FcγRII-Mediated Superoxide Production by Phagocytes Is Augmented by GM-CSF Without a Change in FCγRII Expression

Abstract

Freshly purified neutrophils and monocytes respond to multiple cross-linking of Fc gamma RII with the IgG1 monoclonal antibody, CIKM5, with a rapid rise in Ca(2+)i, but not with a respiratory burst, although superoxide is generated by these cells when stimulated with the chemotactic peptide, FMLP, or phorbol ester (TPA). Incubation in vitro for 30-60 min at 37 degrees C in medium + 0.1% FCS had no effect on the neutrophil superoxide response to CIKM5 but induced a weak monocyte response in 11/13 experiments. However, incubation with rhGM-CSF (10 ng/ml) under similar conditions induced a neutrophil respiratory burst in response to cross-linking Fc gamma RII in 12/14 experiments and enhanced the monocyte response by 181%. GM-CSF also enhanced the response of neutrophils and monocytes to FMLP by 308% and 165%, respectively. The response to TPA was not significantly enhanced by GM-CSF. rhIFN-gamma (100 mu/ml) was ineffective as a priming agent for all agonists tested in short-term incubations but augmented the monocyte response to CIKM5 after 5 d exposure in vitro. Whilst GM-CSF induced neutrophil superoxide production in response to cross-linking Fc gamma RII, there was no concomitant change in Fc gamma RII expression either in in vitro studies of neutrophils from healthy individuals or in in vivo studies of patients receiving GM-CSF. Stimulation of unprimed neutrophils with CIKM5 induced a rapid transient increase in intracellular calcium levels to 181% of resting levels. However, incubation with GM-CSF did not further augment the calcium transients above the stimulated level. The mechanism by which GM-CSF induces an enhanced respiratory burst in response to cross-linking of Fc gamma RII remains to be elucidated, but is not related to receptor expression or increases in receptor mediated calcium mobilization.

Developmental regulation of granulocyte-macrophage colony-stimulating factor production during human monocyte-to-macrophage maturation

“…In addition to its function of cell recruitment, MAC-activating properties of GM-CSF [13,15,24] are well documented. Furthermore, GM-CSF leads to enhanced accessory function for T-cell activation in MAC [16,231, and it is an important survival factor for circulating dendritic cells [19].…”

“…GM-CSF, a glycoprotein of 18-23 kD [31], is a stimulator of stem-cell proliferation, leading to granulocyte colonies, granulocyte-MAC colonies, and some MAC colonies [12]. Similar to M-CSF, it seems to be involved in the modulation and activation of differentiated MO and MAC [13,16,24], in addition to its action on neutrophils. The main source of GM-CSF are activated T-cells, endothelial cells, and fibroblasts [10,22,27].…”

Activation and priming of neutrophil nicotinamide adenine dinucleotide phosphate oxidase and phospholipase A2 are dissociated by inhibitors of the kinases p42ERK2and p38SAPK and by methyl arachidonyl fluorophosphonate, the dual inhibitor of cytosolic and calcium-independent phospholipase A2

“…43 Sterile preparations and procedures were used throughout to minimize contact of cells with endotoxin and to reduce inadvertant priming. Cells were resuspended in PBS supplemented with 0.9 mM calcium, 0.5 mM magnesium, and 5 mM glucose (PBSG).…”

“…43 Purified human neutrophils (1 ϫ 10 6 cells per mL) were incubated in 1-mL cuvettes with or without inhibitors (PD98059 for 10 minutes or SB203580 for 30 minutes) prior to addition of either 500 U/mL TNF-␣, 10 ng/mL GM-CSF, or 0.01% FCS diluent for 30 minutes. The samples were stimulated with 1 M FMLP or 100 ng/mL C5a for 10 minutes, and the reactions were stopped with NEM at a final concentration of 2 mM.…”

Granulocyte‐macrophage colony stimulating factor receptor α and β chain complexes can form both high and intermediate affinity functional receptors

“…The mouse NIH3T3 fibroblast cell line was maintained in Dulbecco's Minimal Essential Medium (DMEM), supplemented with 10% myoclone super plus FCS (Gibco BRL). Peripheral blood granulocytes and monocytes were prepared from healthy subjects using the method of Roberts et al (1990).…”