Ca(2+)-independent F-actin assembly and disassembly during Fc receptor- mediated phagocytosis in mouse macrophages

Phagocytosis of IgG-coated particles by macrophages is presumed to involve the actin-based cytoskeleton since F-actin accumulates beneath forming phagosomes, and particle engulfment is blocked by cytochalasins, drugs that inhibit actin filament assembly. However, it is unknown whether Fc receptor ligation affects the rate or extent of F-actin assembly during phagocytosis of IgG-coated particles. To examine this question we have used a quantitative spectrofluorometric method to examine F-actin dynamics during a synchronous wave of phagocytosis of IgG-coated red blood cells by inflammatory mouse macrophages. We observed a biphasic rise in macrophage F-actin content during particle engulfment, with maxima at 1 and 5 min after the initiation of phagocytosis. F-actin declined to resting levels by 30 min, by which time particle engulfment was completed. These quantitative increases in macrophage F-actin were reflected in localized changes in F-actin distribution. Previous work showed that the number of IgG-coated particles engulfed by macrophages is unaffected by buffering extracellular calcium or by clamping cytosolic free calcium concentration ([Ca2+]i) to very low levels (Di Virgilio, F., B. C. Meyer, S. Greenberg, and S. C. Silverstein. 1988. J. Cell Biol. 106: 657-666). To determine whether clamping [Ca2+]i in macrophages affects the rate of particle engulfment, or the assembly or disassembly of F-actin during phagocytosis, we examined these parameters in macrophages whose [Ca2+]i had been clamped to approximately less than 3 nM with fura 2/AM and acetoxymethyl ester of EGTA. We found that the initial rate of phagocytosis, and the quantities of F-actin assembled and disassembled were similar in Ca(2+)-replete and Ca(2+)-depleted macrophages. We conclude that Fc receptor-mediated phagocytosis in mouse macrophages is accompanied by an ordered sequence of assembly and disassembly of F-actin that is insensitive to [Ca2+]i.     

Calcium transients during Fc receptor-mediated and nonspecific phagocytosis by murine peritoneal macrophages

Studies with populations of macrophages have produced conflicting results concerning the possibility that the concentration of intracellular ionized calcium [( Ca2+]i) may act as an important mediator for phagocytosis. Since asynchronous changes in [Ca2+]i in individual cells undergoing phagocytosis may be averaged to undetectability in population studies, we studied single adhering murine macrophages using fura-2 and our previously described digital imaging system. The proportion of macrophages phagocytosing IgG-coated latex beads was greater than for uncoated beads (percent phagocytosing cells: 71 +/- 7 vs. 27 +/- 7, P less than 0.01). Phagocytosis of IgG-coated and uncoated beads was always associated with a calcium transient that preceded the initiation of phagocytosis. No calcium transients were detected in cells that bound but did not phagocytose beads. Four major differences between Fc receptor-mediated and nonspecific phagocytosis were detected: (a) the duration of calcium transients was longer for nonspecific phagocytosis compared with Fc receptor-mediated phagocytosis (69.9 +/- 10.2 vs. 48.7 +/- 4.7 s, P less than 0.05) and the magnitude of calcium transients was less for nonspecific phagocytosis (178 +/- 43 vs. 349 +/- 53 nM, P less than 0.05); (b) removal of extracellular calcium abolished the calcium transients associated with nonspecific phagocytosis but had no effect on those associated with receptor-mediated phagocytosis; (c) in the absence of extracellular calcium, buffering intracellular calcium with a chelator reduced Fc receptor-mediated phagocytosis but had no additive inhibitory effect on nonspecific phagocytosis; and (d) inhibition of protein kinase C (PKC) with staurosporine inhibited nonspecific phagocytosis but had no effect on receptor-mediated phagocytosis. Our observations suggest that despite both types of phagocytosis being associated with intracellular calcium transients, the role played by intracellular calcium in the signaling pathways may differ for Fc receptor-mediated and nonspecific phagocytosis by elicited murine macrophages.     

Fc-receptor-mediated phagocytosis occurs in macrophages without an increase in average [Ca++]i

The calcium ion has been implicated as a cytosolic signal or regulator in phagocytosis. Using the Ca++-sensitive photoprotein aequorin we have measured intracellular free Ca++ ion concentration ([Ca++]i) in thioglycolate-elicited mouse peritoneal macrophages during phagocytosis and IgG-induced spreading. Macrophages plated on glass were loaded with aequorin and [Ca++]i was then measured from cell populations, both as previously described (McNeil, P. L., and D. L. Taylor, 1985, Cell Calcium, 6:83-92). Aequorin indicated a resting [Ca++]i in adherent macrophages of 84 nM and was responsive to changes in [Ca++]i induced by the addition of Mg-ATP (0.1 mM) or serum to medium. However, during the 15 min required for phagocytosis of seven or eight IgG-coated erythrocytes per macrophage loaded with aequorin, we measured no change in [Ca++]i. Similarly, the ligation of Fc-receptors that occurs when macrophages spread on immune complex-coated coverslips did not change macrophage [Ca++]i. In contrast, a rise in [Ca++]i of macrophages was measured during phagocytosis occurring in a serum-free saline of pH 7.85, and as a consequence of incubation with quin2 A/M. We estimate that had a change in [Ca++]i occurred during phagocytosis, aequorin would have detected a rise from 0.1 to 1.0 microM taking place in as little as 2% of the macrophage's cytoplasmic volume. We therefore suggest that either Ca++ is not involved as a cytoplasmic signal for phagocytosis or that increases in [Ca++]i during phagocytosis are confined to such small regions of cytoplasm as to be below the limits of detection by our cellular averaging method. Our data emphasizes, moreover, the need for well-defined, nonperturbing conditions in such measurements of [Ca++]i.     

Chelation of cytoplasmic Ca2+ increases plasma membrane permeability in murine macrophages

Cytoplasmic free Ca2+ (Ca2+i) was chelated to 10-20 nM in the macrophage cell line J774 either by incubation with quin2 acetoxymethyl ester in the absence of external Ca2+ (Di Virgilio, F., Lew, P.D., and Pozzan, T. (1984) Nature 310, 691-693) or by loading [ethyl-enebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) into the cytoplasm via reversible permeabilization of the plasma membrane with extracellular ATP (Steinberg, T.H., Newman, A.S., Swanson, J.A., and Silverstein, SS.C. (1987) J. Biol. Chem. 262, 8884-8888; Di Virgilio, F., Meyer, B.C., Greenberg, S., and Silverstein, S.C. (1988) J. Cell Biol. 106, 657-666). After removal of ATP from the incubation medium, ATP-permeabilized Ca2+i-depleted macrophages recovered a near-normal plasma membrane potential which slowly depolarized over a 2-4 h incubation at low [Ca2+]i. In both ATP-treated and quin2-loaded cells, depolarization of plasma membrane potential was paralleled by an increase in plasma membrane permeability to low molecular weight aqueous solutes such as eosin yellowish (Mr 692), ethidium bromide (Mr 394), and lucifer yellow (Mr 463). This increased plasma membrane permeability was not accompanied by release of the cytoplasmic marker lactic dehydrogenase for incubations up to 4 h and was likely a specific effect of Ca2+i depletion since it was not caused by: (i) the mere incubation of macrophages with extracellular EGTA, i.e. at near-normal [Ca2+]i; and (ii) loading into the cytoplasm of diethylenetriaminepentaacetic acid, a specific chelator of heavy metals with low affinity for Ca2+. Treatment of Ca2+i-depleted cells with direct (phorbol 12-myristate 13-acetate) or indirect (platelet-activating factor) activators of protein kinase C prevented the increase in plasma membrane permeability. Down-regulation of protein kinase C rendered Ca2+i-depleted macrophages refractory to the protective effect of phorbol 12-myristate 13-acetate. This report suggests a role for Ca2+i and possibly protein kinase C in the regulation of plasma membrane permeability to low molecular weight aqueous solutes.     

Signal transduction by neutrophil immunoglobulin G Fc receptors. Dissociation of intracytoplasmic calcium concentration rise from inositol 1,4,5-trisphosphate.

The signal transduction mechanisms involved in the regulation of phagocytosis are largely unknown. We have recently shown that in neutrophils, when IgG-mediated phagocytosis is stimulated by formyl-methionyl-leucyl-phenyl-alanine (fMLP), the enhanced ingestion is dependent on the increase in [Ca2+]i which results from ligation of Fc receptors by the IgG-coated target (Rosales, C., and Brown, E. (1991) J. Immunol. 146, 3937-3944). Now, we have studied the mechanism by which this rise in [Ca2+]i occurs. Aggregated IgG, the monoclonal antibody 3G8 (which recognizes Fc receptor type III), and insoluble immune complexes caused an increase in [Ca2+]i. The rise in [Ca2+]i induced by Fc receptor ligation was resistant to pertussis toxin. In contrast, fMLP induced a rise in [Ca2+]i which was inhibited by pertussis toxin. fMLP-induced [Ca2+]i was accompanied by an accumulation of inositol 1,4,5-trisphosphate (IP3) which peaked by 15 s, and which was also abolished by pertussis toxin. IP3 accumulation after aggregated IgG, 3G8, or insoluble immune complexes was much less than after fMLP. Unlike [Ca2+]i rise induced by Fc receptor ligation, this small increase in IP3 was inhibited by pertussis toxin. These data demonstrated that the [Ca2+]i increase induced by Fc receptor ligation is not mediated by IP3. Immediate pretreatment of human polymorphonuclear neutrophils with optimal doses of fMLP also reduced subsequent increase in [Ca2+]i rise from thapsigargin, a sesquiterpene lactone tumor promoter that releases intracellular Ca2+ from IP3-sensitive stores without IP3 turnover. Similarly, to its effects on thapsigargin, fMLP inhibited the [Ca2+]i rise upon subsequent immune complex binding. Pretreatment of cells with immune complexes also prevented subsequent [Ca2+]i rise from thapsigargin and fMLP. These data demonstrate that IgG Fc receptor ligation and fMLP activation of human polymorphonuclear neutrophils use distinct signal transduction mechanisms to release Ca2+ from the same thapsigargin-sensitive intracellular pool. In contrast to fMLP, signal transduction for increased [Ca2+]i after Fc receptor stimulation does not involve a pertussis toxin-sensitive G protein, and is independent of IP3.     

Measurement of the cytosolic free calcium ion concentration of individual lymphocytes by microfluorometry using quin 2 or fura-2

The cytosolic free calcium ion concentration ([Ca2+]i) of individual lymphocytes was measured by microfluorometry with dual excitation wavelengths using quin 2 for fura-2. Fura-2 was a more suitable fluorescent Ca2+ indicator than quin 2 for measurements of single cells because of the standard curve calibrated for fura-2 had a good linearity, and the standard deviation (SD) of the value of the intensity ratio of fura-2-loaded cells was much smaller than that of quin 2-loaded cells. The [Ca2+]i in quiescent lymphocytes was about 1 x 10(-7) M, and an increase in the [Ca2+]i was observed within a few minutes of ionomycin, protein A, phorbol myristate acetate (PMA) or concanavalin A (Con A) stimulation. Ionomycin-induced proliferation occurred when the initial [Ca2+]i was approximately 3 x 10(-7) M or greater. The increase in the [Ca2+]i induced by Con A occurred transiently, and another rise in the [Ca2+]i was observed in the stage prior to the S-phase. These results indicate that Ca2+ is necessary for stimulated lymphocytes to enter the cell cycle and S-phase.     

Extracellular nucleotides mediate Ca2+ fluxes in J774 macrophages by two distinct mechanisms

We have studied the effects of extracellular nucleotides on the cytosolic free calcium concentration [( Ca2+]i) in J774 macrophages using quin2 and indo-1 as indicator dyes. Micromolar quantities of ATP induced a biphasic increase in [Ca2+]i: a rapid and transient increase (peak I) which was due to mobilization of Ca2+ from intracellular stores and a second more sustained elevation (peak II) due to influx of extracellular Ca2+. The sustained peak II elevation had two components, a "low threshold" (1 microM ATP) response which saturated at 10-50 microM ATP and a "high threshold" response, apparent at [ATP] greater than 100 microM. The latter component was not seen with nucleotides other than ATP and correlated with an ATP-induced generalized increase in plasma membrane permeability. A variant J774 cell line was isolated which does not demonstrate this ATP-induced increase in plasma membrane permeability; nevertheless, it demonstrated both the release of Ca2+ from intracellular stores and the low threshold component of the Ca2+ influx across the plasma membrane in response to nucleoside di- and triphosphates. Several lines of evidence indicate that the fully ionized (i.e. free acid) forms of nucleoside di- and triphosphates were the ligands that mediated these increases in [Ca2+]i. These data show that extracellular nucleotides mediate Ca2+ fluxes by two distinct mechanisms in J774 cells. In one, the rise in [Ca2+]i is due to release of Ca2+ from intracellular stores and Ca2+ influx across the plasma membrane. This response is elicited preferentially by the free acid forms of purine and pyrimidine nucleoside di- and triphosphates. In the other, the rise in [Ca2+]i reflects a more generalized increase in plasma membrane permeability and is elicited by ATP4- only.     

Thrombin and ionomycin can raise platelet cytosolic Ca2+ to micromolar levels by discharge of internal Ca2+ stores: studies using fura-2

In the presence of 1 mM EGTA, the addition of the calcium ionophore ionomycin to human platelets loaded with 30 microM fura-2 could elevate [Ca2+]i from less than 100 nM to a maximum of greater than 3 microM, presumably by discharge of Ca2+ from internal stores. Under the same conditions thrombin could maximally increase [Ca2+]i to a peak of greater than 1 microM which then declined to near resting levels within 3-4 minutes; by contrast in platelets loaded with 1 mM quin2 thrombin could raise [Ca2+]i to only about 200 nM. In the presence of 1 mM Ca2+ the peak response to thrombin in fura-2-loaded platelets was higher (1.4 microM) than that observed in the presence of EGTA (1.1 microM) and the elevation in [Ca2+] was prolonged, presumably by Ca2+ influx. These results with fura-2-loaded platelets indicate that mobilisation of internal Ca2+ can contribute a substantial proportion of the early peak [Ca2+]i evoked by thrombin directly confirming the deductions from previous work with different loadings of quin2. Under natural conditions the major role of Ca2+ influx may be to prolong the [Ca2+]i rise rather than to make it larger.     

Activation by bacterial lipopolysaccharide causes changes in the cytosolic free calcium concentration in single peritoneal macrophages

Variations in the cytosolic free Ca2+ concentration [( Ca2+]i) upon LPS exposure were studied in single rat peritoneal macrophages loaded with fura-2 under carefully controlled conditions. Of a total of 60 cells examined, 47% responded to LPS (1 microgram/ml) with an increase in [Ca2+]i. Macrophages were heterogeneous with regard to the LPS response, with individual cells exhibiting single rapid and transient increases in [Ca2+]i, multiple transients, or slower and more sustained variations. In 62% of the responding cells, a second exposure to LPS elicited a [Ca2+]i rise, although usually to a slightly lower peak value. Thus, rapid desensitization to LPS does not occur in the majority of these macrophages. EGTA did not abolish the response of those cells that exhibited a single rapid transient in [Ca2+]i, indicating that the source of the initial [Ca2+]i rise was the intracellular stores. There was no obvious correlation between the type of response to LPS and the initial morphologic features (rounded vs polarized) of the cells. Our present work shows unequivocally that LPS induces increases in macrophage [Ca2+]i and, thereby, lends substantial support to the hypothesis that [Ca2+]i is a second messenger in LPS-mediated activation of the macrophage.     

Chemoattractant receptor promotion of Ca2+ influx across the plasma membrane of HL-60 cells. A role for cytosolic free calcium elevations and inositol 1,3,4,5-tetrakisphosphate production

The mechanisms by which the chemotactic peptide formyl-methyl-leucyl-phenyl-alanine stimulates Ca2+ influx across the plasma membrane were investigated in the human promyelocytic cell line HL-60, induced to differentiate with dimethyl sulfoxide. Ca2+ influx was determined: (a) from the initial rate of Mn2+ influx, apparent from the quenching of intracellular quin2 or fura-2 fluorescence; (b) from the rate of the elevation of cytosolic free calcium, [Ca2+]i, upon readdition of Ca2+ to cells previously stimulated in the absence of extracellular Ca2+. [3H]Inositol tris-, tetrakis-, and pentakisphosphates were analyzed by a high performance liquid chromatography procedure which was optimized for the separation of inositol tetrakisphosphates, yielding three predominant isomers: inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), inositol 1,4,5,6-tetrakisphosphate, and inositol 1,3,4, 6-tetrakisphosphate. Both the kinetics and agonist dose dependence of Ca2+ influx stimulation correlated closely with the corresponding receptor-mediated variations of [Ca2+]i either in the presence or in the absence of extracellular Ca2+. Of the different inositol phosphates determined in parallel and under the same conditions, accumulation of [3H]Ins(1,3,4,5)P4 correlated best with Ca2+ influx both temporally and in its dose dependence in the presence or in the absence of extracellular Ca2+; inositol 1,3,4-trisphosphate was also correlated but to a lesser extent. Attenuations of [Ca2+]i elevations by decreasing extracellular Ca2+ or by increasing the cytosolic Ca2+ buffering capacity with quin2 led to parallel inhibition of Ca2+ influx and Ins(1,3,4,5)P4 production. In conclusion: 1) activation of Ca2+ influx by formyl-methionyl-leucyl-phenylalanine depends on the elevation of [Ca2+]i, the latter being initiated by Ca2+ mobilization from intracellular stores; 2) Ins(1,3, 4,5)P4 is a strong candidate for maintaining receptor-mediated activation of Ca2+ influx in differentiated HL-60 cells.     

Cytosolic free calcium increases before and oscillates during frustrated phagocytosis in macrophages

When macrophages and neutrophils are allowed to settle onto an appropriate surface, they attach and spread in a frustrated attempt to phagocytose the substrate. Spreading is associated with extensive rearrangements of the actin cytoskeleton which resemble those occurring during phagocytosis. We have previously shown that spreading in human neutrophils is preceded by an increase in cytosolic-free calcium concentration [( Ca2+]i) (Kruskal, B. A., S. Shak, and F. R. Maxfield. 1986. Proc. Natl. Acad. Sci. USA. 83:2919-2923). To assess the generality of this signal, we measured [Ca2+]i in single thioglycollate- elicited mouse peritoneal macrophages as they spread on an immune complex-coated surface, using fura-2 microspectrofluorometry. A [Ca2+]i increase always precedes spreading. This increase can involve several (up to 8) [Ca2+]i spikes, with an average peak value of 387 +/- 227 nM (mean +/- SD, n = 92 peaks in 24 cells), before spreading is detected. Neither spreading nor the magnitude of these spikes is significantly altered by removal of extracellular calcium. Many of the spreading macrophages exhibit periodic [Ca2+]i increases before and during spreading. The proportion which does so varies among experiments from 0 to 90%, but it is frequently greater than 40%. The largest number of cells (approximately 25%) exhibited only a single peak. In 13 cells that showed more than 10 peaks, the median period was 29 s (range 19-69 s). The average peak [Ca2+]i was 385 +/- 266 nM (mean +/- SD, n = 208 peaks in 14 cells). The calcium producing these increases is derived from intracellular pools. The oscillations occur with spreading on either opsonized or nonopsonized surfaces. The function of these oscillations is not clear, but the large number of cells which exhibit them suggest that they may be important to macrophage function.     

Measurement of the matrix free Ca2+ concentration in heart mitochondria by entrapped fura-2 and quin2.

A method was developed to monitor continuously the matrix free Ca2+ concentration ([Ca2+]m) of heart mitochondria by use of the fluorescent Ca2+ indicators, fura-2 and quin2. The acetoxymethyl esters of fura-2 and quin2 were accumulated in and hydrolysed by isolated mitochondria. An increase of the mitochondrial Ca content from 0.3 nmol/mg of protein to 6 nmol/mg corresponded to a rise of [Ca2+]m from 30 to 1000 nM. The results indicate that physiological fluctuations of the mitochondrial Ca content elicit changes of [Ca2+]m in that range which regulates the matrix dehydrogenases.     

Parallel measurement of oxoglutarate dehydrogenase activity and matrix free Ca2+ in fura-2-loaded heart mitochondria

The entrapment of the Ca2+-sensitive fluorescence indicators fura-2 or quin2 in the matrix space of isolated heart mitochondria renders possible the direct monitoring of the matrix free Ca2+ [( Ca2+]m) [(1987) Biochem J. 248, 609-613]. In this paper the correlation between the [Ca2+]m and the in situ activity of oxoglutarate dehydrogenase (OGDH) in fura-2-loaded mitochondria is shown. At the initial value of [Ca2+]m, 64 nM, which corresponded to 0.36 nmol/mg mitochondrial Ca content, the OGDH activity was 12% of the maximal. Half-maximal and maximal activation were attained at 0.8 and 1.6 microM [Ca2+]m, respectively. The results indicate that an increase of the mitochondrial Ca content in the physiological range enhances the OGDH activity by means of elevation of [Ca2+]m.     

Parasite-induced processes for adenosine permeation in mouse erythrocytes infected with the malarial parasite Plasmodium yoelii.

In fura-2-loaded A10 vascular smooth-muscle cells, 1 nM-vasopressin and 200 nM-endothelin evoked a rapid transient rise in intracellular free Ca2+ concentration [( Ca2+]i), which was then followed by a maintained elevation of [Ca2+]i. The maintained elevation of [Ca2+]i was only partially inhibited by 5 microM-nifedipine, but completely abolished in the presence of 1 mM-EGTA. When extracellular Ca2+ was replaced with 1 mM-Mn2+ (Mn2+ quenches fura-2 fluorescence), both endothelin and vasopressin evoked an Mn2+ quench of the fluorescence from the intracellularly trapped fura-2, even in the presence of 5 microM-nifedipine. These data suggest that both vasopressin and endothelin promote a bivalent-cation influx and provide further evidence for receptor-mediated Ca2+ entry in vascular smooth muscle.     

Macrophage hydrogen peroxide production and phagocytic function are decreased following phagocytosis mediated by Fc receptors but not complement receptors

Previous in vivo and in vitro studies have shown that the phagocytosis of IgG-coated erythrocytes results in a depression of macrophage function. The present study compared the effect of phagocytosis mediated by Fc receptors with that mediated by complement receptors. The phagocytosis of IgG-coated erythrocytes by elicited peritoneal macrophages depressed their capacity to produce hydrogen peroxide as well as phagocytic function. Phagocytosis of erythrocytes coated with IgM and complement had neither of these effects. These results implicate the intracellular signaling that results from Fc receptor mediated phagocytosis in the depression of macrophage function that is caused by phagocytosis.     

Effect of arachidonic and other fatty acids on the intracellular calcium concentration and calcium signaling in peritoneal macrophages

Effects of arachidonic and other fatty acids on the intracellular Ca2+ concentration ([Ca2+]i) in rat peritoneal macrophages was investigated. It has been shown that cis-polyunsaturated arachidonic and linoleic induce a significant and dose-dependent increase in [Ca2+]i, which is due to depletion of thapsigargin-sensitive Ca2+ store and to stimulation of Ca2+ entry from the extracellular medium. Pharmacological characteristics of Ca2+ entry induced by arachidonic acid appeared to be similar to those of store-dependent Ca2+ entry activated by thapsigargin or cyclopiazonic acid; Ca2+ entry is attenuated by the same Ca2+ channel inhibitors, by tyrosine kinase inhibitor genistein and epoxygenase inhibitor proadifen. Cis-monounsaturated oleic and saturated myristic acids appeared to be less effective and induced only a slight increase in [Ca2+]i at much higher concentrations. Arachidonic and other fatty acids can also stimulate Ca(2+)-ATPase in the macrophage plasma membrane. The data are compatible with the important role played by arachidonic and other free fatty acids in the regulation of [Ca2+]i in peritoneal macrophages.     

Mechanisms of host defense against Candida species. I. Phagocytosis by monocytes and monocyte-derived macrophages

We studied the biochemical basis of phagocytosis of Candida albicans, a serious pathogen, and Candida parapsilosis, which is rarely pathogenic, by human monocytes (Mo) and monocyte-derived macrophages (MDM). Optimal phagocytosis of both species by Mo required the presence of extracellular Ca2+ and opsonization through both the classic and alternative complement pathways. Serum-opsonized Candida were ingested equally by Mo and MDM; unopsonized Candida were phagocytosed only by macrophages, and uptake began slowly. This opsonin-independent phagocytosis required Ca2+ and could be blocked by yeast mannan or mannose-BSA conjugate, suggesting a role for the mannose receptor. Opsonized Candida elicited a vigorous increase in the concentration of [Ca2+]i in Mo and MDM, but no Ca2+ transient was detected in MDM stimulated with unopsonized Candida. Pretreatment of MDM with ionomycin to increase [Ca2+]i had no effect on phagocytosis of unopsonized Candida. Addition of 5 mM EGTA completely inhibited changes in [Ca2+]i in Mo and MDM, suggesting that the Ca2+ transient induced by opsonized Candida is due to an influx of extracellular Ca2+. Differences in pathogenicity between the two Candida species could not be explained by differences in any aspect of phagocytosis. Uptake mediated by the macrophage mannose receptor could play a role in clearance of Candida under opsonin-poor conditions.     

Platelet activating factor raises intracellular calcium ion concentration in macrophages

Peritoneal cells from thioglycollate-stimulated mice were allowed to adhere to coverglasses for 2 h to give a dense monolayer of adherent cells greater than 95% of which were macrophages. After incubation with the tetra-acetoxymethyl ester of quin2, coverglasses were rinsed with Ca2+-free saline, oriented at a 45 degree angle in square cuvettes containing a magnetically driven stir bar, and analyzed for changes in quin2 fluorescence in a spectrofluorimeter. Such fluorescence, taken as an indication of intracellular calcium ion concentration ([Ca2+]i), increased as exogenous calcium ion concentration ([Ca2+]o) was raised to 1 mM. At [Ca2+]o approximately equal to 10 microM, [Ca2+]i = 72 +/- 14 nM (n = 26); at [Ca2+]o = 1 mM, [Ca2+]i = 140-220 nM, levels not increased by N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine, a membrane-permeant chelator of heavy metals than can quench quin2. Addition of mouse alpha + beta fibroblast interferon, lipopolysaccharide, thrombin, collagen, vasopressin, ADP, compound 48/80, or U46619 did not change [Ca2+]i. However, addition of platelet activating factor (PAF) (2-20 ng/ml) raised [Ca2+]i by 480 nM within 1 min if [Ca2+]o = 1 mM. In the presence of 5 mM EGTA, PAF raised [Ca2+]i by 25 nM. This suggests that PAF causes influx of exogenous Ca2+, as well as releasing some Ca2+ from intracellular stores. Consistent with these results, when PAF was added to 1 mM Ca2+ in the presence of 100 microM Cd2+ or Mn2+ to block Ca2+ influx, [Ca2+]i increased by only intermediate amounts; at the times of such dampened peak response, [Ca2+]i could be raised within 1 min to normal PAF-stimulated levels by chelation of the exogenous heavy metals with diethylenetriaminepentaacetic acid. Normal PAF responses were observed in the presence of indomethacin. The lowest dose of PAF observed to raise [Ca2+]i was 0.1 ng/ml. Response of [Ca2+]i to 2-20 ng/ml PAF was transient, and second applications had no effect. The PAF response also was seen in cell suspensions. These results suggest that an increase in [Ca2+]i may be an early event in PAF activation of macrophages.     

Intracellular Ca2+ shift and signal transduction from the tubulovesicular portion of gastric parietal cells during gastrin stimulation or Ca2+ ionophore treatment: comparison between luminescent and fluorescent probes, and electron probe X-ray microanalyzer

In gastrin-stimulated, aequorin-loaded parietal cells from guinea pig gastric mucosa, a rapid but transient increase in the cytosolic free Ca2+ concentration ([Ca2+]i), owing to Ca2+ released from the store(s), and a more prolonged Ca2+ entry from outside the cells were observed. However, there was a little increase in [Ca2+]i when similar measurements were assessed by quin 2 or fura-2 in physiological saline. However, depletion or elimination of Na+ from the incubation medium caused a significant increase in the [Ca2+]; response to gastrin as measured by quin 2. These findings suggest that aequorin and quin 2 (or fura-2) provide information about different aspects of Ca2+ homeostasis and that there is an inhomogeneity of [Ca2+]i in the cytoplasm during gastrin stimulation. By the gastrin stimulation, the intracellular Ca2+ gradients were shifted from the unidentified portion(s) to the restricted apical cytoplasm, as determined by electron probe X-ray microanalysis. Therefore, localization and identification of the source of intracellular Ca2+ as a pool were determined by an X-ray microanalyzer. In the resting state, the tubulovesicle had high Ca2+ concentration compared with the level in the apical cytoplasm. Cells treated with the Ca2+ ionophore ionomycin had a decreased tubulovesicular Ca2+ level, followed by a reciprocal increase in area of the canalicular membrane. The secretory canaliculus in stimulated cells had lower Ca2+ or higher K+ and Cl- concentrations than that of tubulovesicles or cytoplasm in the resting state, respectively. These findings suggest that the Ca2+ pool of the parietal cell is in the tubulovesicles and (or) luminal cell membrane and that the Ca2+ released from the store(s) may mediate a flow of K+ or Cl- into the secretory canaliculus.     

Cross-linking of IgG receptors inhibits membrane immunoglobulin- stimulated calcium influx in B lymphocytes

By cross-linking membrane immunoglobulins (mIg), the antigenic stimulation of B lymphocytes induces an increase in intracellular free calcium levels ([Ca2+]i) because of a combination of release from intracellular stores and transmembrane influx. It has been suggested that both events are linked, as in a number of other cases of receptor- induced increase in [Ca2+]i. Conversely, in B lymphocytes, type II receptors for the Fc fragment of IgG (Fc gamma RII) inhibit mIg- mediated signaling. Thus, we have investigated at the level of single cells if these receptors could act on specific phases of mIg Ca2+ signaling. Lipopolysaccharide-activated murine B splenocytes and B lymphoma cells transfected with intact or truncated Fc gamma RII-cDNA were used to determine the domains of Fc gamma RII implicated in the inhibition of the Ca2+ signal. [Ca2+]i was measured in single fura-2- loaded cells by microfluorometry. The phases of release from intracellular stores and of transmembrane influx were discriminated by using manganese, which quenches fura-2, in the external medium as a tracer for bivalent cation entry. The role of membrane potential was studied by recording [Ca2+]i in cells voltage-clamped using the perforated patch-clamp method. Cross-linking of mIgM or mIgG with F(ab')2 fragments of anti-Ig antibodies induced a sustained rise in [Ca2+]i due to an extremely fast and transitory release of Ca2+ from intracellular stores and a long lasting transmembrane Ca2+ influx. The phase of influx, but not that of release, was inhibited by membrane depolarization. The increase in [Ca2+]i occurred after a delay inversely related to the dose of ligand. Co-cross-linking mIgs and Fc gamma RII with intact anti-Ig antibodies only triggered transitory release of Ca2+ from intracellular stores but no Ca2+ influx, even when the cell was voltage-clamped at negative membrane potentials. These transitory Ca2+ rises had similar amplitudes and delays to those induced by cross-linking mIgs alone. Thus, our data show that Fc gamma RII does not mediate an overall inhibition of mIg signaling but specifically affects transmembrane Ca2+ influx without affecting the release of Ca2+ from intracellular stores. Furthermore, this inhibition is not mediated by cell depolarization. Thus, Fc gamma RII represents a tool to dissociate physiologically the phases of release and transmembrane influx of Ca2+ triggered through antigen receptors.