Eosinophil-stimulating Activity Research Articles

Nonpathogenic, Environmental Fungi Induce Activation and Degranulation of Human Eosinophils

Eosinophils and their products are probably important in the pathophysiology of allergic diseases, such as bronchial asthma, and in host immunity to certain organisms. An association between environmental fungal exposure and asthma has been long recognized clinically. Although products of microorganisms (e.g., lipopolysaccharides) directly activate certain inflammatory cells (e.g., macrophages), the mechanism(s) that triggers eosinophil degranulation is unknown. In this study we investigated whether human eosinophils have an innate immune response to certain fungal organisms. We incubated human eosinophils with extracts from seven environmental airborne fungi (Alternaria alternata, Aspergillus versicolor, Bipolaris sorokiniana, Candida albicans, Cladosporium herbarum, Curvularia spicifera, and Penicillium notatum). Alternaria and Penicillium induced calcium-dependent exocytosis (e.g., eosinophil-derived neurotoxin release) in eosinophils from normal individuals. Alternaria also strongly induced other activation events in eosinophils, including increases in intracellular calcium concentration, cell surface expression of CD63 and CD11b, and production of IL-8. Other fungi did not induce eosinophil degranulation, and Alternaria did not induce neutrophil activation, suggesting specificity for fungal species and cell type. The Alternaria-induced eosinophil degranulation was pertussis toxin sensitive and desensitized by preincubating cells with G protein-coupled receptor agonists, platelet-activating factor, or FMLP. The eosinophil-stimulating activity in Alternaria extract was highly heat labile and had an M(r) of approximately 60 kDa. Thus, eosinophils, but not neutrophils, possess G protein-dependent cellular activation machinery that directly responds to an Alternaria protein product(s). This innate response by eosinophils to certain environmental fungi may be important in host defense and in the exacerbation of inflammation in asthma and allergic diseases.

Anti-inflammatory actions of interleukin-13: suppression of tumor necrosis factor-alpha and antigen-induced leukocyte accumulation in the guinea pig lung.

The Th2 cytokine interleukin (IL)-13 is believed to play an important role in the development of allergy, although it has also been ascribed anti-inflammatory roles in several experimental models. In this study, we have examined the effects of human recombinant IL-13 on eosinophilic lung inflammation in the guinea pig. IL-13 (1 to 100 ng, given by intratracheal instillation) did not elicit airway eosinophil recruitment. A pronounced accumulation of eosinophils, as well as monocyte/macrophages, was elicited by intratracheal instillation of guinea pig tumor necrosis factor alpha (gpTNF-alpha). Intratracheal administration of IL-13 (1 to 100 ng) given immediately prior to exposure to gpTNF-alpha resulted in a dose-related suppression of eosinophil and monocyte/macrophage accumulation in the airways, as assessed by bronchoalveolar lavage (BAL) and eosinophil peroxidase activity in whole-lung homogenates. IL-13 treatment also reduced BAL fluid (BALF) leukocyte accumulation induced by subsequent aerosol antigen challenge of sensitized guinea pigs. Antigen challenge also resulted in elevated levels of immunoreactive eotaxin and eosinophil-stimulating activity in BALF, although only the latter was reduced significantly by IL-13 instillation prior to challenge. In contrast to the suppressive effects of IL-13, instillation of human recombinant IL-4 (100 ng) alone elicited an increase in BALF monocyte/macrophage numbers, and IL-4 was unable to inhibit gpTNF-alpha-induced leukocyte accumulation. Hence, IL-13 (but not human IL-4) exhibits an anti-inflammatory action in the airways of gpTNF-alpha- or antigen-challenged guinea pigs, by mechanisms that may involve the decreased generation of eosinophil-stimulating activity in the airways.

Human eosinophil cytotoxicity-enhancing factor. Eosinophil-stimulating and dithiol reductase activities of biosynthetic (recombinant) species with COOH-terminal deletions.

U937 cells produce eosinophil cytotoxicity-enhancing factor (ECEF) polypeptides of 14 and 10 kDa that have identical NH2-terminal amino acid sequences. The 10-kDa form has greater eosinophil-stimulating activity (half-maximal at > 20-fold lower concentration). We considered the hypothesis that there is a precursor-product relationship between the 14- and 10-kDa species. Recombinant 14-kDa 104-amino acid ECEF (rECEF-104) had a slight stimulatory effect on eosinophil cytotoxic function at concentrations of 160 nM and above. In contrast, two species, rECEF-80 and rECEF-84, representing cleavage products of approximately 10 kDa had substantial statistically significant cytotoxicity-enhancing activity at concentrations as low as 10 pM. This evidence demonstrates the potential to generate the high-activity ECEF species by proteolytic cleavage of the 104-amino acid species. Another feature of this cytokine is the sequence from amino acids 31 to 34, which constitutes the conserved and active site of the enzyme thioredoxin. When tested for dithiol reductase enzymatic activity, rECEF-104 was active in a dose- and time-dependent manner, whereas the truncated forms of the molecule had no dithiol reductase activity. Thus the eosinophil-stimulating functions of the molecule do not correlate with its enzymatic activity. The evidence shows that the enzymatic activity is not essential for the initial interaction of ECEF with the eosinophil, and it suggests that the ECEF molecule functions by means of two discrete mechanisms.

Eosinophilia and eosinophil helminthotoxicity in patients treated for Schistosoma mansoni infections

The changes in eosinophil levels and in eosinophilmediated antibody-dependent schistosomular cytotoxicity, following treatment for Schistosoma mansoni infections, have been investigated in 2 similar groups of patients aged 15–50 years. Patients in group 1 were treated with either hycanthone or oxamniquine, and those in group 2 with hycanthone or praziquantel. Eosinophil levels were significantly increased in both groups. In group 1 peripheral blood eosinophil counts rose from a mean of 175/μl before treatment to 745/μl 3 weeks after treatment, and in group 2 from 181/μl to 1066/μl. The increase in eosinophil levels was positively correlated with a rise in circulating anti-adult worm antibodies ( r = −0·587, P < 0·05), whereas a negative correlation was recorded with anti-egg antibodies ( r = −0·727). Despite some enhanced eosinophil helminthotoxicity following treatment in some of the individuals in group 1 ( 7 15 ), the change overall was not significant. In group 2, in which a different standard anti-schistosomular antibody was used, the eosinophil killing capacity recorded at 3 weeks was lower than that before commencement of treatment ( t = 2·89, P < 0·01). The eosinophil stimulating activity, detected in cultured mononuclear cell supernatants (MCS) from individual patients, correlated with eosinophil levels ( r = 0·582, P < 0·02) but there was no association with eosinophil killing. MCS activity did not appear to be boosted by treatment. These studies showed that peripheral blood eosinophil counts were increased following treatment, but their ability to kill schistosome larvae is variable and may depend on the immune serum used as the source of anti-schistosomular antibody.

The presence of eosinophil‐activating mediators in sera from individuals with Schistosoma mansoni infections

IgG antibodies and eosinophils kill schistosomula of Schistosoma mansoni in vitro, and there is now evidence to suggest that the main factor that contributes to the expression of purified IgG effector function is the degree of activation of the donor's eosinophils. This study was designed to identify serum-derived activating factors in sera from individuals infected with S. mansoni. Such activating factors may be responsible for enhancing eosinophil cytotoxicity against schistosomula. Serum-borne mediators were prepared by fractionation of sera from infected individuals by gel filtration high-performance liquid chromatography. The eosinophil-stimulating activity of these mediators was assayed by a new method which depends on the increased expression of the CR3 alpha chain (CD11b) on the surface of activated eosinophils. Sera from infected individuals exhibited different levels of eosinophil activation, and activation appeared to be due to several serum factors, including interleukin 5. In conclusion, our results suggest that eosinophil-activating factors present in infection sera may not only be responsible for enhancing eosinophil cytotoxicity but also be necessary for its expression.

Identification of different charged species of a human monocyte derived neutrophil activating peptide (MONAP)

Lipopolysaccharide-stimulated human monocytes secrete a 10 kD peptide (MONAP) of high neutrophil, not however monocyte or eosinophil stimulating activity. By reversed phase HPLC MONAP could be distinguished from Interleukin 1. Analytic isoelecto-focusing of pure MONAP (single line upon sodiumdodecylsulfate polyacrylamide gel electrophoresis, single peak after RP-18-HPLC), obtained by size exclusion HPLC followed by two different reversed phase HPLC steps revealed charge heterogeneity giving major components with isoelectric points at 4.7, 4.9, 6.4 and 6.9, all of which exhibited chemotactic activity.

Eosinophilia VI, spontaneous synthesis of chemokinetics, chemotactic, complement receptor-inducing activities for eosinophils by bronchial t lymphocytes of asthmatic-bronchitic patients.

T lymphocytes were separated from the bronchial mucus of five patients with extrinsic asthma and chronic and mild bronchitis and who had numerous eosinophils in the bronchial mucus but no significant blood eosinophilia. The bronchial lymphocytes spontaneously, that is without treatment with antigens or mitogens, released into serum-free synthetic medium one or more substances in the molecular weight range of 30 000 to 60 000 daltons. These substances when tested on eosinophils of normal persons stimulated random movement (chemokinesis), attracted them (chemotaxis), enhanced their chemotactic response to activated complement, and increased the expression of C4 and C3b receptors. Chemokinetic and chemotactic activity for neutrophils was weak. No eosinophil-stimulating activity was found in cultures of bronchial lymphocytes treated with puromycin to inhibit synthesis. The blood lymphocytes did not spontaneously synthesize the substance(s). It is not known if the several eosinophil-stimulating activities are due to one or more substance(s), but the nature of the eosinophil responses, molecular weight and other features, indicate similarities with the 'eosinophil stimulation promoter-chemotactic' factor reported to be released from mouse or human lymphocytes treated with antigen. Eosinophil stimulation resulting in increased expression of specific receptors, and potential for non-specific adherence, by trapping or arresting randomly migrating cells, is believed to mediate accumulation of cells in an affected organ. Prolonged synthesis of such products as from activated lymphocytes in the lung, could account for much of the local eosinophilia.

Hydrophobie Adsorption Chromatography of Colony-Stimulating Activities and Erythroid-Enhancing Activity From the Human Monocyte-Like Cell Line, GCT

The human cell line, GCT, secretes hemopoietins into serum-free culture medium. The conditioned medium contains activities that stimulate neutrophil-monocyte, macrophage, eosinophil, and erythroid colony growth in human marrow cultures. We have used hydrophobic adsorption chromatography to separate a neutrophil-monocyte colony-stimulating factor (CSF) from the other colony-stimulating activities. This hydrophobic CSF has no eosinophil-stimulating activity and is virtually devoid of erythroid-stimulating activity.

Hydrophobic adsorption chromatography of colony-stimulating activities and erythroid-enhancing activity from the human monocyte-like cell line, GCT

The human cell line, GCT, secretes hemopoietins into serum-free culture medium. The conditioned medium contains activities that stimulate neutrophil-monocyte, macrophage, eosinophil, and erythroid colony growth in human marrow cultures. We have used hydrophobic adsorption chromatography to separate a neutrophil-monocyte colony-stimulating factor (CSF) from the other colony-stimulating activities. This hydrophobic CSF has no eosinophil-stimulating activity and is virtually devoid of erythroid-stimulating activity.

Granulocyte/macrophage-, megakaryocyte-, eosinophil- and erythroid-colony-stimulating factors produced by mouse spleen cells.

The formation of mature haemopoietic cells is controlled by hormones that specifically stimulate the progenitor cells of the granulocyte/macrophage, eosinophil, megakaryocyte and erythroid pathways. PWMSC medium (pokeweed-mitogen-stimulated spleen-cell-conditioned medium) is known to contain the biological activities that control the clonal proliferation of these four progenitor cells in vitro in semi-solid agar cultures. In this study the molecular properties of these biological activities were characterized, and all four colony-stimulating factors appear to be associated with glycoproteins. These factors were precipitated between 50 and 80%-satd. (NH(4))(2)SO(4) and could be concentrated by ultrafiltration over a 10000-mol.wt.-cut-off hollow-fibre membrane. Megakaryocyte- and erythroid-colony-stimulating factors were lost when the conditioned medium was dialysed at low ionic strength (<0.03m). Neither asialo- nor sialo-erythropoietin was detectable in concentrated PWMSC medium or in the fractions purified from it by gel filtration on Sephadex G-150. The factors bound to concanavalin A-Sepharose were eluted with alpha-methyl-d-glucopyranoside (0.10m). Analysis by gel filtration on Sephadex G-150 indicated that the apparent molecular-weight distributions of all colony-stimulating factors were identical (37000). Treatment with neuraminidase did not alter the biological activities of any of these factors, but when the molecular weights were analysed, after neuraminidase treatment, on Sepharose CL-6B in the presence of guanidine hydrochloride (6m) all were eluted with a mol.wt. of 24000. Although the apparent molecular weights of the different factors were identical, charge differences were detectable by isoelectric focusing on thin-layer granulated gels. There appeared to be considerable charge heterogeneity associated with each factor, as all were focused over 2-4 pH units. The maximum activity of the granulocyte/macrophage-colony-stimulating factor on isoelectric focusing was at pH4.8, whereas the maximum activity for the eosinophil-colony-stimulating factor was at pH5.8. The erythroid- and megakaryocyte-colony-stimulating activities were detected in the pH ranges 4.8-5.8 and 4.6-7.1 respectively. Chromatographic differences between the granulocyte/macrophage- and eosinophil-colony-stimulating factors were also detected by hydrophobic chromatography at low ionic strength (0.15m-NaCl) on Cibacron Blue-Sepharose and at high ionic strength [2m-(NH(4))(2)SO(4)] on phenyl-Sepharose. Eosinophil-colony-stimulating factor bound more strongly than the other factors to both matrices. The megakaryocyte- and erythroid-colony-stimulating activities were always associated with those for granulocytes/macrophages and eosinophils. Preparations highly enriched for eosinophil-colony-stimulating factor were also obtained by DEAE-cellulose chromatography. An overall purification of 100-fold for all of the factors was achieved with the present techniques, and, although differences were observed, only granulocyte/macrophage-stimulating factors and a small proportion of the eosinophil-stimulating factors could be completely separated from the others. Our results are consistent with the existence of separable factors for granulocyte/macrophage and eosinophil stimulation, but the megakaryocyte- and erythroid-stimulating activities were always associated with the granulocyte/macrophage- and eosinophil-stimulating activities. Thus there may be one molecule that is able to stimulate all four colony types or four very similar molecules that are difficult to separate.