Regulation of human neutrophil chemotaxis by intracellular pH

The relationship of N-formyl-methionyl-leucyl-phenylalanine-stimulated Na+/H+ exchange to the chemotactic responsiveness of human neutrophils was investigated. The pHi changes, measured from the equilibrium distribution of 5,5-dimethyloxazolidine-2,4-dione, were correlated with the migratory behavior of the cells as assessed by the leading front method. Exposure of cells to 10 nM FMLP caused activation of Na+/H+ exchange, leading to a rise in pHi from approximately 7.25 to approximately 7.75. This intracellular alkalinization was inhibited by amiloride and by three more potent analogues. All four compounds reduced the chemotactic response to FMLP with apparent Ki values similar to those for inhibition of the pHi transients, thereby suggesting that the blocking effect of the drugs on directed cell migration was related to inhibition of Na+/H+ exchange. The effect was specific for stimulated cell locomotion: FMLP-induced chemotaxis and chemokinesis were inhibited in parallel, whereas random motility was unimpaired. The relationship of pHi to function was also studied as the pHi of FMLP-activated cells was varied between 6.8 and 8.6 by altering the chemical gradients for Na+ and H+ across the cell membrane. There was a direct, positive correlation between the pHi value attained following FMLP-stimulation and the locomotor response to a chemotactic gradient. These results indicate that the motile functions of human neutrophils can be regulated by their pHi.     

The inhibition of neutrophil granule enzyme secretion and chemotaxis by pertussis toxin

Pertussis toxin treatment of rabbit peritoneal neutrophils causes a concentration-dependent inhibition of granule enzyme secretion induced by formylmethionyl-leucyl-phenylalanine, C5a, and leukotriene B4. It also inhibits chemotaxis induced by formylmethionyl-leucyl-phenylalanine. The same toxin treatment, however, has no effect on granule enzyme secretion induced by the calcium ionophore A23187 or phorbol 12-myristate 13-acetate. Moreover, pertussis toxin treatment does not affect either the number or affinity of the formylpeptide receptors on the neutrophil nor does it have any effect on the unstimulated levels of cyclic AMP (cAMP) or the transient rise in cAMP induced by chemotactic factor stimulation in these cells. We hypothesize that pertussis toxin, as in other cells, interacts with a GTP binding regulatory protein identical with or analogous to either Ni or transducin which mediates the receptor-induced inhibition or activation of a target protein or proteins required in neutrophil activation. The nature of the target protein is unknown, but it is not the catalytic unit of adenylate cyclase. The target protein acts after binding of chemotactic factor to its receptor in the sequence that leads to the receptor-induced rise in intracellular Ca2+. It does not affect the responses elicited by the direct introduction of calcium into the cells or the activity of protein kinase C.     

Echinococcus multilocularis: inhibition of murine neutrophil and macrophage chemotaxis

Resident peritoneal neutrophils and macrophages from mice infected with 50 +/- 5 cysts of Echinococcus multilocularis were collected at 2, 4, 6, 8, 10, and 16 weeks postinfection. Their ability to respond and migrate to purified parasite larval antigens or endotoxin-activated mouse serum (EAMS) in comparison to normal peritoneal cells from uninfected mice was tested in vitro using Boyden chambers. Early in the infection, both cell types responded to the specific and nonspecific chemoattractants as the control group. However, at 8 and 10 weeks postinfection, the neutrophils and macrophages lost their response to parasite antigens but retained their ability to migrate to EAMS. Toward the 12th and 16th week postinfection, both cell types lost their ability to migrate to the specific as well as the nonspecific factors. The data presented suggest that the cellular mechanisms of recognition and chemotaxis in mice infected with alveolar hydatid cysts are impaired.     

Effects of C-reactive protein on human neutrophil granulocytes challenged with N-formyl-methionyl-leucyl-phenylalanine and platelet-activating factor

The effects of C-reactive protein (CRP), the prototypical acute-phase reactant were studied on human polymorphonuclear leukocytes (PMNL) challenged with N-formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF). CRP at 8-64 micrograms/ml concentrations inhibited degranulation and superoxide production by PMNL in time-, and dose-dependent manner and stabilized PMNL membranes against the lytic effect of lysophosphatidylcholine. CRP was also capable of binding PAF and in lesser extent fMLP. Furthermore, CRP, 32 micrograms/ml, diminished specific binding of [3H]-fMLP and [3H]-PAF to PMNL. These findings imply that CRP may play an important protective role during the early phase of acute inflammatory reactions.     

Polyvalent cations inhibit human neutrophil chemotaxis by interfering with the polymerization of actin

The effect of a series of di- and trivalent cations on the locomotor response of human neutrophils to the chemotactic tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) was investigated. Migration was assessed by the leading front method. The cations inhibited FMLP-stimulated chemotaxis in the rank order: Ni2+ approximately Co2+ greater than Sr2+ greater than Zn2+ greater than Mn2+ approximately La3+ greater than Cd2+ approximately Ba2+ much greater than Mg2+. Benzamil, which blocks Na+/Ca2+ exchange, did not alter chemotaxis by itself but prevented the suppressive effects of each of the polyvalent cations on motility. The ion selectivity sequence and the lack of activity of benzamil are strikingly different than for O(-2) generation, thereby implying different modes of action in the two functional expressions. The F-actin content of the cells was monitored by the fluorescence of rhodamine-phalloidin. Each of the cations displayed comparable efficacy in blocking the polymerization of actin in FMLP-activated cells. Likewise, benzamil exhibited a protective effect, completely overcoming the inhibitory action of the polyvalent cations. The results indicate that these foreign ions gain access to the cell interior via a benzamil-sensitive pathway, namely Na+/Ca2+ exchange. Upon entry into the cytosol, they then interfere with the formation of filaments from actin monomers. These studies help to shed light on the interaction of divalent cations with cytoskeletal and contractile elements in cell motility.     

The Src family kinases Hck and Fgr regulate neutrophil responses to N-formyl-methionyl-leucyl-phenylalanine

The chemotactic peptide formyl-methionyl-leucyl-phenilalanine (fMLP) triggers intracellular protein tyrosine phosphorylation leading to neutrophil activation. Deficiency of the Src family kinases Hck and Fgr have previously been found to regulate fMLP-induced degranulation. In this study, we further investigate fMLP signaling in hck-/-fgr-/- neutrophils and find that they fail to activate a respiratory burst and display reduced F-actin polymerization in response to fMLP. Additionally, albeit migration of both hck-/-fgr-/-mouse neutrophils and human neutrophils incubated with the Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) through 3-microm pore size Transwells was normal, deficiency, or inhibition, of Src kinases resulted in a failure of neutrophils to migrate through 1-microm pore size Transwells. Among MAPKs, phosphorylation of ERK1/2 was not different, phosphorylation of p38 was only partially affected, and phosphorylation of JNK was markedly decreased in fMLP-stimulated hck-/-fgr-/- neutrophils and in human neutrophils incubated with PP2. An increase in intracellular Ca(2+) concentration and phosphorylation of Akt/PKB occurred normally in fMLP-stimulated hck-/-fgr-/- neutrophils, indicating that activation of both phosphoinositide-specific phospholipase C and PI3K is independent of Hck and Fgr. In contrast, phosphorylation of the Rho/Rac guanine nucleotide exchange factor Vav1 and the Rac target p21-activated kinases were markedly reduced in both hck-/-fgr-/- neutrophils and human neutrophils incubated with a PP2. Consistent with these findings, PP2 inhibited Rac2 activation in human neutrophils. We suggest that Hck and Fgr act within a signaling pathway triggered by fMLP receptors that involves Vav1 and p21-activated kinases, leading to respiratory burst and F-actin polymerization.     

The alterative effect of isobutylmethylxanthine in hypofunctional human neutrophil chemotaxis

The effects of isobutylmethylxanthine on the locomotion of chemotactically normofunctional and hypofunctional human neutrophils were compared. The hypofunctional cells were obtained from the peripheral blood of selected subjects with juvenile periodontitis. The chemically oriented migration (i.e. chemotaxis) of these cells was less than 50% of normal. Isobutylmethylxanthine caused a doubling of the CHO.Met.Leu.Phe.OH-induced chemotactic responsiveness of hypofunctional cells, while no effect of the drug was detected with respect to random migration. Isobutylmethylxanthine had no effect on chemotaxis or random migration in the normofunctional cell populations. Since isobutylmethylxanthine inhibits cAMP catabolism, these data suggest that the cyclic nucleotide plays a role in intracellular transduction mechanisms which affect chemotaxis.     

Intracellular inhibition of human neutrophil elastase by orally active pyrrolidine-trans-lactams

Described are the acylation binding of trans-lactam 1 to porcine pancreatic elastase, the selection of the SO2Me activating group for the lactam N which also confers metabolic stability in hamster liver microsomes, the introduction of aqueous solubility through the piperidine salt 9, the in vivo oral activity of 9 and its bioavailability, and the introduction of 9 as an intracellular neutrophil elastase inhibitor.     

The influence of contact guidance on chemotaxis of human neutrophil leukocytes

Chemotaxis of human neutrophil leukocytes moving on or in aligned 3D fibrin gels is more efficient if the cells are moving along the axis of fibre alignment than if they have to cross the fibres. This was shown by using two assays, one in which the cells were responding to a distant (600 micrometers) gradient source diffusing from a filter paper impregnated with formyl-Met-Leu-Phe and incorporated into the gel, the other in which the cells were responding to nearby (20--30 micrometers) Candida albicans spores in serum. In the former assay, impairment of chemotaxis across the axis of fibre alignment was highly significant. In the latter, cells showed efficient chemotaxis to the spores, but took more irregular paths when crossing the aligned fibres than when running along them. Neutrophils show contact guidance in aligned collagen or fibrin gels (Wilkinson et al., Exp cell res 140 (1982) 55) [1], thus the cells were subjected simultaneously to two directional cues in these experiments, one the chemotactic gradient and the other a contact guidance field. These cues may reinforce or interfere with each other depending on their relative orientation. Since many tissues in vivo show alignment or more complex forms of patterning, tissue architecture is likely to be an important determinant of the efficiency of cellular mobilization in inflamed or infected sites.     

Inhibition of lymphocyte and neutrophil chemotaxis by pertussis toxin

The cells of the mammalian immune system possess special migratory properties within their in vivo environment, a surveillance characteristic that is thought to be important in the protection of the organism from transformants and exogenous pathogens. Pertussis toxin (PT) has been shown to disrupt the intensity of this process by seriously affecting lymphocyte recirculation in vivo. The mechanisms responsible for this inhibition were investigated by using the in vitro model systems of polymorphonuclear leukocyte and lymphocyte chemotaxis. The type of inhibition that was observed in these in vitro assay systems was quite similar to that observed in vivo, because PT could depress chemotaxis in vitro as well as the accumulation of radiolabeled lymphocytes and neutrophils within a peripheral site of inflammation in vivo. The alterations in neutrophil motility were found to be associated with a stimulus-specific inhibition of the triggering of superoxide anion generation and lysosomal secretion. Some inhibition of neutrophil adherence to plastic surfaces was also observed, most notably after augmentation of adherence with the chemoattractant fMLP. The observed alterations in cellular function after PT treatment occurred in the absence of defects in chemoattractant binding to the neutrophil cell surface, or of membrane potential changes stimulated by ligand binding. The effect of PT in this system was found to be associated with an abnormality in the regulation of intracellular free calcium, suggesting that the substrate for PT in neutrophils is involved in the regulation of calcium ion channels.     

Cutting edge: nociceptin stimulates neutrophil chemotaxis and recruitment: inhibition by aspirin-triggered-15-epi-lipoxin A4

The nociceptin receptor (Noci-R) is a G protein-coupled receptor present in neural tissues and its activation by nociceptin is involved in the processing of pain signals. Here, we report that Noci-R is present and functional on peripheral blood polymorphonuclear leukocytes (PMN). Human PMN express mRNA for Noci-R, its nucleotide sequence determined, and specific binding with [(125)I]-labeled nociceptin gave an apparent K(d) approximately 1.5 nM for this PMN opioid receptor. Nociceptin evoked PMN chemotaxis with maximal activity at 100 pM, without intracellular Ca(2+) mobilization. When injected in murine air pouches, nociceptin elicited leukocyte infiltration in a concentration-dependent fashion. Nociceptin-stimulated PMN infiltration was inhibited by treating mice with a synthetic analog of the aspirin-triggered lipid mediator 15-epi-lipoxin A(4). The present results identify nociceptin as a potent chemoattractant and provide a novel link between the neural and immune systems that are blocked by aspirin-triggered lipid mediators and may be relevant in neurogenic inflammation.     

New insights into the inhibition of human neutrophil elastase by heparin

In the normal feedback mechanism of injury and repair in the lung, fragmented heparan sulfate proteoglycans (HSPGs) from damaged extracellular matrix and cells are believed to interact with elastases to limit their activity. An imbalance in the HSPG-elastase response may play an important role in situations where uncontrolled lung injury leads to diseases such as emphysema. To gain insight into this complex process of heparin and heparan sulfate regulation of elastases, an experimental study was undertaken to resolve the mechanism and structural requirements of heparin inhibition of human neutrophil elastase (HNE). Kinetic analyses were completed using in vitro assays with synthetic and insoluble elastin substrates in the presence of HNE and various heparin preparations (14-15 kDa; 17-19 kDa), heparin-derived oligosaccharides (4-22 saccharides), and chemically modified heparins (2-O-, 6-O-, O-, and N-desulfated). Results showed that heparin inhibits HNE by a tight-binding, hyperbolic, competitive mechanism, contrary to previous reports in the literature. A minimum length of at least 12-14 saccharides is required for inhibition, after which inhibitory activity increases with chain length (or molecular mass). Although all N- and O-sulfate groups contribute to inhibition, 2-O-sulfate groups are less critical than either N- or 6-O-sulfate groups, indicating that inhibitory activity is dependent upon the heparin fine structure. Molecular-docking simulations support the kinetic results and provide a plausible model for the size requirement, whereby positively charged, clamp-like regions at the ends of the interdomain crevice (elastase fold) are used by heparin to bridge the active site and inhibit activity.     

Nicotinanilides as inhibitors of neutrophil chemotaxis

A series of 4-fluoronicotinanilides was synthesized and shown to be novel, potent, and selective inhibitors against GRO-alpha-driven human neutrophil chemotaxis. Compounds of this class may be useful for the treatment of inflammatory, autoimmune, and allergic disorders.     

Selected antibodies to leukocyte common antigen (CD45) inhibit human neutrophil chemotaxis.

The CD45 Ag family is a group of high m.w. glycoproteins that are expressed on the plasma membranes of all leukocytes. CD45 has protein tyrosine phosphatase activity and appears to regulate signal transduction and lymphocyte activation by specific association with receptor molecules on T and B lymphocytes. However, little is known about CD45 function in neutrophils (PMN). In this study, PMN were incubated with CD45 mAb and tested for their chemotactic responses to four unrelated chemo-attractants: FMLP, leukotriene B4 (LTB4), recombinant human C5a (C5a), and recombinant human neutrophil-activating protein-1, recently designated IL-8. A panel of CD45 mAb including an IgM mAb, AHN-12.1, and six IgG1 mAb, AHN-12, AHN-12.2, AHN-12.3, AHN-12.4, HLe-1, and KC56(T200), were tested for their effects on PMN chemotaxis. PMN chemotaxis was evaluated with two different membrane assays; one assay quantified the total number of migrating PMN and the other assayed the leading front of migrating PMN. AHN-12.1 and KC56(T200) significantly inhibited PMN chemotaxis to LTB4 and C5a. AHN-12.1 slightly inhibited PMN chemotaxis to FMLP, but KC56(T200) did not. In contrast, AHN-12 and HLe-1 did not significantly inhibit PMN chemotaxis to any of the chemoattractants. None of the CD45 mAb inhibited PMN chemotaxis to neutrophil-activating protein-1/IL-8. None of the CD45 mAb inhibited PMN superoxide production. These results suggest that PMN CD45 epitopes may interact with LTB4 and C5a receptor-associated molecules and regulate chemotactic responses.     

Selective neutrophil desensitization to chemotactic factors

In the presence of extracellular calcium and magnesium, a series of chemotactic oligopeptides and C5a caused aggregation of human polymorphonuclear neutrophils (PMNs). This cellular response developed rapidly and began to reverse 2 min after exposure to the chemotactin. In the absence of the bivalent cations, none of the chemotactins stimulated the aggregation response. If cells were first exposed to a chemotactin and then treated with calcium and magnesium, aggregation was detected only after addition of the cations, and the magnitude of the response fell sharply as the interval between the addition of chemotactin and addition of cations was lengthened: when this interval exceeded 2 min, aggregation was barely detectable. This loss of reactivity persisted even when cells were re-exposed to fresh chemotactic factor and washed between the first and second exposures. In all instances, however, loss of cellular reactivity was highly selective: cells preincubated with any chemotactic oligopeptide were hyporesponsive to subsequent stimulation with an oligopeptide but remained fully responsive to C5a; cells preincubated with C5A were hyporesponsive to C5a but retained their responsitivity to the oligopeptides. Because this selectivity parallels the known specificities of these chemotactic factors for their receptors in or on the neutrophil, desensitization may reflect functional loss of receptors after stimulation. Alternatively, this selectivity may indicate that morphologically identical neutrophils contain subpopulations of cells with varying reactivities to receptor-bound chemotactic factors. In either event, desensitization may be useful in functionally defining chemotactic factors and their respective receptors. The rapidity of development of desensitization suggests that it may operate to limit or moderate various in vitro and in vivo neutrophil responses to chemotactic factors.     

Deactivation of human neutrophil chemotaxis by chemoattractants: effect on receptors for the chemotactic factor f-Met-Leu-Phe

Normal human peripheral blood PMN were exposed to varying concentrations of partially purified chemotactic complement fragments (C5fr) and a chemotactic peptide N-formyl methionylleucylphenylalanine (f-Met-Leu-Phe). This exposure resulted in a decreased chemotactic response termed deactivation of chemotaxis. Deactivation was found to be nonpreferential for the deactivating stimulus when high concentrations of either f-Met-Leu-Phe (10(-6) M) or C5fr (20 micrograms/ml) were used. When PMN were incubated with lower concentrations of C5fr (10 micrograms/ml), there was preferential deactivation towards C5fr. Similarly, preferential deactivation of chemotaxis was observed when PMN were incubated with 10(-6) M f-Met-Leu-Phe, but this was transient and cells were nonpreferentially deactivated 60 min after the initial exposure to f-Met-Leu-Phe. The availability of receptors for tritiated f-Met-Leu-Phe was examined by Scatchard analyses and measurement of reversible f-Met-Leu-[3H]Phe binding to C5fr and f-Met-Leu-Phe-deactivated PMN. When PMN f-Met-Leu-Phe receptors were studied immediately after exposure to concentrations of C5fr causing either preferential or nonpreferential deactivation, there was increased receptor availability compared with control PMN. In contrast, PMN deactivated with high concentrations of f-Met-Leu-Phe 10(-6) M) had a transient decrease in the number of receptors followed 1 hr later by an increase in the number of receptors. This was similar to the functional correlate of preferential deactivation of chemotaxis immediately after incubation with f-Met-Leu-Phe followed by nonpreferential deactivation in these same PMN. The data indicate that preferential deactivation of chemotaxis may be associated with a preferential decrease (down-regulation) of chemoattractant receptors and that nonpreferential deactivation is associated with an increase in chemoattractant receptors.     

Paradoxical effects of adenosine on neutrophil chemotaxis

Chemotaxis of rabbit neutrophils is most sensitive to inhibition by 3-deazaadenosine, followed by 3-deaza-(+/-)aristeromycin, 3-deaza-(+/-)aristeromycinylhomocysteine, 3-deazaadenosylhomocysteine, and adenosylhomocysteine, in that order. Although adenosine by itself had no effect on the chemotaxis of neutrophils, it essentially abolished the inhibitory effects of 3-deaza-adenosine on chemotaxis and the reduction of nitroblue tetrazolium. Paradoxically, adenosine enhanced the inhibition of chemotaxis by 3-deazaadenosylhomocysteine slightly and that of 3-deaza-(+/-)aristeromycin significantly. Adenosine alone unexpectedly inhibited phospholipid methylation to the same extent as 3-deazaadenosine, and reduced protein carboxymethylation to a lesser degree. The inhibition of these two methylation reactions by 3-deazaadenosine was, however, not substantially altered in the presence of adenosine. Drastic changes in the ratio of adenosylmethionine/nucleosidylhomocysteine were observed in the presence of adenosine, 3-deazaadenosine, 3-deaza-(+/-)aristeromycin, or of adenosine in combination with each of the latter compounds. There was no significant effect on the binding of chemotactic peptide to receptors, or on the ratio of ATP/ADP in cells treated by the analogs. These results suggest that the inhibition of methylation reactions per se is not enough to account for the inhibition of both chemotaxis and the reduction of nitroblue tetrazolium by neutrophils.     

Controlling the molecular motor of neutrophil chemotaxis

Our defence against microbes depends largely on the ability of neutrophils to migrate from the blood stream to sites of infection. Although the ability of animal cells to move may be primitive, and also fundamental for a number of phenomena in biology, the cellular mechanism by which neutrophils are able to move rapidly towards the infection remains an enigma. Even though the structures of the receptors involved have been sequenced and many of the molecules involved in neutrophil adherence and traction identified, the essential mechanisms that control and regulate the neutrophil motor remain obscure. Here, an outline of the fundamental inadequacies in our current understanding is given, along with some recent developments that promise to produce some significant advances.