Cellular regulatory role of leukotriene B4: its effects on cation homeostasis in rabbit neutrophils

We have found that exogenous leukotriene B4 modifies calcium homeostasis in rabbit neutrophils in a manner essentially analogous to that of the chemotactic peptide f-Met-Leu-Phe. Leukotriene B4 causes a rapid and dose-dependent increase in membrane permeability to calcium and a release of calcium from previously unexchangeable intracellular pool(s). The net result of these changes is to transiently elevate the intracellular level of exchangeable calcium. A stereoisomer of leukotriene B4 with greatly reduced secretory activity toward neutrophils (5S, 12S-di HETE) is essentially without effect on the rate of 45Ca uptake at concentrations equal to those that produce near maximal enhancement by leukotriene B4. Leukotriene B4, in addition to its effects on calcium metabolism, also increases the rate of 22Na influx into rabbit neutrophils. The relationships between the action of leukotriene B4 on calcium homeostasis and the neutrophil-directed activities of arachidonic acid and its lipoxygenase metabolites are discussed     

Deactivation of the effects of F-Met-Leu-Phe and leukotriene B4 on calcium mobilization in rabbit neutrophils

Preincubation of rabbit neutrophils with the synthetic chemotactic factor f-Met-Leu-Phe has been found to diminish the ability of these cells to mobilize calcium upon subsequent stimulation by f-Met-Leu-Phe or by leukotriene B₄. The preexposure of the neutrophils to leukotriene B₄ on the other hand results in a diminished subsequent response to itself but an unaltered response to f-Met-Leu-Phe. These results demonstrate that deactivation can be observed at the level of calcium mobilization, strengthen the postulated second messenger role of calcium in neutrophils and imply that neutrophil activation by chemotactic factors can bypass the arachidonic acid metabolic pathway.     

Direct demonstration of increased intracellular concentration of free calcium in rabbit and human neutrophils following stimulation by chemotactic factor

An increase in the level of intracellular free calcium concentration in rabbit and human neutrophils stimulated by chemotactic factors has been demonstrated directly using the calcium-sensitive fluorescent probe quin-2. Addition of f-Met-Leu-Phe (10(-9) M), C5a (3 x 10(-9) M) or leukotriene B4 (6 x 10(-8) M) to the neutrophils induces a rapid increase in the intracellular concentration of free calcium that reaches a maximum value 15 seconds following stimulation. At concentrations of f-Met-Leu-Phe less than 10(-8) M the enhancement is dose dependent with an ED50 of 8 x 10(-11) M and is significantly reduced in the presence of EGTA in the suspending medium.     

Similarities in the mechanisms by which formyl-methionyl-leucyl-phenylalanine, arachidonic acid and leukotriene B4 increase calcium and sodium influxes in rabbit neutrophils

The chemotactic factors f-Met-Leu-Phe, arachidonic acid and leukotriene B₄ induce a rapid stimulation of both Ca²⁺ and Na⁺ influx in rabbit neutrophils. In the three cases the stimulation is rapid and the effects are not additive. Furthermore in all cases the stimulation of Na-influx but not of Ca-uptake is inhibited by the potassium-sparing diuretic amiloride. Preincubation with high concentrations of the chemotactic factor f-Met-Leu-Phe followed by washing of rabbit neutrophils reduces significantly the stimulation of calcium uptake induced by arachidonic acid, leukotriene B₄ and f-Met-Leu-Phe. These results strongly suggest that the exogenous addition of arachidonic acid or of leukotriene B₄ leads to the activation of the same permeation pathways as do better defined chemotactic factors.     

Inhibition of chemotactic factor-induced neutrophil responsiveness by arachidonic acid

Arachidonic acid when added simultaneously with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) inhibits the ability of the latter to initiate several but not all of its effects on rabbit peritoneal neutrophils. Stimulated neutrophil aggregation, calcium uptake, and increases in the steady state level of exchangeable calcium are all inhibited by 1-10 microM arachidonic acid. The binding of f-Met-Leu-Phe and the parameters of intracellular calcium redistribution (calcium efflux and changes in the steady state level of exchangeable calcium in the absence of extracellular calcium) and of stimulated sodium uptake are, on the other hand, unaffected by the same concentrations of arachidonic acid. Arachidonic acid, the saturated analog of arachidonic acid, was found not to inhibit f-Met-Leu-Phe-stimulated aggregation and calcium uptake. Arachidonic acid, therefore, in addition to its well-described agonist properties, also possesses antagonist activities toward rabbit neutrophils. These results add a new level of complexity to the study of the role of arachidonic acid in cell activation.     

Pharmacological differentiation between the chemotactic factor induced intracellular calcium redistribution and transmembrane calcium influx in rabbit neutrophils.

Nordihydroguaiaretic acid selectively inhibits the chemotactic factor induced stimulation of calcium influx and increase in the steady-state level of cell-associated ⁴⁵Ca observed in the presence of calcium in rabbit peritoneal neutrophils. On the other hand nordihydroguaiaretic acid does not inhibit the transient decreases in the steady state levels of ⁴⁵Ca observed either in the presence of low extracellular calcium or of low concentrations of chemotactic factors. These results suggest that nordihydroguaiaretic acid does not affect the intracellular calcium redistribution which is induced by chemotactic factors but rather it inhibits the influx of extracellular calcium which accompanies stimulation.     

Effect of arachidonic acid and the chemotactic factor F-Met-Leu-Phe on cation transport in rabbit neutrophils

A detailed examination of the effects of exogenous arachidonate on cation metabolism in rabbit neutrophils was undertaken. Arachidonic acid stimulates the movement of ⁴⁵Ca into and out of the neutrophils with a net result, in the presence of extracellular calcium, of increasing the steady-state level of ⁴⁵Ca. Arachidonate also increases the uptake of ²²Na. These effects of arachidonate are specific to these cations, concentration-dependent, and sensitive to lipoxygenase inhibitors. At the concentrations used in this study arachidonate does not influence the permeability of human erythrocytes to ⁴⁵Ca. Furthermore, both arachidonic acid and F-Met-Leu-Phe release calcium from a previously unexchangeable intracellular pool and the effect of the two stimuli are not additive. Arachidonic acid-dependent, but not F-Met-Leu-Phe-dependent, calcium release is sensitive to lipoxygenase inhibitors. These two stimuli thus appear to release is sensitive to lipoxygenase inhibitors. These two stimuli thus appear to release calcium from the same pool(s) by separate mechanisms. The results summarized above are consistent with the hypothesis that one or more arachidonate metabolites are involved in the mechanism underlying the chemotactic factor induced permeability changes in rabbit neutrophils.     

The chemotactic factors induced movement of calcium and sodium across rabbit neutrophil membranes: effect of densensitization to cytochalasin B

The preincubation of rabbit neutrophils with the chemotactic factor F-Met-Leu-Phe and the subsequent addition of cytochalasin B has previously been shown to induce a time, concentration and calcium dependent loss of secretory responsiveness in neutrophils. This has been termed desensitization. The results reported here first confirm that lysosomal enzyme release from neutrophils will still occur in the absence of extracellular calcium. In addition, a time dependent decrease in the magnitude of the cytochalasin B induced influxes of 45Ca and 22Na was found upon preincubation with F-Met-Leu-Phe. In the presence of extracellular Ca2+, this decrease in ionic responsiveness reaches a maximum by five minutes preincubation with F-Met-Leu-Phe. In the absence of added extracellular Ca2+ an initial and rapid (less than 1 minute) loss of ionic responsiveness is followed by partial recovery as the length of the preincubation with the chemotactic factor is increased from one to five minutes. These changes in ionic responses correspond exactly to the changes in secretory behavior of the neutrophils. Desensitization can thus be explained on the same ionic basis as that underlying the secretory response of the neutrophils. In addition, these results provide information about the sequence of events involved in the cytochalasin B and chemotactic factor induced release of lysosomal enzymes in neutrophils.     

Chemotactic factor causes rapid decreases in phosphatidylinositol,4,5-bisphosphate and phosphatidylinositol 4-monophosphate in rabbit neutrophils

Stimulation of rabbit neutrophils prelabeled with 32P by the synthetic chemotactic peptide f-Met-Leu-Phe induces a rapid decrease in the radioactivity in both phosphatidylinositol, 4,5 bis phosphate and phosphatidylinositol 4-monophosphate. The mean +/- standard error of the mean values of the maximum decrease in phosphatidylinositol, 4,5 bis phosphate occurred at 10 seconds following stimulation and is equal to 19 +/- 3% of the control value. The corresponding value for phosphatidylinositol 4-monophosphate occurred at 60 seconds following stimulation and is equal to 37 +/- 7% of the control value. On the other hand, the radioactivity in phosphatidic acid and lysophospholipids increased continuously with time following stimulation. The relationship of these changes to calcium release and neutrophil activation is discussed.     

Changes in ionic movements across rabbit polymorphonuclear leukocyte membranes during lysosomal enzyme release. Possible ionic basis for lysosomal enzyme release.

Changes in the movements of Na+, K+, and Ca+2 across rabbit neutrophils under conditions of lysosomal enzyme release have been studied. We have found that in the presence of cytochalasin B, the chemotactic factor formyl methionyl leucyl phenylalanine (FMLP) induces within 30 s large enhancements in the influxes of both 22Na+ and 45Ca+2 and an increase in the cellular pool of exchangeable calcium. The magnitude of the changes induced by cytochalasin B and FMLP exceeds that induced by FMLP or cytochalasin B alone, and cannot be explained on the basis of an additive effect of the two agents. However, these compounds either separately or together produce much smaller enhancements in 45Ca efflux. The divalent cation ionophore A23187 also produces a rapid and large increase in the influxes of both 22Na and 45Ca+2 in the presence and absence of cytochalasin B. We have also found an excellent correlation between calcium influx and lysosomal enzyme release. 42K influx is not significantly affected by any of these compounds. On the other hand, a large and rapid increase of 42K efflux is observed under conditions which give rise to lysosomal enzyme release. A flow diagram of the events that are thought to accompany the stimulation of polymorphonuclear leukocytes (PMNs) by chemotactic or degranulating stimuli is presented.     

Stimulus response coupling in the human neutrophil. I. Kinetic analysis of changes in calcium permeability

The relationship between receptor-ligand interaction in human neutrophils and initiation of enhanced Ca permeability ("45Ca uptake") has been correlated with cell function. Different ligands varied in their efficacy in provoking 45Ca permeability changes: chemotactic peptide f-Met-Leu-Phe greater than concanavalin A greater than immune complexes greater than phorbol myristate acetate. Mixtures of stimuli at optimal concentrations elicited no summation of responses, indicating that interaction of f-Met-Leu-Phe, concanavalin A, and phorbol myristate acetate with their respective "receptors" regulates a common site of 45Ca uptake. The onset of Ca uptake was an early event, preceding onset of aggregation, O-2 generation, and degranulation. Enhanced 45Ca permeability during neutrophil activation is dependent on mobilization of intracellular Ca, since 8-(N,N-diethylamino)-octyl:3,4,5-trimethoxybenzoate hydrochloride was a potent inhibitor of the Ca permeability response. In contrast, calmodulin antagonists did not inhibit Ca permeability changes; the requirement for calmodulin in the physiological responses of aggregation, O-2 generation, and degranulation must therefore be subsequent to activation of the "Ca translocator." We propose a role for a "Ca-translocating mechanism" as an amplifying factor in neutrophil activation.     

Biosynthetic human GM-CSF modulates the number and affinity of neutrophil f-Met-Leu-Phe receptors

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) modulates the function of mature neutrophils by priming for enhanced chemotaxis and oxidative metabolism in response to N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe). Our studies establish a relationship between f-Met-Leu-Phe receptor number and affinity and neutrophil chemotaxis and oxidative metabolism. A brief (5- to 15-min) exposure to physiologic concentrations of GM-CSF (10 pM to 100 pM) enhances f-Met-Leu-Phe-induced neutrophil chemotaxis by 85%, correlating with a rapid threefold increase (46,000/cell to 150,000/cell) in high-affinity neutrophil f-Met-Leu-Phe receptors. More prolonged incubation (1 to 2 hr) of neutrophils with GM-CSF is accompanied by a change to low-affinity f-Met-Leu-Phe receptors (Kd = 29 nM to Kd = 99 nM) concomitant with priming for enhanced neutrophil oxidative metabolism. Moreover, enhanced chemotactic responses to f-Met-Leu-Phe are no longer evident after more prolonged incubation of neutrophils with GM-CSF. These results show that a single lymphokine (GM-CSF) induces sequential changes in neutrophil f-Met-Leu-Phe receptor number and affinity that may enhance different physiologic responses.     

The interaction of 3,5-pyrazolidinedione drugs with receptors for f-Met-Leu-Phe on human neutrophil leukocytes: a study of the structure-activity relationship.

The 3,5-pyrazolidinedione (3,5-P) drugs, phenylbutazone and sulfinpyrazone, have been reported to bind to receptors for the chemotactic peptide, f-Met-Leu-Phe, and to behave as functional antagonists of f-Met-Leu-Phe in human and rabbit neutrophils. To explore the structure-activity relationship of this family of drugs for f-Met-Leu-Phe receptor binding, 36 drugs with the 3,5-P structure, a structure related to antipyrine, or an unrelated structure were tested as competitors for the binding of f-Met-Leu-Phe-Lys-fluorescein isothiocyanate on human neutrophils by flow cytometric analysis. Only drugs possessing the 3,5-P ring were significant competitors. The five most potent 3,5-Ps behaved as selective antagonists of f-Met-Leu-Phe-induced superoxide anion release by neutrophils. The potency was not correlated to the pKa or to their capacity to inhibit prostaglandin E2 released from culture fibroblasts but instead appeared to be correlated to their apparent octanol-buffer partition coefficients. The most potent f-Met-Leu-Phe antagonist identified, 1,2-diphenyl-4-(3-(1-naphthyl)-propyl)-3,5-pyrazolidinedione (DPN), may also possess an improved pharmacodynamic specificity compared with phenylbutazone and sulfinpyrazone, as it was less potent than phenylbutazone in the inhibition of prostaglandin synthesis and it was not cytotoxic. DPN may be a prototype for a valuable new class of anti-inflammatory drugs.     

Demonstration of receptor-mediated chemotaxis by human spermatozoa. A novel quantitative bioassay

A novel in vitro technique is described for measuring the chemotactic activity of soluble substances for human spermatozoa. This new bioassay has demonstrated that the synthetic chemotactic peptide N-formyl-Met-Leu-Phe elicits a potent, specific (i.e., receptor-mediated) chemotactic effect on human spermatozoa with an EC50 of 3.2 X 10(-10) M. Quantitative chemotactic studies on human spermatozoa with nine N-formylated-peptide analogs have shown a rank order of peptide potency indistinguishable (p less than 0.001) from that obtained in binding and chemotactic studies with rabbit neutrophils. The competitive antagonist Boc (t-butoxycarbonyl)-Phe-Leu-Phe-Leu-Phe, 10(-6) M, completely inhibited the chemotaxis elicited by f-Met-Leu-Phe, 10(-9) M, and was able to shift by one order of magnitude the molar concentration required by f-Met-Leu-Phe-Phe and f-Met-Leu-Phe to elicit the maximal response. The ability of N-formylated peptides to function as sperm chemoattractants reveals a high degree of correlation with binding, chemotaxis, and lysosomal enzyme release previously employed to define the neutrophil chemotactic receptor. This first unequivocal demonstration of substances having a receptor-mediated chemotactic effect for human male gametes suggests that human spermatozoa may indeed have the ability to respond chemotactically to appropriate environmental signals.     

Coupling of polyphosphoinositide breakdown with calcium efflux in formyl-methionyl-leucyl-phenylalanine-stimulated rabbit neutrophils

Exposure of rabbit neutrophils to formyl-methionyl-leucyl-phenylalanine (FMLP) induced the efflux of 45Ca2+ from pre-labeled cells which was almost complete within 30 s. On the other hand, FMLP-induced 45Ca2+ influx did not become apparent until 60 s after stimulation. When [3H]arachidonic acid-labeled neutrophils were stimulated with FMLP, the radioactivities in phosphatidylinositol 4,5-biphosphate (TPI) and phosphatidylinositol 4-phosphate (DPI) significantly decreased in parallel with the induction of 45Ca2+ efflux. In contrast, degradation of polyphosphoinositides in [3H]glycerol-labeled neutrophils was not significant until 60 s. Taken together, these results indicate that the early degradation of polyphosphoinositides, especially of those rich in arachidonic acid is closely associated with the initial efflux of calcium in FMLP-stimulated rabbit neutrophils. The study of resynthesis of polyphosphoinositides by measuring 32Pi incorporation into these lipids is also presented.     

Neutrophil responsiveness to chemoattractant tripeptide in rheumatoid arthritis

Neutrophils isolated from medication-free rheumatoid arthritis (RA) patients were assayed for responsiveness to the bacterial chemoattractant tripeptide formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe). Rheumatoid arthritis neutrophil preparations contained significantly lower percentages of rapidly migrating cells. This relative hyporesponsiveness of RA neutrophils was related to impaired sensing of chemotactic gradients. Rheumatoid neutrophil abnormalities in sensing of and responding to chemotactic gradients were not associated with resting or f-Met-Leu-Phe-induced changes in arachidonic acid metabolism.     

Transplacental 45Ca and 32P flux in the guinea pig: effect of maternal hypercalcaemia and hypocalcaemia

Transplacental 45Ca and 32P flux was measured across the in situ perfused guinea-pig placenta under conditions of acute maternal hypocalcaemia and hypercalcaemia. Maternal hypercalcaemia induced acutely by calcium gluconate infusion caused an increase in maternal-to-fetal 45Ca flux which was proportional to the increase in maternal plasma ionized calcium concentration. Acute maternal hypocalcaemia was induced by EGTA infusion and resulted in a decrease in maternal plasma ionized calcium concentration proportional to a corresponding decrease in transplacental 45Ca transfer. A bolus of calcium gluconate caused a transient decrease in 32P flux, whereas EGTA administration was without significant effect on transplacental 32P transfer. Calcium transport across the placenta is not saturated under conditions of maternal normocalcaemia and may be altered according to acute changes in maternal plasma calcium concentration. Thus, control of maternal-to-fetal calcium transfer does not appear to be at the placental level. This suggests that fetal calcium homeostasis may be regulated by the fetus itself.     

The effect of various stimuli and calcium antagonists on the fluorescence response of chlorotetracycline-loaded human neutrophils

Chlorotetracycline has been used in neutrophils and other cells as probe of the state of membrane-bound calcium. We report here that human neutrophils treated with chlorotetracycline response to soluble secretagogues by a prompt decrease in chlorotetracycline fluorescence. This response was observed within 2-5 s, making it one of the most immediate reactions in neutrophils to stimulation, and was obtained with three secretagogues studied: a chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine, a tumor promotor (phorbol myristate acetate) and a lectin (concanavalin A). The responses of neutrophils to the three stimuli differed both quantitatively and qualitatively. The calcium EGTA, did not effect the onset of the decrease in chlorotetracycline fluorescence, suggesting that the probe was measuring changes in intracellular calcium pools. The intracellular calcium antagonists, TMb-8, W-7 and trifluoperazine, did not block, but actually augmented, the fluorescence response. All four of these calcium antagonists blocked the recovery of chlorotetracycline fluorescence which was usually observed several minutes after stimulation with N-formyl-methionyl-leucyl-phenylalanine. This suggests that recovery was dependent upon both extracellular calcium and active calmodulin. The results are consistent with the hypothesis that changes in chlorotetracycline fluorescence reflect changes in a pool of membrane-bound 'trigger calcium', the release of which is an essential first step in stimulus-response coupling in human neutrophils.     

Pertussis toxin inhibits the rise in the intracellular concentration of free calcium that is induced by chemotactic factors in rabbit neutrophils: possible role of the "G proteins" in calcium mobilization

The addition of pertussis toxin to rabbit neutrophils inhibits the rise in the intracellular concentration of free calcium induced by the chemotactic factors fMet-Leu-Phe and leukotriene B4. At high concentrations of fMet-Leu-Phe, the inhibitory effect of the toxin is more on the stimulus-induced increase in membrane permeability to calcium than on calcium mobilization from internal stores. These results suggest that the "G protein" system either directly or indirectly is involved in the regulation of the stimulus-induced changes in the calcium mobilization and/or gating systems.     

Characterization of the secretory activity of leukotriene B4 toward rabbit neutrophils

We have described in det ail the secretory activity of leukotriene B4 toward rabbit neutrophils. Leukotriene B4 rapidly and vigorously degranulates rabbit neutrophils. This activity is stereospecific, cytochalasin B-dependent, and is enhanced by extracellular calcium. Pretreatment with leukotriene B4 deactivates rabbit neutrophils, i.e., cells so treated do not respond to stimulation by an additional bolus of leukotriene B4. In addition, the secretory activity of leukotriene B4 is sharply dependent on the simultaneous presence of cytochalasin B. Rabbit neutrophils therefore exhibit the previously described desensitization to the effect of cytochalasin B. In these and other discussed respects the characteristics of the leukotriene B4-induced degranulation of rabbit neutrophils are strikingly similar to those of the chemotactic factors. These results support the hypothesis that leukotriene B4 mediates, at least in part, the secretory, and possibly other, activities of chemotactic factors.