Simultaneous mobilization of Mac-1 (CD11b/CD18) and formyl peptide chemoattractant receptors in human neutrophils [published erratum appears in Blood 1993 Mar 15;81(6):1668]

Abstract

Mobilization of a distinct subset of specific granules provides a physiologically important mechanism to recruit Mac-1 (CD11b/CD18) from an intracellular pool to the external surface of the neutrophil plasma membrane, where the functionally active heterodimer mediates several adherence-dependent processes that are crucial for adequate host defense and cellular inflammatory responses. We observed similar characteristics for translocation of Mac-1 and neutrophil formyl peptide receptors (FPR) and hypothesize that the readily accessible pools of both Mac-1 and FPR are colocalized within this specific granule subset. Plasma membrane levels of both FPR (assessed with 3H- FMLP) and Mac-1 (assessed by fluorescence-activated cell sorter analysis of fluorescein isothiocyanate [FITC]-Mo-1-labeled cells) were markedly downregulated in cells prepared at low temperature from blood cooled to 4 degrees C immediately after removal from the circulation. Levels of both FPR and Mac-1 remained low on cells held at 4 degrees C. Upon warming, spontaneous upregulation of Mac-1 and FPR occurred with similar kinetics and temperature dependency. Translocation of both Mac- 1 and FPR was markedly potentiated by exposure of cells to either fluoride ion (which has been shown by others to specifically elicit exocytosis of gelatinase-rich and vitamin B-12 binding protein-poor granules) or granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine that markedly potentiates the neutrophils' host defense capabilities. Levels of both FPR and Mac-1 on F-- or GM-CSF-treated neutrophils exceeded those present on cells incubated at 37 degrees C for extended time intervals, indicating that stimulated translocation may involve mobilization of an additional granule subset. Scatchard analysis showed that only low-affinity FPR were translocated during spontaneous and stimulus-dependent upregulation. To directly compare FPR levels on the surface of cells displaying varying levels of Mac-1 within a single cell suspension, cells were labeled with FITC-Mo-1 and sorted into subpopulations based on fluorescence intensity. After sorting, the individual populations were held at 4 degrees C to prevent further spontaneous upregulation, concentrated by centrifugation, and assayed for FPR levels. Under a variety of conditions, FPR levels correlated with Mac-1 (CD11b) expression on cell populations selected on the basis of CD11b fluorescence intensity. Analysis of subcellular fractions obtained from disrupted neutrophils before and after upregulation provided additional support for the hypothesis that Mac-1 and FPR are colocalized within a readily accessible subset of neutrophil granules.

Subcellular localization and translocation of the receptor for N-formylmethionyl-leucyl-phenylalanine in human neutrophils

“…Inflammatory stimuli known to mobilize secretory vesicles without major release of other granules are fMLP, PAF, zymosan-opsonized serum, granulocyte-macrophage colonystimulating factor (GM-CSF), tumour necrosis factor (TNF), interleukin 8 and leukotriene B4 (Borregaard et al, 1990(Borregaard et al, , 1992, and all of these will consequently up-regulate fMLP receptors. In accordance, fMLP-receptor up-regulation has been demonstrated for fMLP (Norgauer et al, 1991), TNF and PAF (O'Flaherty et al, 1991), and GM-CSF (English et al, 1988;Graves et al, 1992). Interestingly, Graves et al (1992) described parallel up-regulation characteristics of fMLP receptors and Mac-I (CDl lb/CD18) and concluded that the gelatinase granules were the common housing compartment for these receptors.…”

“…In accordance, fMLP-receptor up-regulation has been demonstrated for fMLP (Norgauer et al, 1991), TNF and PAF (O'Flaherty et al, 1991), and GM-CSF (English et al, 1988;Graves et al, 1992). Interestingly, Graves et al (1992) described parallel up-regulation characteristics of fMLP receptors and Mac-I (CDl lb/CD18) and concluded that the gelatinase granules were the common housing compartment for these receptors. However, the existence of a mobilizable light-membrane compartment in subcellular-fractionation experiments was not considered in that study, although the surface up-regulation of fMLP receptors and Mac-I was far greater than translocation observed in subcellular fractions of the density gradient.…”

“…One freeze-thaw cycle or even storage overnight in the refrigerator enhanced the labelling in the absence of detergent considerably. This granule fragility in the fMLP-receptor assay might explain that previous studies have demonstrated presence of fMLP receptors in fractions containing specific/gelatinase granules, without any solubilization or permeabilization of the granule membrane (Graves et al, 1992;Fletcher and Gallin 1983;Jesaitis et al, 1982). The latency of the fMLP receptors in the y-band disappeared after stimulation with PAF.…”

“…labelling of NGAL illustrates the usefulness of a receptor assay in which the molecular mass of the labelled species can be evaluated. Furthermore, it casts some doubt on the accuracy of the quantification of fMLP receptors done on subcellular fractions by [3H]fMLP binding in previous studies (Fletcher and Gallin, 1983;Graves et al, 1992). This study was supported by grants from The Danish Cancer Society , Danish Hospital Foundation for Medical Research, Anders Hasselbalchs Fund, Emil C. Hertz' Fund, The National Association against Lung Disease, Novo-Nordisk Fund, Amalie Jorgensens Fund, Ane Catrine Plesners Fund, Lundbecks Fund, and The Danish Medical Research Council. N.B.…”

“…the subcellular localization of fMLP receptors. The data presented so far on the subcellular localization have shown fMLP receptors to be present in specific granules (Fletcher and Gallin, 1983;Jesaitis et al, 1982) and possibly gelatinase granules (Graves et al, 1992). Although we have documented the existence of gelatinase granules (Kjeldsen et al, 1993a), the kinetics of up-regulation of fMLP-receptors induced by weak stimulation is in conflict with the mobilization membrane upon stimulation with inflammatory mediators.…”