Antigen-induced eosinophil chemotactic factor (ECF) release by human leukocytes

A complement-independent eosinophil chemotactic factor (ECF) is described which is released from peripheral leukocytes of allergic and normal human volunteers after antigen stimulation and after exposure to anti-IgE. Dose response and time-release curves for ECF and histamine run closely parallel in this system. Histamine by itself is shown to have no effect on chemotaxis at the concentrations present in antigen-induced release, but is inhibitory at very high concentrations. Evidence suggests that the ECF released from human leukocytes is derived from basophils and is similar, or identical, to the ECF released from mast cells.This work was supported by Grants 07290 and 08270 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland.Publication No. 197, O'Neill Research Laboratories.Dr. Czarnetzki is recipient of the Stetler Research Fund for Women Physicians.     

Generation of the eosinophil chemotactic factor (ECF) from various cell types by melittin

The peptide melittin, the main constituent of bee venom is a potent stimulus for the generation of an eosinophil chemotactic factor (ECF) from human polymorphonuclear neutrophils, rat mast cells and rat peritoneal cells depleted in mast cells. Optimal EFC induction required a sublytic activation of the cells. With each cell type the kinetics of ECF generation were similar in that after an early rise in activity a steep fall off occurred at later times of incubation suggesting a mechanism of inactivation. The induction of ECF by melittin is increased in the presence of calcium. The polar portion of the melittin molecule (aminoacids 20–26) is responsible for the generation of the chemotactic activity. Other peptides of honey bee venom such as the mast cell degranulating peptide (MCD) or apamine do not initiate ECF release. It appears that melittin leads to ECF induction via the phospholipase A₂-arachidonic acid dependent pathway of cell activation. Our data suggests that the lipid mediator ECF can be obtained from phagocytes and mast cells thus indicating the interdependence of inflammatory reactions.     

Modulation of eosinophil chemotaxis and eosinophil chemotactic factor release by concanavalin A

Concanavalin A (Con A) is not a chemotactic factor for guinea pig eosinophil leukocytes. It does, however, modulate the response of cells to neutrophil-derived eosinophil chemotactic factor (ECF) or to C5a. This effect occurs by a direct interaction of Con A with the target cell and is reversible by appropriate doses of alpha-methyl-D-mannoside (alpha-MM). The inhibition of chemotaxis at high doses of Con A is due to agglutination of the cells on top of the filter. The mechanism of enhancement at low doses is not understood, but several possible explanations are explored. Careful definition of the effect of Con A on eosinophil chemotaxis makes it possible to exclude an effect of Con A on ECF secretion by neutrophils.     

Modulation of the immunological release of the eosinophil chemotactic factor of anaphylaxis from human lung

The antigen-induced release of preformed eosinophil chemotactic factor of anaphylaxis (ECF-A) from passively sensitized human lung tissue, requires divalent cations, an intact glycolytic pathway and a diisopropyl fluorophosphate (DFP) inhibitable esterase, and appears to be modulated by prototype adrenergic and cholinergic receptors. Increases in intracellular cyclic AMP inhibit the release of ECF-A as shown not only by the effect of exogenous dibutyryl cyclic AMP but also by the kinetic relationship between increases in tissue cyclic AMP and inhibition of the release of ECF-A, histamine and slow reacting substance of anaphylaxis from lung tissue interacted with cholera toxin. Cholinergic enhancement of the antigen-induced release of ECF-A was not associated with measurable alterations in the intracellular content of cyclic AMP and is attributed to elevation of intracellular cyclic GMP, as the introduction of 8-bromo cyclic GMP enhances ECF-A release.     

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.     

Subcellular localization of the eosinophil chemotactic factor (ECF) and its inactivator in human polymorphonuclear leucocytes (PMN).

An eosinophil chemotactic factor (ECF) of low MW can be released from human polymorphonuclear leucocytes (PMN) on stimulation with the Ca-ionophore, arachidonic acid and during phagocytosis. After a rapid rise of ECF activity in the supernatant a steep fall of in its activity occurred at the later times of secretion suggesting a mechanism of ECF inactivation. ECF obtained at the later times of secretion represents a stable biological activity and does not decrease on further incubation. In addition, intact PMN and ECF combined do not lead to its inactivation, while incubation of homogenized PMN with ECF decreased its activity. These data suggest the presence of an inactivator for ECF within human PMN. The purpose of the study was to localize ECF and its inactivator within human PMN. After cell disruption, differential and equilibrium gradient centrifugation, subcellular components of human PMN can be obtained which reveal eosinophilotactic (ECF) or ECF-inactivating activity. ECF activity can be recovered (in a structurally bound state) from the microsomal fraction of unstimulated and stimulated PMNs, while another portion is obtainable as a soluble, low mol. wt ECF. The PMN-derived ECF inactivator can be recovered from the peroxidase positive (azurophilic) granules and has a mol. wt of 60,000 and less. We suggest that low mol. wt ECF is derived from the plasma membrane of PMN which can be inactivated by components of the azurophilic granules. The mechanism of inactivation is still unresolved.     

Generation and release of eosinophil chemotactic factor from human polymorphonuclear neutrophils by arachidonic acid.

This study describes the generation and release of an eosinophil chemotactic factor from human polymorphonuclear neutrophils, rat basophilic leukemia cells, and from a lymphocyte monocyte basophil suspension by arachidonic acid (AA). The eosinophil chemotactic factor (ECF) is highly specific for eosinophils and resembles the ECF activity obtained from human polymorphonuclear neutrophils after stimulation with the Ca ionophore or during phagocytosis. In this regard, AA-induced ECF represents a biological activity distinct from oxidized AA and its conversion products. AA may therefore have a dual function: it represents an important mechanism of cell activation; as AA is converted into prostaglandins, it appears likely that they exert a modulatory and a suppressive role on biological functions, such as chemotaxis and phagocytosis.     

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.     

Production of an eosinophil chemotactic lymphokine by a monocyte-derived factor from patients with hypereosinophilia

Peripheral OKT4-positive T lymphocytes from patients with hypereosinophilia spontaneously and selectively produced an eosinophil chemotactic factor (ECF) with chemokinetic activity. The molecular weight of the ECF was about 45,000 to 70,000. A possible mechanism of its spontaneous production by T lymphocytes was analyzed. Culture supernatants of blood monocytes from the patients showed little or no ECF activity, but they had a potency to induce the ECF production from T lymphocytes from normal donors when the cells were stimulated by the supernatants, which suggests that a monocyte-derived soluble factor (MDF) stimulated T lymphocytes to produce an ECF resembling this spontaneously produced ECF from the patients. MDF seemed to be a synthesized protein by the cells. Gel filtration indicated that molecular weight of MDF ranged between 70,000 and 100,000. MDF activity was stable at 56 degrees C for 30 min but more, supernatants of stimulated monocytes by lipopolysaccharide or silica particles failed to show ECF-producing activity, whereas they showed evident lymphocyte-activation activity. Neither recombinant IL-1 nor IL-2 had ECF and ECF-producing activity. From the present experiments, it was suggested that MDF was at least partly involved in the induction of ECF production by OKT4-positive T lymphocytes in patients with hypereosinophilia.     

Inactivation of polymorphonuclear-neutrophil derived eosinophil chemotactic factor by human serum.

A low-molecular-weight eosinophil chemotactic factor (ECF) can be released from human neutrophils on stimulation with the calcium ionophore (A 23187) and during phagocytosis. The presence of human serum suppresses the chemotaxis of eosinophils by ECF significantly. The inhibition is not cell-directed but affects the ECF either by binding or enzymatic cleavage. The inactivators belong to a heterogeneous class of serum components in the 19s, 7s and 4s molecular weight range. Highly purified human IgM and IgA, but neither IgG nor human serum albumin inhibit eosinophil chemotaxis.     

Neutrophil chemotactic factor of anaphylaxis (NCF-A) release in aspirin-induced asthma

Neutrophil chemotactic activity (NCA) following oral challenge with aspirin (ASA) was determined in ASA-intolerant asthmatic subjects, and in ASA-tolerant asthmatic and normal subjects. There was a statistically significant fall in FEV₁ and a rise in NCA (P <0.01) following challenge in the ASA-sensitive subjects compared with that of the ASA-tolerant subjects and normal controls. No significant difference was observed between the latter two groups. The chemotactic factor identified in this study had a molecular weight greater than 150 000 which is consistent with NCF-A (neutrophil chemotactic factor of anaphylaxis). The ASA-induced fall in FEV₁ and rise in NCA was further studied in three of the ASA-intolerant asthmatic subjects, with and without pretreatment with inhaled sodium cromoglycate. In these subjects, the drug inhibited both the oral ASA-induced bronchoconstriction and the increase in neutrophil chemotactic activity. These results suggest that ASA-induced asthma involves mediator release from mast cells, as shown by the increase in NCA following ASA challenge and the protective effect of sodium cromoglycate which is considered to inhibit mast-cell degranulation.     

Cold urticaria: release into the circulation of histamine and eosinophil chemotactic factor of anaphylaxis during cold challenge.

Patients with idiopathic acquired cold-induced urticaria were evaluated for the release of the preformed mast-cell mediators of immediate-type hypersensitivity during a study in which one arm was immersed in ice water while the other arm remained as a control. Blood specimens were obtained from each arm serially over a one-hour interval, and serum speciments were assessed for histamine, eosinophil chemotactic factor of anaphylaxis, and complement components. Levels of histamine and eosinophil chemotactic factor rose in the arm subjected to cold immersion for three minutes, with peak values occurring between two and five minutes and returning to base line by 30 minutes. No changes occurred in the control arm or in the immersed arm of normal subjects. Assessment of the classical and alternative complement pathways showed no abnormalities. This initial observation of release of eosinophil chemotactic factor of anaphylaxis in vivo along with histamine assigns the mast cell a central role in cold urticaria.     

Histamine release from mast cells of various species induced by histamine releasing factor from human lymphocytes

The aim of our work was to investigate the effect of histamine releasing factor (HRF), producedin vitro by lymphocytes obtained from atopic and non-atopic asthmatics, on mast cells of various species (mouse—peritoneal mast cells, hamster and rat—peritoneal and pleural mast cells, guinea-pig — mesenteric and pulmonary mast cells). We found that human HRF is able to release histamine from the examined mast cell populations in a dose-dependent fashion. Mast cells from various species differed in their susceptibility to the action of HRF; rat pleural and guinea-pig mesenteric and pulmonary mast cells were the most susceptible, while mouse and hamster peritoneal mast cells —the least susceptible. The presence of 50% D₂O in the medium significantly increased HRF-induced histamine release from rat mast cells, while the addition of phosphatidylserine did not change it. HRF-induced histamine release from guinea-pig mesenteric mast cells was not related to sensitization of these cells.We also compared histamine release from guinea-pig pulmonary and mesenteric mast cells induced by human HRF producedin vitro by lymphocytes obtained from atopic and non-atopic asthmatics. We have found that supernatant from atopic asthmatics lymphocyte cultures released significantly more histamine than supernatant from non-atopic asthmatics lymphocyte cultures.Our studies give evidence that human HRF acts across the species barrier and induces histamine release from mast cells of various species. The mechanism of HRF action on mast cells seems to be different from that of allergen.This work was supported by Polish academy of Sciences (Grant CPBP 06.01).     

Identification of an eosinophil chemotactic factor from anopheline mosquitoes as a chitinase family protein

Anopheline mosquitoes play an essential role in malaria transmission. The mosquito salivates copiously when probing for the location of a blood vessel. We found that the saliva of anopheline mosquitoes has chemotactic activity for naive eosinophils or neutrophils. The major eosinophil chemotactic component in saliva was shown to be one of the chitinase family proteins. A similar chitinase family protein was found also in the midgut of the anopheline mosquito. Production of antibodies to the chitinase family protein was generally observed in the sera of residents of a malaria endemic area. Both Plasmodium falciparum-infected and uninfected individuals had antibodies to chitinases. These results suggest that the chitinase family protein in mosquito saliva contributes to eliciting an inflammatory response of eosinophils in the host skin followed by antibody production in the host.     

Eosinophil chemotactic factor release from neutrophils induced by stimulation with Schistosoma japonicum eggs

 The neutrophil is one of the sources of eosinophil chemotactic factor (ECF) in the presence of some stimulants. In the present study we showed that guinea-pig neutrophils could release ECF upon stimulation with Schistosoma japonicum eggs. ECF release from neutrophils began as early as 5 min after the stimulation and reached a peak at 20 min. When homogenate of the eggs was separated into a water-soluble fraction as soluble egg antigen (SEA) and a water-insoluble fraction (eggshell), both preparations possessed a potent neutrophil-stimulating activity to release ECF. The ECF release was dependent on the concentration of eggshells or SEA or on the number of neutrophils. The neutrophil-stimulating activity of eggshells was stable to heat, HCl, or pronase treatment but sensitive to NaOH treatment. When the eggs or eggshells were washed with acetone or Tween-20, they lost the neutrophil-stimulating activity to release ECF, indicating that the neutrophil-stimulating factor (NSF) possesses a lipid nature. The molecular weight of NSF extracted from the eggshells was estimated to be about 1000 Da by gel chromatography on Sephadex G25. The possible role of eggshells in the formation of eosinophil-rich granulomatous lesions in schistosomiasis japonica is discussed. Received: 13 May 1996 / Accepted: 25 July 1996     

Production of eosinophil chemotactic factor by CD8+ T-cells in Toxocara canis-infected mice

Production of eosinophil chemotactic factor by T-lymphocytes (ECF-L) was examined in Toxocara canis-infected mice. When spleen cells from T. canis-infected mice were cultured in serum-free RPMI1640, ECF-L production was detectable in an antigen-specific manner. The ECF-L production peaked at day 9 post-infection and then decreased. Depletion of Thy 1.2⁺ cells or CD8⁺ cells completely abrogated ECF-L production, whereas depletion of CD4⁺ cells did not, indicating that CD8⁺ T-cells are involved in the production of ECF-L. When bone marrow eosinophils obtained from T. canis-infected mice were preincubated with ECF-L, their chemotactic reactivity to parasite-derived ECFs was enhanced, whereas that of peritoneal cavity-derived eosinophils was not. Thus, ECF-L seems to be important not only as a chemoattractant but also as an activator of the chemotactic reactivity of naive eosinophils to the parasite-derived ECF in T. canis infection. Received: 4 June 1997 / Accepted: 11 July 1997