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.     

Endotoxin-induced neutrophilic alveolitis and macrophage chemotaxin production in sheep

The mechanism(s) responsible for endotoxin-induced pulmonary leukostasis has not yet been elucidated. We studied ten unanesthetized sheep by performing serial bronchoalveolar lavages (BAL) before and after infusing Escherichia coli endotoxin to determine whether or not neutrophils appeared in the airways and whether or not endotoxemia stimulated alveolar macrophages to produce neutrophil chemotactic activity. The absolute number and percentage of neutrophils recoverable by BAL increased significantly at 24 hours after infusion of endotoxin. Alveolar macrophages isolated from the BAL of sheep after endotoxemia produced a substance that is chemotactic for neutrophils, a response that is also maximal 24 hours after endotoxin infusion. We conclude that endotoxemia causes migration of neutrophils into lung air spaces and that this response may result from in vivo production of a chemotactic factor(s) by activated alveolar macrophages.     

Modulation of F-met-leu-phe Induced Chemotactic Activity and Superoxide Production by Neutrophils during Chronic Ethanol Intoxication

Chronic alcohol consumption has been associated with increased migration of neutrophils into liver that could contribute to the development of alcoholic liver disease. Mild endotoxemia may be at least partially responsible for this condition since endotoxemia was shown to be present in virtually all chronic alcoholics. This study examines the release of superoxide anion and chemotactic activity by Kupffer cells and sequestered hepatic as well as blood neutrophils during chronic alcohol intoxication (16 weeks) alone, and following an intravenous injection of Escherichia coli lipopolysaccharide (LPS) (1 mg/kg) 3 hr before cell isolation. Chronic ethanol consumption increased the total neutrophil yield per liver, but did not change the f-met-leu-phe induced chemotactic activity by both hepatic and blood neutrophils. However, the combined insults of ethanol and LPS increased the chemotactic activity and superoxide anion generation by these cells. Plasma from ethanol-fed rats was highly chemotactic to syngeneic normal rat neutrophils. This activity was increased 1.75-fold in the plasma obtained from chronic ethanol plus endotoxin-injected rats. The chemotactic activity of Kupffer cells was not significantly modulated during ethanol intoxication plus endotoxin treatment. The f-met-leu-phe-induced superoxide anion release by Kupffer cells was enhanced after LPS treatment. Chronic ethanol consumption did not induce any effect on this parameter. These observations suggest that functional alterations in neutrophils during chronic ethanol intoxication may contribute to hepatic injury.     

Cigarette smoking and lung destruction. Accumulation of neutrophils in the lungs of cigarette smokers

It has been hypothesized that lung destruction in persons with emphysema associated with cigarette smoking is mediated by elastase released by neutrophils that have migrated to the alveolar structures in response to cigarette smoke. To directly evaluate this hypothesis, cell suspensions, isolated from bronchoalveolar lavage fluid and from open lung biopsies of nonsmokers and cigarette smokers with normal lung parenchyma and from open lung biopsies of nonsmokers and cigarette smokers who have sarcoidosis were evaluated for the presence of neutrophils. A significantly increased number of neutrophils was present in the cell suspensions isolated from bronchoalveolar lavage fluid and from open lung biopsies of both normal and sarcoid cigarette smokers compared with that in the nonsmokers (p less than 0.01, each comparison). Evaluation of the alveolar macrophages present in lavage fluid suggested a mechanism by which neutrophils may be attracted to the lungs of cigarette smokers: alveolar macrophages of cigarette smokers release a chemotactic factor for neutrophils, whereas alveolar macrophages of nonsmokers do not. In addition, alveolar macrophages of nonsmokers, after exposure to cigarette smoke, in vitro, are stimulated to release this chemotactic factor. These studies demonstrate that an increased number of neutrophils are present in the lungs of cigarette smokers compared with that in nonsmokers and suggest that cigarette smoke may attract neutrophils to the lung by stimulating alveolar macrophages to release a potent chemotactic factor for neutrophils.     

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.     

Free radicals and inflammation: superoxide-dependent activation of a neutrophil chemotactic factor in plasma.

The intravenous administration of superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) to animals with induced inflammation suppresses the inflammatory response and inhibits leukocyte infiltration into the challenged site, suggesting that neutrophil-generated superoxide reacts with an extracellular precursor to generate a substance chemotactic for neutrophils. Plasma exposed to superoxide in vitro becomes potently chemotactic. The appearance of chemotactic activity is inhibited by superoxide dismutase but not by catalase. The chemotactic factor does not stimulate superoxide production or degranulation in neurtrophils. Intradermal injection of superoxide-treated plasma or of a superoxide-generating system causes heavy infiltration of neutrophils to the injection site but does not cause overt signs of inflammation. The chemotactic factor consists of a chloroform-extractable component bound to serum albumin. The superoxide-dependent chemotactic factor appears to play a major role in communication in neutrophil-mediated inflammatory events. Prevention of production of this factor appears to be the major anti-inflammatory action of superoxide dismutase.     

Lymphokine production by blood or spleen mononuclear cells from patients with schistosomiasis mansoni

Peripheral blood mononuclear cells (PBMN) from individuals with active or former intestinal schistosomiasis mansoni or splenocytes from patients with the hepatosplenic form of the disease were evaluated for their ability to generate chemotactic factors for neutrophils in response to schistosomal antigenic preparations derived from adult worms (SWAP), eggs (SEA), or phytohemagglutinin (PHA). When supernatants from cultures of stimulated PBMN from normal donors were assayed, only those obtained from cells which had been cultured in presence of PHA displayed chemotactic activity for neutrophils. In contrast, supernatants from cultures of SWAP or SEA stimulated PBMN from patients with intestinal or hepatosplenic schistosomiasis were shown to contain chemotactic activity for neutrophils from normal individuals. PBMN from persons who previously had been infected with Schistosoma mansoni but had received chemotherapy years before presented a pattern of response to SWAP or SEA similar to those from patients with active infections. The response of splenocytes from patients with hepatosplenic schistosomiasis was considerably different from PBMN from individuals with active or with treated schistosomiasis. Splenocytes from most of those patients with hepatosplenic disease failed to produce chemotactic factors for neutrophils in response to stimulation with at least 1 of the schistosome antigens tested. These results indicate that the lymphocytes from schistosomiasis mansoni patients are able to recognize and are stimulated by adult worm and egg antigens to produce chemotactic substances for neutrophils, and that this ability persists for many years after chemotherapy with schistosomicidal drugs. At the hepatosplenic stage, immunoregulatory mechanisms, which may prevent the production of chemotactic factors by splenocytes and/or their activity upon neutrophils in vitro, seem to occur.     

Characterization of an activation factor released from human neutrophils after stimulation by triclinic monosodium urate crystals

OBJECTIVE: To determine the presence and characterize the activity of a soluble activation factor rapidly released by human neutrophils after stimulation with monosodium urate (MSU) crystals. METHODS: Supernatants from human neutrophils stimulated by MSU crystals for 5 to 60 min were tested for their ability to stimulate a chemotactic response, induce a mobilization of calcium, and increase the tyrosine phosphorylation levels in naive neutrophils. RESULTS: Supernatant from neutrophils stimulated 相似文献   

Immunologic reactivity of the lung: the in vivo and in vitro generation of a neutrophil chemotactic factor by alveolar macrophages

The generation of a chemotactic factor by guinea pig alveolar macrophages after phagocytosis of heat-killed Staphylococcus aureus was studied. These studies demonstrated that after phagocytosis, alveolar macrophages secrete a small molecular weight (less than 5,000 daltons) chemotactic factor that preferentially attracts neutrophils. The chemotactic factor can be generated in vivo and in vitro, and its chemotactic activity can be detected both in vitro by a chemotactic assay and in vivo by an increase in the absolute number of neutrophils in bronchoalveolar lavage fluid and in lung tissue histologic specimens. Generation of the chemotactic factor was inhibited by 20 microgram of actinomycin D per ml and by 10 microgram of cycloheximide per ml. The factors were stable after incubation at 56 degrees C for 45 min, but not after incubation at 100 degreeg C for 10 min. These studies show that alveolar macrophages can serve as an initiator of pulmonary inflammatory responses by secreting a potent mediator of neutrophil locomotion.     

Transepithelial migration of human neutrophils: an in vitro model system.

An in vitro model system for studying transepithelial migration of human neutrophils has been developed. Canine kidney epithelial cells grown on micropore filters form a confluent, polarized monolayer with an average transepithelial electrical resistance of 181 ohms.cm2. Neutrophils in a chemotactic chamber are stimulated to undergo random migration, chemokinesis, or chemotaxis through the epithelium. When stimulated by a gradient of the synthetic chemoattractant fMet-Leu-Phe, significantly more neutrophils traverse the low-resistance epithelium than do under conditions of random migration or chemokinesis. Transmission and scanning electron microscopy of this process reveal that neutrophils traverse the epithelium through the intercellular space. After leukocyte emigration, lateral epithelial cell membranes reapproximate. Neutrophils undergoing chemotaxis can also traverse the polarized epithelium from the basal epithelial surface, which suggests that the chemotactic gradient and not the apical-basal polarity of the epithelial cells determines the direction of transepithelial migration. The data further suggest that (i) the in vitro model of leukocyte transepithelial migration morphologically simulates the in vivo process, (ii) neutrophils more readily penetrate the epithelium when attracted by a chemotactic factor, and (iii) neutrophils can traverse a low-resistance epithelium in the absence of serum and connective tissue factors.     

Impaired neutrophil chemotaxis in chronic obstructive pulmonary disease

RATIONALE: Neutrophilic airway inflammation is considered to be a major factor in the pathogenesis of chronic obstructive pulmonary disease (COPD), with sputum and bronchoalveolar lavage neutrophil counts broadly correlating with disease severity. The mechanisms responsible for neutrophil accumulation are poorly understood, but they could involve increased influx and/or survival of these cells. OBJECTIVES: To investigate whether neutrophil chemotactic responsiveness and/or chemotactic activity in airway secretions are increased in subjects with COPD. METHODS: Chemotaxis experiments were performed using induced sputum supernatants from subjects with and without COPD as a source of chemotactic activity, and neutrophils from healthy donors as responder cells. In addition, chemotactic responses to N-formyl-Met-Leu-Phe (fMLP) and interleukin-8 (IL-8/CXCL8) were studied using neutrophils from healthy subjects and subjects with COPD. MEASUREMENTS AND MAIN RESULTS: As reported in the literature, sputum neutrophil counts were significantly increased in subjects with COPD compared with healthy subjects. However, this was associated with reduced chemotactic activity in sputum in COPD, as judged by reduced chemotaxis to the fluid phase of sputum from subjects with COPD compared with healthy subjects. Furthermore, whereas neutrophils from subjects with stage I COPD had normal responses to fMLP and IL-8, subjects with more severe stage II-IV COPD showed reduced levels of spontaneous migration and chemotaxis to fMLP and IL-8. CONCLUSIONS: Neither increased chemotactic activity in the airways nor increased chemotactic responsiveness of neutrophils explains the increased number of these cells in subjects with stable COPD. The implications of the observed reduction in neutrophil chemotactic activity remain to be established.     

Identification and characterization of a unique subpopulation (CALLA/CD10/negative) of human neutrophils manifesting a heightened chemotactic response to activated complement

We have previously demonstrated that human neutrophils synthesize the common acute lymphoblastic leukemia antigen (CALLA/CD10). To determine whether CALLA/CD10-positive and -negative neutrophils have similar or distinct functional attributes, we sorted normal peripheral blood neutrophils for CALLA/CD10 expression and compared their chemotactic ability. Surprisingly, the low-frequency (approximately 5%), CALLA/CD10- negative neutrophils displayed a dramatically heightened chemotactic response to activated complement (C') that was (a) specific for C', (b) not observed with other minor subpopulations of neutrophils, (c) not due to previous activation in vivo or in vitro, and (d) apparently not due to an increase in C5a receptors. These results underscore the concept of neutrophil heterogeneity and prompt the hypothesis that CALLA/CD10-negative neutrophils may participate in an inflammatory response to trauma involving complement activation.     

Role of the neutrophil in oral disease: receptor deficiency in leukocytes from patients with juvenile periodontitis

In many diseases in which cellular abnormalities of neutrophil locomotion are found, patients have oral complications. Localized juvenile periodontitis (LJP) is used as an example of a severe periodontal disease that is related to compromised neutrophil function. Studies of chemotaxis and binding of LJP neutrophils in response to chemotactic factors N-formylmethionylleucylphenylalanine (FMLP), a structural analogue of a bacterial product, and complement fragment C5a were carried out to identify the molecular basis of the compromised neutrophil function. The rate of chemotaxis in LJP neutrophils was significantly lower than that of control neutrophils, and LJP neutrophils demonstrated fewer binding sites for these chemotactic factors than did normal neutrophils. The respective numbers of binding sites for FMLP on LJP neutrophils and normal neutrophils were 9,200 and 20,000 and for C5a were 133,000 and 218,000. However, for both chemotactic substances, the dissociation constants for LJP and normal neutrophils were similar. The expression of FMLP receptors was altered in LJP neutrophils, but no modulation abnormality was noted for the C5a receptor.     

Effect of ascorbate on abnormal neutrophil, platelet and lymphocytic function in a patient with the Chediak-Higashi syndrome

A diminished chemotactic response was observed with the neutrophils of a patient with the Chediak-Higashi syndrome, who was not in the accelerated phase of the disease. An abnormally low release of myeloperoxidase from these cells during phagocytosis was also noted; this resulted in a decreased iodination capacity and probably also caused the defect in the intracellular killing of bacteria by the neutrophils. The level of cyclic AMP in these cells was elevated, but decreased after treatment with ascorbate either in vitro or in vivo. During ascorbate therapy, the bactericidal activity of the neutrophils normalized, whereas the chemotactic response remained low. Nevertheless, the patient had significantly less infections during ascorbate therapy. The bleeding tendency, due to a storage-pool disorder of the Chediak-Higashi platelets, was unaffected by treatment with ascorbate. The patient's lymphocytes did not display any activity in antibody-dependent lymphocytotoxicity. This defect was not affected by treatment with ascorbate either.     

Neutrophil Accumulation in Vivo following the Administration of Chemotactic Factors

Intravenously administered ⁵¹Cr-labelled homologous neutrophils accumulated at guinea-pig skin sites prepared by intradermal injection of factors derived from complement-activated serum, possessing in vitro chemotactic activity. There was a strong correlation between the in vitro potency of Sephadex G-100 fractions of the activated serum and their ability to evoke neutrophil accumulation in vivo. These experiments suggest that agents which are chemotactic for neutrophils in vitro also induce the localization of this cell in vivo.     

Effects of benoxaprofen on human neutrophil function

In vitro assays of human neutrophil phagocytosis, adherence, enzyme release, and response to chemotactic factors were utilized to determine the effects of benoxaprofen on these cells. At concentrations of 30 and 300 micrograms/ml benoxaprofen partially inhibited phagocytosis without affecting adherence. High concentrations (300 micrograms/ml) of benoxaprofen, but not aspirin or gold, enhanced the release of the granular enzymes beta-glucuronidase and lysozyme, but not the release of the cytoplasmic enzyme lactic dehydrogenase from both resting and phagocytosing neutrophils. High concentrations (300 micrograms/ml) of benoxaprofen also inhibited the response of neutrophils to chemotactic factors as determined by both the leading front and leukotactic index methods. These unique in vitro effects of this antiinflammatory agent are compatible with recent clinical reports concerning its efficacy and toxicity.     

Patterns of leukocyte chemotaxis to bile after liver transplantation

The role of chemotactic factors in the recruitment of leukocytes to human liver allografts was assessed by studying the effect of bile from transplant recipients on the chemotaxis of cells from normal subjects. Bile samples taken 2-3 days before clinical rejection were more chemotactic for lymphocytes than samples taken during rejection (p less than 0.01), during stable graft function (p less than 0.001), and from nontransplant patients (p less than 0.007). During clinical rejection there was an increase in bile chemotactic activity for both monocytes and neutrophils compared with samples from stable patients (monocytes: p less than 0.001; neutrophils: p less than 0.001) and nontransplant patients (monocytes: p less than 0.001; neutrophils: p less than 0.001). In serial studies chemotactic activity for lymphocytes reached a peak 1-3 days before the onset of clinical rejection, whereas maximum chemotactic activity for monocytes and neutrophils occurred at the time of rejection, when lymphocyte chemotaxis was decreasing. These results suggest that chemotaxis may be important in the recruitment of inflammatory cells to liver allografts and that chemotactic factors for lymphocytes, which appear in bile before clinical rejection, may be critical in the pathogenesis of rejection.     

Chemotactic factors released from hepatocytes exposed to acetaminophen

To clarify the mechanism of neutrophil infiltration in the liver of acetaminophen-induced hepatic injury, chemotactic factor released from hepatocytes exposed to acetaminophen has been investigated. Hepatocytes exposed to acetaminophen release nondialyzable chemotactic factor, although actaminophen in itself inhibits chemotaxis of neutrophils. Chemotactic activity of the nondialyzable chemotactic factor was reduced after treatment with heat (56°C, 30 min) or trypsin. Chemotactic activity was demonstrated at the molecular weights of around 25 and 55 kDa. Chemotactic activity of the conditioned medium was not significantly reduced in the presence of antibody against rat KC/gro protein (interleukin-8-related cytokine in rodent). Chemotactic activity of a 25-kDa factor was reduced by the antibody against KC/gro protein, but that of a 55-kDa factor was not reduced. Immunoblot analysis revealed that the peptide reacted with antibody against rat KC/gro protein was demonstrated at a molecular weight of around 20–25 kDa, but not at around 55kDa, when the conditioned medium of acetaminophen-treated hepatocytes was electrophoresed. These results suggest that hepatocytes exposed to acetaminophen release two types of chemotactic factors for neutrophils and that a major part of the chemotactic factor could be different from a member of interleukin-8 family.     

Chemoattractant-induced changes in surface expression and redistribution of a functional ligand for P-selectin on neutrophils

Adhesion between platelets and neutrophils is mediated through the interaction of P-selectin on activated platelets with a carbohydrate- containing structure on neutrophils, and occurs under both static and shear conditions. Recent studies using flow chambers have shown that neutrophils become activated after binding to surface-adherent platelets expressing P-selectin. The objective of the present study was to investigate the effect of such activation on the interactions of platelet P-selectin with its ligand on neutrophils. Flow cytometric analyses using P-selectin chimeras revealed that activation induced a rapid and marked reduction in chimera binding, with levels of binding decreased by 71% after 15 minutes of stimulation with the chemotactic agent, FMLP. Using a visual assay of platelet-neutrophil rosetting, we showed that the P-selectin ligand was translocated and clustered at the uropod of neutrophils following the shape changes and polarization induced by chemotactic stimulation. Activated neutrophils bound to surface-adherent platelets also displayed the clustering of P-selectin ligand at the uropod, and these neutrophils detached from the platelets when a shear stress (2 dynes/cm2) was applied through the adhesion chamber. These results indicate that chemotactic stimulation of neutrophils induces changes in the surface expression and distribution of a biologically relevant ligand for P-selectin, and that these changes might influence the adhesive interactions occurring between neutrophils and activated platelets.     

The mechanism of neutrophil-induced lung injury--autoregulation of superoxide generation in cells]

Neutrophils which are isolated in the lung adhere to endothelial cells due to chemotactic factors, and release various proteases, superoxide anions and prostanoids in inflammatory processes. However, this host defense mechanism can cause tissue damage. Excessive adherent neutrophils are not always derived from lung injury. We have previously reported that an increase in cell density in human neutrophils attenuates superoxide anion generation by cell to cell communication (autoregulation). Autoregulation of the protein kinase c stimulator, phorbol myristate acetate, and also of the cell membrane receptor stimulator, N-formyl-methionyl-leucyl-phenyl-alanine was observed. The autoregulation was not related to the presence of extracellular Ca2+ not to a change of [Ca2+]i induced by stimulants. These results suggest that neutrophils accumulated in the lung tissue have a built-in defense mechanism induced by membrane to membrane contact of neutrophils, which protects tissues from an excessive inflammatory response.