Chemotactic activity of recombinant human granulocyte colony- stimulating factor

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) induced migration across polycarbonate filters of human polymorphonuclear leukocytes (PMN). rhG-CSF was active in inducing PMN migration at concentrations greater than or equal to 10 to 100 U/mL (7 to 70 ng/mL). rhG-CSF did not contain appreciable levels of endotoxin contamination as assessed by Limulus amebocyte assay, and Polymixin B did not affect the chemotactic activity of rhG-CSF. A monoclonal anti-G- CSF antibody blocked the induction of migration by G-CSF, thus establishing that the cytokine was responsible for the activity of the recombinant preparation. Checkerboard analysis was performed by seeding different concentrations of G-CSF above and/or below the filter and revealed that the migratory response to this cytokine was best observed in the presence of a positive concentration gradient between the lower and upper compartments of the chamber, thus indicating an actual chemotactic effect. When different migrating cells were examined, rhG- CSF was inactive on large granular lymphocytes and endothelial cells under conditions in which appropriate reference attractants were active. In contrast, rhG-CSF elicited a chemotactic response in monocytes inhibited by specific antibody. Thus, G-CSF is a chemotactic signal for phagocytes. This cytokine, when produced at inflammatory sites, may contribute to the recruitment of phagocytes from the blood compartment to amplify resistance against certain noxious agents.     

Regulation of neutrophil migration and superoxide production by recombinant tumor necrosis factors-alpha and -beta: comparison to recombinant interferon-gamma and interleukin-1 alpha

We compared the ability of recombinant human tumor necrosis factor- alpha (rHuTNF-alpha) and tumor necrosis factor-beta (rHuTNF-beta) to stimulate polymorphonuclear neutrophil (PMN) migration and superoxide production. Significant PMN migration occurred across polycarbonate filters after stimulation with rHuTNF-alpha at concentrations ranging from 10(-7) to 10(-10) mol/L and at 10(-7) to 10(-8) mol/L for rHuTNF- beta and N-formylmethionyl-leucyl phenylalanine (FMLP), whereas recombinant human interferon-gamma was only minimally active at 10(-7) mol/L and recombinant human interleukin-1 alpha was inactive at the doses tested. In addition, antibodies to rHuTNF-alpha completely inhibited rHuTNF-alpha but not rHuTNF-beta or FMLP-induced PMN migration. Combinations of rHuTNF-alpha and rHuTNF-beta (at similar molar concentrations) stimulated PMN migration levels comparable to that obtained with rHuTNF-alpha alone. Checkerboard analyses performed by placing different concentrations of rHuTNF-alpha and rHuTNF-beta above and below polycarbonate filters of microchemotaxis chambers demonstrated that rHuTNF-alpha and rHuTNF-beta stimulated both chemotactic and chemokinetic responses by PMN. Additional studies demonstrated that 1 X 10(-8) mol/L rHuTNF-alpha and 3 X 10(-9) mol/L rHuTNF-beta (which represents 10(4) U/mL of each cytokine) were similar in their ability to induce superoxide production by PMNs; however, at ten- to 100-fold lower molar concentrations (10(3) and 10(2) units), rHuTNF-alpha was significantly more active than rHuTNF-beta. At the doses tested, both cytokines were less active than phorbol myristate acetate at stimulating O2- release. The results demonstrate that rHuTNF- alpha and rHuTNF-beta differ quantitatively but not qualitatively in their effects on PMN functions in vitro and suggest that rHuTNF-beta may be less toxic than rHuTNF-alpha in vivo.     

Early pulmonary granulocyte recruitment in response to Streptococcus pneumoniae

Although polymorphonuclear leukocytes (PMN) are a conspicuous histologic feature of clinical and experimental pneumococcal pneumonia, neither the mechanism nor the magnitude of recruitment of these cells to the lung following lesser pneumococcal challenge is known. We have, therefore, investigated the early process of recruitment of PMN to alveolar spaces after pulmonary inoculation of Streptococcus pneumoniae in doses less than those causing pneumonia. We injected Balb/c mice with water and varying inoculums of pneumococci via an endobronchial catheter. Bronchoalveolar lavage (BAL) was performed on the inoculated lung at 0, 2, or 4 h after injection. Cellular response was measured and chemotactic activity was assayed on BAL supernatants at each time interval using the migration of human PMN through 3-micron filters in modified Boyden chambers by the leading front techniques. The BAL of normal and control animals (inoculum of sterile water only used for the control animals) yielded 5.03 +/- 1.51 X 10(2) and 0.17 +/- 0.04 X 10(5) PMN, respectively. The PMN recruitment at 4 h as a function of pneumococcal inoculum was described by the following equation: log PMN = 0.751 log Pn + 1.119 (r2 = 0.82, p less than 0.001). The PMN were, therefore, recruited in a dose-dependent manner. That recruitment may be caused by chemotactic substance(s) was suggested by the significant correlation between the PMN response and the distance of in vitro migration: log PMN = 0.057 micron + 0.52 (r = 0.77, p less than 0.005). We have defined quantitatively the recruitment of PMN to the lung after pneumococcal challenge.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct Chemotaxis and Leucocyte-Induced Chemotaxis of Polymorphonuclear Leucocytes

The present study was designed to discriminate and analyze the presence of direct PMN chemotaxis and leucocyte-induced PMN chemotaxis in filter assay systems of PMN chemotaxis, namely the Wilkinson chamber and the Boyden chamber, which yield a more quantified information on leucocyte chemotaxis than filming of vital cells. The PMN chemotaxis was reduced by approximately 30–35 μm after incubation with vinblastine, 0.01, 0.10 and 1.00 μg/ml respectively, as measured by the leading front method in the Wilkinson chamber. This figure was thought to represent the contribution of the leucocyte-induced antitubulin-sensitive PMN chemotaxis to the casein-induced PMN chemotaxis under the experimental conditions prevailing. The remaining 60 μm antitubulin-insensitive PMN migration into the filter probably represented a combination of direct PMN chemotaxis and stimulated PMN random motility. Since the above-mentioned vinblastine inhibition of PMN chemotaxis was recorded in the absence of serum, complement factors included, the vinblastine-inhibited PMN chemotaxis was thought to be due to the release of a leucocyte-derived cytotaxin. The significance of incubation time and chemotactic parameters was further analyzed in the presence of serum in Boyden chambers, in the intercompartmental filters by means of PMN distribution curves and on the bottom filter by cell numbers. The antitubulin inhibition of PMN chemotaxis was evident in the intercompartmental filter during the initial period of incubation and later by cell counts on the bottom filter. These observations suggested that the antitubulin inhibition of PMN chemotaxis was due to antitubulin inhibition of the direction-finding in a minor proportion of fast-moving PMNs.     

Defective responsiveness to ascorbic acid of neutrophil random and chemotactic migration in Felty''s syndrome and systemic lupus erythematosus.

Polymorphonuclear (PMN) leucocytes from 4 patients with untreated systemic lupus erythematosus (SLE) showed defective random migration (P less than 0-05) and depressed chemotactic responses to C5a and kallikrein (P less than 0-01) compared to PMN leucocytes from normal subjects, or patients with rheumatoid arthritis (4) or Felty's syndrome (4) when examined at a standardized cell concentration with a micropore filter radioassay but not with a conventional Boyden technique. Normal in vitro enhancement of PMN leucocyte random and chemotactic migration by sodium ascorbate was absent in SLE and Felty's syndrome, but sodium ascorbate gave normal stimulation of hexose monophosphate shunt activity in the PMN leucocytes precluding a defect in ascorbate transport.     

Effect of steroid treatment on the migration behaviour of neutrophils in patients with early rheumatoid arthritis

The influence of methylprednisolone on the migratory characteristics of neutrophil granulocytes was investigated in 10 patients with early rheumatoid arthritis (RA) and compared to 12 controls. The migration of neutrophils was measured with a whole-blood membrane filter assay with and without stimulation by the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP). Total migration index (TMI), distribution characteristics (DC) and the product of TMI and DC (neutrophil migratory activity; NMA) served to characterize the migratory behaviour of neutrophils. The data demonstrated an increased polymorphonuclear leucocyte (PMN) migration in patients with early RA, indicating a bystander role of PMNs in inflammatory joint injury. Treatment with methylprednisolone reduced significantly the penetration depth (DC) of neutrophils, but did not influence the number of migrating cells (TMI). The unstimulated NMA was significantly reduced due to the marked DC reduction, whereas steroids did not influence the stimulated NMA of neutrophils. A significant reduction in PMN penetration depth was demonstrated only after a steroid therapy of at least 10 days, suggesting that a longer period of steroid therapy is necessary to provide effective inflammatory control. Received: 24 March 1997 / Accepted: 8 September 1997     

Role of serum in stimulation of polymorphonuclear leukocyte, luminol-dependent chemiluminescence by influenza A

Granulocyte membrane perturbation activates oxidative metabolism with the release of highly reactive species (O2-, H2O2, OH., and 'O2) and emission of light (chemiluminescence (CL)). Using the CL response as a measure of oxidative metabolism, we assayed the effects of influenza A on the granulocyte respiratory burst. Human polymorphonuclear leukocytes (PMNs) were isolated by Ficoll-Hypaque cushioning and dextran sedimentation. The isolated PMNs were incubated with egg-grown influenza A (H3N2) virus, or a medium control, in the presence of 1 microM luminol and fresh autologous serum (10%). No light emission occurred during the incubation of PMNs with the medium control. Influenza A (33 to 50% egg-infective-doses (EID50):1 PMN) stimulated PMN light emission with a maximal response (48,386 +/- 10,764 cpm/10(6) PMN) occurring at 37 degrees CL was dependent on the virus dose with a diminished response (6,041 +/- 3,200 cpm/10(6) PMN) occurring at a lower infectivity of 10 EID50:1 PMN. Chemiluminescence responses were similar with infective and with noninfective virus particles (heat inactivated, 56 degrees C X 2 h). Fresh serum was necessary for the influenza virus to cause a CL response. A significant correlation (p less than 0.01) existed between the level of light emission and the hemagglutination-inhibiting (HI) antibody titer to influenza A of the autologous serum. Virus in the absence of detectable antibody did not stimulate CL. The virus-associated CL was completely inhibited if autologous serum was heated (56 degrees C X 30 min) or if the PMNs were pretreated with cytochalasin B (5 mcg/ml X 5 min). These findings suggest that influenza A-associated PMN CL requires antibody, complement, and phagocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impaired blood polymorphonuclear leukocyte migration and infection risk in severe trauma

OBJECTIVES: We investigated the association of impaired blood polymorphonuclear leukocyte (PMN) migration with the incidence of bacterial infections in patients with severe trauma. METHOD: Twenty-six intensive-care patients with different injury severity scores were enrolled in a prospective study. PMN migration was measured daily using 300 microl fresh whole blood in a membrane filter assay. Migration was evaluated in an automated image analyzer that recorded numbers and distribution of the immigrant PMNs within a filter. The relevant parameter was the percentage of PMNs that migrated from the blood samples into the filters upon f-Met-Leu-Phe stimulation. RESULTS: Nine patients developed posttraumatic infections verified microbiologically. These patients showed a reduced PMN migratory capacity in comparison with the 17 patients without infections. A migrating portion of six per cent or less at least three days in succession preceded infections by one to 19 days and indicated infection in eight true positive versus three false positive cases, and 14 true negative versus one false negative case, i.e. specificity was 82.3% and sensitivity 88.8%, p=0.0008. Trauma severity had no influence on PMN migration. CONCLUSIONS: Trauma patients with impaired PMN migration are at risk for bacterial infections. Whole-blood migration tests can define the infection risk and thus may be useful predictive markers for infections.     

Simplified micropore filter assay of neutrophil migration using whole blood

A new method of micropore filter assay of neutrophil migration requiring only 0.1 ml of whole blood is described and compared with the standard separated polymorphonuclear neutrophil micropore filter assay. Whole blood was added to the upper compartment of a modified Boyden chemotactic chamber, and the neutrophils were allowed to migrate into a cellulose nitrate micropore filter. Acetic acid was used to remove erythrocytes and hemoglobin from the filter. Neutrophil chemotaxis was performed with cells from 19 neonates and 34 adults. The mean neonatal PMN migration was 40% of the adult value (48.1 +/- 12.6 vs 96.8 +/- 16.8 micron) with the whole blood assay and 39% of the adult value (44.7 +/- 10.1 vs 72.9 +/- 22.2 micron) with the separated polymorphonuclear neutrophil assay. The whole blood micropore filter assay, a simple and reliable method of determining neutrophil migration, is especially useful in studying patients when there are difficulties in obtaining large blood samples, such as with neonates and young children.     

Physiologic responses to small emboli and hemodynamic effects of changes in deformability of polymorphonuclear leukocytes in isolated rabbit lung.

We hypothesized that polymorphonuclear leukocytes (PMNs) exposed to lipopolysaccharide (LPS) or chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) would alter the pulmonary hemodynamics of buffer-perfused rabbit lung. Pulmonary arterial pressure (Ppa) was measured at baseline, at peak response, and at 30 min after PMN infusion in the perfusate (Ppa x time, PT product). Infusion of peritoneal-harvested PMNs resulted in a transient increase in both pulmonary vascular resistance (PVR) and lung weight. PVR also increased when glutaraldehyde-treated rabbit PMNs (GPMNs) or beads were infused. Upstream PVR (Pao-Pdo) remained high with the infusion of GPMNs and beads and returned to baseline only when PMNs were infused 30 min thereafter. FMLP-exposed PMNs increased the peak Ppa and PT product. Pretreatment with 3-isobutyl-1-methylxanthine (IBMX) blocked this increase in pressure, suggesting the release of vasoconstrictor(s) or a direct effect of FMLP. PMNs exposed to LPS increased peak Ppa and PT product with and without the addition of IBMX. Cytochalasin D treatment of PMNs prevented the increase in PT product, suggesting that actin polymerization of PMNs is involved. The effects of these agents on PMN rigidity were verified by means of 6.5-microm polycarbonate filters. PMN suspension treated with FMLP or LPS increased filter perfusion pressure and PT product. Cytochalasin D prevented these increases. These results suggest that, initially after injection, PMNs behave like small beads embolizing primarily the small arteries in the lung and that they then move distally through the vasculature. Exposure to FMLP or LPS alters PMN deformability and the ability of PMNs to pass through the pulmonary vasculature, resulting in increased pulmonary vascular resistance.     

Activation of human neutrophils by monoclonal antibody PMN7C3: cell movement and adhesion can be triggered independently from the respiratory burst

Anti-neutrophil monoclonal antibody PMN7C3 (IgG3) recognizes glycoproteins bearing the oligosaccharide lacto-N-fucopentaose III, including the C3bi receptor, LFA-1, and p150,95 on the plasma membrane and a group of granule-associated glycoproteins. We have previously shown that binding of this antibody to polymorphonuclear leukocytes (PMNs) stimulates a transient rise in cytosolic free calcium concentration but does not trigger the neutrophil respiratory burst. We now demonstrate that binding of PMN7C3 (and five other monoclonal antibodies recognizing the same antigen) to human neutrophils activates several other cellular responses. Addition of PMN7C3 to monolayers of neutrophils induces a rapid change in cell shape followed by pseudopod formation and increased migration. With incubation at 37 degrees C, the neutrophils aggregate in clusters (leukoagglutination). Quantitation of cell movement in a multiwell chemotaxis assembly or by migration of PMNs under agarose revealed that PMN7C3 is both chemotactic and chemokinetic. Pretreatment with the antibody inhibits subsequent chemotactic response to other stimuli. Monoclonal antibodies binding to other neutrophil antigens do not mimic these effects. These data suggest that cell movement and adhesion can be triggered independently from the respiratory burst. PMN7C3 may be a useful probe with which to study the events that link receptor-ligand binding to cellular response.     

Inhibition of neutrophil chemotaxis in methotrexate-treated rheumatoid arthritis patients

Summary Polymorphonuclear cell (PMN) chemotaxis was assessed using the in vitro under agarose assay in ten rheumatoid arthritis patients prior to and following a single 10-mg dose of methotrexate (MTX). PMNs obtained from patients after MTX showed a decreased chemotactic migration response to both zymosan activated serum (P<0.005) and N-formyl-L-methionyl-L-phenylalanine (P<0.01). In similar conditions, no significant difference in chemotactic migration could be detected in six rheumatoid arthritis patients not on MTX. In contrast to the in vivo effects of MTX, there was no inhibition of normal PMN chemotactic migration following a 30-min in vitro incubation of the cells with MTX (P<0.99).     

Cultured bovine aortic endothelial cells secrete factor(s) chemotactic for aortic smooth muscle cells

Interactions of vascular endothelial cells (ECs) and smooth muscle cells (SMCs) were studied by testing the ability of cultured bovine aortic ECs to secrete factors influencing the migration of cultured aortic SMCs from the same species. Migration of SMCs was examined in blind-well chambers using gelatin-coated polycarbonate filters. Conditioned culture medium obtained by incubating confluent monolayers of ECs in serum-free RPMI-1640 medium for 48 hours caused a 2.4-fold increase in the migration of SMCs as compared with nonconditioned medium (p less than 0.001). The effect was dependent on the length of conditioning with the ECs and was chemotactic in nature as judged on the basis of checkerboard analysis. Preliminary characterization of the migration stimulating activity indicates that it is sensitive to trypsin, nondialyzable, and stable at 56 degrees C for 30 min. The activity was abolished by heating to 100 degrees C for 20 min but was not significantly inhibited by protamine sulphate, which suggests that most of the activity was not due to platelet-derived growth factor (PDGF)-like proteins. Our results thus show that ECs secrete polypeptide(s) chemotactic for vascular SMCs. Such interactions between ECs and SMCs in vivo might contribute to the migration of medial SMCs into the intima during atherogenesis.     

Changes in normal polymorphonuclear leucocyte motility after ingestion of IgG aggregates.

Changes in normal polymorphonuclear leucocyte (PMN) motility after membrane-binding and internalisation of IgG aggregates (a model of soluble immune complexes) have been studied by the micropore filter assay. The results have confirmed that IgG aggregates stimulate as well as inhibit PMN chemotaxis. These effects are dependent on the size and concentration of the IgG aggregates in solution as well as the length of time of incubation. Stimulated chemotaxis was observed in a small subset of the whole PMN population which was apparent only when cell distribution through the filters was analysed. These results indicate the need for caution when drawing conclusions about PMN function from results obtained by these assay techniques.     

Matrix metalloproteinase and alphavbeta3 integrin-dependent vascular smooth muscle cell invasion through a type I collagen lattice

Smooth muscle cell (SMC) migration from the tunica media to the intima is a key event in the development of atherosclerotic lesions and in restenosis after angioplasty. SMCs require not only migratory but also degradative abilities that enable them to migrate through extracellular matrix proteins, which surround and embed these cells. We used a collagen type I lattice as a coating on top of a porous filter as a matrix barrier in a chamber to test the invasive behavior of SMCs in response to a chemoattractant (invasion assay) and compared that behavior with simple SMC migration through collagen type I-coated filters (migration assay). Inhibitors of matrix metalloproteinase, KB-R8301, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), TIMP-2, and peptide 74, attenuated platelet-derived growth factor-BB (PDGF-BB)-directed SMC invasion across the collagen lattice, whereas no effect was seen with these inhibitors on simple SMC migration through collagen-coated filters. RGD peptide inhibited SMC invasion but did not affect SMC migration. Anti-alphavbeta3 integrin antibody attenuated PDGF-BB-directed SMC invasion, whereas other antibodies against RGD-recognizing integrins, namely alphavbeta5 and alpha5, had no effect. None of these antibodies had any effect on simple SMC migration. RGD peptide and anti-alphavbeta3 antibody inhibited the attachment and spreading of SMCs on denatured collagen but not on native collagen. These findings indicate that there is a difference in the mechanisms between simple SMC migration across a collagen-coated filter and SMC invasion through a fibrillar collagen barrier. A proteolytic process is required for SMC invasion, and the degradation of matrix proteins alters the relationship between matrix protein molecules and SMC surface integrins.     

Characterization of the effect of influenza virus on polymorphonuclear leukocyte membrane responses

Depressed chemotactic activity of polymorphonuclear leukocytes (PMNL) infected with influenza virus could be due to changes occurring at the plasma membrane. The present study examined the effect of unopsonized influenza virus on chemotaxis, adherence, receptor binding, shape change, membrane fluidity, and release of specific granules from PMNL. Chemotactic activity of PMNL under-agarose to the chemoattractants, zymosan-activated serum ( ZAS ) and N-formyl-methionyl-leucyl- phenylalanine (fMLP), and adherence of PMNL to a plastic surface were markedly decreased in virus-treated cells as compared to control cells. The binding of fMLP to the PMNL was increased in virus-treated cells compared with control cells. Exposure of cells to virus, ZAS , or fMLP caused 35%-50% of the cells to become bipolar in shape, whereas less than 5% of the cells exposed to buffer became bipolar. Influenza virus did not alter membrane fluidity as measured by electron spin resonance spectroscopy with the probe 5-doxyl stearate. Virus-treated PMNL stimulated with FMLP or Staphylococcus aureus exhibited a marked decrease in the amount of lactoferrin released into phagosomes, onto the cells' outer membrane, and into the extracellular medium as compared to control cells. The possible relationship between inhibition of lysosomal enzyme degranulation and decreased chemotactic activity and adherence of PMNL is discussed.     

Troxipide, a novel antiulcer compound, has inhibitory effects on human neutrophil migration and activation induced by various stimulants

BACKGROUND: Neutrophils are considered to be involved in the pathogenesis of Helicobacter pylori-associated gastroduodenal diseases on account of their potent biological functions as effector cells. Troxipide, a new antiulcer compound used for patients with gastric ulcer or gastritis, has been shown to inhibit migration and activation of guinea pig neutrophils, but little is known about the pharmacological effects on human neutrophils. AIMS: To study the effects of troxipide on chemotactic migration and superoxide generation by human neutrophils. METHODS: The chemotactic response of neutrophils was determined in a multi-well chamber with a polycarbonate filter and the generation of O2- by neutrophils was measured using a chemiluminescence method. Concentrations of troxipide in gastric mucosa were measured by high-performance liquid chromatography. RESULTS: Incubation of neutrophils with 10(-6) to 10(4) M troxipide caused inhibition of recombinant interleukin-8-induced migration. These concentrations of troxipide also inhibited superoxide generation by neutrophils stimulated by formyl-methionyl-leucyl-phenylalanine or platelet activating factor. These phenomena were not simply due to the direct cytotoxic effects since the above concentrations of troxipide did not induce neutrophil apoptosis. The concentrations of troxipide detected in the gastric mucosa after oral administration were in the range able to inhibit chemotactic migration and superoxide generation by neutrophils in vitro. CONCLUSION: These results suggest that troxipide may exert its therapeutic effect in patients with gastric ulcer or gastritis by inhibiting inflammatory responses and mucosal injury mediated by neutrophils in gastric mucosa.     

Neutrophil-endothelial cell interaction: evidence in vitro for a regulation by endothelial cells of neutrophil functions.

The purpose of this study was to investigate a possible relationship between human umbilical vein endothelial cells (EC) triggered by ionophore A23187 at different doses (0.5-2.5 microM) and polymorphonuclear neutrophils (PMN). EC supernatants were shown to contain neutrophil chemoattractant activity (NCA) and in parallel a factor inducing an inhibition of PMN chemiluminescence (PMN CL). Supernatants obtained from EC triggered by A23187 exhibited a high level of NCA (73 +/- 5 PMN.hpf-1 compared to 21 +/- 4 PMN.hpf-1 in untreated EC supernatants, p less than 0.01). This NCA was independent from arachidonic acid metabolites, since indomethacin and nordi-hydroguaiaretic acid failed to suppress the chemotactic activity. Using gel filtration chromatography (AcA 54) the NCA was recovered in a single peak of apparent molecular weight of 37,000 +/- 4,000 daltons. Checkerboard analysis indicated that NCA exhibited both chemotactic and chemokinetic activities. In addition, supernatants of A23187-stimulated EC, and at a lesser degree, supernatants of unstimulated EC, inhibited PMN CL induced by N-formyl-Methionyl-Leucyl-Phenylalanine (61% inhibition, p less than 0.05), and by A23187 itself (80% inhibition, p less than 0.01), but not that induced by phorbol-myristate-acetate. Indomethacin and protamine sulphate did not modulate this inhibitory activity. By contrast, EC-derived inhibitory activity was inhibited (50%) by an adenosine antagonist (8-phenyltheophylline), indicating a participation of adenosine in this inhibitory activity of PMN CL. These data suggest the possibility that activated endothelial cells could both enhance PMN migration and protect themselves against potential damaging effects of oxygen metabolites produced by PMN, particularly during transvascular migration.     

The effects of ibuprofen and diclofenac on the chemotaxis and adenosine triphosphate level of polymorphonuclear cells in vitro

Investigations were carried out to determine the effect of ibuprofen and diclofenac on the chemotaxis of human polymorphonuclear cells. The experiments were done with the drugs alone as well as in the presence of leukotriene B4 (LTB4). A modified quantitative bioluminescence assay was used to measure chemotaxis, which allowed the calculation of differences in the spontaneous migration in contrast to the effects of the drugs, and simultaneously, also, the evaluation of whether the inhibition or augmentation of the chemotaxis was due to influences on the adenosine triphosphate (ATP) level of the cells. It was found that high concentrations (10 mM) of either ibuprofen or diclofenac destroy the intracellular ATP of polymorphonuclear cells (PMN). Therefore, the reported inhibition of the chemotaxis by ibuprofen at a concentration of 10 mM cannot be understood as a part of the chemotactic process. In the range of 1 microM and 0.1 mM ibuprofen and diclofenac did not statistically affect the intracellular ATP level of PMN cells but at the same time a distinct inhibition of the chemotactic response of PMN cells was observed. This effect occurred even in the presence of a potent chemoattractant (leukotriene B4). Ibuprofen (0.1 mM) reduced chemotaxis to 67% and the same concentration of diclofenac reduced it to 56% of the values of LTB4 alone.     

Platelet-derived growth factor-BB (PDGF-BB) regulation of migration and focal adhesion kinase phosphorylation in rabbit aortic vascular smooth muscle cells: roles of phosphatidylinositol 3-kinase and mitogen-activated protein kinases.

OBJECTIVE: Phosphatidylinositol 3'-kinase (PI3-kinase) is implicated in cell migration and focal adhesion kinase (FAK) phosphorylation. In contrast, it has been proposed that mitogen-activated protein (MAP) kinases are essential for proliferation but may be dissociated from chemotactic signalling. We investigated the roles of PI3-kinase and p42/p44 MAP kinases in cell migration and FAK tyrosine phosphorylation induced by platelet-derived growth factor-BB (PDGF-BB) in rabbit aortic vascular smooth muscle cells (VSMCs). The roles of PI3-kinase and MAP kinase pathways in the chemotactic response to insulin-like growth factor-I (IGF-I) were also examined. METHODS: The roles of PI3-kinase and p42/p44 MAP kinases were assessed using the PI3-kinase inhibitors, wortmannin and LY294002, and an inhibitor of MAP kinase kinase, PD98059. PI3-kinase activity was measured by phosphatidylinositol phosphorylation in anti-phosphotyrosine immunoprecipitates and by thin layer chromatography of phosphorylated products. Phosphorylation was assessed by immunoprecipitation with anti-phosphotyrosine antibodies and Western blotting with FAK-specific antibody. Migration was evaluated in a chemotaxis chamber using polycarbonate filters with an 8-mm pore size. RESULTS: Neither wortmannin nor LY294002 significantly reduced PDGF-BB stimulation of FAK tyrosine phosphorylation, chemotaxis or immunofluorescent staining of focal adhesions in VSMCs. PD98059, a specific inhibitor of MAP kinase activation, did not inhibit FAK tyrosine phosphorylation but markedly inhibited the migratory response of VSMCs to PDGF-BB. IGF-I also stimulated migration of VSMCs, and, relative to the effect of PDGF-BB, induced smaller increases in PI3-kinase and MAP kinase activities. Both wortmannin and PD98059 partially inhibited the migratory response to IGF-I. CONCLUSIONS: PDGF-BB stimulation of both FAK tyrosine phosphorylation and migration in VSMCs are not dependent on activation of PI3-kinase. While PDGF-BB stimulation of FAK tyrosine phosphorylation is not dependent on p42/p44 MAP kinase activation, PDGF-BB and IGF-I both stimulate p42/p44 MAP kinase activity and the chemotactic response to these factors is partially dependent on MAP kinase activation.