Random Migration Of Neutrophils Research Articles

Effects of lead on neutrophil functions in occupationally exposed workers

Chemotactic and intracellular killing activity of neutrophils were examined in 25 male lead-exposed workers from storage-battery plants and compared to 25 healthy males with no history of lead exposure. Lead exposure was assessed using blood lead levels measured by atomic absorption spectrophotometry and zinc protoporphyrin levels assayed by hematofluorometry. Chemotaxis was carried out in Boyden chambers using zymosan activated serum as chemotactic stimulus. Intracellular killing activity of neutrophils was measured using nitroblue tetrazolium reduction test, measured of 515 nm in spectrophotometry. In lead-exposed workers a significant decrease in chemotaxis and random migration of neutrophils ( p<0.001) was observed compared to controls. Intracellular killing activity of polymorphonuclear leukocytes have also seemed to be slightly but not significantly reduced. These results suggest that human chronic exposure to lead may diminish neutrophil function in man.

External calcium-dependent, F-actin-independent and pertussis toxin-insensitive novel neutrophil locomotion induced by a mAb.

We previously demonstrated that a mAb to human neutrophils, designated 3H9, which was established by screening for inhibition of neutrophil adherence to plastic plates containing fetal bovine serum, enhanced both n-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced chemotaxis and random migration of neutrophils. In the present study, we examined the mechanisms of 3H9-induced enhancement of neutrophil locomotion in the phagokinetic track assay. 3H9-induced neutrophil locomotion maintained a straight path which was different from the track resulting from FMLP-stimulated locomotion. This 3H9-induced migration required extracellular Ca2+. beta 2-Integrin activation was a prerequisite for the increase in cytosolic free calcium induced by 3H9 treatment. However, stimulation by 3H9 did not induce an increase in F-actin, even after CD18 activation. Signal transduction after molecular recognition by 3H9 was not mediated by pertussis toxin-sensitive, heterotrimeric G proteins. These results suggest that 3H9 enhances neutrophil migration by mechanisms which are different from those involved in usual chemoattractant-induced migration. Neutrophil surface molecules recognized by 3H9 may play a crucial role in the regulation of transendothelial migration of leukocytes.

Hemodynamic Stress Enhances Neutrophil Responsiveness to Chemotactic Stimuli

Circulating neutrophils are exposed to widely varying levels of hemodynamic stress induced by blood flow conditions. This study examined the effect of hemodynamic stress on the functional responsiveness of neutrophils obtained from healthy humans to chemotactic stimuli. To expose neutrophils to hemodynamic stress in vitro, isolated neutrophils were agitated under artificial flow conditions induced by a rotary tube apparatus. Although such hemodynamic stress produced no spontaneous or random migration of neutrophils, it enhanced neutrophil migration in response to the chemotactic peptide f-methionyl-leucyl-phenylalanine (FMLP) by as much as 200%. Hemodynamic stress also enhanced polarization in response to FMLP, producing a change in shape characteristic of migration. Polarization was reversible when neutrophils were transferred to quiescent conditions after being exposed, to hemodynamic stress. Hemodynamic stress also enhanced O−2 production and granular β-glucuronidase release in response to FMLP and enhanced polarization and O−2 production in response to phorbol myristate acetate (PMA), a direct activator of protein kinase C. Extracellular Ca2+ was not required for the enhancement of chemotactic responsiveness by hemodynamic stress, and the stress produced no detectable change in intracellular Ca2+, intracellular cyclic AMP, or activated protein kinase C levels in neutrophils. The results show that hemodynamic stress enhances the functional responsiveness of neutrophils to chemotactic stimuli and provide insights into interpretation of in vitro data usually obtained from quiescent conditions.

A monoclonal antibody modulates neutrophil adherence while enhancing cell motility.

A monoclonal antibody (MoAb) to human neutrophils, designated 3H9, was established by screening for the inhibition of neutrophil adherence to plastic plates containing a medium supplemented with fetal calf serum (FCS medium). The antigen recognized by 3H9 was shown to be present on human leukocytes and found at the highest levels on granulocytes. On Western blotting, 3H9 reacted with a molecule having a molecular weight of 80 kDa. When this MoAb was added at the same time as a neutrophil stimulant (fMLP), the inhibition of neutrophil adherence to plastic plates in the presence of FCS medium was observed after 60 min incubation. Furthermore, this MoAb enhanced not only fMLP-induced chemotaxis but random migration of neutrophils as well. The mechanisms of these phenomena are discussed.

Neutrophilic migration through capillarylike micropores: influence of pulmonary passage

We report the effect of pulmonary passage on random migration and chemokinesis of neutrophils through capillarylike pores under the in vitro condition of Boyden's test. Neutrophils were isolated either from the left ventricle or from the pulmonary artery of patients who underwent coronary angiography due to suspected angina pectoris or valvular heart disease. In all 14 cases left ventricle neutrophils showed significantly enhanced chemotactic-activated migration compared with pulmonary artery neutrophils. Pulmonary passage also influenced the random migration of neutrophils, except for those derived from five patients suffering from pulmonary hypertension. Our findings might indicate that accumulation of neutrophils in the capillaries of the normal lung is counteracted by a change in neutrophilic migration behavior during pulmonary passage, thus avoiding increased neutrophilic sequestration in pulmonary microcirculation as observed in adult respiratory distress syndrome.

Neutrophil chemotaxis and periodontal status in Down's syndrome patients.

Along with clinical parameters, chemotaxis and random migration of neutrophils were evaluated in 15 patients with Down's syndrome (DS) and 15 healthy subjects. Signs of more severe gingival inflammation were present in the DS group. The random migration and chemotaxis of neutrophils were significantly decreased in comparison with the control group. In DS, the pathological status was attributed to these impaired host defense factors besides the existing bacterial plaque.

Effect of Ascorbic Acid Nutriture on Blood Histamine and Neutrophil Chemotaxis in Guinea Pigs

Histamine suppresses certain immune responses, including neutrophil chemotaxis. The present study examined whether the histamine-lowering effect of ascorbate was accompanied by enhanced chemotaxis in guinea pigs. Animals were fed low ascorbate, adequate or high ascorbate diets (0.5, 2.0 or 50 mg ascorbate.100 g body wt-1.d-1) for 4 wk. Mean liver ascorbate paralleled dietary intake, and these values differed significantly. Blood histamine was significantly depressed in the high ascorbate group compared to the adequate and low ascorbate groups, and liver ascorbate was inversely correlated to blood histamine levels (r = -0.64, P less than 0.001). The random migration of neutrophils was not significantly affected by vitamin dosage. Leukocyte chemotaxis was significantly impaired in low ascorbate animals compared to that of animals with adequate ascorbate nutriture. Leukocyte chemotaxis in high ascorbate animals did not differ significantly from that in the adequate or low ascorbate groups. Furthermore, chemotaxis was significantly lower when cells extracted from animals with adequate ascorbate nutriture were incubated in low ascorbate or high ascorbate serum rather than in autologous serum. These data suggest that the histamine-lowering effect of supplemental ascorbate does not appear to enhance leukocyte chemotaxis and that serum from guinea pigs fed low or high levels of ascorbate appears to contain factors that depress chemotaxis.

Lymphocyte subpopulations and neutrophil function in chronic human Chagas' disease

The absolute numbers of total leukocytes, lymphocytes. T cells, helper/inducer, suppressor/cytotoxic and B cells were decreased in the peripheral blood of patients with chronic Chagas' disease. Since antilymphocyte antibodies were present only in a minority of patients they probably cannot account for the abnormalities in lymphocyte subsets. Patient neutrophils stimulated with endotoxin-treated autologous plasma showed depressed chemotactic activity and this seems to be an intrinsic cellular defect rather than plasma inhibition. Random migration of neutrophils was normal. Reduction of nitroblue tetrazolium by endotoxin-stimulated neutrophils was also decreased. These findings further document the presence of immunosuppression in human Chagas' disease. They may be relevant to autoimmunity, defense against microorganisms and against tumor cells at least in a subset of patients with more severe abnormalities.

In vitro migration responses of neutrophils from cows and calves

SUMMARY The directional (chemotactic) and random migration activities of neutrophils from cows and newborn and 2-week-old calves were determined by use of the chemotaxis-under-agarose assay. Blood samples were stored for 2, 24, or 48 hours and at 4 or 25 C before testing. During the assay, cells were incubated at 17, 27, or 37 C. The assay was found suitable for testing the directional and random migration activities of neutrophils from cattle. Directional migration of neutrophils was diminished (P ≤ 0.05) when cells were incubated at 17 or 27 C, compared with data from incubation at 37 C. Random migration of neutrophils was unaffected by test incubation temperature. Significant (P ≤ 0.05) differences were found between cows and calves regarding the percentage number and viability and the directional and random migration activities of neutrophils. Neutrophils from cows were adversely affected to a greater extent by prolonged sample storage times or low storage temperature than were neutrophils from calves. Results indicate that a sample storage time of up to 24 hours, a sample storage temperature of 25 C, and a test incubation temperature of 37 C provided optimal conditions for testing the migratory activities of neutrophils from cattle.

Pleural mesothelial cells stimulated by asbestos release chemotactic activity for neutrophils in vitro.

The development of the pleural inflammatory response to asbestos remains poorly defined. Importantly, the role of the pleural mesothelial cell in recruitment of neutrophils to the pleural space is not known. We hypothesized that rabbit pleural mesothelial cells stimulated by asbestos fibers release chemotactic factor(s) for neutrophils. Primary cultures of rabbit pleural mesothelial cells were established, and their purity verified by the presence of keratin and hyaluronic acid mucin. Mesothelial cells in serum-free media, in the presence of 30 micrograms/ml of crocidolite asbestos, released chemotaxins for neutrophils. This activity was not dependent on the type of asbestos fiber or fiber length. It was dose-dependent until 30 micrograms/ml of asbestos. The chemotactic fractions had the ability to increase both directed and random migration of neutrophils. The chemotactic activity was not present in sonicated fractions of unstimulated mesothelial cells, nor in supernates of asbestos fibers alone. Characterization of the chemotactic activity showed that it was heat stable (56 degrees C per 30 min) and sensitive to digestion with trypsin and papain. On Sephadex G-50 chromatography, it had a molecular weight between 6,000 and 9,000. Production of the chemotactic activity was inhibitable by cycloheximide. These results demonstrate that pleural mesothelial cells can actively synthesize a protein fraction with chemotactic activity for neutrophils. Production of this mesothelial cell-derived chemotactic activity for neutrophils may play an important role in the initiation of the inflammatory response of the pleura to asbestos.

Botulinum C2 toxin ADP-ribosylates actin and enhances O2- production and secretion but inhibits migration of activated human neutrophils.

The binary botulinum C2 toxin ADP-ribosylated the actin of human neutrophils. Treatment of human neutrophils with botulinum C2 toxin for 45 min increased FMLP-stimulated superoxide anion (O2-) production 1.5-5-fold, whereas only a minor fraction of the cellular actin pool (approximately 20%) was ADP-ribosylated. Effects of botulinum C2 toxin depended on toxin concentrations, presence of both components of the toxin, and incubation time. Cytochalasin B similarly enhanced O2- production. The effects of botulinum C2 toxin and cytochalasin B were additive at submaximally, but not maximally effective concentrations and incubation time of either toxin. Botulinum C2 toxin also enhanced stimulation of O2- production by Con A and platelet-activating factor, but not by phorbol 12-myristate 13-acetate (PMA). Botulinum C2 toxin increased FMLP-induced release of N-acetyl-glucosaminidase by 100-250%; release of vitamin B12-binding protein induced by FMLP and PMA was enhanced by approximately 150 and 50%, respectively. Botulinum C2 toxin blocked both random migration of neutrophils and migration induced by FMLP, complement C5a, leukotriene B4, and a novel monocyte-derived chemotactic agent. The data suggest that botulinum C2 toxin-catalyzed ADP-ribosylation of a minor actin pool has a pronounced effect on the activation of human neutrophils by various stimulants.

Predictive value of skin testing, neutrophil migration and C-reactive protein for postoperative infections in cardiopulmonary bypass patients.

Despite antibiotic cover for cardiac surgery the development of postoperative infection remains a feared complication. The present prospective study was designed to evaluate whether it is possible to identify preoperatively those individuals in whom the risk of a postoperative infection is significantly greater. Skin test response, chemotaxis and random migration of neutrophils, and serum levels of C-reactive protein (CRP) were chosen as indicators of host resistance. Pre-operatively and at defined postoperative time intervals, 80 patients undergoing open-heart surgery with the aid of cardiopulmonary bypass were examined. Of all the immunological variables measured in this study, the state of anergy proved the most reliable predictor of postoperative infection. Six of the nine pre-operatively anergic patients developed infections (P = 0.00026) representing one half of the postoperatively infected patients. No predictive role of CRP level or neutrophil chemotaxis can be derived from our data. The pre-operatively decreased random migration of polymorphonuclear leukocytes (PMN) from postoperatively infected patients, however, along with the significant defects in chemotactic and random PMN motility seen after cardiac surgery may partly explain the predisposition to bacterial infection in these patients. In conclusion, a pre-operative skin test makes a valuable contribution to the assessment of the patient's postoperative infectious risk.

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.

Properties of a high molecular weight neutrophil chemotactic factor, possibly derived from mast cells: evidence for chemokinetic rather than chemotactic activity.

Kay and his colleagues [1] have suggested that the neutrophil high molecular weight chemotactic factor ( NCF ) found in the serum of patients suffering from a variety of allergic diseases is mainly derived from mast cells and is therefore an indicator of mast cell activation. We have studied some of the properties of NCF obtained from patients with atopic extrinsic asthma and compared it with N-formyl-1-methionyl-1-leucyl-1-phenyl-alanine (FMLP), a chemotactic peptide [2]. A number of differences between FMLP and NCF were observed. In contrast to FMLP, checkerboard analysis showed that NCF caused random migration of neutrophils. In addition microscopic analysis of neutrophil locomotion in response to FMLP demonstrated the characteristic pseudopod formation. Furthermore, it was found that in contrast to FMLP, NCF did not cause the release of lysosomal enzymes from cytochalasin B-treated neutrophils. These results suggest that NCF has chemokinetic rather than chemotactic properties.

The effects of ketoconazole on cellular and humoral immune functions.

The effects of ketoconazole at varying concentrations on in-vitro neutrophil random migration, chemotaxis to autologous endotoxin--activated serum and the synthetic chemotactic tripeptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine, phagocytosis and postphagocytic hexose monophosphate shunt activity and myeloperoxidase--mediated protein iodination were investigated. Neutrophil functions, in-vivo mitogen-induced lymphocyte transformation and levels of serum immunoglobulins and complement components were also assessed before and after ingestion of ketoconazole by six individuals. It was found that ketoconazole caused stimulation of neutrophil migration to leucoattractants 2 h after ingestion of a single dose of 400 mg ketoconazole. This stimulation was not sustained. The drug had no effect on the random migration of neutrophils. Likewise serum levels of the immunoglobulins IgG, IgM and IgA, total haemolytic complement and serum levels of the complement components C3 and C4 were unaffected. Ketoconazole in vitro had no effect on the neutrophil functions tested. Ingestion of ketoconazole caused a slight inhibition of lymphocyte mitogen-induced transformation.

Chemotaxis and Random Migration of Polymorphonuclear Leukocytes in Ulcerative Colitis Examined by the Agarose Method

Chemotaxis and random migration of neutrophils under agarose were compared between untreated ulcerative colitis patients (35 women and 36 men) and healthy subjects (25 women and 28 men). The ulcerative colitis patients were divided into two subgroups--those with the active (no. = 41) and those with the inactive (no. = 30) stage of the disease. High spontaneous mobility and subnormal chemotaxis (both aberrations statistically significant) were noted in the ulcerative colitis patients. Polymorphonuclear leukocyte chemotaxis was significantly lower in inactive than in active colitis. Possible causes of the difference in neutrophil chemotaxis between the two disease groups were discussed. No difference in the chemotactic activity between zymosan-stimulated plasma from controls and patients was found. The defect in the chemotaxis of neutrophils in ulcerative colitis might explain the small abscesses persisting in the mucosa of the great bowel.

Inverse correlation between neutrophil microtubule numbers and enhanced random migration.

The random migration of neutrophils under agarose as measured by the number of cells leaving the well, is enhanced when the pH or the osmolality of the medium is reduced or when microtubule agents are used. Concentrations of colchicine above 5 x 10-7 M increased the number of cells migrating and decreased the mean number of centriolar microtubules in a dose-dependent fashion from 16 to 4 per 4 micron 2 at 10-5 M. The distance that colchicine-treated neutrophils migrated from the well was not different from the control. Lowering the pH from 7.4 to 6.0 also increased random migration and decreased pericentriolar microtubules from a mean of 16 to a mean of 10. At pH 6.0, both the number of cells that migrated and the distance the cells forming the leading edge travelled from the well were increased. Since peripheral microtubules may play a greater role in cell migration than centiolar ones, we examined the numerical density of microtubules in the peripheral cytoplasm. Lowering the medium pH reduced the mean number of microtubules per 10 micron 2 from 6 to 2. Colchicine reduced micro-tubules in the same area to I. At the low pH, colchicine reduced even further the numbers of both centriole-associated and peripheral microtubules but the migration pattern was the same as that seen at pH 6.0 without colchicine. Lowering medium osmolality from 280 to 230 m-osmol increased random migration but did not affect microtubule numbers. The addition of colchicine to this system decreased microtubule numbers and increased migration even further. Conditions that enhanced neutrophil migration also affected cell shape. Whereas cells at pH 7.4 were generally fan-shaped with a broad, smooth leading edge, cells at pH 6.0 with or without colchicine were long and narrow. Neutrophils at pH 7.4 but 230 m-osmol had a scalloped edge, which often appeared thickened. This too was not altered by colchicine. The morphology of cells treated with colchicine was similar to controls except for the more frequent presence of long retraction fibres. Each of these treatments thus appears to act on a different aspect of the cell's locomotory apparatus. The mechanisms by which colchicine and lowered ph enhance migration may partially overlap since both significantly decrease peripheral microtubules. The data suggest that microtubules play a constraining role within the cell, limiting the ability of the cell to move and change direction.

Suppression of chemotatic activity of human neutrophils by streptolysin O.

Human neutrophils were treated with streptolysin O in amounts that failed to cause cell lysis or morphologic changes. As a result of this treatment, the neutrophils were unable to respond to chemotactic factors. The inhibition of chemotaxis by streptolysin O was independent of factors in serum and could be prevented by heat inactivation, oxidation, and serum containing antistreptolysin O if used in sufficient titer with prior incubation. In addition to preventing directional mobility of leukocytes, streptolysin O also suppressed the random migration of neutrophils.