The mechanism of macrophage activation induced by Ca2+ ionophore

doi:10.1016/0008-8749(83)90323-4

Cellular Immunology

Volume 75, Issue 2, 1 February 1983, Pages 242-254

https://doi.org/10.1016/0008-8749(83)90323-4 Get rights and content

Abstract

The mechanism of macrophage activation by Ca²⁺ ionophore was studied. Peritoneal exudate macrophages from normal guinea pigs exposed continuously to or pulse treated for 1 hr with the ionophore, A23187, were activated, manifesting increased glucose consumption and inhibition of migration. Highly purified macrophages were also activated as effectively as crude macrophage preparations, and the culture supernatant of spleen lymphocytes treated with A23187 lacked a macrophage activating effect, showing that the macrophage activation resulted from the direct effect of A23187 on macrophages, not via lymphokines produced by lymphocytes. The macrophage activation by A23187 was suppressed in the presence of EGTA, but the suppressive effect was overcome by the addition of Ca²⁺, but not of Mg²⁺. A dilution experiment with Ca²⁺ and Mg²⁺ during the pulse treatment of cells with A23187 revealed that the activating effect of A23187 was more dependent on Ca²⁺ content than Mg²⁺. In addition, the Ca²⁺ antagonist, nicardipine, was found to suppress the activating effect of A23187. The Ca²⁺ uptake into macrophages was increased by treatment with A23187. These results indicate that Ca²⁺ influx into cells is primarily important in the macrophage activating effect of A23187. Trifluoperazine (TFP: a specific inhibitor of calmodulin that is an intracellular Ca²⁺ receptor protein) was found to inhibit the activating effect of A23187. Further, the cyclic nucleotides, dibutyryl-cAMP and −cGMP, did not activate macrophages. Therefore, macrophage activation was presumed not to be directly mediated by cyclic nucleotides. All these findings show that macrophage activation with the ionophore proceeds by the following scheme: Ca²⁺ influx → activation of Ca²⁺ receptor protein, calmodulin → activation of calmodulin-regulated enzymes → metabolic changes, activation. TFP was found to suppress the macrophage activation with highly purified guinea pig macrophage activation factor/macrophage migration inhibitory factor (MIF/MAF) or lipopoly-saccharide (LPS), suggesting that calmodulin also played an important role in macrophage activation with MIF/MAF or LPS.

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Our findings indicated that the nano/micro-topographies of bioceramics resulted in a differential integrin expression, which might trigger the intracellular signaling cascade to modulate macrophage polarization and subsequently affect the repair effects. The Ca2+ concentration has a big influence on macrophage behaviors [51]. Chen et al. found that a decrease in Ca2+ concentration inhibits the Wnt/Ca2+ signaling pathway, leading to anti-inflammatory effects [52].
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Determination of the oxidative burst chemiluminescent response of avian and murine-derived macrophages versus corresponding cell lines in relation to stimulation with Salmonella serotypes
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In contrast to mammalian systems, avian species lack a resident or harvestable macrophage population in the abdominal exudate. Peritoneal macrophages in the chicken can be elicited if an inflammatory agent such as sephadex is injected. This study examines the kinetics of different macrophage populations, derived by different methods of isolation and from different hosts, with respect to the elicited oxidative burst upon infection with host-adapted Salmonella serotypes.
The nature of the oxidative burst elicited by murine and avian-derived and cell line macrophages was determined after stimulation with phorbol myristate (PMA), zymosan A, and Salmonella serotypes. Both murine and chicken peritoneal macrophages, chicken blood monocytes and corresponding cell lines, J774A.1 and HD-11, were unable to produce a detectable chemiluminescent (CL) response after interaction with Salmonella using the luminescent probe luminol. However, both PMA and zymosan A induced a CL response in all cell types, with PMA eliciting a higher and earlier peak response (pkH) than zymosan A. Lucigenin-enhanced CL in both murine and chicken macrophages was achieved with PMA, zymosan A and Salmonella serotypes. In this case, zymosan A induced higher responses than PMA. In the peritoneal macrophages of both hosts, there were no significant differences in the oxidative burst induced by the different Salmonella serotypes. However, the J774A.1 (murine) cells demonstrated significant differences, with S. enterica serotype Choleraesuis (S. choleraesuis and S. gallinarum producing the highest response. In the HD-11 (chicken) cells, S. choleraesuis and S. dublin elicited the higher CL. With both cell lines, S. abortusovis failed to induce an appreciable CL response.
In these experiments it was demonstrated that oxidative burst was not detectable in monocytes/macrophage populations using luminol, which suggests a link to the lack of a myeloperoxidase system in these cells. Lucigenin-enhanced CL appeared independent from the myeloperoxidase system, indicating production of another oxidative species compared with luminol. No discernable effect of host specificity with regard to Salmonella serotype and respective host was seen in host-derived or cell line macrophages, and cell line macrophages displayed altered functional characteristics with regard to oxidative burst in comparison with their primary counterparts.
Induction of the respiratory burst in turtle peritoneal macrophages by Salmonella muenchen
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Peritoneal macrophages were collected from juvenile turtles 72 h after intraperitoneal inoculation with a 3% Sephadex suspension. The macrophages were assayed for their chemiluminescent (CL) properties, reflecting their respiratory burst activity, after stimulation with Zymosan A, phorbol 12-myristate 13-acetate (PMA), N-formyl-methionyl-leucyl-phenylalanine (fMLP), and calcium ionophore A23187. Except for fMLP, all triggering agents induced a marked CL response. Luminol was used as the chemiluminescent probe. When comparing CL responses in temperatures ranging from 15 to 35°C, lower assay temperatures induced lower and slower CL responses. Stimulation with viable Salmonella muenchen resulted in a distinct response. Bacteria, inactivated by means of heat or acetone, induced a faster and stronger oxidative burst. Opsonization of either viable or heat-inactivated S. muenchen with non-inactivated anti-S. muenchen serum, prepared in turtles, induced faster and higher CL responses. On the other hand, opsonization of acetone-inactivated S. muenchen caused CL responses to be slower and weaker. S. muenchen, opsonized with heat-inactivated turtle anti S. muenchen serum, induced higher responses than non-opsonized bacteria, but slower and weaker responses than bacteria opsonized with native turtle antiserum. No response was recorded after stimulation with LPS and the supernatant of heat-inactivated bacteria.
Low-calcium peritoneal dialysis fluid should not impact peritonitis rates in continuous ambulatory peritoneal dialysis
1996, American Journal of Kidney Diseases
It has been suggested that reducing the calcium content of peritoneal dialysis fluid (PDF) to 2.5 mEq/L decreases peritoneal macrophage (PMO) function and increases the incidence of peritonitis (especially Staphylococcus epidermidis peritonitis) in continuous ambulatory peritoneal dialysis patients. We studied the uptake and killing of S epidermidis and Escherichia coli by PMOs and peripheral blood leukocytes incubated in control buffer (Hank's balanced salt solution containing 0.1% gelatin [GHBSS]) and PDF containing varying concentrations of calcium (0 to 3.5 mEq/L) and magnesium (0 to 1.5 mEq/L) using ether diamine tetraacetic acid and ethylenediaminetetraacetic acid chelation, respectively. In addition, interleukin-1β-induced interleukin-6 production by human mesothelial cells was measured in the presence of concentrations of calcium increasing from 0 to 3.0 mmol/L. Fc receptor-mediated uptake of S epidermidis by PMO in the complete absence of Ca++ was comparable to that by PMO incubated in GHBSS with calcium. In contrast, the complement-dependent uptake of E coli was significantly lower in GHBSS devoid of Ca++ (46% ± 5% v 24% ± 3%; 0.05 < P < 0.02). No effect on intracellular killing of either microorganism by PMO was observed. The same held true for the phagocytic and killing capacity of polymorphonuclear granulocytes and monocytes obtained from healthy donors. Using Ca++ (2 to 3.5 mEq/L) and Mg++ (0.5 to 1.5 mEq/L) concentrations as applied in commercial PDFs, however, phagocytes performed as well as in control buffer. Interleukin-6 production by stimulated human mesothelial cells also required a small amount of Ca++ only, being normal above the 0.1 to 3 mmol/L Ca++ range tested. Thus, complement-dependent uptake of bacteria by phagocytes is calcium dependent, whereas antibody-dependent uptake of S epidermidis is not. The concentrations of calcium in the current PDFs, however, will not compromise human mesothelial cells and leukocyte functions, and therefore should not impact the peritonitis rate.
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We demonstrated that a 65-kDa cytosolic protein (pp65) was most heavily phosphorylated in murine macrophages in response to bacterial lipopolysaccharide (J. Immunol. 146:3617,1991). The pp65 phosphorylation closely correlated with cellular responses in the macrophages. We have thus performed amino acid sequence analysis of the two pp65-derived peptide fragments in order to elucidate its structure and function. The results indicated that pp65 is a novel protein existing almost exclusively in hemopoitetic cells and has the strong homology with human L-plastin, a substrate which has recently been identified as a novel protein transformation-dependently expressed in neoplastic human cells. Our pp65 is apparently the first purified protein whose phosphorylation has been augmented by stimulation with bacterial lipopolysaccharide and whose primary structure has been clarified. Because L-plastin possesses the unique functional domains, detailed investigation of the pp65 phosphorylation should lead to progress in our understanding of the mechanisms of macrophage activation.
Intracellular protein phosphorylation in murine peritoneal macrophages in response to bacterial lipopolysaccharide (LPS): Effects of kinase-inhibitors and LPS-induced tolerance
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Intracellular protein phosphorylation is thought to be the initial step in cell activation. Bacterial lipopolysaccharide (LPS) induces a special set of the protein phosphorylation in the murine peritoneal macrophages, including p65 (molecular mass of 65 kDa) which is a substrate of serine kinase and the most dominant phosphorylated cytosolic protein. This article deals with the relation between the LPS-induced protein phosphorylation in the murine peritoneal macrophages and their productions of IL-1β and TNF-α. LPS-induced p65 phosphorylation seems to be dependent on protein kinase C (PKC) and calmodulin (CaM), because it diminishes in the presence of inhibitors to PKC or CaM. Tyrosine kinase inhibitors do not affect the p65 phosphorylation. The PKC inhibitors also affect the mRNA expressions and the productions of active molecules of IL-1β and TNF-α. Though the CaM inhibitor inhibits the mRNA expression and the active molecule production of IL-1β, it does not affect those of TNF-α. These results suggest that LPS-induced p65 phosphorylation is closely related to PKC and CaM, and that IL-1β production depends on PKC and CaM, while the TNF-α production is not dependent on CaM. These findings indicate the existances of multiple pathways and different regulatory mechanisms for transduction of LPS signal in the macrophages. Furthermore, LPS-induced phosphorylation is not observed in endotoxin tolerant macrophages after re-stimulation with LPS, suggesting that the LPS-stimulus signal is blocked at a site in the signal transduction-pathway before the point of phosphorylation of proteins in the tolerant macrophages.

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The mechanism of macrophage activation induced by Ca2+ ionophore

Abstract

Cell. Immunol

Cell. Immunol

Cell. Immunol

Biochem. Pharmacol

Biochem. Biophys. Res. Commun

Cell. Immunol

Biochem. Biophys. Res. Commun

Biochem. Biophys. Res. Commun

Cell. Immunol

Cell. Immunol

Advan. Immunol

Science

J. Exp. Med

The mechanism of macrophage activation induced by Ca²⁺ ionophore