Inhibition of platelet-activating factor (PAF)-induced chemotaxis and PAF binding to human eosinophils and neutrophils by the specific ginkgolide-derived PAF antagonist, BN 52021

We have investigated the effects of BN 52021 (a specific PAF antagonist derived from Ginkgo biloba) on PAF-induced human eosinophil and neutrophil chemotaxis. In response to an optimal concentration of PAF (10(-6) mol/L), the drug was significantly more potent (p less than 0.001) in inhibiting eosinophil as compared to neutrophil locomotion. These inhibitory effects were observed in a dose-dependent manner with a concentration of drug required to produce 50% inhibition of 7.0 (+/- 2.2) X 10(-6) mol/L and 2.3 (+/- 0.2) X 10(-5) mol/L for eosinophils and neutrophils, respectively. Sodium cromoglycate, nedocromil sodium, salbutamol, and dexamethasone (preincubated with cells up to 6 hours) had no effect over a wide dose range (10(-3) to 10(-9) mol/L). BN 52021 was significantly more effective in inhibiting chemotaxis when the cells were preincubated with the compound for up to 1 hour before commencement of the locomotion assay, whereas washing the cells completely abolished this effect. Inhibition by BN 52021 was specific for PAF in that it had no effect on chemotaxis induced by either leukotriene B4, N-formyl-methionyl-leucyl-phenylalanine, or a purified human mononuclear cell-derived neutrophil chemotactic factor. BN 52021 also inhibited the specific binding of [3H]-PAF (10(-8) mol/L) to eosinophils and neutrophils in a concentration-dependent fashion with a concentration of drug required to produce 50% inhibition of 1.5 (+/- 0.3) X 10(-6) mol/L and 9.1 (+/- 2.5) X 10(-7) mol/L, respectively. These results suggest that BN 52021 has potential as an anti-inflammatory agent in conditions associated with PAF-induced accumulation of neutrophils and eosinophils.     

Modulation of neutrophil chemokine receptors by Staphylococcus aureus supernate

In a previous study, we showed that Staphylococcus aureus supernate (SaS) is a potent agonist for both neutrophils and mononuclear cells. To further investigate the immunomodulating effects of SaS, the effect on different neutrophil receptors was studied. Expression of various neutrophil receptors, before and after treatment with SaS, was quantified by flow cytometry. We found that SaS treatment of neutrophils resulted in a specific and total downregulation of the C5a and the fMLP receptor, both serpentine receptors, while other receptors were totally unaffected. Since these two receptors are both involved in chemotaxis, we tested the effect of SaS in calcium flux and chemotaxis assays. We showed that preincubation with SaS abrogated the rise in intracellular calcium concentration upon triggering with fMLP and C5a. We also showed that SaS is a potent inhibitor of neutrophil chemotaxis towards fMLP and C5a, but does not interfere with chemotaxis towards interleukin-8. These findings indicate that S. aureus produces a virulence factor extracellularly, which impairs chemotaxis towards the infected site.     

In vitro and ex vivo effect of RU41740 on human polymorphonuclear leukocyte function

We investigated the effect of RU41740, a glycoprotein extracted from Klebsiella pneumoniae and possessing immunomodulating properties, on human neutrophil functions in vitro and ex vivo. Our in vitro results showed that RU41740 increased complement- and Fc receptor-dependent phagocytosis. Moreover, the drug enhanced the oxidative metabolism (assessed by chemiluminescence) both in resting and stimulated cells; in the latter case the RU41740-induced enhancement was observed when neutrophils were stimulated with opsonized particles of N-formyl-methionyl-leucyl-phenylalanine (FMLP) but not when phorbol myristate acetate was used. Using otherwise effective experimental conditions, RU41740 did not affect spontaneous or FMLP-induced neutrophil migration. For the ex vivo experience we tested neutrophils of ten elderly subjects with a previously demonstrated phagocytic defect. These subjects were treated orally with RU41740 at a daily dose of 2 mg for 1 week during the first month, and of 1 mg for 1 week in the second month. In this population, RU41740 was able to restore the impaired phagocytic activity and to induce a significant increase of spontaneous chemiluminescence (CL); stimulated CL was also positively influenced. These effects on neutrophils provide new explanatory bases for the immunostimulatory activity of RU41740.     

Inhibition of neutrophil shape change by an inhibitor of chemotaxis

Human mononuclear cells exposed to staphylococcal peptidoglycan in serum-free culture rapidly produce an inhibitor of neutrophil chemotaxis which we have previously described. We found that this inhibitor of chemotaxis has its most potent effect on the inhibition of neutrophil shape change from a spherical to a polarized configuration. In order to quantify this shape change inhibition, we developed an assay using flow cytometric techniques. Neutrophils exposed to a chemoattractant simultaneously change their shape and decrease their forward angle light scattering intensity (delta FLS) with a correlation coefficient of 0.886 (p less than 0.001). In 51 experiments, neutrophils pretreated with the inhibitor of chemotaxis decreased their FLS by only 6.8 +/- 1.3 channels, while neutrophils pretreated with medium or control culture supernatants decreased theirs by 26.4 +/- 1.9 and 20.5 +/- 3.0 channels respectively (p less than 0.001). The factor which causes inhibition of shape change was indistinguishable from the inhibitor of chemotaxis by physical properties and chromatography. We conclude that this inhibitor of chemotaxis may act by inhibiting a physiologic step at or before shape change.     

Modulation of human neutrophil and monocyte chemotaxis and superoxide responses by recombinant TNF-alpha and GM-CSF

The effects of recombinant TNF and GM-CSF on human peripheral blood neutrophil and monocyte chemotaxis and the superoxide response were studied. TNF exhibited a slight chemotactic activity for both cell types. Preincubation of neutrophils with as little as 40 units/ml strongly inhibited the neutrophil chemotaxis towards f-Met-Leu-Phe. The inhibition of monocyte chemotaxis required higher concentrations of TNF (greater than 400 units/ml). TNF at concentrations higher than 500 units/ml enhanced the generation of superoxide anions by neutrophils stimulated with f-Met-Leu-Phe. In contrast, TNF even at 2,000 units/ml did not prime monocytes for enhanced superoxide response. GM-CSF alone did not exhibit any chemotactic activity for any of the cell types tested. Preincubation of cells with GM-CSF inhibited chemotaxis of neutrophils but not of monocytes. GM-CSF was as potent as TNF in enhancing the generation of superoxide response by neutrophils. However, GM-CSF did not have any effect on monocyte superoxide response. The priming ability of TNF and GM-CSF on neutrophils was heat-sensitive. We conclude that TNF and GM-CSF play a more pronounced regulatory role on neutrophils than on monocytes. Inhibition of neutrophil chemotaxis followed by enhancement of the superoxide response by TNF and GM-CSF may provide an attractive mechanism by which these cytokines assist in fighting invading microorganisms.     

Lysophosphatidic acids: III. Enhancement of neutrophil chemotaxis.

1-Palmitoyl-lysophosphatidic acid (LPA) was studied for its influence on the chemotaxis and ultrastructure of human neutrophils. By itself, LPA had no effect on the indices of chemotaxis or random migration of neutrophils. However, LPA on either the cellular or attractant side of Boyden chambers significantly enhanced the chemotactic responses of neutrophils to suboptimal concentrations of formyl-methionyl-phenylalanine. The enhancement of chemotaxis was achieved with concentrations of LPA (120-240 microM) that had no effect alone on neutrophil ultrastructure. The results, taken together with recent advances in knowledge of the role of the phosphatidylinositol turnover response in mediating effects of stimulating agents on cells, may provide a novel concept for understanding neutrophil chemotaxis.     

Effect of pentoxifylline on polarization and migration of human leukocytes

Leukocyte polarization has a key role in the induction and effector phases of immune response. We assessed the effect of pentoxifylline (PTX) on the polarization and migration of human lymphocytes and neutrophils. A dose-dependent, inhibitory effect on the polarization of lymphoid cells induced by chemokines or IL-15 was found. In addition, PTX interfered with the chemotaxis of peripheral blood T cells and T lymphoblasts. A similar effect was observed on the transendothelial migration of these cells. In addition, the polarization of neutrophils, its adherence to endothelium, and their transendothelial migration, induced by different stimuli, were inhibited by PTX. By contrast, this drug had only a mild effect on endothelial cells and a partial inhibition on the induction of ICAM-1 expression by TNF-alpha. The inhibitory effect of PTX on leukocyte polarization and extravasation may contribute significantly to the anti-inflammatory and immunoregulatory activity of this drug.     

Effects of theophylline on human eosinophil functions: comparative study with neutrophil functions

The understanding of theophylline as a bronchodilator has been reconsidered in recent years. We undertook to determine its immunomodulatory actions in granulocytes and elucidate their mechanism. Preincubation of neutrophils with theophylline (10(-5) to 5 x 10(-3) M) had a biphasic effect on O2(-) production stimulated with N-formyl-methionyl-leucyl-phenylalanine or C5a. Theophylline potentiates O2(-) production via adenosine A(2A) receptor antagonism induced by receptor-linked agonists from neutrophils, but not from eosinophils. The addition of theophylline caused a significant decline in neutrophil chemotaxis at lower concentrations than those for eosinophil motility. Theophylline reduces neutrophil chemotaxis via adenosine A1 receptor antagonism. At high concentrations, with an intracellular cAMP accumulation as a result of phosphodiesterase (PDE) inhibition, theophylline also exerts an inhibitory effect on the O2(-) production and chemotaxis of both types of cells. The difference in theophylline's effect on neutrophils and eosinophils appears to depend on the existence of specific adenosine receptors. Theophylline thus modulates granulocyte functions in association with specific adenosine receptor antagonism and cAMP-PDE inhibition.     

The Effects of Bruton Tyrosine Kinase Inhibition on Chemotaxis and Superoxide Generation in Human Neutrophils

Purpose

The role of the Bruton tyrosine kinase (Btk) protein in neutrophil function has been evaluated using neutrophils from healthy volunteers after incubation with a Btk inhibitor, leflunomide metabolite analog (LFM-A13), suggesting an important role for Btk in neutrophil function. We sought to determine the role of Btk protein on neutrophil superoxide generation and chemotaxis stimulated by N-formyl-methionine-leucine-phenylalanine (fMLP).

Methods

Chemotaxis was assayed on agarose gel and superoxide generation by cytochrome C reduction. The affects of LFM-A13 on chemotaxis and superoxide generation in unstimulated and fMLP stimulated neutrophils were studied in Btk deficient neutrophils from XLA patients compared with matched controls analyzed simultaneously.

Results

Chemotaxis and stimulated superoxide production were similar in the normal and Btk deficient neutrophils and were similarly inhibited by LFM-A13. In one patient, LFMA13 had no effect on superoxide generation in Btk deficient neutrophils up to a concentration of 25 microM, while inhibited superoxide production by control neutrophils.

Conclusions

Our results suggest that Btk does not have a specific role in neutrophil fMLP-stimulated superoxide generation and chemotaxis since these activities were similarly inhibited by LFM-A13 in Btk deficient and normal neutrophils. The lack of superoxide generation following Btk inhibition by LFM-A13 in Btk deficient neutrophils from one patient may suggest some heterogeneity in the role of Btk in fMLP induced neutrophil superoxide generation.     

Interaction of Pseudomonas aeruginosa alkaline protease and elastase with human polymorphonuclear leukocytes in vitro.

Little is known about the interaction of Pseudomonas aeruginosa extracellular products and human polymorphonuclear leukocytes. The present study was designed to examine the effect of alkaline protease and elastase purified from P. aeruginosa on human neutrophil function. Neutrophil chemotaxis, oxygen consumption, glucose oxidation, superoxide production, and nitro blue tetrazolium reduction were studied. It was found that alkaline protease and elastase at fairly low concentrations (0.05 and 0.0025 micrograms/ml, respectively) inhibited chemotaxis. The inhibitory effect of both enzymes was increased at higher concentrations. The chemotaxis of preincubated and washed cells was also inhibited. Alkaline protease but not elastase inhibited opsonized zymosan-stimulated neutrophil oxygen consumption, whereas neither of the enzymes had any effect on glucose oxidation and nitro blue tetrazolium-reducing activity of stimulated neutrophils. The data on superoxide production ability of the cells indicated that the cells preincubated with enzyme and washed were capable of producing superoxide equal to the amount produced by untreated cells when they were stimulated with phorbol myristate acetate or zymosan. However, when elastase was present in the reaction mixture, the reduction of cytochrome c as a measure of superoxide production was inhibited. Inhibition of neutrophil function, particularly chemotaxis, will have important bearing on the escape of the microorganism from the phagocytic defense system of the host. The role of these products in localized infections and avascular areas such as skin burns, cornea, and, at least initially, in chronic lung colonization in cystic fibrosis patients becomes important.     

The effect of bovine serum albumin on the in vitro inhibition of chemotaxis by anti-inflammatory agents

The anti-inflammatory agents, phenylbutazone, naproxen and niflumic acid inhibited in vitro rabbit peritoneal neutrophil chemotactic responsiveness toEscherichia coli derived chemotactic factor/s when added to cells suspended in 0.1% bovine serum albumin (BSA). Inhibition of chemotaxis by these drugs was markedly diminished as the BSA concentration was increased to 2%. Experiments with phenylbutazone demonstrated that inhibition of chemotaxis could be observed using neutrophils suspended in 2% BSA by either increasing the drug concentration or by preincubating the cells suspended in 0.1% with the drug prior to the addition of the additional BSA. These data suggest that protein binding can prevent in vitro inhibition of neutrophil chemotaxis by anti-inflammatory agents.     

Inhibitory effects of human neutrophil functions by the 45-kD glycoprotein derived from the parasitic nematode Trichinella spiralis

AIM: We evaluated the effect of the 45-kD protein of Trichinella spiralis (gp45), purified by affinity chromatography, on random migration and chemotaxis, the oxidative metabolism of human neutrophils and on the CD11b upregulation induced by formyl-methionyl-leucyl-phenylalanine (f-MLP). METHODS: Donor neutrophils incubated with different amounts of gp45 (0.5, 1, 1.5, 2 microg/ml) or buffer and the random migration and chemotaxis, evaluated by means of a special technique of image analysis, and the chemiluminescence response to f-MLP or phorbol myristate acetate (PMA) were analyzed. The effect on CD11b upregulation was assessed incubating cells with the protein, when activating them with f-MLP. RESULTS: The results showed that gp45 inhibited both random and stimulated migrations, and reduced the response to f-MLP and PMA. Furthermore, gp45 significantly reduced the upregulation of the CD11b induced by f-MLP. CONCLUSION: The results show that gp45 inhibits PMN in different functions, suggesting an anti-inflammatory action.     

Normal chemotactic migration of polymorphonuclear leukocytes stimulated with mononuclear-derived chemotactic factor in ulcerative colitis

The migration of polymorphonuclear leukocytes preincubated with autologous or heterologous serum was examined in 100 patients with untreated ulcerative colitis (UC) and in 100 age- and sex-matched healthy controls. The activity of complement-derived chemotactic factors and mononuclear-derived chemotactic factor (MDCF) was also investigated in the same group of patients. No significant difference was found in random and chemotactic migration of patients or control leukocytes preincubated in different concentrations (10, 50 and 100%) of autologous or heterologous serum. Defective chemotaxis of leukocytes stimulated with complement-derived chemotactic factors was found in UC and was more marked in patients in remission than with active UC independently of whether complement was activated by the alternative or the classical pathway. However, the random migration of neutrophils was enhanced in both groups of UC patients. The leukocytes of patients stimulated with MDCF (mononuclear cells were activated with lipopolysaccharide from Escherichia coli O55:B5) show normal chemotaxis. Our data suggest an impairment of neutrophil receptors for complement-derived chemotactic factor in UC. The decreased response of neutrophils to these factors and normal response to MDCF suggest that the main way in which cells are attracted to the site of inflammation in UC may be a factor produced by stimulated mononuclear cells.     

Neutrophil chemotaxis by Propionibacterium acnes lipase and its inhibition.

The chemoattraction of Propionibacterium acnes lipase for neutrophils and the effect of lipase inhibitor and two antibiotic agents on the chemotaxis were evaluated. Of the various fractions tested, partially purified lipase (fraction 2c) was the most active cytotaxin produced by P. acnes. Serum mediators were not required for the generation of chemotaxis by lipase in vitro. Diisopropyl phosphofluoridate at low concentration (10(-4) mM) completely inhibited lipase activity as well as polymorphonuclear leukocyte chemotaxis generated by lipase. Tetracycline hydrochloride and erythromycin base at concentrations of 10(-1) mM and 1 mM, respectively, caused 100% inhibition of PMN migration toward lipase or zymosan-activated serum. The inhibiting activity of the antibiotics was directed against cells independently of any effect on lipase. Chemotaxis by P. acnes lipase suggests a wider role for this enzyme in the inflammatory process and the pathogenesis of acne vulgaris.     

Regulation of neutrophil functions by elastase-generated IgG fragments.

IgG is split by neutrophil elastase into Fc and Fab fragments. These IgG fragments influence the functions of stimulated neutrophils such as chemotaxis, oxidative burst, and enzyme release. FMLP stimulated leukocyte chemotaxis is specifically inhibited by the elastase generated Fc fragments. Seven nmol Fc/10(6) PMN totally inhibit the chemotaxis stimulated by 16 to 125 nM FMLP. Native IgG and Fab fragments show no effect. FMLP-stimulated superoxide anion generation is specifically inhibited by Fc fragments with half maximal inhibition by 1.2 nmol/10(6) PMN. The generation of hydrogen peroxide is concomitantly stimulated, resulting in a superoxide dismutase-like effect. FMLP-stimulated elastase and myeloperoxidase release are enhanced by Fab fragments (10 nmol/10(6) PMN) to 206 and 155%, respectively, of reference values by 25 nM FMLP, while Fc and native IgG stimulate to a less extent. Consequently, elastase-generated Fc fragments have an inhibitory effect on inflammation by reducing chemotaxis and oxidative burst of stimulated neutrophils. The release stimulating activity of Fab fragments results in an up-regulation of elastase induced IgG degradation.     

Studies in vitro on the effects of rhein on the chemotaxis of human leukocytes

Rhein (R: 1,8-dihydroxy-3-carboxyanthraquinone) is the active metabolite of the drug diacetylrhein (DAR), an anthraquinone molecule which has recently been proposed for the long-term treatment of osteoarthrosis. Its action mechanism in rheumatic pathology has not been fully explained. It is known that DAR, while not inhibiting the formation of prostaglandins, inhibits certain proteolytic enzymes, and acts on phlogistic cells by lysosomal enzymic and superoxide-anion modifications. Moreover DAR modifies phagocytic functions and the motility of cells. This paper is a contribution to the clarification of the last point, namely the effect of rhein on cell motility. It reports that in vitro no effect of R on random migration was found, but instead a double inhibiting effect on chemotaxis (i.e. a low-dosage and a high-dosage effect). Furthermore, R did not modify the inhibition or induce modification of chemotaxis by vinblastine. Finally R cancelled the stimulating effect of ionic potassium. The results thus indicate that R acts on the chemotaxis of the leukocytes with a complex action at different doses. The action mechanism is probably due to a membrane effect, since rhein (R) did not modify the chemotaxis-inhibiting activity of vinblastine but did interfere with the stimulating effect of K+.     

Streptolysin O Inhibition of Neutrophil Chemotaxis and Mobility: Nonimmune Phenomenon with Species Specificity

The effects of streptolysin O (SO) (1 to 4 hemolytic units) on the mobility of neutrophilic leukocytes from humans, baboons, sheep, and rabbits were compared. After SO treatment, chemotaxis and random mobility of human neutrophils were markedly suppressed, baboon and sheep neutrophils were partially suppressed, and rabbit neutrophils were unaffected and demonstrated normal chemotaxis and mobility. The amounts of SO used in the mobility studies caused no leukocyte lysis or trypan blue uptake by human, baboon, or sheep cells, and minimal lysis or trypan blue uptake by rabbit cells. The possible involvement of immune mediators in the observed inhibition of human neutrophils was considered and excluded by the following studies. White blood cells from humans with humoral or cellular immune deficiencies responded in a manner similar to normal human cells; supernatant solutions from SO-treated human white blood cells did not contain a chemotactic suppressor; preincubation of SO with cholesterol (an inhibitor of SO hemolytic activity) caused loss of the chemotactic suppressive effect of the toxin on human leukocytes; and leukocytes from rabbits preimmunized with SO remained refractory to chemotactic suppression.     

The effect of histamine on the oxidative burst of HL60 cells before and after exposure to reactive oxygen species

During an inflammation neutrophils are stimulated to produce reactive oxygen species (ROS). These ROS induce the release of histamine from mast cells, which are also present at the inflammation site. In this study dibutyryl cAMP differentiated HL60 cells are used as a model for human neutrophils. The effect of histamine on formyl-methionyl-leucyl-phenylalanine (fmlp) stimulated cells is examined. Except for histamine also an accumulation of ROS takes place at the inflammation site and we investigated if ROS can influence the response of the stimulated HL60 cells. It is found that 10^–3 M histamine can inhibit the fmlp induced superoxide anion radical production. This occurs partly via an H₂ receptor because H₂ antagonists like famotidine, mifentidine and ranitidine could partially antagonize this effect of histamine. When HL60 cells are exposed to hydrogen peroxide or hypochlorous acid (20 min), an increased fmlp response is found while the inhibiting effect of histamine remains unchanged.     

Stimulation of neutrophil locomotion by inosiplex

Experiments were performed to investigate the in vitro effects of inosiplex on the locomotion and oxidative metabolism of human neutrophils. The drug, when used at the concentration of 500 micrograms/ml, significantly increased neutrophil random migration and chemotaxis. Enhancement of chemotaxis was due to an increased rate of locomotion as well as toan increased true chemotactic response. Inosiplex appeared to exert its effect on the whole migrating cell population. No significant effect on the oxidative metabolism of both resting and phagocytizing neutrophils was found. The results suggest that the effect of inosiplex may be related chiefly to its effect on neutrophil locomotion.