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Astronauts live and work in relatively crowded, confined environments on the Space Shuttle and the International Space Station. They experience a unique set of stressors that contribute to a diminishment of many immune responses. This study investigated the ability of the shuttle crew members' monocytes to respond to gram-negative endotoxin that they could encounter during infections. Blood specimens were collected from 20 crew members and 15 control subjects 10 days before launch, 3 to 4 h after landing, and 15 days after landing and from crew members during their annual medical examination at 6 to 12 months after landing. When challenged with gram-negative endotoxin, the crew member's monocytes collected at all three time points produced lower levels of interleukin-6 (IL-6) and IL-1β and higher levels of IL-1ra and IL-8 compared to those of control subjects. Cytokines were assessed by measuring the number of cells positive for intracellular cytokines. These values returned to normal 6 to 12 months after landing, except for IL-1ra, which was still higher (five- to sixfold) than in controls. This phenomenon was accompanied by an increased expression of Toll-like receptor 4 and decreased expression of CD14 on the crew members' monocytes at all time points. There were also increased levels of the lipopolysaccharide binding protein in the plasma of the crew members 3 to 4 h and 15 days after landing. This study shows that spaceflight-associated factors (in-flight and preflight) modulate the response of monocytes to gram-negative endotoxins.

Human polymorphonuclear neutrophils (PMN) chemotax to a foreign entity. When the chemoattractants’ origins are reached, specific receptors bind to the invader's 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+]iand ΔpHi) and of bactericidal entities (oxidative species and elastase) exposed toN-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+]iand 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+]iis restored, 10−7M fMLP, previously shown to elicit maximal Δ[Ca2+]ibut no bactericidal functions, did not prevent the cells’ responses with Δ[Ca2+]ito a subsequent high dose of fMLP or IC; conversely, cells first exposed to 120 μg/ml IC, previously shown to elicit maximal Δ[Ca2+]iand bactericidal functions, exhibited no subsequent Δ[Ca2+]ior ΔpHito either stimulus. While exposure to 10−7M fMLP, which saturates the PMN high-affinity receptor, did not elicit bactericidal release from these naïve unprimed PMN in suspension, 10−5M fMLP did, presumably via the low-affinity receptor, using a different Ca2+source.

The activation of human phagocytic leukocytes by immune complexes (IC) or opsonized microbes via their Fc and complement receptors has been well-described (for reviews, see refs. [1–3]). The mechanisms involved in this process are complex and depend on the receptors involved. The biochemical events that lead to the destruction of invading organisms in turn display varying degrees of interdependence, but the controlling elements that lead to the ultimate killing of ingested organisms within phagosomes by lysosomal enzymes and reactive oxygen intermediates are still not completely understood. We have addressed these mechanisms by following and correlating the kinetics of responses by individual cells, using multiparameter flow cytometry [3, 4]. Using nonopsonized IC as stimuli, we document here the presence of a novel Ca2+/H+ voltage-independent channel in human neutrophils, which helps to control their cytoplasmic pH.

We have previously demonstrated the antifungal activity of the weak bases chloroquine and quinacrine against Cryptococcus neoformans. Quinacrine, being fluorescent, was seen to be concentrated within a complex vacuolar structure within the cryptococcal cell. Here we determined the pH of this compartment using the pH-sensitive fluorescent dye, 5-(and 6-) carboxy-2',7'-dichlorofluorescein diacetate (carboxy-DCFDA). Carboxy-DCFDA was concentrated within the cryptococcal vacuole, giving a pattern of fluorescence similar to that previously observed with quinacrine. For each experiment, a standard curve of fluorescence ratio against pH was generated using buffers of defined pH containing a mixture of ionophores and inhibitors to equilibrate vacuolar pH with that of the medium. The pH of the cryptococcal vacuole of five strains was calculated to range from 5.3 to 5.9 with a mean of 5.6. This acidic pH is consistent with a model in which weak bases such as chloroquine and quinacrine are accumulated, by ion trapping within the fungal vacuole. Antifungal activity may result from the consequent disruption of pH-dependent processes as well as effects on other as yet undefined fungal targets.

THE recently published Technical Note by Vines et al., ‘‘A Flow-Cytometric Method for Continuous Measurement of Intracellular Ca Concentration,’’ Cytometry Part A 77A:1091 1097, 2010, demonstrated that such measurements are possible in an Accuri C6 flow cytometer. Their contribution demonstrates that rapid measurements can be achieved when stimuli are added to cells (in an open tube) which then flow via a pump mechanism, yielding observations within 5 s, judging from their figures, albeit without thermostating or stirring the sample. Intracellular kinetic measurements of cytoplasmic Ca (and other events) using other flow cytometers (FACS 440, MoFlo, Aria) have been previously reported by a number of laboratories, including ours and extensively reviewed (1 9). Since our own first report in 1986 (10), we have published numerous flow cytometric measurements of cytoplasmic Ca fluxes ([Ca]in) in various blood cells, using a modified FACS 440 (BD) or MoFlo (Cytomation), injecting the compounds into a stirred thermostated flowing cell suspension. These flow cytometers, and the LSRII and FACSAria to which we are now adapting the sample handling system we described, use N2 pressure to move the cell stream, which permits the sample to be stoppered and reduces risk from potential biohazards. Thermostating and stirring assure physiologic conditions and rapid mixing. Injecting into pressure-driven, flowing cells a very short (

Neutropenia is considered a significant risk factor for invasive aspergillosis but is almost always associated with concurrent thrombocytopenia. Studies determined that platelets, like neutrophils, attached to cell walls of the invasive hyphal form of Aspergillus fumigatus . Organisms were damaged as shown by loss of cell wall integrity in scanning laser confocal microscopy and release of defined hyphal surface glycoproteins. Rapid expression appearance of surface antigen CD63 and release of markers of platelet degranulation confirmed activation during attachment to hyphae. Optimal platelet activation required opsonization of hyphae with fresh or heat-inactivated whole plasma. These effects of opsonization with whole plasma could not be duplicated by pooled human serum, immunoglobulin G, or fibrinogen, whether used separately or combined. Thus, platelets in the presence of whole plasma have the potential to play an important role in normal host defenses against invasive aspergillosis.

Proteolytic cleavage of the amyloid precursor protein (A beta PP) results in the generation of the amyloidogenic fragment known as amyloid beta peptide (A beta). Deposition of A beta in the brain parenchyma and cerebrovasculature is a feature of Alzheimer's disease (AD). To date, the process whereby A beta is generated and deposited remains unclear. We have previously established that activated platelets from AD patients retain more A beta PP on their surface than control platelets. We report here that an endothelial cell-derived enzyme can cleave this surface platelet A beta PP. Human blood brain barrier endothelial cells from brains of AD patients were assayed for potential A beta PP-cleaving enzymes using synthetic peptide substrates encompassing the A beta N-terminus cleavage site. A protease activity capable of cleaving A beta PP on the surface of AD platelets was noted. The A beta PP cleavage is partially inhibited by EDTA, by ZincOV, as well as by a specific inhibitor of the Zn metalloprotease E.C.3.4.24.15. Furthermore, the protease is recognized by an antibody directed against it, using immunohistochemistry, Western blot analysis and flow cytometry. The protease is not secreted, but rather resides intracellularly as well as on the surface of the endothelial cells. The data suggest that E.C.3.4.24.15 synthesized by brain endothelial cells may process the platelet-derived A beta PP, yielding fragments which could contribute to cerebrovascular A beta deposits.

Neutrophils phagocytize high-valency immune complexes (HIC) by an Fcγ receptor-mediated mechanism, activating an oxidative burst and initiating degranulation. In contrast, neutrophils exhibit chemotaxis to N-formylated peptides [e.g., N-formylmethionyl-leucyl-phenylalanine (fMLP)] and secrete far fewer oxidants or granule contents than neutrophils activated by HIC. However, if neutrophils are treated with cytochalasin B (CB) or permeabilized with streptolysin O, chemoattractant-induced neutrophil secretion is increased to a level beyond that observed in response to HIC. Because priming neutrophils with CB, or permeabilizing them, also augments activation of phospholipase D (PLD) in response to fMLP, we reasoned that, in intact (i.e., nonpermeabilized) unprimed neutrophils, PLD may participate in a signaling pathway specific to phagocytic stimuli such as HIC and hence may contribute to degranulation control. PLD activity in response to HIC and fMLP correlated closely with stimulus-induced azurophilic degranulation under a wide variety of experimental conditions, including compounds that abrogated or augmented stimulus-induced PLD action. PLD activation preceded, and appeared to be necessary for, azurophilic degranulation. These results suggest that PLD may play a central role in controlling azurophilic degranulation and provide signaling specificity between pathways activated by fMLP and HIC in intact neutrophils.

We previously reported that platelets from advanced sporadic Alzheimer's disease (AD) patients exhibit two defects: first, an aberrant signal transduction presenting as a thrombin-induced hyperacidification, which is more severe for donors with the apolipoprotein E4 allele (apoE4), and second, an AD-specific Amyloid Precursor Protein (APP) processing defect that presents as retention of APP on the activated platelets' surface and in independent of the apo E allele. This retention of membrane APP correlates with decreased release of soluble APP. To determine at what stage in the disease progression these defects appear, we performed signal transduction and secretion studies on moderate AD patients. Thrombin-activated platelets from these patients do not exhibit either hyperacidification or APP retention; their APP processing and secretion are normal by Western blotting, suggesting that the two platelet defects appear in the advanced stages of AD.

The Rho subfamily of small guanosine triphosphate (GTP)-binding proteins, through their role in cytoskeletal organization, is involved in diverse cellular functions, including cell motility and morphologic changes during differentiation. Rac also has a special role in the production of superoxide, a key component in phagocytic antimicrobial function. Guanosine diphosphate (GDP)-dissociation inhibitors (GDIs) belong to one of three classes of proteins that regulate the critical cycling of GTP-binding proteins between the inactive and active states. Two homologous GDIs for the Rho subfamily have been identified. GDID4 is preferentially expressed in hematopoietic cells, while RhoGDI is ubiquitously expressed. Whether different physiologic functions are subserved by the two GDIs is unknown. We have derived embryonal stem (ES) cells with targeted disruption of both alleles of the GDID4 gene and examined hematopoiesis and phagocytic functions of macrophages derived from in vitro ES-cell differentiation. GDID4-/- ES cells develop like wild-type cells into colonies that contain heterogeneous populations of progenitor cells and differentiated erythromyeloid cells. GDID4-/- cells express no GDID4 protein, but have normal levels of RhoGDI. GDID4-/- macrophages phagocytose yeasts and antibody-opsonized erythrocytes as effectively as wild-type macrophages. However, a slight but consistent reduction in their capacity to generate superoxide was observed, which suggests new insight into the cellular role of GDID4. The minimal phenotypic effect of a loss of function of GDID4 also indicates a significant redundancy of function between GDID4 and RhoGDI. Their functional repertoire may be better revealed by a disruption of both genes. The use of hematopoietic cells derived in vitro from genotypically altered ES cells avoids the difficulties inherent in generating knockout animals and is a useful complementary approach for evaluating the gene function.

Platelets carry over 95% of the circulating Alzheimer's 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 Alzheimer's 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 Pick's 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.

Two Fc gamma receptor (Fc gamma R) subclasses on human neutrophils, Fc gamma RII and Fc gamma RIII, activate different cellular functions. To examine the involvement of each receptor subtype in polymorphonuclear leukocyte activation, Fab and F(ab')2 fragments of subclass-specific monoclonal antibodies ([mAbs] mAb IV.3 against Fc gamma RII and mAb 3G8 against Fc gamma RIII, respectively) were used to block the binding of low valency immune complexes (LICs) and high valency immune complexes (HICs). Flow cytometry then permitted the simultaneous quantitation of antibody and ligand binding, the elicited intracellular Ca2+ concentration (delta[Ca2+]int), initiation of the oxidative burst, and/or the phospholipase A activation in the same cell. We have previously demonstrated that subsaturating dosages of HIC bind uniformly to all the cells but elicit an "all-or-none" (i.e., dose independent) maximal delta[Ca2+]int in a dose-dependent subpopulation of the cells. In contrast, both the proportion of cells responding and the magnitude of the delta[Ca2+]int transient depend on the subsaturating dose of LIC, even though it too binds uniformly to all the cells, nonresponding as well as responding. These earlier findings have here been extended by single cell flow cytometric analysis to demonstrate that F(ab')2 Fc gamma RIII is the major Fc gamma R involved in HIC binding (and [Ca2+]int mobilization), as well as in oxidative burst and phospholipase A activation. In contrast, both receptor subclasses must be available for LIC-elicited delta[Ca2+]int, as blockage by either of the mAb Fab or F(ab')2 fragments abrogates this response, even though LIC binding to the receptors is not decreased. Furthermore, LIC elicited little oxidative burst activity and failed to activate phospholipase A but cross-linking to achieve multivalency, previously shown to induce [Ca2+]int and oxidative burst responses, elicited phospholipase A activity via Fc gamma RIII. Fc gamma RII's role appears to be modulation of the small, late Ca2+ influx observed at > 1 min, whereas Fc gamma RIII modulates all the earlier larger events. Thus, simultaneous observation of receptor identity, receptor occupancy, and consequent activation parameters in the same cell by flow cytometry permits use to demonstrate that Fc gamma RII is necessary for the small signal transduction elicited by LIC; it plays a relatively small role in polymorphonuclear leukocyte stimulation by HIC. Fc gamma RIII is the main receptor responsible for immune complex-elicited polymorphonuclear leukocyte responses; its efficacy is greatly enhanced when the receptors are cross-linked, either by preequilibrated multivalent complexes or by in situ cross-linking of bound LIC with excess antibody.

It has been suggested that the multifunctional protein, calreticulin, is a major calcium sequestering protein in the inositol 1,4,5-trisphosphate receptor-containing endoplasmic reticulum subcompartment. In neuroblastoma X glioma NG-108-15 cells, bradykinin can effectively stimulate the release of inositol 1,4,5-trisphosphate and cause a cytosolic calcium transient. To explore the function of calreticulin as an intracellular calcium sequestering protein, we investigated calcium dynamics in NG-108-15 cells after treatment with an antisense oligonucleotide against calreticulin, CrtAS1. Cells treated with either CrtAS1 or the corresponding sense oligonucleotide CrtPS1 were examined for their calreticulin content by Western blotting, the amplitude of their calcium transient in response to bradykinin, and their sensitivity toward the calcium ionophore, ionomycin. Treatment with CrtAS1 decreased the amount of calreticulin in comparison to CrtPS1-treated and untreated control cells. At the same time, CrtAS1-treated cells had a significantly reduced calcium response to bradykinin and were more sensitive to ionomycin-induced cell death. These data show that the level of calreticulin expression is directly related to the calcium storage capacity of the inositol 1,4,5-trisphosphate-sensitive calcium pool and indicate a direct relationship between the level of calreticulin and the protection against cytotoxic calcium overload.

Alzheimer's Disease(AD), characterized by a deposition of beta-amyloid peptide (beta/A4) in the brain and in the cerebral microvasculature of affected individuals, is derived from its precursor protein (beta APP) via proteolytic processing by enzyme(s) which have not yet been characterized or localized. Since platelets carry APP in one of their granules, they have been implicated as a source of the beta/A4 deposits in the microvasculature of AD patients, attributable to either an abnormality in the platelets' stimulus response, in the quantity or nature of the APP they release upon activation and/or in the processing of that protein. We show here that platelets from patients with severe AD have abnormal stimulus responses to alpha-thrombin. Specifically, these cells hyperacidify. While it is not clear why this abnormality occurs, it may contribute to aberrant granule secretion since we have demonstrated earlier that release of platelet granule contents is partially controlled by the cytoplasmic pH.

To more closely examine the role of the cell surface in transmembrane signal transduction in human neutrophils, sealed right-side-membrane vesicles free of organellar membrane components were used as models of the plasma membrane. These vesicles, incubated with a fluorescent analogue of the chemotactic peptide fMLP, bound this ligand similarly in extent and kinetics to intact neutrophils. Vesicles responded to this stimulation with a slow increase in internal [Ca++] which was inhibited by EGTA but not by verapamil; the cytosolic Ca++ transient seen in intact cells within 10 sec of stimulation was absent in vesicles. The vesicles also maintained a transmembrane potential (ψ) and were depolarized by the K+ ionophore valinomycin. However, unlike intact cells which hyperpolarized and then depolarized in response to fMLP, the vesicles demonstrated only a sustained hyperpolarization. Vesicles also differed from intact cells by not producing superoxide (O2−) in response to fMLP. Finally, fMLP caused dramatic alterations in membrane vesicle lipid metabolism: at early time points (within 5–10 sec), there was a transient production of diacylglycerol (DAG) concomitant with inositol lipid breakdown, with no apparent hydrolysis of non-inositol phospholipids. For up to 5 min after stimulation, there was no increase in the levels of phosphatidic acid or of inositol lipids. Thus, a significant portion of the signalling pathway in neutrophils is located at the cell surface or in the plasma membrane and functions independently of intracellular components. Furthermore, the plasma membrane is intimately involved in events occurring during both the early (DAG generation) and late (slow, prolonged rise in [Ca++]) phases of cellular response. In contrast, several of the responses to fMLP (the Ca++ transient, depolarization, generation of O2−, recycling of lipid metabolites) involve signalling machinery not constitutively resident on the neutrophil surface. © 1993 Wiley-Liss, Inc.

Phagocytes are the most important early components of the immune response, programmed to recognize, engulf, and destroy immune complexes (formed when antibodies recognize their specific antigens), foreign particles, bacteria, mycobacteria, apoptotic cells, etc. Neutrophils, monocytes, macrophages, and dendritic cells all participate in this process. Flow cytometry permits observation of phagocytes that have responded and, with the appropriate fluorescent probes, of the environment in the phagosome that has enclosed the foreign matter. This unit gives the background and the protocols for performing such studies. Curr. Protoc. Cytom. 51:9.31.1-9.31.10. © 2010 by John Wiley & Sons, Inc. Keywords: phagocytosis; phagosome; phagocytes; flow cytometry

The interrelationships between activation of phospholipases and neutrophil stimulus-induced Ca2+ responses remain unclear. We report here that immune complexes activate a phosphatidylcholine-specific phospholipase A in a neutrophil only after the cytoplasmic Ca2+ transient has been initiated in the same cell, while chemotactic peptide activation does not proceed via such a phospholipase A-mediated mechanism. Measurements of [Ca2+] changes and of phosphatidylcholine-specific phospholipase A activity were made by flow cytometry, using Indo-1 for Ca2+ indication, and a new fluorescent probe, bis-BODIPY-phosphatidylcholine, localized in the inner leaflet of the plasma membrane, to measure phospholipase A activation. Both 100 nM formyl-methionyl-leucyl-phenylalanine (with or without cytochalasin B) and 60 micrograms/ml insoluble immune complexes elicited cytoplasmic Ca2+ transients, but only insoluble immune complexes stimulated phospholipase A activation in a subpopulation of cells exhibiting an elevation of [Ca2+]in. Phospholipase A activation followed the Ca2+ transient, starting, in each cell, after [Ca2+]in had begun to decrease as Ca2+ redistributed in the activated cell. The products of this phospholipase activation were confirmed by thin layer chromatography. We conclude that neutrophils respond to immune complexes with an elevated cytoplasmic Ca(2+)-requiring phosphatidylcholine-specific phospholipase A activation and to chemotactic peptides by a different mechanism.

Insoluble immune complexes (IIC) stimulate human neutrophils through Fc gamma receptors. Freshly isolated human neutrophils express two FcR subclasses, FcRII and FcRIII. We explored the role of FcRII and FcRIII in this activation process by selectively binding each FcR subclass with the Fab fragments of the respective anti-FcR monoclonal antibodies (MFab) before exposure to IIC. Correlation among liganded FcR subclass, IIC binding, and ensuant IIC stimulation was achieved with multiparameter flow cytometry. We utilized rhodamine-labeled anti-FcRIII and fluorescein-labeled IIC to study binding and observed the change in [Ca2+]i in the same cell with a Ca2+ indicator, Indo-1. Treatment with either anti-FcRII (IV.3) or anti-FcRIII (3G8) MFab decreased both the fraction of cells exhibiting a Ca2+ transient and the magnitude of that transient, although only anti-FcRIII but not anti-FcRII significantly inhibited the subsequent IIC binding. In addition, cells treated with anti-FcRII and then stimulated with IIC exhibited a decrease in both the intracellular Ca2+ transient and the later Ca2+ influx, whereas anti-FcRIII totally abolished the mobilization of intracellular Ca2+ without affecting the Ca2+ influx. Treatment with either anti-FcR MFab decreased the IIC-stimulated transmembrane potential change, oxidative burst, and elastase release. These studies indicate that freshly isolated neutrophils' Fc receptor subclasses have unique roles in the IIC-initiated stimulation and that full activation can only be achieved when both FcR subclasses are available.

Microorganisms are recognized by specific phagocyte surface receptors. Liganded receptors then signal a series of events leading to phagocytosis and destruction of the organism by oxidative, lytic, and associated processes. Some organisms, such as Mycobacterium tuberculosis (Mtb), Cryptococcus neoformans (Cf), and others, evade such destruction, surviving and sometimes multiplying within the phagosome to later cause disease. To study such evasion, we developed protocols which permit simultaneous kinetic measurement of early cytoplasmic signaling and of phagosomal pH (pHp) and oxidative burst, on a cell-by-cell basis, of polymorphonuclear (PMN) leukocytes exposed to fluorescently labeled, nonpathogenic Staphylococcus epidermidis (Se). The availability of a new, highly sensitive pH probe, pHrodo™, permits observation of increasing pHp. Simultaneous labeling of the organism, applicable to any phagocyte target, with a probe insensitive to pH and oxidative species, such as AlexaFluor350™, permits distinction between binding and functional responses to it by ratioing fluorescences. Addition of an extracellular-specific quencher (Trypan blue) permits distinction between bound and phagosome-enclosed targets, so that conditions within the closed phagosome can be studied. We found that opsonization is required for functional activation of PMN by Se, that the organism causes early alkalinization of the phagosome (in contrast to Cf which hyperacidifies it), and that extracellular Ca2+ is not required for cytoplasmic Ca2+ signaling but contributes markedly to binding of Se to PMN and to ensuant bactericidal functions. These findings lead to a new approach to the study of select organisms, like Cf and Mtb, which evade killing by manipulating the phagosomal environment. © 2009 International Society for Advancement of Cytometry

Using three experimental approaches, we have addressed the questions of whether the presence of saturably bound thrombin plays a role in potentiating the activation of platelet phospholipase C (PLC) and/or accumulation of the 3-phosphorylated phosphoinositides (3-PPI), i.e. phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, and whether the generation of tethered ligand (Vu, T-K.H., Hung, D. T., Wheaton, V. I., and Coughlin, S. R. (1991) Cell 64, 1057-1068) by thrombin can account fully for thrombin's proteolytic effects in activating platelets, as gauged by the above parameters. We have 1) measured PLC activation or 3-PPI after we have exposed platelets to thrombin for various periods and either blocked thrombin's proteolytic activity without interrupting its binding or blocked both binding and proteolytic activity of thrombin; 2) attempted to potentiate 3-PPI accumulation, using combinations of protein kinase C stimulation, Ca2+ elevation, and saturating but proteolytically inactive thrombins; and 3) compared the activation of platelets by thrombin with activation by the "thrombin" receptor-directed peptide, SFLLRNPNDKYEPF (SFLL; a portion of the tethered ligand created by thrombin's proteolytic activity), and examined the effect of thrombin on this latter activation. We conclude that the initial and sustained effects of thrombin in stimulating PLC and the accumulation of 3-PPI are completely attributable to thrombin's proteolytic activity. Further, thrombin's effects in promoting these responses can be accounted for by the actions of SFLL peptide, and by implication, formation of tethered ligand.

Immune complexes (ICs) induce an initial transient increase in cytosolic intracellular calcium [( Ca2+]in) levels in human neutrophils (PMN). Changes in PMN [Ca2+]in were measured with the fluorescent calcium indicator Indo-1 ( [1-[2-amino-5-(6-carboxylindol-2-yl]-phenoxyl]-2-(2'-amino-5 '- methylphenoxy]ethane-N,N,N'N'-tetraacetic acid), at the level of individual cells by flow cytometry. Two kinds of immune complexes (ICs) were used in this study: an insoluble (IIC) and a more soluble less valent immune complex (SIC) with fewer available Fc receptor binding ends per molecule of SIC than IIC. Simultaneous binding and activation studies performed on the flow cytometer with fluoresceinated IIC or SIC demonstrated that a majority of the cells bound each stimulus uniformly. However, only an IC dose-dependent proportion of those IC-bound cells responded with an increase in [Ca2+]in. Analysis of Indo-1 fluorescence signals from neutrophils exposed to IIC, corrected for the contribution of the nonresponding population, indicated that every dose of IIC elicited a similar maximum [Ca+2]in within the responding population. In contrast, the magnitude of the increase in [Ca2+]in elicited by low doses of SIC did become dependent on dose. Cells treated with pertussis toxin and exposed to IIC exhibited a normal [Ca2+]in response both in magnitude and expression. Therefore, [Ca2+]in responses induced by immune complexes are expressed by subpopulations of PMN, in a response which is dependent on the valency of the stimulus. In addition, pertussis toxin sensitive G protein(s) appear not to have a major role in IIC-induced [Ca2+]in changes, membrane potential changes, production of superoxide anions, and elastase release.

Mastoparan, a tetradecapeptide component of wasp venom, is a potent activator of secretion in a variety of cell types, and has been shown to activate purified G-proteins reconstituted into phospholipid vesicles with a preferential activation of Gi over Gs (Higashijima, T., Uzu, S., Nakajima, T., and Ross, E. R. (1988) J. Biol. Chem. 263, 6491-6494). To identify the biochemical activities of mastoparan in a cellular system, we characterized the effects of mastoparan on signal transduction pathways in rat pulmonary alveolar type 2 epithelial cells, which synthesize and secrete pulmonary surfactant. Mastoparan inhibited adenylylcyclase activity in a manner that was dose-dependent (IC50 = 30 microM), but sensitive to neither guanine nucleotide nor pertussis toxin (PT). Mastoparan induced a PT-sensitive increase in cellular inositol trisphosphate and a rapid rise in cytosolic calcium released from intracellular stores; the time to onset of the calcium rise, but neither the rate nor the amplitude of the rise, were PT-sensitive. Mastoparan also caused a dose- (EC50 = 16 microM) and time-dependent activation of arachidonic acid release that was completely insensitive to pretreatment with PT. Secretion of pulmonary surfactant was increased by mastoparan approximately 8-fold over constitutive levels at 1 h with an EC50 = 20 microM, and mastoparan-stimulated secretion was partially sensitive to PT at late time points and to inhibitors of arachidonic acid metabolism, but not to the protein kinase C inhibitor H7. These findings are consistent with the activation of Gi proteins in type 2 cells by mastoparan, although the lack of predicted triphosphoguanine nucleotide and PT sensitivity for some activities indicates that mastoparan does not act in a manner strictly analogous to liganded receptors or that some activities are not mediated by activation of Gi. While mastoparan is a potent secretagogue in several cell types, its secretory activity appears to have only a limited dependence on the activation of Gi proteins in type 2 cells.

After human platelet and leucocyte isolation, and Indo-1 acetoxymethyl ester loading, the fluorometric technique of flow cytometric measurments has been used to analyse changes in cytosolic calcium. Measurments in cells suspensions and in individual cells.

Elkan R. Blout's laboratory at the Children's Cancer Research Foundation and Harvard Medical School pioneered many approaches to the synthesis, conformation and structural studies of polypeptides, biopolymers and selected proteins. Here the early days (1957-1972) of his research group are remembered.

The chemotactic peptide formylmethionyl-leucyl-phenylalanine (fMLP), at concentrations below 10(-9) M, elicits a sustained increase in the human neutrophil's membrane potential within 10 s of its addition. This hyperpolarization, detected with the fluorescent cationic potentiometric probes, 3,3'-dipentyloxacarbocyanine (diO-C5-(3)), and 1,1'-dipropyl-3,3,3',3'-tetramethylindocarbocyanine iodide (diI-C3-(3)), and with the anionic probe bis-(1,3-diethylthiobarbituric)trimethine oxonol (bis-oxonol), is immediately followed by a large depolarization when [fMLP] greater than 10(-9) M. By extracellular substitution of sodium ions with potassium ions or choline or by pretreatment of the cells with ionophores, we report here that the hyperpolarization is primarily dependent on an intact potassium ion gradient and is accompanied by a concurrent acidification of the cytoplasm (approximately 0.05 pH unit) Although the latter occurs simultaneously with a large, transient increase in cytosolic Ca2+ at [fMLP] greater than 10(-10) M, it occurs without a detectable increase in cytosolic Ca2+ at [fMLP] less than 10(-10) M. The hyperpolarization is neither affected nor initiated by the chemotactic peptide antagonist tert-butyloxycarbonyl-methionyl-leucyl-phenylalanine, whereas the depolarization is completely inhibited. Neutrophils isolated from patients with X-linked chronic granulomatous disease exhibit normal hyperpolarizations and cytosolic Ca2+ increases in response to chemotactic peptides but exhibit no depolarization or oxidative burst. The hyperpolarization appears earlier in the ontogeny of differentiating myeloid precursor cells than either the rise in cytosolic Ca2+ or the depolarization response. Together, these findings indicate that an increase in transmembrane potential is one of the earliest events in the neutrophil response to chemotactic peptides, coinciding temporally with increases in cytoplasmic Ca2+ and H+ concentrations but preceding detectable oxidative burst activity.

Human platelets exhibit an extremely rapid increase in cytoplasmic Ca2+ concentrations ((Ca2+]in) and a dose-dependent cytoplasmic pH change ((pH]in) upon thrombin stimulation. A cytoplasmic alkalinization, maximal by 60 s, is preceded by a very rapid acidification, which is masked by the alkalinization when saturating thrombin doses are used. Using the pH probe 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein we report here the kinetics of simultaneous cytoplasmic pH and Ca2+ changes in thrombin-stimulated platelets, measured in single cells by flow cytometry. This permits analysis of the responding subpopulation. Maximal thrombin stimulation (greater than or equal to 4.5 nM) induces a dose-dependent increase in pHin from approximately 7.0 to 7.30 and a maximal [Ca2+]in transient of up to 800 nM. The Ca2+ transient coincides temporally with the rapid initial acidification, while the alkalinization is maximal considerably later. The Ca2+ transients occur maximally in each responding cell, but occur only in a subpopulation of the platelets at subsaturating (less than 4.5 nM) thrombin doses; in contrast, the dose-dependent cytoplasmic acidification appears to occur uniformly in all platelets. The rapid increase in [Ca2+]in is not dependent on the alkalinization, and the former occurs maximally in amiloride treated, Na+/H+ exchange inhibited human platelets. These results indicate that the acidification and the rise in [Ca2+]in may be interrelated, whereas the cytoplasmic alkalinization (maximal considerably later than either the acidification or the [Ca2+]in rise) may be independent of these earlier, temporally correlated increases in H+ and Ca2+ concentrations.

Treatment of human leukemic HL-60 cells with N,N-dimethylformamide (DMF) induces them to mature until they reach granulocytoid morphology 3–6 d later. We have reported a maturation-dependent ability of these cells to respond to phorbol myristate acetate (PMA), as evaluated by membrane depolarization and by oxidative burst product formation (Newburger et al.: J. Biol. Chem. 259, 3771, 1984). More recently we have attempted to develop techniques for simultaneous evaluation of these parameters during HL-60 cell maturation. Here, we compare the cytoplasmic [Ca+ +] and membrane potential changes elicited by the chemotactic peptide fMLP via simultaneous measurement of individual cells in a fluorescence-activated cell sorter (FACS), as done previously for mature granulocytes (Lazzari et al.: J. Biol. Chem. 261,9710, 1986). The stimulus-induced [Ca++]in changes are detected with the fluorescent probe lndo-1 and reproducibly increase in magnitude for a subpopulation of cells as the cells mature into granulocytes. Ca+ + responsiveness to formyl peptide is restricted to a subpopulation of HL-60 granulocytes which expresses receptors for chemotactic peptide and consistently increases in magnitude (in response to the same concentration of agonist) with maturation. In contrast, there is less consistency in the direction or magnitude of membrane potential changes elicited under the same circumstances from the same maturing HL-60 cells.

In view of the reports that polymorpho- nuclear leukocytes (PMN) of patients with localized aggressive periodontitis (LAP) exhibit hyper-re- sponsiveness to stimulation, it has been suggested that such abnormalities could lead to PMN-medi- ated tissue damage during inflammation. To deter- mine whether these abnormalities include signal transduction, we compared cytoplasmic calcium concentration ((Ca 2 )i) and cytoplasmic pH (pH i ) changes, early stimulus responses to chemo- tactic agents, of LAP versus control (C)-PMN and explored whether these could be modulated by sensitizing cytokines or calcium channel-blocking agents. PMN responses of LAP patients were compared with age- and gender-matched con- trols. (Ca 2 )i and pHi were measured fluori- metrically using 1H-indole-6-carboxylic acid, 2-(4-(bis(2-((acetyloxy)methoxy)-2-oxoethyl)- amino)-3-(2-(2-(bis(2-((acetyloxy)methoxy)-2- oxoethyl)amino)-5-methylphenoxy)ethoxy)phenyl)-1 and 2,7-bis-(carboxyethyl)-5(6)-carboxyfluores- cein as respective probes. Not only was the maxi- mal calcium response to chemoattractants higher in LAP-PMN, but also their subsequent intracellu- lar calcium redistribution was significantly slower. The slower calcium redistribution of LAP-PMN, but not their higher maximal calcium response, was successfully mimicked in C-PMN treated with Nifedipine™ or 1-(b-(3-(4-methoxyphenyl)propoxy)- 4-methoxyphenethyl)-1H-imidazole-HCl, both known to be inhibitors of membrane-associated calcium influx, but this redistribution was not af- fected when inhibitors of other calcium influx mechanisms, Diltiazem™ or Verapamil™, were used. Taken together, our findings indicate that certain early stimulus responses are aberrant in LAP-PMN, that internal redistribution of cytoplas- mic-free calcium is compromised, and, addition- ally, that a membrane-associated Ca 2 transport defect may be present. J. Leukoc. Biol. 78: 612-619; 2005.

As part of the systematic evaluation of the innate immune system for long duration missions, this study focused on the antimicrobial functions of monocytes in astronauts participating in spaceflight. The study included four space shuttle missions and 25 astronauts. Nine non-astronauts served as controls. Blood specimens were collected 10 days before launch, within 3h after landing, and again 3 days after landing. The number of monocytes did not differ significantly over the interval sampled in both the astronaut or control groups. However, following 5-11 days of spaceflight, the astronauts' monocytes exhibited reductions in ability to engulf Escherichia coli, elicit an oxidative burst, and degranulate. The phagocytic index was significantly reduced following spaceflight when compared to control values. This reduction in phagocytosis was accompanied by changes in the expression of two surface markers involved in phagocytosis, CD32 and CD64. Levels of cortisol, epinephrine, and norepinephrine after spaceflight did not increase over preflight values.

It has long been appreciated that polymorphonuclear leukocytes (PMN) killCryptococcus neoformans, at least in part via generation of fungicidal oxidants. The aim of this study was to examine the contribution of nonoxidative mechanisms to the inhibition and killing ofC. neoformans. Treatment of human PMN with inhibitors and scavengers of respiratory burst oxidants only partially reversed anticryptococcal activity, suggesting that both oxidative and nonoxidative mechanisms were operative. To define the mediators of nonoxidative anticryptococcal activity, PMN were fractionated into cytoplasmic, primary (azurophil) granule, and secondary (specific) granule fractions. Incubation ofC. neoformanswith these fractions for 18 h resulted in percents inhibition of growth of 67.4 ± 3.4, 84.6 ± 4.4, and 29.2 ± 10.5 (mean ± standard error,n= 3), respectively. Anticryptococcal activity of the cytoplasmic fraction was abrogated by zinc and depletion of calprotectin. Antifungal activity of the primary granules was significantly reduced by pronase treatment, boiling, high ionic strength, and magnesium but not calcium. Fractionation of the primary granules by reverse phase high-pressure liquid chromatography on a C4column over an acetonitrile gradient revealed multiple peaks with anticryptococcal activity. Of these, peaks 1 and 6 had substantial fungistatic and fungicidal activity. Peak 1 was identified by acid-urea polyacrylamide gel electrophoresis (PAGE) and mass spectroscopy as human neutrophil proteins (defensins) 1 to 3. Analysis of peak 6 by sodium dodecyl sulfate-PAGE revealed multiple bands. Thus, human PMN have nonoxidative anticryptococcal activity residing principally in their cytoplasmic and primary granule fractions. Calprotectin mediates the cytoplasmic activity, whereas multiple proteins, including defensins, are responsible for activity of the primary granules.

We report here the discovery of two novel human platelet and megakaryocytic DAMI cell enzymes that have β-secretase-like activity. These activities could potentially effect cleavage of the amyloid precursor protein (APP) at the β-amyloid peptide N-terminus, by an EC 3.4.24.15-like metalloprotease, and the N terminus-1 position, by a serine protease. Thus both enzymes may generate the amyloidogenic β-peptide. Studies of intact and Triton X-100-lysed DAMI cells, as well as intact versus subcellular fractions of platelets, demonstrate the presence of these proteolytic activities. The resting platelet has (1) a surface serine protease, demonstrated by its ability to cleave a β-secretase substrate and by its inhibitor sensitivity; and (2) a metalloprotease, recognized by an antibody to EC 3.4.24.15, which resides intracellularly in the α-granule membrane, is translocated to the surface on activation, and shows β-secretase-like activity by cleaving the same substrate. This metalloprotease can also cleave recombinant APP to a potentially amyloidogenic fragment. Surface metalloprotease was identified in DAMI cells by flow cytometry and Western blotting with a specific anti-EC 3.4.24.15 monoclonal antibody, while activity was identified by using two β-secretase substrates. This article is the first to document two previously unknown endoproteinases with β-secretase-like activity in platelets and DAMI cells. These proteases are capable of effecting cleavage of APP and could therefore contribute to Aβ deposition in the cerebrovasculature. (J Lab Clin Med 1999;133:507-15)

Recently, we demonstrated that human monocyte-derived macrophages (MDM) treated with chloroquine or ammonium chloride had markedly increased antifungal activity against the AIDS-related pathogen Cryptococcus neoformans . Both of these agents raise the lysosomal pH, which suggested that the increased antifungal activity was a function of alkalinizing the phagolysosome. Moreover, there was an inverse correlation between growth of C. neoformans in cell-free media and pH. These data suggested that C. neoformans was well adapted to survive within acidic compartments. To test this hypothesis, we performed studies to determine the pH of human MDM and neutrophil phagosomes containing C. neoformans . Fungi were labeled with the isothiocyanate derivatives of two pH-sensitive probes: fluorescein and 2′,7′-difluorofluorescein (Oregon Green). These probes have pK a s of 6.4 and 4.7, respectively, allowing sensitive pH detection over a broad range. The phagosomal pH averaged approximately 5 after ingestion of either live or heat-killed fungi and remained relatively constant over time, which suggested that C. neoformans does not actively regulate the pH of its phagosome. The addition of 10 and 100 μM chloroquine resulted in increases in the phagosomal pH from a baseline of 5.1 up to 6.5 and 7.3, respectively. Finally, by immunofluorescence, colocalization of C. neoformans and the MDM lysosomal membrane protein LAMP-1 was demonstrated, establishing that fusion of C. neoformans -laden phagosomes with lysosomal compartments takes place. Thus, unlike many other intracellular pathogens, C. neoformans does not avoid fusion with macrophage lysosomal compartments but rather resides and survives in an acidic phagolysosome.

We have previously demonstrated that thrombin-activated platelets from patients with advanced Alzheimer's disease (AD) retain significantly more surface membrane-bound amyloid precursor protein (mAPP) than platelets from non-demented age-matched individuals (AM). We have studied interactions between these platelets and the cerebrovascular endothelium to which activated platelets adhere in a model system, investigating their involvement in the formation of amyloid beta peptide (Abeta) deposits in AD patients. We report here that there appear to be alpha and beta secretase-like activities in primary human blood brain barrier endothelial cell (BEC) cultures from both AD patients and AM control subjects (AD-BEC and AM-BEC, respectively) as well as a gamma secretase-like activity that appears only in AD-BEC. No such activities were observed in human umbilical vein endothelial cells (HUVECs). Furthermore, there is more penetration of the platelet-released products platelet factor 4 and soluble APP through the BEC layer grown from AD patients than that grown from AM individuals, whereas none penetrate through a HUVEC layer. Thus the interaction between platelets, the APP they have retained or released, and cerebral vascular endothelial cells may be at least partially responsible for amyloidogenic deposits around the cerebral vasculature of AD patients.

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.

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.

CD14 is a signaling receptor for both gram-negative bacterial lipopolysaccharide (LPS) and mycobacterial lipoarabinomannan (LAM) that lacks terminal mannosyl units (AraLAM). In contrast, terminally mannosylated LAM (ManLAM) binds the macrophage mannose receptor (MMRc), although the ability of the MMRc to serve as a signaling receptor has not been previously reported. We compared the abilities of AraLAM and ManLAM to induce distinct responses in two monocytic cell populations, freshly isolated human peripheral blood monocytes (PBM) and monocyte-derived macrophages (MDM). The responses examined were chemotaxis and transient changes in free cytosolic calcium ([Ca 2+ ] in ). We found that AraLAM but not ManLAM was chemotactic for both PBM and MDM. Migration of these cells in vitro to AraLAM was specifically blocked by an anti-CD14 monoclonal antibody, suggesting that CD14 mediates the chemotactic response to AraLAM. Subsequently, we found that AraLAM induced a transient rise in [Ca 2+ ] in levels within a subpopulation of PBM but not MDM. This response was blocked by anti-CD14 antibodies. In contrast, ManLAM induced a transient rise in [Ca 2+ ] in levels within a subpopulation of MDM but not PBM. This response was blocked by either anti-CD14 or anti-MMRc antibodies. These data suggest that the MMRc can serve as a signaling receptor and that coligation of both CD14 and the MMRc is required to elicit a specific response. Thus, one response to LAM (chemotaxis) can be elicited solely by engaging CD14, whereas a different response (changes in [Ca 2+ ] in levels) depends on both the differentiation state of the cells and concomitant engagement of CD14 and the MMRc.

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.

Platelets, when released as anuclear cells by their precursor megakaryocytes, already carry soluble proteolytic fragments of the amyloid precursor protein (APP) within their alpha-granules and intact APP in the alpha-granule membranes. In response to activation signals elicited by physiologic stimuli such as thrombin, platelets release their granules' soluble contents and translocate granule membrane-bound proteins to the plasma membrane. Because platelets carry >90% of the circulation's APP, activated platelets have been implicated as origins of the beta-amyloid peptide fragment of APP (A beta), whose deposition in the cerebrovasculature is characteristic of Alzheimer's disease. We have therefore studied the APP contents and proteolytic processing in resting DAMI human megakaryocytic cells, along with the consequences of the activation of these cells by thrombin, comparing the results in each case to those with human platelets. Resting and PMA-differentiated DAMI cell contents were examined by Western blotting, immunoprecipitation, or metabolic labeling with sulfur 35-labeled methionine during culture, while plasma membrane-bound APP was evaluated by flow cytometry. Activation was followed by changes in cytoplasmic calcium concentration ((Ca++)in) and in membrane potential. Like platelets, DAMI cells exhibited a thrombin dose-dependent delta(Ca++)in, and membrane potential change; in contrast to the surface of a platelet, the surface of an agranular resting DAMI cell expresses granule-membrane proteins (APP and CD63) that appear on platelets only after activation. DAMI cell culture with 35S-labeled methionine confirmed that megakaryocytes synthesize large amounts of APP, of slightly higher molecular weight, and degrade their APP extensively before platelets are formed.

Upon activation, platelet alpha-granules' soluble contents are secreted and membrane-bound contents are translocated to the plasma membrane. Membrane-bound proteins include the beta-amyloid precursor protein (APP) from which the beta-amyloid (A beta) deposits found surrounding the cerebrovasculature of patients with Alzheimer's Disease (AD) may originate. We show here that activated platelets from AD patients exhibit less APP processing, retain more of the protein on their surface, and secrete less as soluble fragments than do controls. Surface labeling demonstrated that there is little APP or CD62 on the surface of resting platelets. Upon activation, control platelets exhibited more of both proteins on their surface, while advanced AD patients exhibited similar amounts of CD62 as controls, but retained significantly more surface APP. AD platelets secreted similar amounts of most soluble alpha-granule contents as controls, but less APP fragments. Together these results suggest a processing defect that may account for greater deposition of A beta-containing products in the vasculature to which activated platelets adhere.

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.

Publisher Summary Flow cytometers were initially designed to permit identification of cells by means of their individual, highly specific, and therefore uniquely identifying surface proteins that served as antigens to well-characterized monoclonal or polyclonal antibodies. These, in turn, were detected either by their own fluorescent markers or by appropriately labeled second antibodies. The usual labels remain fluorescein, rhodamine, and phycoerythrin derivatives. The light scattering profiles of certain cells (e.g., those of the circulating blood), were also found to be fairly consistent and specific, thus permitting some cell characterization based on differences in cell size, with right angle and forward light-scatter profiles pragmatically related to the granularity and the size of the cell, respectively. All of these applications utilized equilibrated cells, whether fixed, permeabilized, or native. More recently, the ability to introduce samples and obtain data rapidly, as well as improvements in instrumentation, in fluorescent probe technology, and in antibody development, have permitted flow cytometry to advance from its initial application, and have enabled flow cytometers to be used for kinetic measurements and therefore to follow the responses of living and fully functional cells. Because some are equipped to measure up to six parameters simultaneously using four photomultipliers, several parameters of cell function can be correlated, for any given cell, with occupancy of its receptors.

Stimulation of phagocytic leukocytes with chemotactic factors results in transient acidification, followed by alkalinization of the cytosol. Human monocytes are known to alter their functional responses to the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP) in a complex fashion as they mature in vitro to macrophages. To examine the evolution of the cytoplasmic pH (pHi) response of monocytes to fMLP as they mature into macrophages, we incubated cells for 0, 24, 48, and 96 h (Medium-199 + 10% fetal bovine serum; 37°C) and examined pHi using the fluorescent probe 2′, 7′-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF; 1 μΜ) and a Perkin-Elmer 650/10 spectrofluorimeter (λem = 530 nm, λ_ex = 500, 450 nm) as previously described. The resting pH; of fresh (0 h) monocytes was 7.07 ± 0.16 (SD) and was unchanged after incubation for 24, 48, or 96 h (7.09, 7.11, 7.05, respectively). Cells exhibited an fMLP dose-dependent cytoplasmic acidification, with maximal δpHi occurring 30-60 s after exposure to 10−7 M fMLP. The response to fMLP did not change with the duration of incubation and, as with neutrophils, cytoplasmic realkalinization was blocked by dimethyl- amiloride (20 μΜ). Incubation with 2-deoxyglucose (10 min, 5 mM), sufficient to inhibit by more than 90% the formyl peptide-stimulated superoxide generation by monocytes, slowed fMLP-induced acidification and abrogated the alkalinization. In addition, monocytes isolated from the blood of a patient with X-linked chronic granulomatous disease (CGD) underwent fMLP-induced acidification that was unmasked further by coincubation with di- methylamiloride, in a manner quantitatively similar to that of normal monocytes, despite the inability of the CGD cells to produce superoxide. The chemotactic factor-induced cytoplasmic pH responses of mono- cytes/macrophages remained constant as the cells matured in vitro and exhibited a dimethylamiloride-independent acidification and dependent alkalinization, as did the response in neutrophils. The cytoplasmic acidification of these cells thus did not correlate with the cells’ production of superoxide and with the concomitant hexose monophosphate shunt activation, as has been suggested for other leukocyte types. J. Leukoc. Biol. 53: 673–678; 1993.

Immune complex stimulation of human neutrophils elicits, among other events, the formation of phagocytic vacuoles into which the products of the stimulus activated oxidative burst and degranulation are released. In order to monitor burst activity in the phagocytic vacuole, we have developed a fluorochrome-coupled derivative of this neutrophil agonist. The fluorochrome 2',7'-dichlorodihydrofluorescein (DCFH) (the nonfluorescent, reduced form of 2',7'-dichlorofluorescein (DCF] has been covalently linked to bovine serum albumin (BSA), which can be used to form an immune complex with anti-BSA immunoglobulin. The resultant complex is an effective agonist for stimulating all immune complex-mediated neutrophil responses, as compared to nonderivatized controls. Upon exposure to hydrogen peroxide the stimulus-linked probe is converted to its oxidized, fully fluorescent form, the fluorescence of which is linearly related to the extent of probe oxidation. Using flow cytometry, we have demonstrated that the probe-stimulus complex is capable of monitoring the kinetics of the production of activated oxygen species by the membrane bound NADPH-oxidase enzyme, presumably within the phagocytic vacuoles of immune complex-activated neutrophils. We have found that the immune complex-mediated activation of the oxidative burst within the phagocytic compartment is preceded by a lag of approximately 30 s followed by a large sustained release of superoxide dependent hydrogen peroxide. Neutrophils from patients with chronic granulomatous disease, however, demonstrated no sustained increase in probe fluorescence, a finding consistent with the lack of oxidative burst activity in these cells. The DCFH-immune complex conjugate therefore provides an effective probe for monitoring the kinetics of the localized release of oxidative products within the forming phagocytic vacuoles of activated neutrophils, and may be used to further examine both the activation and activity of human neutrophils in response to 'physiologic' host defense agonists such as immune complexes.

It is common experience that watches run down, bouncing balls stop bouncing, and even the most perfectly designed and crafted engine will eventually cease operating. This universal tendency toward stasis is the essence of the second law of thermodynamics. There are multiple possible statements of the second law, some useful and some needlessly confusing. Some of the more useful paraphrases are included in the following list; of these, the first two expressions are familiar from the discussion in the first chapter and are the more traditional representations of the second law: 1) No process is possible where heat is transferred from a colder to a hotter body without causing a change in some other part of the universe. 2) No process is possible in which the sole result is the absorption of heat (from another body) with complete conversion into work. 3) A system will always move in the direction that maximizes choice. 4) Systems tend toward greater disorder. 5) The macroscopic properties of an isolated system eventually assume constant values.