Interaction of chemotactic factors with human polymorphonuclear leukocytes: Studies using a membrane potential-sensitive cyanine dye

Summary Changes in the fluorescence intensity of the dye 3-3 dipentyloxacarbocyanine were measured in suspensions of purified human peripheral blood polymorphonuclear leukocytes (PMNs) during exposure to the chemotactic factors N-formyl-methionylleucyl-phenylalanine (f-met-leu-phe) and partially purified C5a. Incubation of PMNs with dye resulted in a stable fluorescence reflecting the resting membrane potential of the cell. Exposure of PMNs to dye did not affect stimulated chemotaxis or secretion. The mechanism of cell-associated dye fluorescence involved solvent effects from partitioning of the dye between the aqueous incubation medium and the cell and not dye aggregation, Chemotactically active concentrations of f-met-leu-phe (5×10^–9 m or greater) produced a biphasic response characterized as a decrease followed by an increase in fluorescence. No fluorescence response was seen in lysed PMNs, and no response was elicited by an inhibitor of f-met-leu-phe binding (carbobenzoxy-phenylalanyl-methionine). The ability of several other synthetic peptides to elicit a fluorescence response corresponded to their effectiveness as chemotactic agents. Although the first component of the response suggested a depolarization, it was not influenced by variation in the external concentration of sodium, potassium, chloride, or calcium, and could not be characterized as a membrane potential change. The second component of the response, which was inhibited by both Mg²⁺ (10mm)-EGTA (10mm) and high external potassium, was compatible with a membrane hyperpolarization. The data indicate that chemotactic factors produce changes in dye fluorescence which can, at least in part, be attributed to a hyperpolarizing membrane potential change occurring across the plasma membrane.Presented in part at the 17th Annual Cell Biology Meeting.Cell Biol. 75:103a, 1977.     

Plasma membrane potential modulates chemotactic peptide-stimulated cytosolic free Ca2+ changes in human neutrophils

The relationship between fMet-Leu-Phe-induced changes in the cytosolic free Ca2+ concentration [( Ca2+]i), plasma membrane potential depolarization, and metabolic responses was studied in human neutrophils. Receptor-activated depolarization occurred both at high and resting [Ca2+]i, but was inhibited at very low [Ca2+]i. Phorbol 12-myristate 13-acetate-induced plasma membrane depolarization, on the contrary, was independent of [Ca2+]i. The threshold fMet-Leu-Phe concentration for plasma membrane depolarization (10(-8) M) was at least 1 log unit higher than that for [Ca2+]i increases (5 X 10(-10) M) and coincident with that for NADPH oxidase activation. Nearly maximal [Ca2+]i increases were elicited by 3 X 10(-9) fMet-Leu-Phe in the absence of any significant plasma membrane potential change. This observation allowed us to investigate the effects of artificially induced plasma membrane depolarization and hyperpolarization at low fMet-Leu-Phe concentrations (10(-9) to 3 X 10(-9) M) which did not perturb plasma membrane potential. Depolarizing (gramicidin D at 10(-7) to 10(-6) M or KCl at 50 mM) and hyperpolarizing (valinomycin at 4 microM) treatments had little influence on unstimulated [Ca2+]i levels, whereas fMet-Leu-Phe-induced transients were significantly altered. Gramicidin D and KCl decreased the fMet-Leu-Phe-induced [Ca2+]i increases in Ca2+-containing or in Ca2+-free media. Valinomycin, on the contrary, increased receptor-stimulated [Ca2+]i increases, and the effect was larger in the presence of extracellular Ca2+. Valinomycin also strongly potentiated secretion. It is suggested that plasma membrane depolarization in human neutrophils is a physiological feedback mechanism inhibiting receptor-dependent [Ca2+]i changes.     

Prostaglandin modulation of N-formylmethionylleucylphenylalanine-induced transmembrane potential changes in rat neutrophils

Prostaglandins of the E-series (PGEs) and PGI2 will inhibit formylmethionylleucylphenylalanine-(f-Met-Leu-Phe) induced lysosomal enzyme release and superoxide-anion (O2-) production by neutrophils. The inhibitory effects of PGEs and PGI2 on neutrophil functional responses have been correlated with their ability to increase intracellular cAMP. In this study we have examined the effects of PGEs and PGI2 on f-Met-Leu-Phe- and phorbol-myristate-acetate-induced rat neutrophil membrane potential changes using an optical probe of membrane potential 3,3-dipropylthiodicarbocyanine iodide. 15-(S)-15-methyl-PGE1 (15-methyl-PGE1), a stable analogue of PGE1 and PGI2 inhibited f-Met-Leu-Phe-induced transmembrane potential changes in a dose-dependent manner. This inhibition was correlated with the ability of these agents to increase intracellular cAMP levels and inhibit O2- production and degranulation. In contrast, 15-methyl-PGE1 and PGI2, did not inhibit phorbol-myristate-acetate-induced transmembrane potential changes and O2- production. These results suggest independent mechanisms of activation of neutrophils by phorbol myristate acetate and f-Met-Leu-Phe, and they also suggest that the inhibitory effects of prostaglandins and cAMP on f-Met-Leu-Phe-stimulated cells is at a step or steps prior to activation of those processes involved in effecting changes in transmembrane potential, which are common to both stimuli.     

Low-temperature induced transmembrane potential changes in the liverwort Conocephalum conicum

Intracellular microelectrode measurements revealed that the liverwort Conocephalum conicum generates all-or-none action potentials (APs) in response to a sudden temperature drop. In plants with anion and potassium conductance blocked, dose-dependent voltage transients (VTs) were evoked by cold stimuli. These VTs did not propagate. When the external concentration of Ca(2+) was decreased or calcium channel inhibitors (La(3+), Gd(3+), verapamil, Mg(2+), Mn(2+)) were used, inhibition of VTs was observed. Amplitudes of both APs and VTs grew when Sr(2+) ions, known to release calcium from internal stores, were added to the medium. Neomycin, which suppresses phospholipase C and indirectly affects inositol triphosphate formation, caused substantial inhibition of both APs and VTs. It is concluded that a temperature drop elucidated membrane potential changes due to calcium influx both from external and internal stores.     

Interaction of LDL with human arterial proteoglycans stimulates its uptake by human monocyte-derived macrophages

The aim of this work was to investigate the possible mechanisms for uptake by human monocyte-derived macrophages (HMDM) of low density lipoprotein (LDL) pretreated with human arterial chondroitin-6-SO4-rich proteoglycan (LDL-PG). HMDM were incubated with 125I-labeled tyramine cellobiose-labeled LDL-PG, native LDL, and acetylated LDL (Ac-LDL). The results showed that two to four times more LDL-PG than LDL was bound and internalized by the HMDM. Competition experiments showed that LDL-PG competed with native LDL for the apoB,E (LDL) receptor, but not for the Ac-LDL scavenger receptor. Both the LDL and LDL-PG uptake were reduced after preincubation of the macrophages with unlabeled native LDL, though to a lesser extent with LDL-PG. The specific binding of 125I-labeled LDL and 125I-labeled LDL-PG at 4 degrees C was both saturable and concentration-dependent. The dissociation constant (Kd) for binding was 8.6 x 10(-9) M for LDL and 9.4 x 10(-9) M for LDL-PG, but the maximum binding (Bmax) was 1.5-times higher for LDL-PG. Cholesterol derived from LDL-PG was less effective than native LDL in suppressing HMG-CoA reductase activity. The results indicate that the uptake of LDL-PG is mediated not only by the LDL-receptor, but also by another unspecific pathway, which may not be subjected to regulation. These results provide further support for the hypothesis that LDL modifications induced by arterial PG may contribute to the formation of foam cells.     

Production of cyclooxygenase products and superoxide anion by macrophages in response to chemotactic factors

Mononuclear phagocytes are known to play a key role in various phlogistic reactions by synthesizing and releasing products that may potentiate or inhibit inflammatory processes. The expression of these products appears to be dependent on the source of the macrophage population as well as the stimulus employed. We have studied superoxide anion (O-2) production as well as the generation of PGE2, PGF2 alpha, and TXB2 from resident, oil-elicited and thioglycollate-induced peritoneal macrophages in mice in the presence and absence of chemotactic peptides. Production of O-2, occurred only in elicited macrophages stimulated with high concentrations of FMLP or C5a; resident cells stimulated with either of the chemotactic peptides were completely unresponsive. Although resident peritoneal macrophages incubated with chemotactic peptides did not generate O-2, these cells did secrete significant levels of PGE2, PGF2 alpha, and TXB2 in response to C5a. FMLP had no stimulatory effect. Elicited macrophages generated increased levels of PGE2 and PGF2 alpha when incubated with C5a. However, production of TXB2 was not stimulated. FMLP was inactive in stimulating PGE2, PGF2 alpha, and TXB2 in all types of macrophages studied. These studies indicate a heterogeneity in the production of inflammatory mediators from various macrophage populations in response to chemotactic factors.     

Production of cyclooxygenase products and superoxide anion by macrophages in response to chemotactic factors

Mononuclear phagocytes are knwon to play a key role in various phlogistic reactions by synthesizing and releasing products that may potentiate or inhibit inflammatory processes. The expression of these products appears to be dependent on the source of the macrophage population as well as the stimulus employed. We have studied superoxide anion (O₂⁻) production as well as the generation of PGE₂, PGF_2α, and TXB₂ from resident, oil-elicited and thiogylcollate-induced peritoneal macrophages in mice in the presence and absence of chemotactic peptides. Production of O₂⁻, occurred only in elicited macrophages stimulated with high concentrations of FMLP or C5a; resident cells stimulated with either of the chemotactic peptides were completely unresponsive. Although resident peritoneal macrophages incubated with chemotactic peptides did not generate O₂⁻, these cells did secrete significant levels of PGE₂, PGF_2α, and TXB₂ in response to C5a. FMLP had no stimulatory effect. Elicited macrophages generated increased levels of PGE₂ and PGF_2α when incubated with C5a. However, production of TXB₂ was not stimulated. FMLP was inactive in stimulating PGE₂, PGF_2α, and TXB₂ in all types of macrophages studied. These studies indicate a heterogeneity in the production of inflammatory mediators from various macrophage populations in response to chemotactic factors.     

Experimental and functional changes in transmembrane potential and zeta potential of single cultured cells

Experiments were performed on single cells to investigate the relations between the total bioelectrical potential difference (PD) across the cell membrane (so-called transmembrane potential) and the net negative surface charge of the cell (zeta potential). The experiments were carried out on FL-cells, leucocytes and ovarian tumour cells. The PD was measured electrophysiologically by means of intracellular glass microelectrodes; the surface charge or the zeta potential was determined using cell electrophoresis. Both measuring methods are critically discussed.Under different conditions (hypothermia, hyperthermia, mitotic blocking agent, cell cycle), the transmembrane potential and zeta potential showed changes in an identical direction and often the response of transmembrane potential was found to be quicker and more intensive than that of the zeta potential. In other experiments (e.g. changing the extracellular Cl^? ion concentration) the reactions of both potentials showed no coincidence. Depending on the type of functionally or experimentally borne changes on the cytoplasmatic membrane, either both potentials or only one of them may be altered.     

Interaction of Candida albicans with macrophages

Study of the in vitro interaction of mouse peritoneal macrophages with C. albicans has revealed that these macrophages, though easily phagocytizing C. albicans blastospores, are incapable of destroying the fungus. The phagocytic, but not candidostatic, activity of these macrophages has been found to depend on the conditions on their cultivation, as well as on the age and viability of fungal cells. The representatives of 7 C. albicans strains, obtained from various sources and stored at the museum for different spans of time, have shown practically no difference in the character of their interaction with mouse peritoneal macrophages.     

Interaction of leishmania metacyclics with macrophages

Metacyclics of L. major and putative metacyclics of L. m. mexicana survived better in explanted murine macrophages than promastigotes from mid-log phase cultures. The latter forms, however, attached in greater numbers to macrophages and, in the case of L. major, also more became intracellular. Only a small percentage of the macrophages infected with amastigotes exhibited a respiratory burst (as detected by nitroblue tetrazolium (NBT) reduction), whereas this occurred with most of the macrophages infected with either metacyclic or non-infective promastigotes of L. major. Approximately half of the macrophages infected with L. m. mexicana promastigotes reduced NBT. The results suggest that avoidance of the oxygen metabolite arm of the host cell's microbicidal activity is not the main survival strategy for metacyclics entering macrophages. Metacyclics of L. major, however, were found to be less sensitive than mid-log phase promastigotes to hydrogen peroxide and also human serum; properties which may aid their survival.     

Interaction of T-activin with peritoneal macrophages

The action of T-activin on peritoneal macrophages of CBA mice after its introduction into the animals has been studied. In intact mice the phagocytic activity of macrophages and their resistance to the cytopathogenic action of Salmonella typhimurium live cells remains unchanged. The injection of corpuscular pertussis vaccine into mice leads to a decrease in the resistance of macrophages to the action of salmonellae. The simultaneous injection of T-activin into mice in doses of 0.1 and 1.0 microgram per animal abolishes the damaging action of the vaccine. The analysis of the in vitro action of T-activin on macrophages of intact mice revealed that the preliminary incubation of cells with the preparation sharply increases their resistance to the action of salmonellae, while its introduction simultaneously with bacteria or after them rapidly leads to the death of macrophages. The action of T-activin is supposed to be linked with triggering the biosynthetic processes mediating the resistance of macrophages to the cytopathogenic action of salmonellae.     

Fluorimetric determination of the resting potential changes associated with the chemotactic response in Paramecium

1. (1) Evidence is presented which indicates that the carbocyanine dye (3,3′ dipropyl thiadicarbocyanine) can be used as a spectroscopic probe for monitoring the resting potential across the plasma membrane of the ciliated protozoan Paramecium.

2. (2) The dye at low concentrations ( 1 μM) does not affect either the viability or the motility of the cells, nor does it induce a chemotactic response.

3. (3) The fluorescence of the dye bound to the cells alters as the potential across the membrane is changed by increasing the external cation concentration.

4. (4) The absorbance of the bound dye also changes in response to an alteration of the membrane potential.

5. (5) The membrane potential changes as measured by the fluorescence method have been correlated with the measurements of the potential estimated by microelectrode methods.

6. (6) Both cations which induce a negative chemotactic response in Paramecium (K⁺, Na⁺, Ba²⁺) and several non-toxic cations bring about a rapid depolarization of the plasma membrane. The significance of these rapid changes in relation to the swimming behaviour of the ciliate is discussed.

Endotoxins of enteric pathogens are chemotactic factors for human neutrophils

Early activation of human peripheral blood polymorphonuclear neutrophils is characterized by their morphological changes from spherical to polarized shapes. The endotoxins from enteric pathogens (S. dysenteriae type 1, V. cholerae Inaba 569B, S. typhimurium, and K. pneumoniae) were assessed by their ability to induce morphological polarization of the neutrophils as measures of early activation. Phagocytic activity, adhesion, chemokinetic locomotion, and nitroblue tetrazolium (NBT) dye-reduction ability measured the later activation of the cells. Neutrophils showed distinct morphological polarization in suspension over a wide range of concentrations of these endotoxins when were compared with those that were induced by the standard chemotactic factor, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP). It was discovered that all of the endotoxins induced locomotor responses in neutrophils in suspension that were dose- and time-dependent. The optimum concentration for the endotoxins of S. dysenteriae, V. cholerae, and K. pneumoniae was 1 mg/ml in which 71, 69, and 66% of the neutrophils were polarized. However, the S. typhimurium dose was 2 mg/ml in which 50% of the cells responded. Neutrophils that were stimulated with endotoxins also showed increased random locomotion (p<0.005) through cellulose nitrate filters, but an enhanced adhesion of the cells to glass surfaces (p<0.03). These are important functions of these cells to reach and phagocytose damaged cells, as well as invading microorganisms. Interestingly, the endotoxins had a highly-significant inhibitory effect upon the proportions of neutrophils phagocytosing opsonized yeast (p<0.01) with a small number of yeast that were engulfed by the cells (p<0.02). Further, endotoxin-treated cells showed an enhanced ability to reduce NBT dye (p<0.03). Therefore, we concluded that endotoxins of enteric pathogens are neutrophil chemotactic factors.     

Transmembrane potential changes during phagocytosis in rat alveolar macrophages

Studies were carried out to measure changes in the transmembrane potential of rat alveolar macrophages during exposure of the cells to zymosan particles or to the membrane perturbant, phorbol-12-myristate-13-acetate (PMA), and to determine if changes in membrane potential are related to superoxide anion release. Exposure of the cells to either zymosan or PMA leads to membrane depolarization, which precedes superoxide anion release. Furthermore, the magnitude of the depolarization is dependent upon the concentration of either zymosan or PMA. During exposure of the alveolar macrophages to increasing levels of zymosan, there is an increase in the amount of superoxide released as well as an increase in the magnitude of the depolarization. Incubation of the cells in medium containing 150 mM K+, a medium which causes membrane depolarization, leads to superoxide release from resting cells and a decrease in the amount of superoxide released from cells exposed to zymosan. These results indicate that release of superoxide anion from rat alveolar macrophages is related to membrane depolarization and suggest that the transmembrane potential change may act as a signal to initiate the phagocytotic responses of the cells.     

Light-scattering changes during chemotactic stimulation of human neutrophils: kinetics followed by flow cytometry

The light-scattering properties of human neutrophils were compared on a cell-by-cell basis before and after stimulation with chemotactic peptide using flow cytometry. Between 20 and 180 sec after peptide addition, side (90 degrees) scatter declined by up to 4% and forward scatter increased up to 6%. Between 3 and 15 min, side scatter increased up to 15% and forward scatter decreased up to 5%. Association of a fluorescence chemoattractant with neutrophils was most rapid during the initial phase of increasing forward and decreasing side scatter, and association saturated before the maximum increase in side scatter. Evidence is presented that the observed changes in scatter were not a consequence of chemoattractant-induced cell-cell adhesion or neutrophil degranulation. Rather, the early phases of light-scattering changes are interpreted to represent membrane ruffling by the stimulated neutrophil; the later phases polarization of the neutrophil morphology.     

Atorvastatin inhibits angiotensin-converting enzyme induction in differentiating human macrophages

Statins are effective drugs in the prevention of cardiovascular disease. Recent studies suggested that statins have additional beneficial effects on the vascular wall independent of their cholesterol-lowering effects. We investigated whether atorvastatin influences angiotensin-converting enzyme (ACE) production in differentiating human macrophages. Human peripheral blood monocytes (PBM) were isolated from fresh buffy coats. The cells were allowed to differentiate for 0-8 days in macrophage serum-free medium with 5 ng/ml granulocyte-macrophage colony-stimulating factor. Atorvastatin (0.005-0.5 microM), mevalonate (200-400 microM), geranylgeranyl pyrophosphate (1.25-2.5 microM), and/or farnesylpyrophosphate (FPP; 1.25-2.5 microM) was added on the second day of differentiation and then every other day. After incubation time, the ACE amount in intact macrophages was measured. ACE amount in PBM was low. A marked time-dependent ACE induction was noticed during differentiation of monocytes to macrophages. Atorvastatin treatment inhibited ACE induction during differentiation. In the presence of mevalonate, atorvastatin failed to downregulate ACE production. Cotreatment of the cells with atorvastatin and FPP reversed the suppressive effect of atorvastatin on ACE. In conclusion, atorvastatin inhibited ACE upregulation, normally occurring in differentiating human macrophages. This effect was mediated via the mevalonate pathway, and inhibition of FPP was probably involved. The finding that atorvastatin inhibited ACE upregulation may represent a novel pleiotropic action and an additional beneficial effect of statins in treatment of cardiovascular disease.