2+]i at 2 min, HIC‐stimulated BAPTA‐treated cells were able to elicit an oxidative burst (33% of control) and degranulation (67% of control). Therefore, we conclude that δ[Ca2+]i modulates but is not required for oxidative burst or degranulation. J. Leukoc. Biol. 62: 329–340; 1997.

Neutrophil functional responses depend on immune complex valency

Ligand‐induced cross‐linking of Fcγ receptors (FcγR) on neutrophils plays a significant role in their stimulation, shown here by contrasting the responses induced by low valency immune complexes (LICs) and high valency immune complexes (HICs) and by cross‐linking LICs in situ (L/Ab) after their addition to the cells. Multiparameter flow cytometry was used to measure immune complex (IC)‐elicited changes in cytoplasmic Ca2+ concentration and initiation of the oxidative burst simultaneously in the same cell and to correlate these with FcγR occupancy. We have previously shown that subpopulations of neutrophils respond maximally to subsaturating concentrations of HIC; saturating dosages stimulate the entire population. This discrepancy was not due to differences in receptor occupancy. The magnitude of the transient Ca2+ increase was independent of the dose of HIC but depended on the dose when an LIC was used. As shown here, L/Ab cross‐linking elicited Ca2+ responses similar to those observed in HIC‐stimulated cells. In contrast, LIC elicited only minimal intracellular ΔpH and no oxidative burst or membrane potential changes at all unless FcγR was cross‐linked, accomplished by HIC or by L/Ab. However, azurophilic degranulation, as determined by elastase release, was not observed in cells stimulated by the in situ cross‐linking method, whereas the HIC preparation triggered azurophilic degranulation. Thus, some FcγR‐mediated neutrophil effector functions such as azurophilic degranulation and oxidative burst initiation have an absolute requirement for FcγR cross‐linking, whereas signaling functions such as changes in membrane potential, intracellular pH, and intracellular Ca2+ concentration can occur, albeit more slowly and to a lesser extent, if single FcγR are occupied.

Adherence-dependent calcium signaling in monocytes: induction of a CD14-high phenotype, stimulus-responsive subpopulation

Isolation of monocytes by plastic adherence alters cell morphology and function. In order to study the effects of cell isolation procedures and subsequent culture on monocyte function, we examined cytoplasmic calcium concentration changes (delta[Ca2+]in) in human monocytes isolated by either negative (magnetic bead) or positive (plastic adherence) selection then stimulated with formyl-Met-Leu-Phe (fMLP), either immediately after isolation, or after 48 h in culture. We have previously shown that fresh adherence-isolated monocytes respond to fMLP with small delta[Ca2+]in and oxidative burst responses, exhibiting larger responses following 48 h of incubation. We now demonstrate that fresh monocytes, prevented from adhering by negative selection, exhibit an even smaller fMLP-induced delta[Ca2+]in, which does not increase during 48 h in culture if cells are kept nonadherent, in Teflon. Calcium responses of adherent, fresh monocytes do not increase if cells are subsequently placed into suspension and maintained nonadherent, but increase if nonadherent cells are permitted to adhere to plastic. Furthermore, augmented fMLP-[Ca2+]in and oxidative burst responses in plastic-adherent cells are restricted to a CD14-high phenotype subpopulation. The CD14-high phenotype also describes a subpopulation of cells that responds to CD4 crosslinking with a rapid delta[Ca2+]in. Induction of a subpopulation of CD14-high expressing cells by adherence may explain in part maturation-induced response changes observed in macrophage but not in monocyte in vitro systems.

Platelets from patients with Alzheimer's disease or other dementias exhibit disease-specific and apolipoprotein E correlatable defects

Platelets carry over 95% of the circulating Alzheimers β-amyloid precursor protein (AβPP), and release soluble and hydrophobic proteolytic fragments of AβPP upon activation. These cells may be the source of cerebrovascular amyloid peptides, a part of Alzheimers disease (AD) pathology. Our previous studies showed that platelets from patients with advanced AD exhibit both signal transduction (hyperacidification) and AβPP processing defects. Here, we show further that a similar hyperacidification also exists in patients with Picks disease (a dementia with AD-like symptoms but a different amyloid pathology) or Down syndrome (trisomy and hence overproduction of AβPP), while the AβPP processing defect and consequent AβPP retention on the membrane is absent and is thus likely to be AD-specific. The hyperacidiftcation defect correlates with all three dementias and with the presence of apolipoprotein E4 which has been implicated as a risk factorial-AD.

Sequential Chemotactic and Phagocytic Activation of Human Polymorphonuclear Neutrophils

ABSTRACT Human polymorphonuclear neutrophils (PMN) chemotax to a foreign entity. When the chemoattractants’ origins are reached, specific receptors bind to the invaders surface, initiating phagocytosis, phagosome formation, and fusion with granule membranes, generating the bactericidal oxidative burst, and releasing lytic enzymes, specific peptides, and proteins. We explored the initial signaling involved in these functions by observing naïve, unprimed PMN in suspension using fluorescent indicators of cytoplasmic signals (Δ[Ca2+]i and ΔpHi) and of bactericidal entities (oxidative species and elastase) exposed to N-formyl-methionyl-leucyl-phenylalanine (fMLP) and/or multivalent immune complexes (IC). fMLP and IC each initiate a rapid transient rise in [Ca2+]i, mostly from intracellular stores, simultaneously with a drop in pHi; these are followed by a drop in [Ca2+]i and a rise in pHi, with the latter being due to a Na+/H+ antiport. The impact of a second stimulation depends on the order in which stimuli are applied, on their dose, and on their nature. Provided that [Ca2+]i is restored, 10−7 M fMLP, previously shown to elicit maximal Δ[Ca2+]i but no bactericidal functions, did not prevent the cells’ responses with Δ[Ca2+]i to a subsequent high dose of fMLP or IC; conversely, cells first exposed to 120 μg/ml IC, previously shown to elicit maximal Δ[Ca2+]i and bactericidal functions, exhibited no subsequent Δ[Ca2+]i or ΔpHi to either stimulus. While exposure to 10−7 M fMLP, which saturates the PMN high-affinity receptor, did not elicit bactericidal release from these naïve unprimed PMN in suspension, 10−5 M fMLP did, presumably via the low-affinity receptor, using a different Ca2+ source.