Chemotaxis Of Human Polymorphonuclear Leukocytes Research Articles

Medium-chain Fatty Acid-sensing Receptor, GPR84, Is a Proinflammatory Receptor

G protein-coupled receptor 84 (GPR84) is a putative receptor for medium-chain fatty acids (MCFAs), whose pathophysiological roles have not yet been clarified. Here, we show that GPR84 was activated by MCFAs with the hydroxyl group at the 2- or 3-position more effectively than nonhydroxylated MCFAs. We also identified a surrogate agonist, 6-n-octylaminouracil (6-OAU), for GPR84. These potential ligands and the surrogate agonist, 6-OAU, stimulated [(35)S]GTP binding and accumulated phosphoinositides in a GPR84-dependent manner. The surrogate agonist, 6-OAU, internalized GPR84-EGFP from the cell surface. Both the potential ligands and 6-OAU elicited chemotaxis of human polymorphonuclear leukocytes (PMNs) and macrophages and amplified LPS-stimulated production of the proinflammatory cytokine IL-8 from PMNs and TNFα from macrophages. Furthermore, the intravenous injection of 6-OAU raised the blood CXCL1 level in rats, and the inoculation of 6-OAU into the rat air pouch accumulated PMNs and macrophages in the site. Our results indicate a proinflammatory role of GPR84, suggesting that the receptor may be a novel target to treat chronic low grade inflammation associated-disease.

Influences of aflatoxin B1 on reactive oxygen species generation and chemotaxis of human polymorphonuclear leukocytes

The effects of aflatoxin (AF), a hepatotoxic agent and the strongest carcinogen in nature, on polymorphonuclear leukocyte (PMN) chemotaxis and chemiluminescence (CL) were studied. Luminol-dependent CL activity, which reflects the production of reactive oxygen species (ROS) from PMNs, was up-regulated to approximately 150% when PMNs were treated with 0.05 ng/ml of AFB1 upon stimulation with N-formyl-methionine-leucine-phenylalanine (fMLP) or zymosan. On the other hand, PMN CL activity was down-regulated to approximately 25% of the control when PMNs were treated with 50 ng/ml of AFB1 upon stimulation with zymosan. The effect of AFB1 on PMN chemotaxis was also investigated using the Boyden chamber method. The chemotactic ability of PMNs when interleukin (IL)-8 was used as a chemoattractant was inhibited in a dose-dependent manner at AFB1 concentrations of >10 ng/ml. In reverse transcriptase (RT)-PCR analysis, gene expression levels of CXC chemokine receptor (CXCR)1/2, whose ligands are IL-8, granulocyte chemotactic protein (GCP)-2, neutrophil attractant-activation protein (NAP)-2, and epithelial neutrophil-activating protein (ENA)-78, which regulate PMN chemotaxis, were also down-regulated in a dose-dependent manner by 50 ng/ml AFB1. mRNA expression levels of CXCR1/2 were down-regulated to approximately 85% of that in the controls when PMNs were treated with 100 ng/ml AFB1. These results suggest that a high concentration of AFB1 reduces the chemotactic ability of PMNs via the CXCR1/2 cascade indirectly.

Antiinflammatory effect of topically applied propolis extract in carrageenan‐induced rat hind paw edema

The antiinflammatory effect of an ointment containing propolis extract (3%-7%) was examined using carrageenan-induced hind paw edema in rats. Treatment with the ointment inhibited the edema moderately, and the inhibition was significant at 5% and 7%. Additionally, the effect of the ointment on chemotaxis of human polymorphonuclear leukocytes (PMNs) was investigated using the agarose plate method. Migration of PMNs toward zymosan-treated serum was inhibited in the presence of 5% propolis ointment. These results demonstrate that topical application of propolis extract is effective in inhibiting carrageenan-induced rat hind paw edema, and its inhibitory effect on the chemotaxis of PMNs may also contribute to the antiinflammatory effect.

The differential ability of IL-8 and neutrophil-activating peptide-2 to induce attenuation of chemotaxis is mediated by their divergent capabilities to phosphorylate CXCR2 (IL-8 receptor B).

IL-8 and neutrophil-activating peptide-2 (NAP-2) are two closely related C-X-C chemokines that differ in their abilities to induce chemotaxis of human polymorphonuclear leukocytes (PMN). Although two IL-8R types are expressed by PMN, only CXCR2 binds NAP-2 and IL-8 with equally high affinity. By using enriched CXCR2-transfected 293 cells, we show that high doses of IL-8 induce attenuation of chemotaxis, while equivalent doses of NAP-2 do not. Phosphorylation analysis shows that IL-8 induces higher levels of phosphorylation of the carboxyl terminus of CXCR2 than does NAP-2, suggesting that the level of phosphorylation contributes to the ability of the chemokines to attenuate the chemotactic response. To directly evaluate this difference, we analyzed the ability of receptors mutated to delete regions that highly express potential phosphorylation sites to be phosphorylated and to mediate chemotactic attenuation. We found that a carboxyl terminus-truncated mutant of CXCR2 was not phosphorylated by high doses of IL-8, as determined by in vivo phosphorylation assays and by analysis of the electrophoretic mobility of the receptors on SDS-PAGE gels. This mutated receptor had a significantly lower ability to attenuate IL-8-induced chemotaxis, indicating that the attenuation of chemotaxis is mediated by chemokine-induced receptor phosphorylation. In conclusion, the data show that the greater ability of IL-8 to induce receptor phosphorylation contributes to its more potent attenuation of chemotaxis as compared with NAP-2. This differential phosphorylation by IL-8 and NAP-2 of CXCR2 provides a basis for the divergent outcome of PMN-induced inflammation in response to these two closely related C-X-C chemokines.

Potent anti-inflammatory activity of pheophytin a derived from edible green alga, Enteromorpha prolifera (Sujiao-nori)

Recently, a chlorophyll-related compound, pheophytin a, has been identified from an edible green alga, Enteromorpha prolifera (Sujiao-nori in Japanese) as a potent suppressive substance against genotoxin-induced umu C gene expression in a tester bacteria (Okai and Higashi-Okai, 1997, J. Sci. Food Agricul. 71, 531–535). In the present study, anti-inflammatory effects of pheophytin a from Enteromorpha prolifera have been analyzed using in vitro and in vivo experiments. 1. 1. Pheophytin a suppressed the production of superoxide anion (O 2) in mouse macrophages induced by 12- O-tetradecanoylphorbol-13-acetate (TPA) using the cytochrome C reduction method. 2. 2. Pheophytin a caused a suppressive effect against formyl-Met-Leu-Phe (FMLP)-induced chemotaxis of human polymorphonuclear leukocytes (PMNs) in Boyden's chamber experiment. 3. 3. Pheophytin a exhibited a significant suppression against TPA-induced inflammatory reaction such as edema formation in BALB/c mouse ear. These results suggest that pheophytin a from Enteromorpha prolifera has a potent anti-inflammatory activity.

Cleavage of human complement component C5 by cysteine proteinases from Porphyromonas (Bacteroides) gingivalis. Prior oxidation of C5 augments proteinase digestion of C5.

Since severe periodontitis is characterized by an acute inflammatory response with cellular infiltration and microbial overgrowth, plasma proteins could be exposed to both proteinases and oxidants released from the granulocytes, as well as to proteinases from the microorganisms. When human complement component C5 was digested by cysteine proteinases (i.e. gingipain-R and gingipain-K) from Porphyromonas gingivalis, limited cleavage of the C5 molecule was observed. If C5 was first oxidized by hydroxyl radicals, these gingipains converted modified C5 to fragments that exhibited significantly greater pro-inflammatory activity than did digests of unmodified C5. After cleavage of oxidized C5 by gingipain-R, the digest exhibited measurably greater neutrophil enzyme release and chemotaxis of human polymorphonuclear leukocytes (PMNs) compared with the activities of unoxidized C5 digests. Gingipain-K generates virtually no polarization or chemotactic activity of human PMNs from C5, nor is enzyme release stimulated by these C5 digests. However, when oxidized C5 was digested by gingipain-K, human PMNs were stimulated for polarization, chemotaxis and enzyme release indicating that an active fragment had been generated. Proteolysis of oxidized C5 evokes greater neutrophil activation than does proteolysis of unoxidized protein, a fact which supports the hypothesis that oxidation and proteolysis may be coupled to enhance the destructive effects of the inflammatory process. These results, in which digests of both oxidized and unmodified complement component C5 were evaluated, support the general concept that oxidation and proteolysis may participate cooperatively in amplifying both the severity and duration of the inflammatory reaction.

The Use of the Vitality and Chemotaxis of Human Polymorphonuclear Leukocytes for the In Vitro Estimation of the Acute Toxicity of the First Ten Chemicals from the MEIC List

The acute cytotoxicity of the first ten MEIC chemicals has been estimated by others in various cell lines. In the present investigation, isolated human polymorphonuclear leukocytes (PMN) from ten healthy non-smoking laboratory personnel were used to assess in vitro toxicity of the same chemicals. The cells were treated with different concentrations of the respective chemicals for three hours and their vitality and chemotaxis were tested. Vitality was measured by fluorescence microscopy after the addition of fluorescein diacetate and ethidium bromide. Living cells which took up and hydrolysed fluorescein diacetate, and dead cells, stained by ethidium bromide, were counted and the percentage of live cells was calculated. Locomotion stimulated by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (F-MLP), was measured in blind-well Boyden chambers and a chemotactic index was calculated. The results were mathematically transformed to produce a linear curve, and then fitted by the linear least squares procedure, from which LC50 and IC50 values were obtained by interpolation. All the chemicals decreased the vitality and inhibited the chemotaxis of the PMN. Obviously the chemotactic test was more sensitive than the vitality one. A correlation (r = 0.933) was found between vitality and chemotaxis inhibition. Spearman rank correlation analysis revealed significant correlations between our results and those from in vitro experiments conducted in other laboratories, as well as with data concerning mouse, rat and human lethal doses.

New Antiasthmatic Drugs from Traditional Medicine?

Several plants are used in traditional medicine for the treatment of bronchial asthma. We are trying to identify the active compound(s) and their mode of action. For the isolation and identification of the active principles, different chromatographic methods, HPLC, MPLC, elementary analysis, UV, mass, 1H- and 13C-NMR spectroscopy are used. Whole plant extracts, fractionated extracts and pure compounds are tested in the following pharmacological systems: cyclooxygenase and lipoxygenase pathway of arachidonic acid metabolism, bronchial obstruction of guinea pigs after inhalation of allergens, platelet-activating factor (PAF), histamine or acetylcholine, PAF-induced bronchial hyperreactivity of guinea pigs, histamine release, chemoluminescence and chemotaxis of human polymorphonuclear leukocytes as well as thromboxane biosynthesis of human platelets. As active compounds in onion extracts, thiosulfinates and cepaenes could be identified. They exert a wide spectrum of pharmacologic activities, both in vitro and in vivo. Tetragalloyl quinic acid from Galphimia glauca, suppressed allergen- and PAF-induced bronchial obstruction, PAF-induced bronchial hyperreactivity (5 mg/kg orally) in vivo and thromboxane biosynthesis in vitro. Hitherto unknown alkaloids from Adhatoda vasica showed pronounced protection against allergen-induced bronchial obstruction in guinea pigs (10 mg/ml aerosol). Androsin from Picrorhiza kurroa prevented allergen- and PAF-induced bronchial obstruction (10 mg/kg orally; 0.5 mg inhalative). Histamine release in vitro was inhibited by other compounds of the plant extract yet to be identified. Pharmacological effects of plant extracts and pure compounds in man are under investigation.

Characterization of leukocyte chemotactic activity of bacteriocin fromStreptococcus mutans Rm-10

Bacteriocins have several biological activities in addition to their antibacterial effect. We investigated the chemotactic properties and mode of action of purified streptococcal bacteriocin. Bacteriocin purified from a culture supernatant of Streptococcus mutans (S. mutans) Rm-10 induced chemotaxis of human polymorphonuclear leukocytes and monocytes. Following purification, leukocyte migration appeared in one bacteriocin fraction, and this migration was dependent on the concentration gradient in dilution ranges from 1/960 to 1/15. Chemotactic activity of the bacteriocin was heat labile and trypsin sensitive. Moreover, preincubation of bacteriocin with varying dilutions of its antiserum prepared in rabbits resulted in a significant loss of the chemotactic activity.

Anti-Inflammatory Effects of Onions: Inhibition of Chemotaxis of Human Polymorphonuclear Leukocytes by Thiosulfinates and Cepaenes

Seven different synthetic thiosulfinates, and cepaene- and/or thiosulfinate-rich onion extracts were found to inhibit in vitro the chemotaxis of human granulocytes induced by formyl-methionine-leucine-phenylalanine in a dose-dependent manner and at a concentration range of 0.1-100 microM. Diphenylthiosulfinate showed the highest activity and was found to be more active than prednisolone. The anti-inflammatory properties of onion extracts are related, at least in part, to the inhibition of inflammatory cell influx by thiosulfinates and cepaenes.

Inhibitory Effect of SV-IV, a Major Protein Secreted From the Rat Seminal Vesicle Epithelium, on Phagocytosis and Chemotaxis of Human Polymorphonuclear Leukocytes

The effect of SV-IV, one of the major proteins secreted from the rat seminal vesicle epithelium, on phagocytosis and chemotaxis of human polymorphonuclear leukocytes (PMNs) has been studied. Various cytological, biochemical, metabolic, and physical correlates of both biological activities have been found to be markedly reduced by the presence in the medium of micromolar concentrations of protein SV-IV. Moreover, the Scatchard analysis of the labeled SV-IV binding to PMN cell surface has demonstrated that such binding is specific. The binding sites contain only saturable components, completely displaceable by unlabeled SV-IV. The number of the specific sites has been calculated to be 87,000/cell, with a Kd of 1.72 X 10(-7) M. The molecular mechanism of the inhibitory effect is discussed along with the possible biological and clinical implications of the experimental findings.

Formyl peptide-induced chemotaxis of human polymorphonuclear leukocytes does not require either marked changes in cytosolic calcium or specific granule discharge. Role of formyl peptide receptor reexpression (or recycling).

We examined the role of intracellular and extracellular calcium on the ability of human polymorphonuclear leukocytes to migrate chemotactically and reexpress (or recycle) formyl peptide receptors when challenged with the synthetic chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP). Extracellular calcium was not required for either optimal chemotactic responses or receptor reexpression. Depletion and chelation of intracellular calcium resulted in significant diminution in the ability of polymorphonuclear leukocytes to release the specific granule constituents lactoferrin and vitamin B12-binding protein during the process of chemotaxis, but had no effect on the capability of these cells to respond chemotactically. Similarly, chelation of intracellular calcium did not affect the ability of these cells to reexpress a population of formyl peptide receptors. Inhibition of receptor reexpression, by a nonagglutinating derivative of wheat-germ agglutinin, was associated with inhibition of chemotactic responses to FMLP. Thus, it appears that large changes in cytosolic free calcium are not necessary for formyl peptide-induced polymorphonuclear leukocyte chemotaxis. In contrast, continuous reexpression (or recycling) of formyl peptide receptors is required for polymorphonuclear leukocyte chemotactic responses to FMLP, a process that appears to be independent from specific granule fusion with plasma membrane.

Interaction between Chlamydia spp. and human polymorphonuclear leukocytes in vitro.

Chlamydia psittaci and Chlamydia trachomatis elementary bodies (EB) incubated in the presence of complement or specific antibody or both caused chemotaxis of human polymorphonuclear leukocytes (PMN) in vitro. Reticulate bodies and culture supernatants had no effect on these cells. The ability of chlamydiae to enter and survive in PMN under nonopsonizing conditions was investigated by measuring the association of 3H-labeled EB and of inclusion-forming units with these phagocytes. Both assays indicated that C. psittaci as well as C. trachomatis EB are efficiently internalized. The mechanism by which this is accomplished is distinct from classical phagocytosis in that it is not dependent upon the presence of complement or antibody. Furthermore, uptake of at least C. psittaci appeared to be rapid, with no additional increase occurring after 15 min. The majority of cell-associated chlamydiae were rendered acid soluble or noninfectious within 1 h. Subsequently, there was a small but steady loss of infectivity for up to 10 h, which may have been due to the conversion of EB to the noninfectious reticulate-body form of the organism. However, even at 10 h after entry a small percentage of bacteria was still capable of infecting a second target cell. This is noteworthy in that PMN are relatively short-lived cells, and after lysis, intracellular organisms may be free to infect adjacent tissue. Electron microscopic observations were consistent with the data on uptake and persistence. The ability of a small percentage of infecting chlamydiae to maintain infectivity in PMN for at least several hours may enable these organisms subsequently to establish productive infection in permissive host cells.

Impairment of human polymorphonuclear leukocyte chemotaxis by 2,5-hexanedione

The effect of n-hexane metabolites on human polymorphonuclear leukocyte chemotaxis and luminol-dependent chemiluminescence was investigated. No effect was detected when 2-hexanol, 2-hexanone and gamma-valerolactone were used; 2,5-hexanedione at 75 micrograms/ml inhibited chemotaxis and a direct correlation between increasing the xenobiotic concentration and the degree of inhibition was found. Chemotactic peptide-induced chemiluminescence was not affected by 2,5-hexanedione. In order to clarify the phenomenon, plasma membrane fluidity was investigated by fluorescence polarization of the fluorescent probe trimethylammonium diphenylhexatriene. 2,5-hexanedione increased the membrane fluidity, while the other n-hexane metabolites did not change the degree of fluorescence polarization. Results suggest that the cellular functions modulated by membrane-cytoskeletal organization are affected by 2,5-hexanedione also at the low concentrations.

Anthrax toxin components stimulate chemotaxis of human polymorphonuclear neutrophils.

Effects of the three-component toxin of Bacillus anthracis on chemotaxis of human polymorphonuclear leukocytes (PMN) were investigated in an effort to determine the basis of the reported antiphagocytic effect of the toxin. The three toxin components, edema factor (EF), protective antigen (PA), and lethal factor (LF), were tested alone and in various combinations for their effect on PMN chemotaxis under agarose to formyl peptides and zymosan-activated serum. No component was active alone; combinations of EF + PA, LF + PA, and EF + LF + PA markedly stimulated chemotaxis (directed migration), but had little or no effect on unstimulated random migration. The toxin components were not themselves chemoattractants. EF in combination with PA had previously been identified as an adenylate cyclase in Chinese hamster ovary (CHO) cells. We found that EF + PA produced detectable cyclic adenosine 3'-5'monophosphate (cAMP) in PMN, but the level of cAMP was less than 1% of that produced in CHO cells by EF + PA, and in PMN by other bacterial adenylate cyclases. LF + PA (which stimulated chemotaxis to an equivalent extent) had no effect on cAMP levels. Thus, the enhancement of chemotaxis by anthrax toxin (at least by LF + PA) does not seem to be related to adenylate cyclase activity.

Chemotaxis of human polymorphonuclear leukocytes under agarose: lack of requirement for media protein and differential effects of buffer and agarose type on locomotion.

AbstractMigration and chemotaxis of human polymorphonuclear leukocytes (PMN) under agarose was studied in the presence and absence of various proteins. Contrary to previous reports, chemotaxis could be elicited in a protein-free medium using both complement-derived factors and fMet - Leu - Phe. Cell movement was critically dependent on the choice of agarose and buffer system. Addition of human serum or albumin produced marked chemokinesis while gelatin had no effect. These results indicate that media protein is a methodological requirement of some in vitro test systems but not a physiological requisite for PMN chemotaxis. The described procedures provide a valuable control for studying protein effects on cell migration as well as a means of avoiding unwanted cell stimulation and inactivation of test substances by media protein. This should facilitate studying chemokinetic and chemotactic materials, helper factors, and pharmacological agents. Agarose types were found to differ in the occurrence of a concen...

Chemotactic factor-induced effects upon deformability of human polymorphonuclear leukocytes

We have compared the effects of the synthetic chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe), on the deformability and chemotaxis of human polymorphonuclear leukocytes (PMNs). By utilizing the technique of cell elastimetry (deformability), which measures the negative pressure required to aspirate cells into a micropipet, we have shown a correlation between f-Met-Leu-Phe concentrations and PMN deformability which parallels the effects of that chemoattractant on filter chemotaxis. At concentrations as low as 10(-11) M, a change in the deformability characteristics of a population of neutrophils was demonstrated. The increase in deformability became most marked at peptide concentrations in the range of 1-5 x 10(-9) M, corresponding to the optimal concentration range in the filter chemotaxis assay. Concentrations of 10(-8) M and greater were associated with a significant increase in the negative pressure required to aspirate the cells into a micropipet. Alterations in PMN deformability, therefore, occur as a natural consequence of stimulation of a human PMN with a chemotactic factor and may be a prerequisite to efficient movement.

Chemotaxigenesis and Complement Fixation by Listeria Monocytogenes Cell Wall Fractions

Abstract Data reported here show that a crude cell wall fraction of Listeria monocytogenes (LCWF) and its purified derivative (PF) induce chemotaxis of human polymorphonuclear leukocytes in the presence of normal rabbit serum. In addition, both LCWF and PF can fix rabbit and guinea pig complement. The purified cell wall preparation is more active than LCWF in both chemotaxigenesis and complement fixation, indicating that these activities are due primarily to the nonprotein portion of LCWF. Complement fixation by PF was demonstrated in the presence of ethylene glycol tetraacetic acid (EGTA) and in C4-deficient serum, which is evidence that PF activates complement by the alternative pathway. Therefore, chemotaxigenesis by PF and LCWF seems to involve fixation of complement via the alternative pathway.

Effects of cigarette smoke components on in vitro chemotaxis of human polymorphonuclear leukocytes.

Some ciliostatic components of cigarette smoke were studied as inhibitors of in vitro chemotaxis of human polymorphonuclear leukocytes (PMNs). In comparison to their concentration in an inhibitory level of cigarette smoke, the unsaturated aldehydes acrolein and crotonaldehyde were the most potent inhibitors, whereas nicotine, cyanide, acetaldehyde, and furfural were the next strongest inhibitors. In contrast, sulfide, propionaldehyde, butyraldehyde, and the phenols (phenol and o-, m-, and p-cresol) were relatively weak inhibitors of PMN chemotaxis. Acrolein and crotonaldehyde mimicked whole cigarette smoke in their effects on PMNs by not causing loss of PMN viability, yet their effects were prevented by the addition of cysteine. On the other hand, addition of nicotine, cyanide, acetaldehyde, and furfural to PMN suspensions resulted in a limited loss of cellular viabilities, and their effects on PMNs were not prevented by cysteine. Of the tested components, only cyanide significantly altered PMN glucose metabolism by increasing carbon flow via the glycolytic and hexose monophosphate pathways in a manner similar to that observed with whole cigarette smoke. The results of this study suggest that the unsaturated aldehydes, including acrolein and crotonaldehyde, are major contributors to the inhibitory properties of cigarette smoke. The inhibitory effects of these unsaturated aldehydes are probably due to a direct interaction of these oxidants and/or thiol-alkylating agents with PMNs, yet the glucose metabolism of these cells is unaffected. One interpretation of these data is that PMN chemotaxis is dependent upon particular cellular proteins containing one or more essential thiol group(s) but that these proteins are unrelated to glucose metabolism.

Effects of tobacco smoke on chemotaxis and glucose metabolism of polymorphonuclear leukocytes.

The effect of tobacco smoke on in vitro chemotaxis of human polymorphonuclear leukocytes (PMN) was determined. Whole tobacco smoke, gas phase of smoke, and water-soluble fraction were potent inhibitors of PMN chemotaxis. The results indicated that PMN chemotaxis was inhibited in a dose-dependent manner by water-soluble fraction and that this suppression was not a result of cytotoxicity. In an attempt to determine the mechanism of chemotaxis inhibition, the effect of tobacco smoke on glucose metabolism of PMN was studied. Exposure of PMN to whole smoke, gas phase, or water-soluble fraction resulted in an increase (twofold) in glucose catabolism via both glycolysis and the hexose monophosphate shunt, with no apparent effects on the metabolism of glucose via the tricarboxylic acid cycle. These results suggest that the inhibitory effects of tobacco smoke on PMN chemotaxis were not directly attributable to effects on glucose metabolism of these cells. Further, the inhibitory effects of water-soluble fraction on PMN chemotaxis were shown to be largely irreversible and preventable in the presence of cysteine. Thus, the major inhibitory effects of tobacco smoke probably result from the direct action of oxidants and/or thiol-reactive substances on PMN.