The anti-infective peptide, innate defense-regulator peptide, stimulates neutrophil chemotaxis via a formyl peptide receptor

The anti-infective peptide, innate defense-regulator peptide (IDR-1), has been selectively reported to modulate the innate immune response. We found that IDR-1 stimulates the chemotactic migration in human neutrophils. Moreover, IDR-1-induced neutrophil chemotaxis was completely blocked by pertussis toxin, suggesting the importance of the G_i protein in this process. The mechanism governing the IDR-1-induced neutrophil chemotaxis was found to be completely inhibited by the formyl peptide receptor (FPR) antagonist; cyclosporin H. IDR-1 was also found to induce chemotactic migration in FPR but not in vector-expressing HCT116 cells. Meanwhile, IDR-1 failed to stimulate superoxide anion generation and intracellular calcium increase in human neutrophils. Furthermore, IDR-1 was found to inhibit fMLF (an FPR agonist)-induced superoxide generation and calcium signaling in human neutrophils and FPR-expressing HCT116 cells. Taken together, the results demonstrate that IDR-1 is a partial agonist for FPR and further, stimulates neutrophil chemotaxis without inducing calcium signaling and superoxide generation.     

Alternagin-C, a nonRGD-disintegrin, induces neutrophil migration via integrin signaling.

Recently, a new protein containing a disintegrin domain, alternagin-C (Alt-C), was purified from Bothrops alternatus venom. Unlike other disintegrins, in Alt-C an ECD amino acid mogif takes the place of the RGD sequence. Most disintegrins contain an RGD/KGD sequence and are very potent inhibitors of platelet aggregation, as well as other cell interactions with the extracellular matrix, including tumor cell metastasis and angiogenesis. The present study investigated the effects of Alt-C on human neutrophil chemotaxis in vitro and the activation of integrin-mediated pathways. Alt-C showed a potent chemotactic effect for human neutrophils when compared to N-formyl-methionyl-leucyl-phenylalanine peptide (fMLP), a classic chemotactic agent. Moreover, preincubation of neutrophils with Alt-C significantly inhibited chemotaxis toward fMLP and itself. In addition, a peptide containing an ECD sequence presented a chemotactic activity and significantly inhibited chemotaxis induced by Alt-C and fMLP. A significant increase of F-actin content was observed in cells treated with Alt-C, showing that the chemotactic activity of Alt-C on neutrophils is driven by actin cytoskeleton dynamic changes. Furthermore, this protein was able to induce an increase of phosphotyrosine content triggering focal adhesion kinase activation and its association with phosphatidylinositol 3-kinase. Alt-C was also able to induce a significant increase in extracellular signal-regulated kinase 2 nuclear translocation. The chemotactic activity of Alt-C was partially inhibited by LY294002, a specific phosphatidylinositol 3-kinase inhibitor, and by PD98056, a Map kinase kinase inhibitor. These findings suggest that Alt-C can trigger human neutrophil chemotaxis modulated by intracellular signals characteristic of integrin-activated pathways and that these effects could be related to the ECD mogif present in disintegrin-like domain.     

Divergent effects of alpha1-antitrypsin on neutrophil activation, in vitro

alpha1-Antitrypsin (AAT) is a major circulating serine proteinase inhibitor in humans. The anti-proteinase activity of AAT is inhibited by chemical modification. These include inter- or intramolecular polymerisation, oxidation, complex formation with target proteinases (e.g., neutrophil elastase), and/or cleavage by multi-specific proteinases. In vivo, several modified forms of AAT have been identified which stimulate biological activity in vitro unrelated to inhibition of serine proteinases. In this study we have examined the effects of native and polymerised AAT and C-36 peptide, a proteolytic cleavage product of AAT, on human neutrophil activation, in vitro. We show that the C-36 peptide displays striking concentration-dependent pro-inflammatory effects on human neutrophils, including induction of neutrophil chemotaxis, adhesion, degranulation, and superoxide generation. In contrast to C-36 peptide, native and polymerised AAT at similar and higher concentrations showed no effects on neutrophil activation. These results suggest that cleavage of AAT may not only abolish its proteinase inhibitor activity, but can also generate a powerful pro-inflammatory activator for human neutrophils.     

S1P stimulates chemotactic migration and invasion in OVCAR3 ovarian cancer cells

OVCAR3 ovarian cancer cells express three sphingosine 1-phosphate (S1P) receptors, S1P(1), S1P(2), and S1P(3), but not S1P(4). Stimulation of OVCAR3 cells with S1P induced intracellular calcium increases, which were partly inhibited by VPC 23019 (an S1P(1/3) antagonist). S1P-induced calcium increases were mediated by phospholipase C and pertussis toxin (PTX)-sensitive G-proteins in OVCAR3 cells. S1P stimulated extracellular signal-regulated kinase, p38 kinase, and Akt which were inhibited by PTX. S1P-stimulated chemotactic migration of OVCAR3 cells in a PTX-sensitive manner, indicating crucial role of G(i) protein(s) in the process. S1P-induced chemotactic migration of OVCAR3 cells was completely inhibited by LY294002 and SB203580. Pretreatment of VPC 23019 (an S1P(1/3) antagonist) completely inhibited S1P-induced chemotaxis. S1P also induced invasion of OVCAR3 cells, which was also inhibited by VPC 23019. Taken together, this study suggests that S1P stimulate chemotactic migration and cellular invasion, and VPC 23019-sensitive S1P receptor(s) might be involved in the processes.     

MAPK-activated protein kinase-2 participates in p38 MAPK-dependent and ERK-dependent functions in human neutrophils

Many neutrophil responses, including chemotaxis, exocytosis, respiratory burst activity and chemokine synthesis, are mediated by p38 MAPK. MAPK-activated protein kinase-2 (MK2) is activated by p38 MAPK in human neutrophils. The present study tested the hypothesis that MK2 mediates multiple p38 MAPK-dependent responses in human neutrophils by comparing the effect of the p38 MAPK inhibitor, SB203580, with an MK2 inhibitory peptide. Both SB203580 and MK2 inhibitory peptide attenuated respiratory burst activity, exocytosis, and chemotaxis. Lipopolysaccharide (LPS)-induced IL-8 production was inhibited by SB203580, but not by the MK2 inhibitory peptide. Inhibition of chemotaxis and respiratory burst activity by SB203580 was less than that of MK2 inhibitory peptide. Inhibition of extracellular signal-regulated kinase (ERK) activity by PD98059 attenuated superoxide release and chemotaxis, and simultaneous treatment with SB203580 and PD98059 demonstrated additive inhibition. ERK phosphorylated MK2 in vitro and activated MK2 in f-methionyl-leucyl-phenylalanine (FMLP)-stimulated neutrophils. These data suggest that MK2 mediates both ERK- and p38 MAPK-dependent neutrophil responses.     

Calcium modulation and chemotactic response: divergent stimulation of neutrophil chemotaxis and cytosolic calcium response by the chemotactic peptide receptor

Stimulation of neutrophils by chemoattractants is followed by a rapid, transient rise in cytosolic calcium concentration. The role of calcium in activation of cell movement and related responses was examined by selectively chelating extracellular or both extra- and intracellular calcium. Removal of calcium from the extracellular medium did not alter the cytosolic calcium concentration (Quin 2 fluorescence, 110 to 120 nM) of unstimulated neutrophils and did not dramatically affect the rise induced by formyl peptide. Despite the intact Quin 2 response, depletion of extracellular calcium partially inhibited chemotaxis, adherence to substrate, and polarization (increased forward light scatter) in response to formyl peptide. Loading neutrophils with Quin 2 in the absence of calcium depressed cytosolic Ca2+ to 10 to 20 nM and abrogated a detectable rise with formyl peptide stimulation. Depletion of intracellular calcium further inhibited chemotaxis and polarization, although neutrophils still demonstrated significant directed migration and shape change to formyl peptide (30 to 40% of control) without an increase in Quin 2 fluorescence. Other neutrophil responses related to chemotaxis (decreased right-angle light scatter, actin polymerization) were minimally affected by depletion of calcium from either site. The data indicate that neutrophil chemotaxis and related responses to formyl peptide may be activated by intracellular signals not detectable with Quin 2.     

CD38 cleavage in fMLP- and IL-8-induced chemotaxis is dependent on p38 MAP kinase but independent of p44/42 MAP kinase

In this study, we examined the mechanism by which CD38 cleavage is regulated through the mitogen-activated protein (MAP) kinases after stimulation by fMLP and interleukin-8 (IL-8) in neutrophils. Both fMLP and IL-8 increased chemotaxis and decreased CD38 protein in neutrophils, but did not change CD38 mRNA levels. Both fMLP and IL-8 increased CD38 in supernatants, which was inhibitable with PMSF. fMLP stimulation resulted in phosphorylation of p38 MAP kinase and p42/44 MAP kinase (ERK). SB20358, a p38 MAP kinase inhibitor, down-regulated neutrophil chemotaxis. Conversely, PD98059, an ERK inhibitor, did not influence chemotaxis to either agonist. The addition of SB20358 blocked the decrease of CD38 on neutrophils and the increase in supernatants induced by fMLP or IL-8, whereas PD98059 did not. These findings suggest that CD38-mediated chemotaxis to fMLP or IL-8 is characterized by proteolytic cleavage of CD38 and signaling through p38 MAP kinase. Activation of the protease for cleavage appears to be a postreceptor event that is dependent on p38 MAP kinase signaling.     

Neutrophil activation by inflammatory microcrystals of monosodium urate monohydrate utilizes pertussis toxin-insensitive and -sensitive pathways

The activation of leukocytes by particulates is a critical event in certain inflammatory syndromes, including acute gout associated with microcrystals of monosodium urate monohydrate. In this study we have evaluated mechanisms of human neutrophil activation by urate crystals. Both N-formyl-nor-leu-leu-phe-nor-leu-tyr-lys and uncoated urate crystals (0.5 to 5 mg/ml) but not urate crystals coated with human low density lipoprotein (which suppresses crystal-induced neutrophil responsiveness), stimulated pertussis toxin (PT)-sensitive GTPase activity in purified preparations of human neutrophil membranes. Hydroxyapatite crystals (up to 5 mg/ml) were inactive. Pretreatment of neutrophil membranes with cholera toxin also inhibited crystal-induced and formylated peptide-induced GTPase activity. Pretreatment of whole neutrophils with PT resulted in nearly complete inhibition of formylated peptide-induced cytosolic calcium mobilization, release of superoxide and release of the azurophil granule constituent alpha-mannosidase. In contrast, identical pretreatment of whole neutrophils with PT only partially suppressed urate crystal-induced alpha-mannosidase and superoxide release and failed to inhibit crystal phagocytosis and increases in cytosolic free calcium. Mechanisms of neutrophil activation by monosodium urate crystals appear to be heterogeneous in comparison with activation by formylated peptides and there is no absolute requirement for PT-sensitive membrane G proteins in neutrophil responsiveness to urate crystals.     

Priming of normal human neutrophils by tachykinins: tuftsin-like inhibition of in vitro chemotaxis stimulated by formylpeptide or interleukin-8.

Tachykinins have priming effects on polymorphonuclear neutrophils, since they may activate the neutrophils to exhibit an exaggerated inflammatory response to phlogistic mediators. In order to investigate mechanisms involved in this action, we determined the influence of substance P and neurokinin A on chemotaxis of human neutrophils towards gradients of formymethionyl-leucyl-phenylalanine or recombinant human interleukin-8. As seen with other neutrophil-priming agents such as tumor necrosis factor-alpha, exposure of neutrophils to substance P or neurokinin A had an inhibitory effect upon a stimulated migration, with effective concentrations being in the nanomolar range. Tuftsin, a known neutrophil activating peptide, similarly inhibited stimulated migration. Analysis of structure-activity relationships revealed that activity of tachykinins is located in amino-terminal, tuftsin-like sequences. The inhibition of stimulated migration was partly reversed by (Pro1)-tuftsin, a partial tuftsin receptor antagonist, which suggests that the effects of amino-terminal tachykinins involves activation of tuftsin receptors of neutrophils.     

Suppressive effect of rebamipide, an antiulcer agent, against activation of human neutrophils exposed to formyl-methionyl-leucyl-phenylalanine

Rebamipide, an antiulcer agent, has been shown to be able to prevent gastric mucosal injury resulting in part from activation of neutrophils. The mechanism of its suppressive action, however, remains to be established. The present study aimed to determine the effect of rebamipide on activation of isolated human neutrophils and to identify the signal transduction pathway involved in its regulation. In unstimulated cells, alkaline phosphatase activity was found residing in short rod-shaped intracellular granules. Upon stimulation with a chemotactic peptide formyl-methionyl-leucyl-phenylalanine, the granules fused to form elongated tubular structures and spherical vacuoles. Rebamipide inhibited reorganization of alkaline phosphatase-containing granules along with upregulation of alkaline phosphatase activity and CD16, a marker of the granules. It also suppressed chemotaxis, an increase in intracellular calcium ion concentration, and NADPH oxidase activation in cells stimulated with formyl-methionyl-leucyl-phenylalanine. In contrast, the drug showed no inhibitory action toward upregulation of alkaline phosphatase activity and CD16, and activation of NADPH oxidase in cells stimulated with phorbol myristate acetate, an activator of protein kinase C. These findings demonstrate that rebamipide exerts a broad spectrum of suppressive actions toward biological functions of human neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine, but not with phorbol myristate acetate, and suggest that the upstream point of protein kinase C is the signal transduction pathway involved in its regulation.     

The chymotrypsin inhibitor carbobenzyloxy-leucine-tyrosine-chloromethylketone interferes with the neutrophil respiratory burst mediated by a signaling pathway independent of PtdInsP2 breakdown and cytosolic free calcium.

The effects of carbobenzyloxy-leucine-tyrosine-chloromethylketone (zLYCK), an inhibitor of chymotrypsin, were investigated on the activation pathways of the human neutrophil respiratory burst. At 10 microM zLYCK, a parallel inhibition was observed of superoxide production stimulated with the chemo-attractant FMLP and of chymotrypsin-like activity of human neutrophils. By contrast, superoxide production induced by PMA was minimally affected by zLYCK. The known transduction pathways triggered by FMLP were analyzed. zLYCK did not affect either the FMLP-induced cytosolic free calcium transient, inositol 1,4,5 trisphosphate formation, nor the PMA-induced phosphorylation of the 47-kDa substrate of protein kinase C. zLYCK did not affect the activity of protein kinase C extracted from neutrophils. In Ca(2+)-depleted cells, in which phosphatidylinositol 4,5-biphosphate breakdown does not occur, zLYCK inhibited the FMLP-induced respiratory burst in cells primed by low doses of PMA. The activity of the NADPH oxidase tested with active membranes from stimulated neutrophils or in a cell-free system was not inhibited by zLYCK. We conclude that: 1) zLYCK inhibits superoxide production through the inhibition of a chymotrypsin-like protease of the neutrophil, 2) zLYCK inhibits FMLP-induced activation of NADPH oxidase through a pathway independent of PtdInsP2 breakdown and cytosolic free calcium, and 3) zLYCK may prove a useful probe for the characterization of its target protease in neutrophil activation.     

Selective inhibition of human neutrophil chemotaxis to N-formyl-methionyl-leucyl-phenylalanine by sulfones

The therapeutic efficacy of the sulfones, dapsone, and sulfoxone in neutrophilic dermatoses may be related to the effects of these drugs on neutrophil function. Therefore we determined whether neutrophil chemotactic migration to various chemoattractants could be inhibited by sulfones in vitro. The chemotactic responses of human neutrophils from healthy donors were tested by using N-formyl-methionyl-leucyl-phenylalanine (F-met-leu-phe), purified human C5a, and leukocyte-derived chemotactic factor (LDCF). Therapeutic concentrations of sulfones selectively inhibited neutrophil chemotaxis to F-met-leu-phe, but did not affect neutrophil chemotaxis to LDCF or C5a. Inhibition of neutrophil chemotaxis to F-met-leu-phe was induced by both dapsone and sulfoxone at a concentration of 10 micrograms/ml without affecting random migration, and the inhibition was reversed by washing the neutrophils. When dapsone- and sulfoxone-treated neutrophils (100 micrograms/ml) were stimulated with F-met-leu-phe, neutrophil superoxide generation was not inhibited. Sulfapyridine (10 micrograms/ml) also selectively inhibited neutrophil chemotaxis to F-met-leu-phe; however, sulfamethoxazole and sulfisoxazole did not affect chemotaxis. The inhibitory effects of dapsone, sulfoxone, and sulfapyridine could not be demonstrated with granulocytes from rabbits or guinea pigs nor with human monocytes. Experiments with radiolabeled dapsone showed rapid, nonspecific, and reversible binding of dapsone to human neutrophils. These data suggest that a mechanism of action of sulfones in neutrophilic dermatoses may be a selective inhibition of neutrophil migration to as yet undefined chemoattractants in the skin.     

Lysophosphatidylserine stimulates leukemic cells but not normal leukocytes

In this study, we observed that lysophosphatidylserine (LPS) stimulated intracellular calcium ([Ca(2+)](i)) increase in leukemic cells but not in normal human peripheral blood mononuclear cells. LPS also stimulated [Ca(2+)](i) increase in human leukemic THP-1 cells. LPS-stimulated [Ca(2+)](i) increase was inhibited by U-73122 but not by U-73343. LPS also stimulated inositol phosphates formation in THP-1 cells, suggesting that LPS stimulates calcium signaling via phospholipase C activation. Moreover, pertussis toxin (PTX) completely inhibited [Ca(2+)](i) increase by LPS, indicating the activation of PTX-sensitive G-proteins. We also found that LPS-induced [Ca(2+)](i) increase was completely inhibited by suramin, suggesting G-protein coupled receptor activation. Since LPS specifically stimulates PTX-sensitive G-proteins, phospholipase C-dependent [Ca(2+)](i) increase in leukemic cells but not normal peripheral blood leukocytes, LPS receptor may be associated with leukemia.     

Akt phosphorylates p47phox and mediates respiratory burst activity in human neutrophils

Respiratory burst activity and phosphorylation of an NADPH oxidase component, p47(phox), during neutrophil stimulation are mediated by phosphatidylinositol 3-kinase (PI-3K) activation. Products of PI-3K activate several kinases, including the serine/threonine kinase Akt. The present study examined the ability of Akt to regulate neutrophil respiratory burst activity and to interact with and phosphorylate p47(phox). Inhibition of Akt activity in human neutrophils by an inhibitory peptide significantly attenuated fMLP-stimulated, but not PMA-stimulated, superoxide release. Akt inhibitory peptide also inhibited hydrogen peroxide generation stimulated by bacterial phagocytosis. A direct interaction between p47(phox) and Akt was shown by the ability of GST-p47(phox) to precipitate recombinant Akt and to precipitate Akt from neutrophil lysates. Active recombinant Akt phosphorylated recombinant p47(phox) in vitro, as shown by (32)P incorporation, by a mobility shift change detected by two-dimensional gel electrophoresis, and by immunoblotting with phospho-Akt substrate Ab. Mutation analysis indicated that 2 aa residues, Ser(304) and Ser(328), were phosphorylated by Akt. Inhibition of Akt activity also inhibited fMLP-stimulated neutrophil chemotaxis. We propose that Akt mediates PI-3K-dependent p47(phox) phosphorylation, which contributes to respiratory burst activity in human neutrophils.     

Guanine nucleotides stimulate NADPH oxidase in membranes of human neutrophils

In the chain of events by which chemotactic peptides stimulate NADPH oxidase-catalyzed superoxide formation in human neutrophils, the involvements of a pertussis toxin-sensitive guanine nucleotide-binding protein (N-protein), mobilization of intracellular calcium and protein kinase C stimulation have been proposed. Superoxide formation was studied in membranes from human neutrophils; NADPH oxidase was stimulated by arachidonic acid in the presence of neutrophil cytosol. Fluoride and stable GTP analogues, such as GTP gamma S and GppNHp, which all activate N-proteins, enhanced NADPH oxidase activity up to 4-fold. GDP beta S inhibited the effect of GTP gamma S. These data suggest that NADPH oxidase is regulated by an N-protein, independent of an elevation of the cytoplasmic calcium concentration.     

Diacylglycerol, 1-oleoyl-2-acetyl-glycerol,stimulates superoxide-generation from human neutrophils

1-Oleoyl-2-acetyl-glycerol which activates Ca²⁺-activated phospholipid-dependent protein kinase, induced the superoxide-production of human neutrophils, while other diacylglycerols did not. The induction was independent of extracellular calcium and did not accompany the increase of the intracellular free calcium. The superoxide-release by the diacylglycerol was inhibited by retinal, the inhibitor of the protein kinase. The diacylglycerol stimulated the phosphorylation of at least 4 proteins in intact neutrophils, the phosphorylation of which was stimulated by phorbol 12-myristate 13-acetate, the activator of the protein kinase. These observations indicate the possible involvement of the kinase in the induction process.     

A stable aspirin-triggered lipoxin A4 analog blocks phosphorylation of leukocyte-specific protein 1 in human neutrophils

Lipoxins and their aspirin-triggered 15-epimers are endogenous anti-inflammatory agents that block neutrophil chemotaxis in vitro and inhibit neutrophil influx in several models of acute inflammation. In this study, we examined the effects of 15-epi-16-(p-fluoro)-phenoxy-lipoxin A(4) methyl ester, an aspirin-triggered lipoxin A(4)-stable analog (ATLa), on the protein phosphorylation pattern of human neutrophils. Neutrophils stimulated with the chemoattractant fMLP were found to exhibit intense phosphorylation of a 55-kDa protein that was blocked by ATLa (10-50 nM). This 55-kDa protein was identified as leukocyte-specific protein 1, a downstream component of the p38-MAPK cascade in neutrophils, by mass spectrometry, Western blotting, and immunoprecipitation experiments. ATLa (50 nM) also reduced phosphorylation/activation of several components of the p38-MAPK pathway in these cells (MAPK kinase 3/MAPK kinase 6, p38-MAPK, MAPK-activated protein kinase-2). These results indicate that ATLa exerts its anti-inflammatory effects, at least in part, by blocking activation of the p38-MAPK cascade in neutrophils, which is known to promote chemotaxis and other proinflammatory responses by these cells.     

A new staphylococcal anti-inflammatory protein that antagonizes the formyl peptide receptor-like 1

Bacteria have developed mechanisms to escape the first line of host defense, which is constituted by the recruitment of phagocytes to the sites of bacterial invasion. We previously described the chemotaxis inhibitory protein of Staphylococcus aureus, a protein that blocks the activation of neutrophils via the formyl peptide receptor (FPR) and C5aR. We now describe a new protein from S. aureus that impaired the neutrophil responses to FPR-like1 (FPRL1) agonists. FPRL1 inhibitory protein (FLIPr) inhibited the calcium mobilization in neutrophils stimulated with MMK-1, WKYMVM, prion-protein fragment PrP(106-126), and amyloid beta(1-42). Stimulation with low concentrations of fMLP was partly inhibited. Directed migration was also completely prevented toward MMK-1 and partly toward fMLP. Fluorescence-labeled FLIPr efficiently bound to neutrophils, monocytes, B cells, and NK cells. HEK293 cells transfected with human C5aR, FPR, FPRL1, and FPRL2 clearly showed that FLIPr directly bound to FPRL1 and, at higher concentrations, also to FPR but not to C5aR and FPRL2. FLIPr can reveal unknown inflammatory ligands crucial during S. aureus infections. As a novel described FPRL1 antagonist, it might lead to the development of therapeutic agents in FPRL1-mediated inflammatory components of diseases such as systemic amyloidosis, Alzheimer's, and prion disease.     

Involvement of leukotriene B4 receptor 1 signaling in platelet-activating factor-mediated neutrophil degranulation and chemotaxis

Platelet-activating factor (PAF) is a potent lipid mediator of inflammation that can act on human neutrophils. When neutrophils are stimulated with PAF at concentrations greater than 10 nM, a double peak of intracellular calcium mobilization is observed. The second calcium peak observed in PAF-treated neutrophils has already been suggested to come from the production of endogenous leukotriene B4 (LTB4). Here we demonstrate the involvement of endogenous LTB4 production and subsequent activation of the high affinity LTB4 receptor (BLT1) in this second calcium mobilization peak observed after stimulation with PAF. We also show that the second, but not the first peak, could be desensitized by prior exposure to LTB4. Moreover, when neutrophils were pre-treated with pharmacological inhibitors of LTB4 production or with the specific BLT1 antagonist, U75302, PAF-mediated neutrophil degranulation was inhibited by more than 50%. On the other hand, pre-treating neutrophils with the PAF receptor specific antagonist (WEB2086) did not prevent any LTB4-induced degranulation. Also, when human neutrophils were pre-treated with U75302, PAF-mediated chemotaxis was reduced by more than 60%. These data indicate the involvement of BLT1 signaling in PAF-mediated neutrophil activities.     

Effects of aminopeptidase inhibitors actinonin and amastatin on chemotactic and phagocytic responses of human neutrophils

Actinonin and amastatin are low-molecular-weight inhibitors of aminopeptidases associated with cell surfaces. The purpose of this study was to determine their effects on human neutrophil functions such as chemotaxis and phagocytosis. Both actinonin and amastatin enhanced chemotaxis to the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine. On the other hand, the effects of both agents on neutrophil phagocytosis were varied. Bacterial attachment to neutrophils was slightly affected by these agents. However, actinonin enhanced the internalization of bacteria by neutrophils. Neutrophil leucine aminopeptidase activity was also determined and was found to be weakly inhibited by these agents.