Eosinophil granulocytopoiesis in hepatic periovular granulomas during the chronic phase of experimental murine Schistosomiasis mansoni

We have observed in hepatic periovular granulomas of C3H mice infected with Schistosoma mansoni, in the chronic phase of the disease (12-19 weeks of infection), groups of early precursors and immature eosinophil granulocytes corresponding, at the ultrastructural level, to promyelocytes and myelocytes. Mitosis was also seen in eosinophil myelocytes. These eosinophil myeloid foci were observed in close contact with macrophages and epithelioid cells, and they were surrounded by an extracellular matrix, rich in collagen fibres. These morphological observations give support to the concept of a peripheral proliferation of eosinophils in chronic schistosomiasis, mediated by a factor secreted by macrophages present in granulomas.     

Type-specific collagen degradation by eosinophils.

Despite the association of eosinophils with wound-healing and fibrotic processes, their collagenolytic ability has been poorly defined. By using highly purified eosinophil preparations (greater than 95% eosinophils) obtained from guinea-pigs by peritoneal lavage, we have examined type-specific collagen degradation by eosinophils using sensitive detection methods. The results show that eosinophils contain a metalloprotein that degrades types I and III collagens. This activity was apparent only after the addition of 4-aminophenylmercuric acetate to the reaction mixture, a finding similar to that for latent collagenases described from other sources. No collagenolytic activity against types IV and V collagens could be detected. Thus eosinophils may play a role in the alterations in connective-tissue matrices seen in physiological and pathological states.     

Analysing the eosinophil cationic protein - a clue to the function of the eosinophil granulocyte

Eosinophil granulocytes reside in respiratory mucosa including lungs, in the gastro-intestinal tract, and in lymphocyte associated organs, the thymus, lymph nodes and the spleen. In parasitic infections, atopic diseases such as atopic dermatitis and asthma, the numbers of the circulating eosinophils are frequently elevated. In conditions such as Hypereosinophilic Syndrome (HES) circulating eosinophil levels are even further raised. Although, eosinophils were identified more than hundred years ago, their roles in homeostasis and in disease still remain unclear. The most prominent feature of the eosinophils are their large secondary granules, each containing four basic proteins, the best known being the eosinophil cationic protein (ECP). This protein has been developed as a marker for eosinophilic disease and quantified in biological fluids including serum, bronchoalveolar lavage and nasal secretions. Elevated ECP levels are found in T helper lymphocyte type 2 (atopic) diseases such as allergic asthma and allergic rhinitis but also occasionally in other diseases such as bacterial sinusitis. ECP is a ribonuclease which has been attributed with cytotoxic, neurotoxic, fibrosis promoting and immune-regulatory functions. ECP regulates mucosal and immune cells and may directly act against helminth, bacterial and viral infections. The levels of ECP measured in disease in combination with the catalogue of known functions of the protein and its polymorphisms presented here will build a foundation for further speculations of the role of ECP, and ultimately the role of the eosinophil.     

Effect of cartilage proteoglycans on human collagenase activities.

No significant inhibition of purified rheumatoid synovial collagenase was found when this enzyme was assayed in the presence of porcine or human cartilage proteoglycans. Reaction mixtures containing up to twice the amount of proteoglycan compared to that of collagen, w/w, had little effect on collagen degradation as judged by the reconstituted [4C]collagen fibril assay and polyacrylamide gel electrophoresis. Proteoglycans were not degraded by the synovial collagenase preparation. Although the human collagenases derived from rheumatoid synoviam, gastric mucosa, skin and granulocytes showed some reduction in activity when exposed to aggregated proteoglycans at high concentrations, disaggregated proteoglycans had no inhibitory effect. It is concluded that cartilage proteoglycans do not directly inhibit human collagenases in vitro, but in vivo they may provide some physical barriers which might limit the accessibility of the enzyme to its collagen substrate.     

A fluorescent screening assay for collagenase using collagen labeled with 2-methoxy-2,4-diphenyl-3(2H)-furanone

This report describes the use of the compound 2-methoxy-2,4-diphenyl-3(2H)-furanone to label collagen as a substrate for the detection of mammalian collagenase in a fluorescent assay which is suitable for screening large numbers of samples. The compound 2-methoxy-2,4-diphenyl-3(2H)-furanone presents distinct advantages over other fluorophores, since both the unbound reagent and its hydrolysis products are nonfluorescent. The labeling procedure uses commercially available collagen, is fast and simple, and gives a 90% yield of labeled substrate. The fluorescent collagen substrate is stable and retains fluorescence over a wide range of pH. The assay detects, reproducibly, metal-dependent collagenase activity in microliter volumes of conditioned media from cultured neoplastic cells or in chromatographic fractions from such media.     

Effect of cartilage proteoglycans on human collagenase activities

No significant inhibition of purified rheumatoid synovial collagenase was found when this enzyme was assayed in the presence of porcine or human cartilage proteoglycans. Reaction mixtures containing up to twice the amount of proteoglycan compared to that of collagen, w/w,, had little effect on collagen degradation as judged by the reconstituted [¹⁴C] collagen fibril assay and polyacrylamide gel electrophoresis. Proteoglycans were not degraded by the synovial collagenase preparation. Although the human collagenases derived from rheumatoid synovium, gastric mucosa, skin and granulocytes showed some reduction in activity when exposed to aggregated proteoglycans at high concentrations, disaggregated proteoglycans had no inhibitory effect. It is concluded that cartilage proteoglycans do not directly inhibit human collagenases in vitro, but in vivo they may provide some physical barriers which might limit the accessibility of the enzyme to its collagen substrate.     

Composition and ultrastructure of elasmobranch granulocytes. II. Rays (Rajiformes)

The blood granulocyte composition of seven species of ray is given together with ultrastructural observations made on the epigonal organ and blood of Pavoraja spinifera and the spleen of a deepwater rajid skate. Under the light microscope three granulocyte types, eosinophils, eosinophilic granulocytes and neutrophilic granulocytes could be distinguished. At the EM level two granulocyte types were apparent, one with elongated granules containing longitudinal fibrils that consolidated to form an axial rod-like inclusion, and the other with large, spherical, uniformly electron-dense granules. Correlation of light and electron microscope observations indicated that the neutrophilic granulocytes with weakly basophilic, elongated granules become weakly eosinophilic, as eosinophilic granulocytes, and these in turn develop to eosinophils with granules containing axial rods. The other granulocyte type forms another population of eosinophils with spherical granules.
The inter-relationship of these granulocytes, the identification of eosinophilic granulocytes, or heterophils, as immature eosinophils, and the co-existence of two morphologically distinct eosinophil forms are discussed.     

Biological activity of leukotriene B4 analogs: inhibition of guinea pig eosinophil migration in vitro by the 2,6-disubstituted pyridine analogs U-75,302 and U-75,485.

A "late phase" antigen-induced bronchoalveolar eosinophilia has been demonstrated in ovalbumin sensitized guinea pigs (1,2). This in vivo response to antigen inhalation can be inhibited by a 2,6-disubstituted pyridine analog of LTB4, U-75,302(2) (3). In the present study, the mechanism of the drug action was studied by assessing the activity of U-75,302 and a second analog, U-75,485 to displace [3H]-leukotriene B4 binding at the guinea pig eosinophil membrane, as well as their action as chemoattractants or inhibitors of the directional migration of guinea pig eosinophils in vitro. Radioligand competition experiments demonstrated that both analogs interacted strongly with the high affinity LTB4 binding sites on guinea pig eosinophil membrane. Both analogs are powerful chemoattractants for guinea pig eosinophils since they induced directional migration of guinea pig eosinophils when administered alone. In addition, when the cells were treated with either analog and their chemotaxis response was measured in response to a natural chemoattractant, both U-75,302 and U-75,485 at concentrations of 0.1 to 100 microM dose dependently inhibited the LTB4 induced chemotaxis response. The EC50s obtained for U-75,302 and U-75,485 as inhibitors of LTB4 induced guinea pig eosinophil chemotaxis were estimated to be 11.5 +/- 5.5 microM and 5.4 +/- 2.5 microM respectively. Under the same conditions, they had no significant effect upon eosinophil migration induced by zymosan activated plasma at concentrations below 100 microM. We suggest that the inhibition of antigen-induced eosinophil infiltration in guinea pig airway in vivo by U-75,302 or U-75,485 may be a result of partial antagonism or desensitization at the LTB4 receptor level of guinea pig eosinophils.     

Activated human eosinophils synthesize new proteins

In order to study the biochemical consequences of prolonged in vitro activation of human blood eosinophils, aqueous whole cell lysates, cell-free supernatants from resting eosinophils, and cells activated with opsonized zymosan, calcium ionophore (A23187), N-formylmethionylleucylphenylalanine (fMet-Leu-Phe), and phorbol 12-myristate 13-acetate (PMA) were analyzed by polyacrylamide gel electrophoresis (PAGE). In comparison to resting eosinophils, opsonized zymosan-activated eosinophil extracts demonstrated altered protein composition on both the native PAGE and sodium dodecyl sulfate (SDS) -PAGE. Three new polypeptides of apparent molecular mass 24 kDa, 43 kDa and 60 kDa appeared on SDS-PAGE gels when opsonized zymosan-activated eosinophil extracts were electrophoresed. In contrast, extracts from fMet-Leu-Phe, A23187, and PMA-activated eosinophils demonstrated neither altered polypeptide composition nor new polypeptides. Opsonized zymosan also induced the incorporation of L-[35S]methionine into eosinophil proteins and this was completely blocked by pretreating the cells with cycloheximide. This finding suggests that eosinophils activated by certain stimuli synthesize new proteins. These newly synthesized proteins, which are freely secreted into the medium during cell activation, may possess important immunological functions.     

Induction of collagenase production in Vibrio B-30.

The inducible nature of an extracellular collagenase produced by a marine Vibrio (Vibrio B-30, ATCC 21250) was demonstrated by observing the increase in extracellular collagenase activity after the addition of collagen to cell cultures in the latter part of the exponential growth phase. When collagenase-hydrolyzed collagen was added, the lag time required before collagenase production was detected decreased significantly compared with cultures receiving collagen. Cells preinduced to synthesize collagenase did not produce the enzyme when collagen was removed from the culture medium. Incorporation of penicillin G had no effect on final collagenase activity levels in suspensions of Vibrio B-30 in complete medium supplemented with collagen. However, chloramphenicol and tetracycline inhibited collagenase production, indicating that de novo protein synthesis was necessary for the appearance of activity. Attempts to isolate the inducing substance(s) involved filtering hydrolyzed collagen through a series of ultrafiltration membranes. The lowest-molecular-weight fraction of collagen hydrolysate with inducing ability was between 1,000 and 10,000. Gel filtration of this fraction on Sephadex G-50 resulted in the appearance of three protein peaks, two of which were capable of inducing collagenase production. Results from amino acid composition and N-terminal amino acid analysis suggest that the inducing substance originates from the polar helical portion of the collagen molecule.     

Angiostrongylus cantonensis: role of eosinophils in the neurotoxic syndrome (Gordon-like phenomenon)

The role of eosinophils in the pathophysiology of Angiostrongylus cantonensis infections was investigated in nonpermissive (guinea pig) and permissive (rat) hosts. Neurological symptoms similar to the Gordon phenomenon (ataxia, tremor, paralysis) together with a loss of Purkinje cells in the cerebellum were observed after intracraneal injection of human eosinophil extracts or after infection with A. cantonensis, only in guinea pigs and not in rats. Blood eosinophilia as well as eosinophil numbers present in the cerebellum and in the cerebrospinal fluid were higher in guinea pigs than in rats, at all times after infection with A. cantonensis. Increased levels of cytotoxicity toward L3 larvae in vitro were obtained in the presence of guinea pig eosinophils and IgE antibodies, rather than with the corresponding rat effector system. The detection of one eosinophil granule component, the eosinophil peroxidase, in the cerebrospinal fluid from infected guinea pigs but not from rats suggested that in nonpermissive hosts, neurological disorders, similar to the previously described Gordon phenomenon, might be due to eosinophil neurotoxins released after interaction of eosinophils with the parasites.     

Giant eosinophil colonies from cultures of bone marrow cells

Summary To date, the small size and slow growth of eosinophil colonies in vitro has hampered study of cloned eosinophils. We found enhanced eosinophil colony size and numbers in methylcellulose cultures of bone marrow cells utilizing defined supplemented bovine calf serum (DSBCS) in combination with EL4 conditioned medium (EL4-CM). At days 9, 16 and 23 significantly more eosinophil colonies and more cells/colony were present in cultures incubated with DSBCS/EL4-CM than in cultures incubated with fetal calf serum/EL4-CM. The ability to generate large numbers of eosinophils in vitro should facilitate study of cloned eosinophils. Supported in part by a grant from the National Institutes of Health, AI 20416, and by the Mayo Foundation. Editor's statement Previous approaches to in vitro culture of eosinophils generally have achieved, at best, mixed cultures of colonies of these cells and granulocyte-macrophage colonies. The improved culture methods described in this report produce more homogeneous eosinophil cultures and larger colonies of these cells. The procedure employs EL4 murine thymoma-conditioned medium, which apparently contains eosinophil colony-stimulating activity in the absence of granulocyte-macrophage colony-stimulating activity. David W. Barnes     

Demonstration of collagenase activity in adult Strongyloides ratti (Nematoda:Strongyloididae) and its absence in the infective larvae

Larvae and adults of Strongyloides ratti were examined for collagenolytic activity on 14C proline-labelled, native, guinea-pig skin collagen substrate. The activity was measured by determining either the amount of hydroxyproline released or the amount of radioactivity in the solubilized fraction of the collagen substrate. Bacterial collagenase was used for enzyme control and trypsin served as substrate control. No collagenolytic activity was found in living larvae, their extracts or metabolites. The collagenolytic activity of the metabolites of adult worms appeared weak, whereas that of the extracts of the adults was pronounced. It is suggested that collagenase is active in the adult females at the time of migration in the intestinal mucosa during oviposition.     

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.     

Variations in protein expression related to human eosinophil heterogeneity

In hypereosinophilic patients, eosinophil heterogeneity has been assessed mainly according to morphologic and biologic criteria. In order to investigate the molecular basis of such heterogeneity, biochemical analysis was performed on various eosinophil subpopulations fractionated on metrizamide gradients. Whole cell extracts from purified eosinophils disrupted with a nonionic (NP-40) detergent were successively analyzed by SDS-PAGE and two-dimensional electrophoresis (isoelectric focusing or nonequilibrium pH gradient electrophoresis in the first dimension). Hypodense eosinophils that sediment in the lightest density gradients (18 to 22% metrizamide solution) differed from other purified eosinophils (intermediate and normodense eosinophils respectively collected in 22 to 23% and 23 to 25% metrizamide solutions). Comparative analysis of protein patterns on both monodimensional and bidimensional electrophoresis showed that a basic protein of Mr 51 kDa, present on normodense or intermediate eosinophils, was poorly detected in the case of hypodense eosinophils. In contrast, two other proteins with apparent Mr of about 23 kDa and 41 kDa were exclusively or predominantly identified in these latter cell fractions. Immunochemical analysis with polyclonal antibodies against eosinophil basic proteins and enzymatic assays revealed that the 51-kDa polypeptide could be related to an eosinophil peroxidase-like molecule. In addition, the two proteins detected only in hypodense eosinophils might be related to proteins newly synthesized by in vivo activated eosinophils. Our results suggest that variations in protein expression might represent a good marker of in vivo activation.     

Mechanism of eosinophil induced signaling in cholinergic IMR-32 cells

Eosinophils interact with nerve cells, leading to changes in neurotransmitter release, altered nerve growth, and protection from cytokine-induced apoptosis. In part, these interactions occur as a result of activation of neural nuclear factor (NF)-kappaB, which is activated by adhesion of eosinophils to neural intercellular adhesion molecule-1 (ICAM-1). The mechanism and consequence of signaling after eosinophil adhesion to nerve cells were investigated. Eosinophil membranes, which contain eosinophil adhesion molecules but not other eosinophil products, were coincubated with IMR-32 cholinergic nerve cells. The studies showed that there were two mechanisms of activation of NF-kappaB, one of which was dependent on reactive oxygen species, since it was inhibited with diphenyleneiodonium. This occurred at least 30 min after coculture of eosinophils and nerves. An earlier phase of NF-kappaB activation occurred within 2 min of eosinophil adhesion and was mediated by tyrosine kinase-dependent phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1). Coimmunoprecipitation experiments showed that both extracellular signal-regulated kinase 1/2 and IRAK-1 were recruited to ICAM-1 rapidly after coculture with eosinophil membranes. This was accompanied by an induction of ICAM-1, which was mediated by an IRAK-1-dependent pathway. These data indicate that adhesion of eosinophils to IMR-32 nerves via ICAM-1 leads to important signaling events, mediated via IRAK-1, and these in turn lead to expression of adhesion molecules.     

Glucocorticoids inhibit eosinophil responses to granulocyte-macrophage colony-stimulating factor

The role of the eosinophil as an active proinflammatory cell in asthma and other allergic disorders has been well established. Glucocorticosteroids have long been used therapeutically as antiinflammatory agents in a variety of disease states where eosinophilia is a prominent feature. Although glucocorticoids are known to reduce tissue and circulating eosinophil numbers, the mechanisms by which they do so have not been clearly elucidated. Culture of eosinophils in vascular endothelial cell supernatants (VEC SUP) induces phenotypic and functional changes and prolongs the survival of the eosinophils. The survival-promoting activity in VEC SUP was shown to be granulocyte-macrophage CSF (GM-CSF) by neutralization with specific antibody. The potent glucocorticosteroid, dexamethasone (DEX), inhibited the prolongation of eosinophil survival caused by culture in either VEC SUP or human rGM-CSF. DEX (10(-6) M) exerted a direct survival-inhibitory effect on the eosinophil by the 4th day in culture in VEC SUP. This survival-inhibitory effect was dependent on the concentration of DEX (10(-10)-10(-6) M). Other glucocorticoids, including prednisolone (10(-7), 10(-6) M) and hydrocortisone (10(-7), 10(-6) M) also inhibited survival. The rank order of potency of the steroids indicates that this effect is mediated by a glucocorticoid receptor. This conclusion is supported by the failure of the sex steroids testosterone (10(-8)-10(-6) M) or beta-estradiol (10(-6) M) to inhibit eosinophil survival in the presence of VEC SUP. The effect of glucocorticoids on eosinophils is not a simple direct toxic effect because it was reversed by higher concentrations of GM-CSF. DEX shifted the GM-CSF dose-response curve for survival approximately fivefold to the right. GM-CSF induced a shift in eosinophil buoyant density which was partially blocked by DEX. These results suggest that glucocorticoids may inhibit elements of cytokine "priming" of eosinophils and that the eosinophilopenic effects of glucocorticoids may result in part from a direct effect on the eosinophil within a regulatory system involving cytokines.     

IL-5 drives eosinophils from bone marrow to blood and tissues in a guinea-pig model of visceral larva migrans syndrome

This study was undertaken to evaluate the role of IL-5 in eosinophil migration and in the maintenance of eosinophilia in a guinea-pig model of visceral larva migrans syndrome. The results show that the infection of animals with Toxocara canis induced an early increase in serum IL-5 levels that might be essential for eosinophil differentiation and proliferation and for the development of eosinophilia. When infected guinea-pigs were treated with mAb anti-IL-5 (TRFK-5) given at the same time or 1 or 3 days after infection, there was a high percentage of reduction of eosinophil counts 18 days after infection. However, when the mAb was administered during the peak of eosinophilia, there was high inhibition in blood, no inhibition in bronchoalveolar lavage fluid (BALF) or peritoneum and an increase in eosinophil numbers in bone marrow. Thus, a basic level of IL-5 may be essential to drive eosinophils from bone marrow to blood and tissues, and for the maintenance of eosinophilia in infected animals. We may also conclude that when eosinophils have already migrated to the lungs, TRFK-5 has no power to inhibit eosinophilia, which is also under control of local lung cells producing IL-5. In this way, only one later TRFK-5 treatment may not be sufficient to modify the lung parenchyma microenvironment, since T. canis antigens had already stimulated some cell populations to produce IL-5.     

Human eosinophil peroxidase: purification and characterization

Human eosinophil peroxidase (EPO) was isolated from granules from granulocytes of a patient with hypereosinophilia. The granules were extracted by means of 0.2 M NaAc, pH 4.0. The purification steps included gel filtration chromatography on Sephadex G-75 superfine and ion-exchange chromatography on CM-Sephadex G-50. The purified protein showed one band on agarose-electrophoresis, a high peroxidase activity, and a 415-nm/280 nm ratio of 1.15. After reduction, EPO showed two bands on SDS-PAGE of m.w. 52,000 and 15,000, respectively. On gel filtration, the unreduced protein had a m.w. of approximately 77,000. Amino acid analyses showed a high content of arginine and aspartic acid. Monospecific antibodies to EPO were prepared in rabbits, and a specific radioimmunoassay was developed. There was an almost linear correlation between the content of EPO measured by the radioimmunoassay and the number of eosinophils in a mixed cell extract from reference material, indicating the eosinophil origin of EPO. The content of EPO was estimated to be 15.0 micrograms/10(6) eosinophils.