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 withN-formyl-1-methionyl-1-leucyl-1-phenylalanine (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.     

Identification of a low molecular weight, macrophage-derived chemotactic factor for fibroblasts

Fibroblasts migrate in vitro in response to a variety of chemoattractants including cellular, humoral, and connective tissue components. This report describes a low molecular weight, macrophage-derived chemotactic substance for fibroblasts (MDCF-F). Thioglycolate-elicited peritoneal exudate cells were collected from mice 4 days postinjection. Platelets were removed and glass-adherent macrophages were selected in the absence of fetal calf serum. The macrophage culture medium was then tested for chemotactic activity for 3T3 mouse fibroblasts using a modified Boyden chamber. Chemotactic activity was observed in the macrophage medium after a 48-hr culture period and increased during the next 24 hr. The activity was sensitive to heat treatment at 56 degrees C for 30 min, could be effectively absorbed out of the culture medium by preincubation with 3T3 fibroblasts, and was not recognized by human fibroblasts. Molecular-sieve chromatography indicated a molecular weight below 10,000. This low molecular weight, macrophage-derived chemoattractant for fibroblasts may represent a rapidly diffusable substance involved in the recruitment of fibroblasts to sites of inflammation.     

The initiation of the neutrophil chemotactic response in filters.

The fluid gradient chamber was used to study the migration of human neutrophils in preformed gradients of N-formyl-methionyl-leucyl-phenylalanine. After 60 min, the chemotactic gradient was replaced by a new one of identical steepness but opposite direction. In a control group of experiments, the first gradient was retained. Migration was assessed from cell distributions in filter sandwiches after 30, 60, and 90 min. Filters obtained after 5, 15, and 30 min of migration were stained with fluorescent phalloidin for microscopic evaluations of cell polarity. At 30 min most cells had polarized in vertical directions and invaded the filters. The distance of chemotactic migration was similar during the second and the third 30-min periods (although the direction of migration was reversed in the new gradient) and significantly greater than during the first 30 min. In conclusion, the initial slow response to the chemotactic gradient represents an adaptation of the cells that later respond promptly to changes in gradient direction.     

Convergent alteration of granulopoiesis,chemotactic activity,and neutrophil apoptosis during mouse selection for high acute inflammatory response

Neutrophil homeostasis was investigated in two mouse lines, AIRmax and AIRmin, genetically selected for high or low acute inflammatory response (AIR) and compared with unselected BALB/c mice. Mature neutrophil phenotype and functions appeared similar in the three mouse lines. However, an unprecedented phenotype was revealed in AIRmax animals characterized by a high neutrophil production in bone marrow (BM), a high number of neutrophils in blood, a high concentration of chemotactic agents in acrylamide-induced inflammatory exudates, and an increased resistance of locally infiltrated neutrophils to spontaneous apoptosis. In vitro, BM production of neutrophils and eosinophils was accompanied by an unusual high up-regulation of cytokine receptors as assessed by antibodies to CD131, which bind the common beta chain of receptors to interleukin (IL)-3, IL-5, and granulocyte macrophage-colony stimulating factor. An accelerated neutrophil maturation was also observed in response to all-trans retinoic acid. Several candidate genes can be proposed to explain this phenotype. Yet, more importantly, the results underline that genetic selection, based on the degree of AIR and starting from a founding population resulting from the intercross of eight inbred mouse lines, which display a continuous range of inflammatory responses, can lead to the convergent selection of alleles affecting neutrophil homeostasis. Similar gene combinations may occur in the human with important consequences in the susceptibility to inflammatory or infectious diseases and cancer.     

A superoxide-activated chemotactic factor and its role in the inflammatory process

Superoxide dismutase or derivatives thereof have been shown to be potent anti-inflammatory agents in several models of induced inflammation. In all cases, the anti-inflammatory effects included a marked suppression of the accumulation of inflammatory cells at the site of the potential lesion, suggesting a role for the superoxide radical in the chemotaxis process. The exposure of normal plasma to a source of superoxide in vitro resulted in the formation of a powerful chemotactic factor for human neutrophils. The factor is activated by reacting specifically with superoxide, and was found to consist of a complex of serum albumin and an unidentified lipid. The complex may be resolved and reconstituted. Both components are required for expression of biologic activity. The major mechanism for the anti-inflammatory activity of superoxide dismutase appears to be the prevention of the formation of this plasma-derived superoxide-dependent chemotactic factor.     

哮喘中性粒细胞趋化因子性质的初步研究

测定哮喘发作和缓解期患者的中性粒细胞趋化因子活性,对中性粒细胞趋化因子(NCF)的理化性质进行初步研究。发现哮喘发作期NCA显著升高,RNCF对热较稳定,对蛋白酶敏感。脂溶性NCF活性仅占总NCF活性的4.2%。Sephadex G-200层析表明有两种NCF,一种分子量大于500000,另一种分子量约10000,前者是哮喘发作时的主要NCF。     

Isolation of Schistosoma japonicum egg-derived neutrophil stimulating factor: its role on eosinophil chemotactic factor release from neutrophils

Neutrophil stimulating factor (NSF), which can stimulate polymorphonuclear neutrophils (PMNs) to release eosinophil chemotactic factor (ECF), was isolated from Schistosoma japonicum soluble egg extract (SEA). The release of ECF from PMNs began as early as 5 min after stimulation and reached a peak at 40 min, and was dependent on the concentration of SEA. After Sephadex G150 gel filtration, toluene extraction, Sephadex G25 gel filtration and Dowex 1 X 8 anion exchange chromatography, NSF was identified as a hydrophilic and negatively charged component with a molecular weight of about 1,000 daltons. It was heat-stable at 100 degrees C for 60 min. NSF was easily separable from SEA-derived neutrophil chemotactic factor or from SEA-derived ECF. PMNs are suggested to be one of the sources of ECF in the eosinophil accumulation of granulomatous lesions around the deposited eggs in schistosomiasis japonica.     

CD28 signaling in neutrophil induces T-cell chemotactic factor(s) modulating T-cell response

We previously reported that human peripheral blood neutrophils express CD28 and interact with macrophage B7 to generate CD28 signaling through PI-3 kinase. Here, we demonstrate that crosslinking of CD28 on neutrophils results in the release of IFN-gamma, which restricts amastigote growth and modulates CD4+ T cells cytokine secretion. CD28 crosslinking also induces a T-cell chemotactic factor (TCF) that induces chemotactic migration of CD4+ T cells. Based on our previous and the current set of data, we propose an operational model explaining how neutrophils are involved in Leishmania infection and how the reported effect of neutrophils on the control of infection is mediated by alteration of T-cell function.     

Studies of neutrophil chemotactic factor of anaphylaxis in metabisulfite sensitivity

The mechanism(s) of adverse reactions to sulfites remains unclear. To determine whether mast cell degranulation is involved in sulfite sensitivity, we performed single-blind, placebo-controlled oral aqueous challenges with potassium metabisulfite in 13 patients with histories suggestive of sulfite sensitivity. Ten patients were also skin tested with potassium metabisulfite at 10 mg/mL and all had negative reactions. Serum samples were obtained from all the patients before the challenge and for 180 minutes after the challenge. The samples were tested for the presence of neutrophil chemotactic factor of anaphylaxis, using both a 51chromium microchamber chemotaxis assay and a leukocyte polarization technique. Six of 13 patients had positive challenges as defined by a fall equal to or greater than 20% in their forced expiratory volume in one second. No significant increase in neutrophil chemotactic factor activity was detected in the postchallenge serum samples from patients who experienced positive or negative challenges. We conclude that sensitivity to aqueous metabisulfite is not associated with mast cell degranulation in metabisulfite skin test-negative patients.     

Production of a neutrophil chemotactic factor by endotoxin stimulated alveolar macrophages in vitro

Alveolar macrophages (AM) isolated from normal guinea-pigs and from those chronically exposed to endotoxin (LPS) were cultured in the presence of various concentrations of LPS (from 0.5 ng to 5 micrograms/ml). The presence of a neutrophil chemotactic factor (NCF) in culture supernatants was tested in migrations chambers. Contamination of all reagents has been tested using LAL test (Limulus Amoebocyte Lysate). The results indicate a production of NCF with low LPS concentrations (0.5 and 5 ng/ml) within the first 6 h of incubation: when larger doses are used the response decreases and a significant inhibition is observed with 5 micrograms LPS/ml (P less than or equal to 0.05). When contaminated medium was used, all responses observed were three times higher than with LPS-free medium (P less than or equal to 0.01). However, the response pattern was the same. AM from chronically exposed animals exhibit the same response patterns: the magnitude of NCF production was higher than with normal AM but not significantly. The data suggests that initial conditions of AM in vitro or in vivo with reference to LPS contamination have to be determined as they are of importance when AM NCF production has to be tested.     

Recombinant gamma interferon causes neutrophil migration mediated by the release of a macrophage neutrophil chemotactic factor

A dose-dependent neutrophil migration was observed following the injection of purified (Hu IFN-gamma) or recombinant (rIFN-gamma) human gamma interferon into rat peritoneal cavities. This finding contrasts with their inability to cause chemotaxis in vitro in the Boyden chamber. Neutrophil migration into peritoneal cavities and subcutaneous air pouches induced by both preparations of interferon was abolished by pretreatment of the animals with dexamethasone. IFN-gamma-induced neutrophil migration was enhanced when the macrophage population of the peritoneal cavities was increased by previous injection of thioglycollate and reduced by peritoneal lavage. Macrophage monolayers pretreated either with rIFN-gamma or with lipopolysaccharide from E. coli release into the supernatant a factor that stimulates neutrophil recruitment in animals treated with dexamethasone. Dexamethasone blocked this release but did not affect the neutrophil recruitment induced by this factor. These results suggest that IFN-gamma-induced neutrophil migration in vivo may be mediated by the release from resident macrophages of a neutrophil chemotactic factor and that dexamethasone blockade of neutrophil recruitment by IFN-gamma is due to inhibition of the release of this factor.     

Functional properties of a novel neutrophil chemotactic factor derived from human monocytes

Neutrophilic granulocytes (PMN) are attracted to sites of inflammation by chemotactic factors, the most potent of which are the complement split product C5a, the leukotriene B4 and the bacterial chemotactic factor-related tripeptide formyl-methionyl-leucyl-phenylalanine (FMLP). In addition to inducing directed migration, these agents increase the adherence of PMN to synthetic surfaces and endothelial cells; some stimulate an oxidative burst and the production of reactive oxygen derivatives, and they may be involved in the release of granule constituents. Here, we describe studies on the activities stimulated by a novel monocyte-derived chemotaxin (MOC). Human MOC attracted human PMN, but not monocytes or eosinophils. Like all chemotactic agents, it increased the adherence of PMN on nylon fibers. In contrast to other chemotactic factors it did not stimulate the release of superoxide anion regardless whether the cells were in suspension or adherent on nylon fibers. There was no release of the primary granule enzyme glucosaminidase or the secondary granule component vitamin B12-binding protein in the absence or presence of cytochalasin B. The results suggest that MOC is a unique chemotactic agent with properties different from the most potent chemotactic factors C5a, LTB4 and FMLP. The delayed release from macrophages suggests its involvement in protracted and chronic inflammatory reactions.     

Role of plasma histamine and neutrophil chemotactic factor in exercise-induced asthma

The mechanism of exercise-induced asthma (EIA) is still controversial, although the role of chemical mediator is strongly suspected. In the present study, 50 asthmatic patients were observed after exercise on bicycle ergometer during dry air breathing, and changes of plasma histamine and neutrophil chemotactic factor (NCF) were measured and effect of anti-allergic drugs was examined. 31 patients developed postexertional bronchoconstriction and their % reduction of FEV1 after exercise correlated significantly with the degree of airway hyperresponsiveness to inhaled methacholine determined by Astograph. Plasma histamine levels were examined in 20 EIA positive cases and 13 EIA negative cases, but no significant changes were observed between pre- and post-histamine levels in either group. On the other hand, NCF was elevated significantly after exercise in both EIA positive and negative cases, but postexertional NCF levels were significantly higher in EIA positive than in EIA negative cases. The relationship between % increase of NCF and the % reduction of FEV1 after exercise was significant (r = 0.472, p less than 0.05). DSCG and Azelastine protected the development of EIA in 14 out on 19 cases and 7 out of 12 cases, respectively. Pretreatment with DSCG significantly reduced the increase of NCF after exercise. These results indicates that one of the chemical mediator, NCF, may play an important role in producing postexertional bronchoconstriction in asthmatic patients.     

Exercise-induced release of histamine and neutrophil chemotactic factor in atopic asthmatics

Concentrations of plasma histamine and serum neutrophil chemotactic factor (NCF) were measured in seven atopic asthmatics who developed exercise-induced asthma (EIA) after a treadmill task. The results were compared with those obtained after inhalation of specific antigen or methacholine. Plasma histamine concentrations were measured with a novel double-isotope radiometric assay, and NCF was identified by its elution in the void volume fractions of Sephadex G-200 and as a single peak of activity at approximately 0.20 molar NaCl after anion exchange chromatography on diethylaminoethyl-Sephacel (pH 7.8). After exercise or antigen challenge, the time courses of appearance of both mediators were virtually identical and accompanied the increase in airways obstruction. There was a statistically significant correlation between the concentrations of histamine or NCF and the magnitude of airflow obstruction after exercise and antigen challenge. This suggested that there may be a direct association between mediator release and EIA or antigen-induced bronchoconstriction. In contrast, there were no significant elevations in circulating histamine and NCF after inhalation of methacholine, at concentrations giving a fall in FEV1 comparable to that induced by exercise or antigen. The prior administration of cromolyn to three asthmatics inhibited both their EIA and the release of histamine and NCF. When four asthmatics were exercised for periods of 1, 3, and 6 min, the release of NCF and fall in peak expiratory flow rate were directly related to the duration of the exercise. The rise of NCF activity in subjects with EIA was fivefold greater than that observed in asthmatics who did not experience airways obstruction when subjected to the same exercise task. These results provide further evidence that mediators of hypersensitivity are released during EIA.     

A low molecular weight factor is a significant mediator of non-opsonic neutrophil activation by Helicobacter pylori

Helicobacter pylori is believed to trigger neutrophil activation through several factors, including the H. pylori neutrophil-activating protein (HpNAP). The aim of this study was to characterise the factors within H. pylori cell-free extracts that stimulate neutrophil activation. Neutrophil activation was found to be dose-dependent and exhibited considerable variation between different clinical isolates. Activity was attributable to more than one protein factor. A low mol. wt fraction of <3 kDa was found to contribute a large proportion of the neutrophil-stimulating activity within H. pylori cell-free extract. Additional activity was provided by a high mol. wt fraction, possibly representing HpNAP. An inhibition ELISA and neutralisation experiments failed to identify or exclude formyl methionyl leucyl phenylalanine as the active factor within the low mol. wt fraction. The importance of the putative, low mol. wt neutrophil-activating factor may have been overlooked by those studies that have used concentrated H. pylori extracts.     

Production of a neutrophil chemotactic factor by endotoxin stimulated alveolar macrophages in vitro.

Alveolar macrophages (AM) isolated from normal guinea-pigs and from those chronically exposed to endotoxin (LPS) were cultured in the presence of various concentrations of LPS (from 0.5 ng to 5 micrograms/ml). The presence of a neutrophil chemotactic factor (NCF) in culture supernatants was tested in migrations chambers. Contamination of all reagents has been tested using LAL test (Limulus Amoebocyte Lysate). The results indicate a production of NCF with low LPS concentrations (0.5 and 5 ng/ml) within the first 6 h of incubation: when larger doses are used the response decreases and a significant inhibition is observed with 5 micrograms LPS/ml (P less than or equal to 0.05). When contaminated medium was used, all responses observed were three times higher than with LPS-free medium (P less than or equal to 0.01). However, the response pattern was the same. AM from chronically exposed animals exhibit the same response patterns: the magnitude of NCF production was higher than with normal AM but not significantly. The data suggests that initial conditions of AM in vitro or in vivo with reference to LPS contamination have to be determined as they are of importance when AM NCF production has to be tested.     

Low molecular weight eosinophil chemotactic factor (ECF) in the serum of murine schistosomiasis japonica

Eosinophil chemotactic activity was detected in the serum obtained at an acute stage of murine schistosomiasis japonica. Gel filtration of the dialyzable fraction of the serum on Sephadex G25 showed that the chemotactic component had an apparent molecular weight of less than 1,000. It was stable to heating, but was sensitive to pronase or carboxypeptidase A digestions, indicating its peptide nature. Eosinophil chemotactic activity of the dialysate of the serum from mast cell-deficient W/Wv mice infected with Schistosoma japonicum was far less than that from normal littermate +/+ mice, although the titers of specific IgE antibody to soluble egg antigen in the serum measured by passive cutaneous anaphylaxis was comparable between them. These results suggest that at least some part of low molecular weight ECF in the circulation seems to be a ECF-A derived from mast cells. Possible biological significance of circulating ECF in schistosomiasis has been discussed.     

Spiracular indices in Anopheles stephensi: a taxonomic tool to identify ecological variants

Thoracic spiracle length and its index was examined for their ability to discriminate two ecological variants, type form and mysorensis, of Anopheles stephensi in the adult stage. The type form is exclusively domestic in all seasons, whereas the mysorensis variant occupies the outdoor niche during monsoon and postmonsoon seasons, with spillover into domestic sites during summer ecological stress periods. A statistically significant co-relation was established between the ridge count of the egg and two adult measurements, the thoracic spiracle length, and the spiracular index. In An. stephensi type form, average spiracle length was 0.11-0.12 mm and average spiracular index was 8.09-9.23, whereas in mysorensis, the corresponding figures were 0.09-0.10 mm and 6.82-7.60. These parameters showed consistent variations in population of mosquitoes that emerged during monsoon and summer season. The thoracic lengths in both variants remained constant, and only spiracular lengths showed fluctuations in three seasonal populations. These measures provide discrimination of adult variants--identifications that are essential in malaria control programs.