Deactivation of guinea pig pulmonary alveolar macrophage responses to N-formyl-methionyl-leucyl-phenylalanine: chemotaxis, superoxide generation, and binding

Incubation of pulmonary alveolar macrophages (PAM) with the synthetic chemotactic tripeptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP) results in deactivation of PAM chemotaxis. The chemotactic response to 10(-8) M FMLP was inhibited 85% after 30 min of preincubation with 10(-6) M FMLP and 48% by 10(-8) M FMLP. Only the higher dose of FMLP (10(-6) M) caused deactivation of the chemotactic response to C5a (20%). Preincubation with partially purified C5a at a concentration of 100 microliter/ml produced a 32% inhibition of the PAM response to 10(-8) M FMLP. In contrast, preincubation with FMLP had no significant effect on superoxide generation, either at baseline or after stimulation. Levels of intracellular cyclic adenosine-3',5'-monophosphate (cAMP) increased in response to PGE1 in the presence of 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, but FMLP failed to induce a change in cAMP levels. Studies of 3H-FMLP binding were consistent with two populations of membrane receptors with different affinities. Preincubation of PAM with FMLP did not result in a reduction of maximal binding. We conclude that FMLP induces deactivation of PAM chemotaxis, but cross-deactivation occurs only after high dose treatment. Unlike the PMN, macrophage chemotactic activation is not accompanied by an elevation in cAMP levels. These observations suggest that PAM chemotaxis is influenced by prior exposure to chemotactic stimuli, but other aspects of the PAM response diverge from that of PMN. The mechanism of deactivation of PAM does not appear to result from a shift in the dose-response curve or decreased availability of membrane receptors, but may involve uncoupling of post-receptor cellular responses.     

Inhibition of human neutrophil chemotaxis by the protein kinase inhibitor, 1-(5-isoquinolinesulfonyl) piperazine

The protein kinase inhibitor, 1-(5-isoquinolinesulfonyl) piperazine (C-I), inhibits superoxide release from human neutrophils (PMN) stimulated with phorbol myristate acetate or synthetic diacylglycerol, without inhibiting superoxide release from PMN stimulated with the chemoattractants C5a or N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe). In this study, we investigated the effect of C-I on human PMN chemotaxis to C5a, f-Met-Leu-Phe, leukotriene B4 (LTB4), and fluoresceinated N-formyl-methionyl-leucyl-phenylalanine-lysine (f-Met-Leu-Phe-Lys-FITC). PMN, preincubated for 5 min at 37 degrees C with 0 to 200 microM C-I, were tested for their migratory responses to the chemoattractants. C-I (greater than or equal to 1 microM) significantly inhibited PMN chemotaxis to f-Met-Leu-Phe, f-Met-Leu-Phe-Lys-FITC, and C5a without affecting random migration. Maximal inhibition of chemotaxis to these attractants occurred with greater than or equal to 50 microM C-I, at which chemotaxis was inhibited by 80 to 95%. The C-I inhibition was reversible. In contrast, 200 microM C-I did not inhibit the number of PMN migrating to LTB4, although, the leading front of PMN migration to LTB4 was inhibited by C-I. C-I inhibited PMN orientation to C5a and f-Met-Leu-Phe without affecting orientation to LTB4. C-I did not inhibit the binding of radiolabeled f-Met-Leu-Phe or f-Met-Leu-Phe-Lys-FITC to PMN. These findings suggest that the chemotactic responses of PMN to f-Met-Leu-Phe and C5a involve a protein kinase-dependent reaction which is inhibited by C-I.     

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.     

Development of a new method of analysing chemotactic deactivation of human neutrophil granulocytes

Measurement of chemotactic migration of human neutrophil granulocytes (PMN) induced by chemotaxins serves as a simple and reliable method for assessing the expression of chemotaxin receptors. Incubation of PMN with a certain chemotaxin leads to a diminished chemotactic migration towards this chemotaxin. This is called chemotactic deactivation. We developed a new deactivation chamber to determine chemotaxis and chemotactic deactivation of human PMN. This novel chamber is a modification of the commercially available acrylic 48-well microchemotaxis chamber consisting of an upper block with wells drilled all the way through the block and a blind-well lower block. Both blocks are separated by a polycarbonate membrane. PMN from the wells in the upper block migrate through the pores of the membrane into the wells of the lower block containing the chemoattractants. Migrated PMN on the lower side of the PC membrane were quantified after staining by measuring specific light absorbance. The chemotactic activity is quantified as a ratio of stimulated migration and random migration (chemotactic index=CI). For our novel chamber, only the upper blocks of this commercial chamber were connected like a sandwich, including a polyvinylpyrrolidone-free polycarbonate membrane with a pore size of 3 microm. The wells in the upper compartment were filled with 5 x 10(4) PMN and deactivating chemotaxin. The lower block was then filled with the chemotactic stimulus and the chamber was then incubated in humidified air with 5% CO2 atmosphere at 37 degrees C. The influence of cell concentration, incubation time, chemotactic factor concentration, pore size and alkaline treatment of polycarbonate membranes on migrational activity of PMN have been investigated. The technique was rigorously standardized in order to optimize the assay conditions. The method is relatively simple, sensitive and fast. The determination of chemotaxis and deactivation are performed in the same chamber, thus avoiding cell loss due to nonspecific adherence in other incubation tubes. The chamber can be used to characterize the chemotactic activity of chemoattractants of unknown structure via known and unknown receptors. This new chamber can be very helpful in detecting unknown chemotactic stimuli, which are not detectable by, for example, antibodies.     

The combined action of hyaluronic acid and fibronectin stimulates neutrophil migration

Previous investigations have demonstrated that the chemotactic response of polymorphonuclear leukocytes (PMN) was stimulated by hyaluronic acid (HA) when serum was present. The aim of the present investigation was to identify the serum factor necessary for the stimulation of PMN chemotaxis by HA. By means of gel filtration, the m.w. of the serum component was shown to be greater than 350,000. Immunoprecipitation of serum with anti-fibronectin, but not with anti-IgM and anti-alpha 2-macroglobulin, inhibited the stimulation of PMN chemotaxis by HA. Preincubation of PMN with HA (10 to 500 micrograms/L) and isolated fibronectin (0.1 to 100 mg/L) significantly stimulated the chemotactic response of PMN. Also, random migration of PMN was significantly increased by preincubation of the cells with HA (10 to 500 micrograms/L) and isolated fibronectin (50 to 200 mg/L). Additionally, PMN preincubated with HA (10 to 50 micrograms/L) and with fibronectin (10 to 50 mg/L) added afterwards, and PMN preincubated with fibronectin (10 mg/L) and with HA (5 to 10 micrograms/L) added after the preincubation, showed a significant stimulation of the chemotactic response. PMN preincubated with serum and chondroitin sulfate, or with fibrinogen and HA, demonstrated no stimulation of the chemotactic response. The present investigation suggests that the combined action of HA and fibronectin, which probably takes place at the cellular membrane, is a major mechanism in the HA-mediated stimulation of PMN migration.     

Laminin promotes the oxidative burst in human neutrophils via increased chemoattractant receptor expression

The extracellular matrix component, laminin, enhances the chemotactic responsiveness of polymorphonuclear leukocytes (PMN) in vitro, and low doses of chemoattractant substances augment the expression of PMN cell surface receptors for laminin. This study determined whether laminin acts in concert with chemoattractants to activate PMN. Laminin (5 to 100 micrograms/ml) stimulated lysozyme release and superoxide production in response to the chemoattractant, FMLP by as much as 69%. These results could be explained by changes in cell surface chemoattractant receptor expression in that incubation of normal PMN with laminin (5 to 75 micrograms/ml) increased the binding of 19 nM FML[3H]P by 35 to 80%. This corresponded to as much as a 2.5-fold increase in the number of chemoattractant receptors/cells which had a lower average affinity. Laminin did not change the number or affinity of FML[3H]P receptors present on organelle-depleted PMN cytoplasts, and the laminin-induced increase in FML[3H]P receptors expressed on PMN from a patient with a specific granule deficiency was only 11 to 21% of that seen in normal PMN. These findings suggest that chemoattractants augment the expression of laminin receptors which mediate PMN attachment to basement membranes, followed by laminin-induced increases in the expression of cryptic chemoattractant receptors contained in intracellular granules, with resultant augmentation of the oxidative burst.     

Human neutrophil laminin receptors: activation-dependent receptor expression

Activated human polymorphonuclear leukocytes (PMN) isolated from peripheral blood specifically bind 125I-laminin after stimulation with phorbol 12-myristate 13-acetate (PMA) or f-Met-Leu-Phe (FMLP) at 37 degrees C. Changes in laminin receptor expression are stimulus dose dependent at both chemotactic (10(-10) M to 10(-6) M) concentrations of FMLP, and secretory (greater than 5 ng/ml) levels of PMA. In the presence of cytochalasin B (5 micrograms/ml), 10(-7) M FMLP activation stimulates specific laminin binding, with an apparent Kd = 3.9 X 10(-9) M and 6.47 X 10(5) binding sites/cell, reaching equilibrium within 10 min at 4 degrees C. This observed activation-dependent change in laminin receptor expression is not due to interference by endogenous laminin, because no fluorescein-visualized anti-laminin antibody bound to cells without added glycoprotein, regardless of the level of activation. Levels of neutrophil lysozyme release, which show a PMA dose dependence similar to that of receptor binding activity, suggest that granule-plasma membrane fusion may be significant during increases in receptor expression. A lack of receptor stimulation by PMA from a granule-deficient patient or in granule-depleted cytoplasts from normal donors additionally supports this hypothesis. Electroblot transfer and autoradiography of subcellular fractions from unstimulated PMN reveals the presence of a 68,000 dalton laminin-binding component in the secondary/tertiary granule (beta) fraction, which may represent an intracellular laminin receptor pool.     

Amphotericin B alters the affinity and functional activity of the oligopeptide chemotactic factor receptor on human polymorphonuclear leukocytes

Leukocyte chemotaxis is initiated by the binding of chemotactic factors to specific, high-affinity receptors. Amphotericin B, a polyene antibiotic that binds to membrane cholesterol, inhibits human neutrophil (PMN) chemotaxis. We examined the effects of this drug on PMN functions mediated by the oligopeptide chemotactic factor receptor. The antibiotic irreversibly inhibited chemotaxis and depressed the binding of the radiolabeled chemoattractant, fMet-Leu-[3H]Phe, to its receptor without affecting the receptor's specificity. The drug lowered the binding affinity of the receptor by up to fivefold and slightly increased its number. Doses of amphotericin B that depressed receptor affinity and inhibited chemotaxis did not diminish lysosomal enzyme secretion or superoxide anion production. Nystatin, a less potent polyene antibiotic, also diminished chemotactic factor binding, but to a lesser degree than amphotericin B did. A chemically unrelated antifungal agent had no effect on either binding or chemotaxis. Thus, pharmacologic manipulation can alter the affinity of the chemotactic factor receptor on human PMN; this alteration is associated with a change in receptor function. The data suggest that receptor affinity regulates or at least reflects its functional state, and that the transduction mechanisms for various biologic responses mediated by the chemoattractant receptor are heterogeneous. By pharmacologic alterations of receptor affinity, one may be able to modulate specific biologic responses elicited by chemoattractant receptor-ligand interactions.     

Specific binding of leukotriene B4 to receptors on human polymorphonuclear leukocytes

Leukotriene B4 (5(S),12(R)-di-hydroxy-eicosa-6,14-cis-8,10-trans-tetraenoic acid [LTB4]) is a product of the 5-lipoxygenation of arachidonic acid, which elicits human PMN leukocyte chemotactic responses in vitro that are 50% of the maximal level at concentrations of 3 X 10(-9) M to 10(-8) M and are maximal at 2 X 10(-8) M to 10(-7) M. The specific binding of highly purified [3H]LTB4 to human PMN leukocytes was assessed both by extracting the unbound and weakly bound [3H]LTB4 with acetone at -78 degrees C and by centrifuging the PMN leukocytes through cushions of phthalate oil to separate the unbound from bound [3H]LTB4. The levels of total binding of [3H]LTB4 and of nonspecific binding of [3H]LTB4, in the presence of a 1500-fold molar excess of nonradioactive LTB4, were approximately two times higher with the phthalate oil method. Scatchard plots of the concentration dependence of the specific binding (total - nonspecific binding) of [3H]LTB4 to PMN leukocytes were linear for the acetone extraction and phthalate oil methods and revealed dissociation constants of 10.8 X 10(-9) M and 13.9 X 10(-9) M, respectively, and mean of 2.6 X 10(4) and 4.0 X 10(4) receptors per PMN leukocyte. The 5(S),12(S)-all-trans-di-HETE analog of LTB4 and 5-HETE competitively inhibited by 50% the binding of [3H]LTB4 to PMN leukocytes at respective concentrations that evoked half-maximal chemotactic responses, whereas neither N-formyl-methionyl-leucyl-phenylalanine nor chemotactic fragments of C5 inhibited the binding. Human erythrocytes exhibited no specific binding sites for [3H]LTB4. Human PMN leukocytes possess a subset of receptors for LTB4 that are distinct from those specific for peptide chemotactic factors.     

Stimulus-specificity of the chemotactic deactivation of human neutrophils by lipoxygenase products of arachidonic acid

Preincubation of human neutrophils with chemotactic concentrations of 5(S)-hydroxy-eicosatetraenoic acid (5-HETE) or 5(S), 12(R)-dihydroxy-6,14 cis-8,10 trans-eicosatetraenoic acid (leukotriene B4) induces a state of preferential chemotactic unresponsiveness to the homologous factor, termed deactivation, and less suppression of the responses to other chemotactic stimuli. The ratio of the concentrations required for maximal chemotactic deactivation of neutrophils to that which stimulates chemotaxis optimally is greater for 5-HETE and leukotriene B4 than for peptide and protein factors. In contrast to other chemotactic factors, 5-hydroperoxy-eicosatetraenoic acid (5-OOHETE) induces neutrophil chemotactic deactivation that is independent of the nature of the subsequent stimulus and is more slowly reversible after elimination of the fluid-phase deactivating factor. The unique characteristics of the chemotactic deactivation of human neutrophils by 5-OOHETE may be attributable in part to its endogenous metabolism to potent deactivating factors or to covalent derivatization of subcellular structures of the neutrophils by the highly reactive 5-OOHETE.     

Transduction by leukotriene B4 receptors of increases in cytosolic calcium in human polymorphonuclear leukocytes

The uptake of Quin-2 by human polymorphonuclear (PMN) leukocytes permitted accurate fluorimetric quantification of the cytosolic concentration of intracellular calcium [( Ca+2]in), without altering the expression of the two subsets of leukotriene B4 (LTB4) receptors, as assessed by the binding of [3H]LTB4. Chemotactic concentrations of LTB4 elicited a rapid increase in [Ca+2]in, which reached a peak within 0.6 to 1 min and then decayed back to baseline levels by 6 to 10 min. The maximal increase and the half-maximal increase in [Ca+2]in were achieved by LTB4 at mean concentrations of 5 X 10(-10) M and 2 X 10(-10) M, respectively, where the binding of LTB4 to high-affinity receptors predominates. A rank order of potency of LTB4 greater than 5(S),12(S)-6-trans-LTB4 greater than 12(S)-LTB4 was established for the elicitation of increases in [Ca+2]in, which reflects the binding of the isomers to low-affinity receptors. PMN leukocytes were preincubated with 10(-8) M LTB4 to induce chemotactic deactivation, which eliminates the expression of high-affinity receptors without altering the expression of the low-affinity receptors for LTB4. LTB4 elicited an increase in [Ca+2]in in the deactivated PMN leukocytes with an EC50 of 3 X 10(-8) M, which is similar to the Kd for LTB4 binding to the low-affinity receptors. Two lines of cultured human leukemic cells, IM-9 and HL-60, did not bind LTB4 specifically and did not show any change in [Ca+2]in upon the addition of 3 X 10(-8) M LTB4. The HL-60 human promyelocytic leukemia cell line was induced to differentiate in 1% dimethyl sulfoxide to leukocytes with more mature myelocytic characteristics. Differentiated HL-60 cells expressed an average of 54,000 low-affinity receptors for LTB4 per cell with an average dissociation constant of 7.3 X 10(-8) M and concurrently developed the capacity to respond to LTB4 with an increase in [Ca+2]in. The binding of LTB4 to either high-affinity or low-affinity receptors appears to be sufficient to initiate an increase in [Ca+2]in in human PMN leukocytes and differentiated HL-60 cells. The specificity of LTB4 receptors in transducing maximum increases in [Ca+2]in is determined by the subset of receptors that predominate as a result of the concentration of LTB4 and the state of the responding cells.     

Studies of leukotriene B4-specific binding and function in rat polymorphonuclear leukocytes: absence of a chemotactic response

Rat PMN isolated from peripheral blood show a small amount of high-affinity (specific) binding of [3H]-LTB4 at nanomolar concentrations. This binding is reversible and has a stereospecificity similar to rat PMN aggregation in response to several LTB4 analogs. This population of binding sites shares many characteristics with a population of high-affinity binding sites in human PMN; however, human PMN bind a significantly greater amount of [3H]-LTB4 to a second population of specific binding sites that is not present in rat PMN. The aggregation responses of human and rat peripheral blood PMN to LTB4 are similar in magnitude and specificity, but unlike human PMN, LTB4 fails to elicit a chemotactic response in rat PMN at concentrations from 10(-10) M to 10(-6) M. Rat PMN also fail to metabolize exogenous LTB4 when compared with human PMN. These data suggest that different PMN functions, such as chemotaxis and aggregation, may involve different classes of specific receptors. The finding that rat PMN do not exhibit chemotaxis to LTB4 calls for a reevaluation of the relevance to inflammation in humans of studies of inflammation performed in rat models.     

Stimulus-specificity of the chemotactic deactivation of human neutrophilis by lipoxygenase products of arachidonic acid

Preincubation of human neutrophils with chemotactic concentrations of 5(S)-hydroxy-eicosatetraenoic acid (5-HETE) or 5(S), 12(R)-dihydroxy-6, 14 cis-8, 10 trans-eicosatetraenoic acid (leukotriene B₄) induces a state of preferential chemotactic unresponsiveness to the homologous factor, termed deactivation, and less suppression of the responses to other chemotactic stimuli. The ratio of the concentration required for maximal chemotactic deactivation of neutrophils to that which stimulates chemotaxis optimally is greater for 5-HETE and leukotriene B₄ than for peptide and protein factors. In contrast to other chemotactic factors, 5-hydroperoxy-eicosatetraenoic acid (5-OOHETE) induces neutrophil chemotactic deactivation that is independent of the nature of the subsequent stimulus and is more slowly reversible after elimination of the fluid-phase deactivating factor. The unique characteristics of the chemotactic deactivation of human neutrophils by 5-OOHETE may be attributable in part to its endogenous metabolism to potent deactivating factors or to covalent derivatization of subcellular structures of the neutrophils by the highly reactive 5-OOHETE.     

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.     

Generation of biologically active, complement-(C5) derived peptides by cathepsin H

The thiol proteinase cathepsin H, isolated and purified from rat liver lysosomes, provokes acute inflammation characterized by the accumulation of polymorphonuclear leukocytes (PMN) when injected intracutaneously into newborn rats. We have examined the possibility that the accumulation of PMN at skin sites injected with cathepsin H is due, in part, to generation locally of C-derived chemotactic factors. We have found that cathepsin H acts in a concentration- and time-dependent fashion in whole human (and rat) EDTA-plasma to generate C5-derived peptides with chemotactic activity for PMN. Chemotactic activity was not generated in EDTA-plasma by either heat-inactivated cathepsin H or by a combination of active enzyme and a thiol proteinase inhibitor isolated from rat epidermis. Cathepsin H also acted in a concentration- and time-dependent fashion on isolated (functionally pure) human C5 to yield chemotactic activity for PMN as well as PMN lysosomal enzyme-releasing activity. Whereas 10 ng/ml cathepsin H generated significant chemotactic activity from isolated C5 (1000 CH50 U/ml), 7 to 10 micrograms/ml were required to generate chemotactic activity in whole EDTA-plasma. Cathepsin H not only was capable of generating biologically active, C5-derived peptides, but also was capable of degrading these peptides. Incubation of either whole EDTA-plasma or isolated C5 with high concentrations of cathepsin H (e.g., 25 micrograms/ml and 100 ng/ml, respectively) caused the rapid appearance of chemotactic activity followed by an equally rapid disappearance. PMN accumulated more rapidly in the skin of newborn rats injected with cathepsin H-treated C5 than in the skin of animals injected with cathepsin H alone. These data suggest that generation by cathepsin H of C-derived chemotactic activity contributes to the ability of this enzyme to induce dermal inflammation.     

Polymorphonuclear leukocytes cap a derivative of wheat germ agglutinin upon stimulation with formyl peptide and C5a but not leukotriene B4

Previously, we reported that a derivative of wheat germ agglutinin (termed WGA-D) specifically inhibits human polymorphonuclear leukocyte (PMN) chemotaxis to FMLP by blocking reexpression (or recycling) of formyl peptide receptors. WGA-D (? formyl peptide receptor probe) binds to a protein on the PMN membrane that exhibits the same m.w. as the formyl peptide receptor. Since clustering (i.e., capping) of ligand-receptor complexes most likely precedes their internalization, we examined the ability of normal and stimulated PMN to cap fluoresceinated WGA-D. We found that, in contrast to capping of fluoresceinated Con A, PMN cap WGA-D in a chemotactic factor-specific fashion. Fluoresceinated WGA-D (5.0 to 20 micrograms/ml) alone did not induce either PMN shape changes (i.e., activation) or capping. Both FMLP (1 to 1000 nM) and human C5a (0.1 to 1.0 nM) induced PMN to polarize and to cap bound WGA-D, in a concentration-dependent fashion. Interestingly, leukotriene B4 (LTB4) (5.0 nM), while inducing the same degree of PMN polarization as FMLP (100 nM) and C5a (0.5 nM), failed to induce PMN to cap bound WGA-D. In contrast, FMLP (100 nM), C5a (0.5 nM), and LTB4 (5.0 nM) induced PMN to cap bound fluoresceinated Con A (10 micrograms/ml) to the same extent. The effect of suboptimal concentrations of FMLP and C5a on capping of WGA-D by PMN was additive. LTB4 did not enhance either FMLP or C5a-induced capping of WGA-D by PMN. Also, FMLP and C5a (but not LTB4) were capable of inducing both desensitization and cross-desensitization of WGA-D capping by PMN. Studies using rhodamine-labeled WGA-D and a fluoresceinated analog of FMLP revealed that both capped to the same place on the PMN membrane. Thus, the data suggest that WGA-D binds to a site on the PMN membrane that is either the FMLP receptor or very closely associated with it.     

Recombinant human tumor necrosis factor alpha lacks chemotactic activity for human peripheral blood neutrophils and monocytes

Preparations of recombinant human tumor necrosis factor alpha (rhuTNF alpha) free of aminoterminal methionine were tested for human neutrophil granulocyte (PMN) and monocyte (MO) chemotactic activity using the Boyden chamber system. Over a wide range of concentrations (10(-7)-10(-15) M) rhuTNF alpha of two different sources failed to elicit chemotactic responses in PMN or MO, whereas strong PMN and MO chemotactic activity could be detected using the tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). In addition, rhuTNF alpha containing 62% aminoterminal methionine failed to induce PMN and MO chemotaxis. It is concluded that rhuTNF alpha may not be a chemotaxin for human PMN and MO in vitro.     

Chemotactic peptides modulate adherence of human polymorphonuclear leukocytes to monolayers of cultured endothelial cells

We have used a new centrifugation assay to examine the effects of highly purified human C5a and C5a des Arg, as well as effects of N-formyl-methionyl-leucyl-phenylalanine (FMLP), on both the extent and strength of human polymorphonuclear leukocyte (PMN) adherence to monolayers of cultured human umbilical vein endothelial cells. At concentrations that were chemotactic for PMN, C5a (0.1 nM), C5a des Arg (5.0 nM), and FMLP (1.0 nM) significantly reduced the percentage of PMN that adhered to endothelial monolayers. Adherence also was reduced by C5a des Arg that was generated by incubating (37 degrees C, 30 min) fresh human serum with either zymosan or purified C5a. High concentrations of C5a (greater than 1.0 nM) and FMLP (greater than 50 nM) that diminished PMN chemotaxis significantly enhanced the percentage of PMN that adhered tightly to endothelial cells (adherent cells resisted a dislodgment force of 1200 X G). Tight adherence of PMN to endothelial cells also was increased by high concentrations of C5a that were added to human serum in which carboxypeptidase N activity was destroyed by heating (56 degrees C, 30 min), and by C5a that was generated by incubating (37 degrees C, 30 min) fresh human serum with zymosan in the presence of the carboxypeptidase N inhibitor, epsilon-aminocaproic acid. High concentrations of C5a des Arg (up to 80 nM) neither enhanced adherence of PMN to endothelial cells nor decreased PMN migration. Thus, a reciprocal relation exists between PMN migration and PMN adherence to endothelial cells in response to chemotactic factors. At concentrations that are chemotactic for human PMN, C5-derived peptides and FMLP reduce the adherence of PMN to endothelial monolayers. Only at concentrations that decrease PMN migration do C5a and FMLP augment PMN adherence.     

Localization of submembranous cations to the leading end of human neutrophils during chemotaxis

Potassium pyroantimonate was used to localize sites of bound cations in human neutrophils under conditions of random migration, stimulated random migration (chemokinesis), and directed migration (chemotaxis). The cells were placed in a standard chamber in which 0.45-micron micropore filters separated the cells from the stimulus (buffer, Escherichia coli endotoxin-activated serum or the synthetic chemotactic peptide N-formyl-Met-Leu-Phe). The small pore filters permitted pseudopod formation but impeded cell imgration through the filter. Cells examined under all conditions had electron-dense precipitates of antimonate salts in some granules. However, antimonate deposits were localized in the condensed chromatin of the nucleus during random migration and associated to a large extent with the uncondensed nuclear chromatin during chemokinesis and chemotaxis. Under conditions of chemokinesis deposition of antimonate procipitates appeared on the cytoplasmic side of the plasma membrane of neutrophils whereas under conditions of chemotaxis cation deposits beneath the cell membrane were localized to the pseudopods which were directed toward the chemoattractant. In addition to endotoxin-activated serum, concentrations of N-formyl-Met-Leu-Phe which caused neutrophil chemotaxis (10(-8) M) also caused cation deposition beneath the cell membrane at the leading end of the cell regardless of whether albumin was present in the incubation media. However, with higher concentrations of the synthetic peptide (10(-5) M) which caused granule release and were not chemotactic, submembranous cation deposition was not seen. EDTA (10 mM) and EGTA (10 mM) removed nuclear, granular, and submembranous cation deposits from neutrophils examined under conditions of chemotaxis. X-ray microprobe analysis of antimonate deposits revealed the possible presence of calcium but did not detect sodium or magnesium. The data indicate that chemotactic factors induce submembranous deposition of cations, most likely Ca++, which localize to the leading edge of cells exposed to a gradient of chemoattractant.     

Independent regulation of human neutrophil chemotactic receptors after activation

The fluoresceinated chemotactic factors, C5a, formyl-methionyl-leucyl-phenylalanyl-lysine (FMLPL), and casein were used in conjunction with flow cytometry to examine chemotactic factor receptor expression on polymorphonuclear leukocytes (PMN) activated with phorbol myristate acetate (PMA), C5a, or formyl-methionyl-leucyl-phenylalanine. Activation with PMA resulted in a dose-dependent increase in binding of fluorescein-labeled (FL)-casein and (FL-FMLPL) over the range of PMA concentrations from 0.5 to 50 ng/ml. In contrast, activation of PMN with PMA resulted in a dose-dependent decrease in FL-C5a binding, and activation with concentrations above 5 ng/ml resulted in a complete loss of binding. This loss of binding was not caused by inactivation of the ligand or prevented by the addition of superoxide dismutase and catalase or protease inhibitors. Furthermore, incubation of PMN with supernatants from PMN stimulated to degranulate did not reduce the availability of C5a receptors. This pattern of increased FMLPL and casein binding with decreased C5a binding was also observed with cytochalasin B-pretreated PMN that were stimulated with chemotactic factors. Parallel studies of superoxide anion generation demonstrated that PMA-treated PMN were still responsive to formyl-methionyl-leucyl-phenylalanine, but not to C5a. These data demonstrate that the activation of PMN up-regulates formyl peptide and casein receptors whereas C5a receptors are down-regulated under similar conditions.