Cytosolic acidification as an early transductory signal of human neutrophil chemotaxis.

The inflammatory reaction of human neutrophils consists of two successive phases. In the first, designated chemotaxis, the cells home in on a foreign intruder. In the second, the cells attempt to eliminate the intruder by secreting lysosomal enzymes and superoxide anions. The initiation of chemotaxis involves prompt morphological changes that are manifested by a sharp biphasic drop in light scattering, accompanied by a transient cytosolic acidification. In a search for a causal relation between these two events, the neutrophil cytoplasm was abruptly acidified by the application of sodium propionate. This evoked a pulse of decreasing light-scattering, the time course and amplitude of which were practically identical to the rapid response induced by chemoattractants such as N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Both fMLP- and sodium propionate-induced responses were unaffected by amiloride, but were inhibited with a similar dose-dependence by a series of proton uncouplers. The initial phase of the cytosolic acidification seems, therefore, to fulfill the criteria for a second messenger for the initiation of chemotaxis.

Rapid changes in light scattering from human polymorphonuclear leukocytes exposed to chemoattractants. Discrete responses correlated with chemotactic and secretory functions.

A platelet aggregometer was adapted for the simultaneous measurement of perpendicular light scattering in addition to light transmission. The addition of chemoattractants to polymorphonuclear leukocyte suspensions evoked a single wave of increased light transmission, whereas the perpendicular scattering measurement demonstrated a previously unrecognized biphasic response. The first perpendicular scattering response had no detectable latency and peaked at 10 +/- 1 s, then decayed rapidly. The second response peaked at 40 +/- 5 s, and decayed over several minutes. The dose-response curve of chemoattractants for inducing the rapid (10 +/- 1 s) perpendicular scattering peak corresponded to that which initiated chemotaxis. Initiation of the slow (40 +/- 5 s) peak required 10-fold higher amounts of chemoattractants, and the dose-response curve correlated with the induction of lysosomal enzyme secretion and superoxide anion production. Low doses of aliphatic alcohols, which have been shown to enhance chemotaxis but to inhibit secretion and superoxide anion production, abolished the slow perpendicular light-scattering response but left the fast response intact. Stimulants of secretion induced only slow and prolonged responses that were best observed in transmission measurements. In an attempt to resolve the origin of the light-scattering responses, the morphological changes of polymorphonuclear leukocytes were examined microscopically. Neither aggregation nor morphological whole cell polarization could be correlated with changes in light transmission or perpendicular scattering, which suggested that the source of scattering is of subcellular dimensions. The rapid perpendicular light-scattering response of polymorphonuclear leukocytes to chemoattractants appears to record an initial event in the stimulus-response coupling, and its measurement should provide a useful new tool for the study of leukocyte function. The biphasic nature of the light-scattering responses to chemoattractants, moreover, correlates with the dual regulation of the chemotactic and secretory responses of leukocytes.

Temporal segregation in signalling: a novel mechanism in human neutrophils.

The ability of neutrophils to carry out chemotaxis in response to low chemoattractant concentrations, but arrest their motility when exposed to higher concentrations of the same substance, has fascinated investigators for years. By analyzing the temporal characteristics of the morphological responses, corresponding to chemotaxis and cell arrest, we have recently discovered that the choice between them is made by transduction of the continuous binding process into either single or multiple stimuli within defined time intervals, initiating chemotaxis or cell arrest, respectively. Both experimental and theoretical lines of evidence are presented to support the validity of this unique mechanism.

Instability of leukocyte aggregation: lack of evidence for leukoembolization during various states of inflammation.

This study centers on the question of whether the phenomenon of leukocyte aggregation, which is typical to inflammatory conditions, is pathogenic per se. We examined patients and laboratory animals in whom the presence of aggregated leukocytes in the peripheral blood was documented by direct visualization and where, despite the presence of aggregated leukocytes, neither the patients nor the laboratory animals showed clinical or pathological evidence for leukoembolization. Our in vitro findings about the reversibility of the phenomenon of leukocyte aggregation help to explain the above-mentioned observations as well as the well-known daily clinical experience that, despite complement activation and other aggregatory stimuli, there is no clinical or pathological evidence for leukoembolization.

Light scattering by polymorphonuclear leukocytes stimulated to aggregate under various pharmacologic conditions.

Enhancement of light transmission has been widely accepted as an empirical measure of cell aggregation in suspension. Several years ago, this measurement was introduced to the study of polymorphonuclear leukocyte (PMN) aggregation by adapting a hypothesis originally developed for platelets. Accordingly, an increase in light transmission is attributed to cell aggregation and a decrease in transmission below baseline level is indicative of increased cell symmetry. We tested this hypothesis for human PMNs by comparing the whole cell shape or the cells' aggregation state with the light transmission under particular experimental conditions. The PMN light response to the chemoattractant, f-Met-Leu-Phe, in the presence of low doses of aliphatic alcohols was associated with transient enhanced transmission, followed by a rapid decrease below baseline. In contrast to the platelet hypothesis, the below-baseline effect coincided with a decrease in PMN symmetry from spheres to wedge-shaped (polarized) cells. PMNs fixed mildly with various doses of formaldehyde (0.1% to 0.3%) were completely aggregated by the addition of 50 micrograms/mL phytohemagglutinin (PHA). Despite the complete aggregation of the PMNs, there was a dose-dependent inhibition of the above-baseline level transmission response by the fixative, demonstrating a clear dichotomy between aggregation and increased light transmission. However, PMN aggregation could be monitored by observing the pattern of enhanced light transmission coupled with decreased perpendicular light scattering immediately after the stirring of the cell suspensions was stopped. PMNs aggregated by PHA cleared from suspension very rapidly (t1/2 less than or equal to one minute), whether or not they were formalin-fixed. In contrast, unaggregated cells revealed constant transmission and perpendicular scattering intensities for as long as five minutes after the stirring was stopped. The clearance patterns of f-Met-Leu-Phe-stimulated PMNs initiated even at the time of maximally increased light transmission were indistinguishable from those of the unstimulated cells, indicating the absence of aggregation. The lack of correlation between light output and changes in cell shape or degree of aggregation of PMNs causes us to reject the hypothesis that attributes enhanced light transmission to PMN aggregation. We suggest that modulation of light transmission by PMNs stimulated with chemoattractants is due to changes in light output from subcellular objects.

Extensive hydrolysis of N-formyl-L-methionyl-L-leucyl-L-[3H] phenylalanine by human polymorphonuclear leukocytes. A potential mechanism for modulation of the chemoattractant signal.

Chemoattractant receptors on human polymorphonuclear leukocytes (PMNs) stimulate a series of important biological responses. In an attempt to better understand the mechanism of stimulus response coupling of chemoattractant receptors, the kinetics of N-formyl-L-methionyl-L-leucyl-L-[3H]phenylalanine (fMet-Leu-[3H]Phe) binding to PMNs was evaluated. Unexpectedly, extensive degradation of the ligand was found to occur rapidly at 37 degrees C. Exposure of 10(7) cells/ml to 10 nM fMet-Leu-[3H]Phe led to the specific uptake of approximately 1% of the ligand within 10 min, while approximately 50% of the extracellular chemoattractant was hydrolyzed to free amino acids. Under the same conditions, isolated plasma membranes equivalent to 2.5 X 10(7) PMNs/ml bound specifically approximately 1% of the ligand and degraded about one-half of it primarily to Leu-[3H]Phe. The fMet-Leu-[3H]Phe hydrolysis commenced with no apparent latency, yet disobeyed first order kinetics as a 100-fold excess unlabeled ligand enhanced the initial consumption rate of 10 nM fMet-Leu-[3H]Phe by 500-fold, yielding an enhancement of the relative hydrolysis to approximately 70%. 1-butanol at 0.25%, which accelerates chemotaxis but inhibits superoxide anion and lysosomal enzyme secretion, reduced the hydrolysis to about 15% independent of the fMet-Leu-[3H]Phe specific activity. Lysosomal secretion could not mediate the hydrolysis process, since the supernatants of PMNs exposed either to 10 nM of 1 microM fMet-Leu-Phe reveal no degradation capacity toward fMet-Leu-[3H]Phe. These data indicate that the hydrolysis of the chemoattractant occurs at the cell surface and is dependent on the plasma membrane physical state. This phenomenon may well modulate the chemotactic signal due to its ability to profoundly alter the level of the chemoattractant proximate to the cell surface.

Neutral endopeptidase activity in the interaction of N-formyl-L-methionyl-L-leucyl-L-phenylalanine with human polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes (PMN) hydrolyze the synthetic chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe) at nanomolar concentrations in an autocatalytic-like manner that deviates from classical Michaelis-Menten kinetics [Yuli, I. & Snyderman, R. (1986) J. Biol. Chem. 261, 4902-4908]. By using inhibitors of distinct classes of endoproteases, this particular fMet-Leu-Phe degradation was attributed exclusively to an exoplasmic metalloendoprotease that matches the ubiquitous neutral endopeptidase (NEP). Membrane-bound NEP hydrolyzes non-chemotactic substrates according to a classic Michaelis-Menten mechanism. By competitive inhibition with non-chemotactic substrates, fMet-Leu-Phe was found to interact with membrane NEP through a single active site, in a non-cooperative mode with an apparent Km in the order of 1 mM. The discrepancy between the ordinary hydrolysis of the micromolar and millimolar concentrations of fMet-Leu-Phe, reported by others, and the particular degradation of the nanomolar fMet-Leu-Phe, could not be accounted for by any coherent correlation between NEP activity/inhibition and modulation of fMet-Leu-Phe binding to its receptor, and/or induction of fMet-Leu-Phe-receptor-mediated inflammatory responses. Based on these and previously reported results, a novel model is proposed in which the fMet-Leu-Phe-induced inflammatory stimulation of PMN involves both NEP and the fMet-Leu-Phe receptor. By this model, NEP and the fMet-Leu-Phe receptor are distinct membrane entities which can form dynamic binary and tertiary complexes; thus accounting for the unusual kinetic features of fMet-Leu-Phe degradation, as well as the two receptor states. The complex of NEP and the fMet-Leu-Phe receptor might be conceived as a chemotactic-perception mechanism that combines the high affinity of the receptor and the rapid turnover of NEP.

Chemoattractant receptor functions in human polymorphonuclear leukocytes are divergently altered by membrane fluidizers.

The chemotactic factor receptor on leukocytes initiates several cellular responses including chemotaxis, lysosomal enzyme secretion, and O2- production. The latter two responses require approximately 10-100 times more chemoattractant than is required for chemotaxis. We determined the effects of membrane fluidizers on the binding characteristics and the functional activities of the oligopeptide fMet-Leu-Phe chemotactic factor receptor on polymorphonuclear leukocytes. Fluidization was induced by aliphatic alcohols and monitored by diphenylhexatriene fluorescence polarization. Low doses of n-butanol (0.25%) and n-pentanol (0.1%) were nontoxic to the leukocytes yet reduced their diphenylhexatriene-induced polarization, indicating increased membrane fluidity. At these doses of alcohols, the affinity of the fMet-Leu-Phe receptor was enhanced from Kd = 25.5 +/- 7.6 nM to Kd = 5.2 +/- 0.9 nM and Kd = 6.0 +/- 0.9 nM, respectively. Chemotaxis was also increased, as indicated by the decrease, by a factor of approximately 1/3 in the ED50 for fMet-Leu-Phe, as well as by a 1.5-fold increase in the maximal distance of migration in the presence of 0.25% butanol or 0.1% pentanol. In contrast to chemotaxis, the alcohols depressed fMet-Leu-Phe stimulation of O2- production by 90% although they had no effect on phorbol 12-myristate 13-acetate-induced O2- production. Secretion of lysozyme was also inhibited. Thus, the affinity of the fMet-Leu-Phe receptor can be modulated by membrane fluidizers. The higher affinity state of the receptor induced by the alcohols is more efficient in transducing chemotactic signals but is deficient in mediating O2- production or secretion. Thus, the transduction mechanisms for the various biological activities of the chemotactic factor receptor are heterogeneous and can be differentially manipulated by membrane fluidizers.

Glucose transport through cell membranes of modified lipid fluidity.

Carrier-mediated transport of glucose in human erythrocytes and 3T3 mouse fibroblasts was examined at different lipid viscosities of the cell membrane. Rigidification of the membrane lipid layer was accomplished by incorporation of cholesterol or one of the hydrophilic esters, cholesteryl hemisuccinate or cholesteryl betainate, whereas fluidization was accomplished by cholesterol depletion. In both cells the dependence of the maximal rate of glucose transport at 37 degrees C, Vmax, on the lipid microviscosity of the cell plasma membrane, eta, is of a similar pattern which does not obey simple diffusion considerations. When the eta value of untreated cells is slightly increased (10-20%), Vmax increases to a peak value, beyond which a further increase in eta progressively reduces it. Decrease of the natural eta is also accompanied by a progressive decrease of Vmax. This general pattern was also observed for the transport of alpha-aminoisobutyric acid in 3T3 fibroblasts (unpublished results). A theoretical analysis of the dependence on eta of the transport turnover number and of the accessibility of carrier sites was carried out in order to account for this behavior. On the basis of this analysis, a general expression for the dependence of Vmax on eta, which fits reasonably well with the experimental data, was derived. This expression is also valid for the dependence on eta of the overt activity of membrane-bound enzymes and receptors.

Medium viscosity regulates the activity of membrane-bound and soluble phospholipase A2.

Medium viscosity is a regulator of very low density lipoprotein production by cultured hepatocytes; their secretion and synthesis are inversely proportional to the extracellular fluid viscosity. The possibility that the mechanism of this extracellular effect on cell function involves modulation of cell membrane component(s) was considered. Along with this assumption, we studied the effect of medium viscosity on the activity of phospholipase A2 (PLA2), an enzyme present in the cell surface membrane, and the activity has been correlated with cellular secretion. We have found that culture medium viscosity inhibits the activity of PLA2 in the plasma membrane of cultured liver cells, concomitantly with the inhibition of lysosomal enzyme and lipoprotein secretion. It was also found that the degradation of liposomal phosphatidylcholine by soluble snake venom PLA2 is inversely proportional to the solvent viscosity. The possibility that the effect of medium viscosity on the enzymatic reaction involves the modulation of dynamic properties of membrane phospholipids was then considered. This hypothesis was examined by monitoring the fluorescence depolarization of fluorophores incorporated into phospholipid vesicles. No significant effect of the solvent viscosity on the phospholipid bilayer was observed. It is proposed that the regulation of cellular secretion by extracellular fluid viscosity involves modulation of the cell membrane PLA2 activity.

Studies on the amino acid-incorporating activity of native rat liver rough membrane and that reconstituted in vitro.

The amino acid-incorporating activities of free polyribosomes, rough membranes and rough membranes reconstituted in vitro, derived from rat liver, were compared. The amino acid-incorporating activity of the two membrane fractions were very similar in their response towards changes in pH, Mg2+ concentration and temperature, but differed from the response of the amino acid-incorporating activity of free polyribosomes. Free polyribosomes irreversibly lost part of their amino acid-incorporating capacity after they had become bound to rough membrane, from which the original ribosomes had been removed. Ribonuclease activity present in the membrane fraction may be responsible for this loss.

Neonatal neutrophil inflammatory responses: parallel studies of light scattering, cell polarization, chemotaxis, superoxide release, and bactericidal activity.

Neutrophil dysfunction among newborn infants, especially those born prematurely, is well recognized, but the mechanism responsible for this phenomenon is yet to be clarified. In this study, we evaluated the stimulus response coupling in neutrophils from 90 healthy newborns and 96 healthy adults in an effort to establish whether defective neonatal neutrophil function is a result of impaired signal perception or immature responsiveness. Measurement of rapid- and slow-light scattering responses (LSR) to 1 microM FMLP stimulation revealed that neonatal neutrophils have about one-half the corresponding responsiveness of adult cells (rapid-LSR: 6.1 +/- 3.1 arbitrary light intensity units vs. 12.0 +/- 2.8, P < .001; and slow-LSR: 5.0 +/- 2.5 vs. 9.1 +/- 2.0; P < .001). The same markedly reduced activity was observed in newborn neutrophil chemotaxis and bactericidal activity in comparison with adult cells. Nevertheless, low FMLP concentrations (less than 1 nM) induced no difference in cell polarization between newborn and adult neutrophils, yet at higher FMLP concentrations, the newborn revealed significantly reduced cell polarization. Our data suggest that newborn infants bear a fully functional FMLP signal perception but lack the full capacity of inflammatory responsiveness.

Altered cell-averaged microviscosity of murine peritoneal macrophages undergoing activation in vivo or in vitro.

The cell-averaged microviscosity of intact murine peritoneal mononuclear phagocytes in various stages of activation was assessed by quantifying fluorescent depolarization of 1,6-diphenyl-1,3,5-hexatriene. Macrophages activated in vivo with Mycobacterium bovis, strain BCG, were significantly more fluid than resident peritoneal macrophages, responsive macrophages elicited with thioglycollate broth, proteose peptone broth, or fetal bovine serum, or primed macrophages elicited with pyran copolymer, MVE-2. Specifically, the cell-averaged microviscosity decreased from a mean of 3.47 +/- .07 eta 25 degrees C (poise) (range of 3.32 to 3.67 p) to 2.62 eta 25 degrees C. Exposure of responsive macrophages in vitro to bacterial endotoxin plus hybridoma supernatants containing macrophage-activating factor or purified recombinant interferon gamma resulted in decreased microviscosity; the largest effect was seen after 24 hr. Macrophages primed in vivo with MVE-2 and treated in vitro with endotoxin also developed decreased microviscosity. Similar changes in microviscosity were observed in a plasma membrane-enriched fraction isolated from macrophages activated in vitro with interferon gamma and endotoxin, thus suggesting that the cell-averaged measurements reflected changes in membrane viscosity. The optimum concentration of MAF-inducing decreased overall microviscosity was identical to that for inducing tumoricidal capacity. Taken together, the data indicate activation of lytic capacity in murine macrophages is closely associated with decreased cell-averaged microviscosity and that this change reflects, at least in part, decreased microviscosity of the plasma membrane of these cells.

Chemotaxis and chemokinesis of human spermatozoa to follicular factors.

Human spermatozoa accumulate in vitro in diluted follicular fluids obtained from follicles from which the eggs have been fertilized. Using capillary assays under a variety of experimental conditions (ascending or descending gradients of follicular fluid, or no gradient at all) and microscopic assays in which individual spermatozoa could be followed, we found that the sperm accumulation in follicular fluid was the result of both sperm chemotaxis and chemokinesis and eventually hyperactivation-like motility. We determined the optimal conditions for sperm accumulation, which involved sperm preincubation (possibly to induce sperm capacitation) and proper dilution of follicular fluid. In all the assays, the net accumulation was low, probably reflecting the chemotactic responsiveness of only a small fraction of the sperm population at any given time. We partially fractionated follicular fluid in a Centricon microconcentrator (Amicon, Danvers, MA) and by acetone precipitation, and found that at least one of the chemotactic factors is a small (< 10-kDa) molecule that is probably nonhydrophobic. This is the first time that sperm chemotaxis and chemokinesis in response to a follicular factor(s) in mammals has been established and has been distinguished from other processes that might cause sperm accumulation. The physiological significance of these findings is discussed.