Chemotactic peptide-induced cytoplasmic pH changes in incubated human monocytes.

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 degrees C) and examined pHi using the fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF; 1 microM) and a Perkin-Elmer 650/10 spectrofluorimeter (lambda em = 530 nm, lambda ex = 500, 450 nm) as previously described. The resting pHi 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 delta pHi 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 dimethylamiloride (20 microM). 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 dimethylamiloride, 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 monocytes/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.

Simultaneous flow cytometric measurements of cytoplasmic Ca++ and membrane potential changes upon FMLP exposure as HL-60 cells mature into granulocytes: using [Ca++]in as an indicator of granulocyte maturity.

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 Indo-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.

NMR chemical shift imaging.

Chemical shift imaging combines the spatial information provided by a conventional nuclear magnetic resonance (NMR) image with the chemical shift spectral information provided by NMR spectroscopy. In order to preserve the chemical shift information and provide a spatial map simultaneously, new NMR imaging methods have been developed. In general, these methods have taken two forms: (a) three-dimensional techniques which add an extra axis of information--chemical shift spectral data--to a planar (2-D) image; and (b) two-dimensional techniques which, in certain circumstances, allow one to use techniques only slightly different from conventional ones to obtain high-resolution images of particular chemical shift species. Each of these methods offers unique challenges to the imager, as well as special advantages. In particular, three-dimensional techniques offer the opportunity to visualize the chemical shift spectra explicitly, while two-dimensional techniques allow for rapid imaging times and high spatial resolution. Most of the work in chemical shift imaging to date has focused on 1H, 31P, and 23Na. The high concentration of water and lipids in biological tissue has made the proton especially amenable to study, and the ability to sample other proton-containing compounds (such as lactate) in the face of high concentration lipid and water is now being explored. The potential use of chemical shift imaging techniques in the research and clinical settings is currently under investigation.

Time dependence of transmembrane potential changes and intracellular calcium flux in stimulated human monocytes.

An important characteristic of the functional differentiation of the blood monocyte is the development of its capacity to recognize and respond to stimuli. This ability is mediated to a large extent by specific receptor glycoproteins located on the cell surface. Stimulation of mononuclear phagocytes via these receptors results in a rapid rise in intracellular Ca++ concentration, accompanied or followed by a change in membrane potential, generation of oxidative products, degranulation, and effector functions such as phagocytosis, aggregation, or locomotion. While the development of these characteristics is difficult to characterize in vivo, several investigators have demonstrated in vitro changes in these cells that correlate with the development of effector function. To examine the mechanisms of specific membrane-stimulus interactions of monocytes as they differentiate into macrophage-like cells, we studied the responses of human monocytes and of monocytes incubated in serum-containing medium for up to 96 hr to the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP). Freshly isolated monocytes exhibited little change in transmembrane potential following stimulation with an optimal concentration of peptide and underwent a significant increase only after 48 hr in culture. While constant resting intracellular Ca++ concentrations were maintained during the culture period, intracellular Ca++ levels following fMLP stimulation increased with with incubation in serum, for up to 96 hr. In contrast, fMLP-induced respiratory burst activity increased from 0 to 24 hr in culture; it remained elevated at 48 hr but declined again by 96 hr. Incubation of the cells for 24 hr increased their random (unstimulated) motility in modified Boyden chambers but did not alter the cells' directed (chemotactic) response to fMLP in comparison to the response of freshly isolated monocytes. Peptide binding to the cells did not increase during the incubation period, indicating that an increase in receptor number or in affinity for fMLP was not responsible for the enhanced responsiveness to fMLP as incubation time increased. These studies indicate that incubation of monocytes in serum-containing medium leads to a complex, altered series of responses to fMLP that correlate with the differentiation of the original monocytes in vitro and may relate to the in vivo differentiation of monocytes to macrophages.