Leukotrienes and other products of the 5-lipoxygenase pathway. Biochemistry and relation to pathobiology in human diseases.
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Arachidonate 5-Lipoxygenase↗
Biomedical subjects
K F Austen
Publications and source records attributed to K F Austen.
Mast cell heterogeneity. Differential synthesis and expression of glycosphingolipids by mouse serosal mast cells as compared to IL-3-dependent bone marrow culture-derived mast cells before or after coculture with 3T3 fibroblasts.
The synthesis and intracellular expression of glycosphingolipids by mouse serosal mast cells (SMC) have been characterized by radiolabeling and TLC and by immunodetection in situ. Chromatographic analysis of purified glycosphingolipids from SMC intrinsically labeled with [14C]galactose and [14C]glucosamine hydrochloride revealed the predominant synthesis of only the simplest neutral glycosphingolipid and ganglioside, glucosylceramide and ganglioside GM3, respectively. Intracellular indirect immunofluorescence staining of permeabilized SMC demonstrated the absence of the more complex neutral glycosphingolipids lactosylceramide, globotriosylceramide, globotetraosylceramide, and globopentaosylceramide, the absence of ganglioside GM1, and the presence of ganglioside GM3. By contrast, permeabilized mouse IL-3-dependent bone marrow culture-derived mast cells (BMMC) and mast cells recovered after 21 days of coculture of BMMC with mouse 3T3 fibroblasts expressed lactosylceramide, globotriosylceramide, globotetraosylceramide, ganglioside GM1, and ganglioside GM3, but not globopentaosylceramide intracellularly as determined by immunofluorescence. The findings indicate a loss of biosynthetic capacity and epitope maintenance for glycosphingolipids with in vivo differentiation of SMC from IL-3-dependent BMMC progenitors. Thus, although mast cells derived after coculture of these progenitors for 21 days with fibroblasts assume multiple SMC-like properties in terms of their histochemical staining and their secretory granule proteoglycan and neutral protease constituents, they do not lose the ability to express complex glycosphingolipids. The finding that glycosphingolipid composition does not change coordinately with other secretory granule markers defines a new stage of mouse mast cell development between the BMMC and SMC and provides evidence that mast cell development is more complex than previously appreciated.
Animals↗
The mechanism of leukotriene B4 export from human polymorphonuclear leukocytes.
Recently, we characterized the export of leukotriene (LT) C4 from human eosinophils as a carrier-mediated process (Lam, B. K., Owen, W. F., Jr., Austen, K. F., and Soberman, R. J. (1989) J. Biol. Chem. 264, 12885-12889). To determine whether a similar mechanism regulates the release of leukotriene B4 (LTB4), human polymorphonuclear leukocytes (PMN) were preloaded with LTB4 by incubation with 25 microM leukotriene A4 (LTA4) at 0 degrees C for 60 min. PMN converted LTA4 to LTB4 in a time-dependent manner as determined by resolution of products by reverse-phase high performance liquid chromatography and quantitation by integrated optical density. When PMN preloaded with LTB4 were resuspended in buffer at 37 degrees C for 0-90 s, there occurred a time-dependent release of LTB4 but little formation or release of 20-hydroxy-LTB4 and 20-carboxy-LTB4. When PMN were preloaded with increasing amounts of intracellular LTB4 by incubation with 3.1-50.0 microM LTA4 and were then resuspended in buffer at 37 degrees C for 20 s, there occurred a concentration-dependent and saturable release of LTB4 with a Km of 798 pmol/10(7) cells and a Vmax of 383 pmol/10(7) cells/20 s. The release of LTB4 was temperature-sensitive with a Q10 of 3.0 and an energy of activation of 19.9 kcal/mol. The rate of LTB4 release at 37 degrees C is about 50 times the rate of 20-carboxy-LTB4 release. PMN preloaded with LTB4 and resuspended at 0 degree C for 1-60 min in the presence of 30 microM LTA5 progressively converted LTA5 to LTB5. The rate of LTB4 release at 0 degree C was inhibited over the entire time period, peaking at about 50% at 30 min. These results indicate that the release of LTB4 from PMN is a carrier-mediated process that is distinct from its biosynthesis.
Chromatography, High Pressure Liquid↗
Characterization of the human gene that encodes the peptide core of secretory granule proteoglycans in promyelocytic leukemia HL-60 cells and analysis of the translated product.
Based upon the deduced amino acid sequence of a cDNA (cDNA-H4) that had been proposed to encode the peptide core of an eosinophil and a HL-60 cell secretory granule proteoglycan, a 16-amino acid peptide was synthesized. This peptide was then used to elicit rabbit antibodies for study of the translation and post-translational modification of this gene product in hematopoietic cells. When HL-60 cells were radiolabeled for 2 min with [35S]methionine, a protein that migrated in a sodium dodecyl sulfate-polyacrylamide electrophoresis gel with a Mr of 20,000 was immunoprecipitated with the IgG fraction of the anti-peptide serum. Kinetic experiments revealed that within 10 min this radiolabeled precursor protein was converted in HL-60 cells into an Mr approximately 150,000 chondroitin sulfate proteoglycan intermediate. After a 20-min to 1-h chase, this [35S]methionine- or [35S]sulfate-labeled proteoglycan intermediate lost its antigenicity, presumably due to proteolysis of its N terminus. A human genomic library was probed under conditions of high stringency with cDNA-H4 to isolate genomic clones that contain the gene that encodes this proteoglycan peptide core. This gene spans approximately 15 kilobases and consists of three exons. The first exon encodes the 5'-untranslated region of the mRNA transcript, as well as the entire 27-amino acid signal peptide of the translated molecule. The second exon encodes a 49-amino acid region of the peptide core, predicted to be the N terminus of the molecule after its proteolytic processing in the endoplasmic reticulum. The third exon encodes the remainder of the molecule, including its glycosaminoglycan attachment, serine-glycine repeat region. As assessed by S1 nuclease mapping and primer extension analysis, the transcription-initiation site in HL-60 cells for this gene resides 53 base pairs upstream from the translation-initiation site.
Amino Acid Sequence↗
Identification and molecular cloning of a novel mouse mucosal mast cell serine protease.
A novel 28,000 Mr serine protease, designated mouse mast cell protease-2 (MMCP-2), that is stored in the secretory granules of Kirsten sarcoma virus-immortalized mouse mast cells (KiSV-MC) has been identified and its NH2-terminal amino acid sequence has been determined. Analysis of a 953-base pair cDNA that encodes MMCP-2 revealed that this serine protease is a basically charged protein, possessing the histidine-aspartic acid-serine charge relay system that is characteristic of other serine proteases. DNA blot analysis using the full-length MMCP-2 cDNA indicated the existence of a family of highly related serine protease genes in the mouse genome. When the same DNA blot was probed with the 149-base pair KpnI----3' fragment of the cDNA, the probe hybridized to a single DNA fragment, thereby demonstrating that this 3' fragment could be used as a gene-specific probe. The presence of high levels of the MMCP-2 mRNA transcript in the intestines of nematode-infected mice, and its absence in mouse bone marrow-derived mast cells and peritoneal cavity-derived connective tissue mast cells, suggest that this member of the mouse mast cell protease family is preferentially expressed late in the differentiation of mucosal mast cells.
Amino Acid Sequence↗
Inhibition of leukotriene B4 synthesis in neutrophils from patients with rheumatoid arthritis by a single oral dose of methotrexate.
We studied the effects of a single, oral dose of methotrexate (MTX) on arachidonic acid metabolism in neutrophils from 6 patients with rheumatoid arthritis, which were obtained 1 day before and 1 day after their usual weekly MTX dose. The 6 patients had received a mean weekly MTX dose of 9.6 mg (range 5-15) for a mean of 61.7 months (range 58-64), and none received concomitant corticosteroids. Total generation of leukotriene B4 (LTB4) in neutrophils stimulated ex vivo with 10 microM calcium ionophore A23187 for 20 minutes was significantly suppressed, by a mean of 53%, after the MTX dose compared with the predose levels (mean +/- SEM 13.0 +/- 1.4 ng/10(6) cells versus 6.0 +/- 0.9 ng/10(6) cells; P = 0.0019), reflecting a comparable suppression of both released and cell-retained LTB4. A 49% decrease in omega-oxidation products of LTB4 demonstrates that decreased LTB4 synthesis, rather than increased degradation, is responsible for the decrease in LTB4 generation. The absence of a significant change in either 3H-labeled arachidonic acid release or platelet-activating factor generation indicates that the observed decrease in LTB4 synthesis was apparently not caused by diminished phospholipase A2 activity. A 28% decrease in the total formation of the 5-lipoxygenase products 5-hydroxyeicosatetraenoic acid and the 6-trans-LTB4 diastereoisomers, and a 48% suppression of production of LTB4 plus its omega-oxidation metabolites after the MTX dose suggest inhibition of 5-lipoxygenase activity and possible suppression of leukotriene A4 epoxide hydrolase activity.
Aged↗
Cromolyn sodium in the management of systemic mastocytosis.
A multicenter, double-blind, placebo-controlled trial of the efficacy of oral cromolyn sodium (200 mg orally four times per day) was conducted in 11 patients with systemic mastocytosis who had been maintained with the drug on an individualized compassionate-need basis. Efficacy was measured by physician assessment of overall disease severity based on history and physical examination at specified intervals and by the average daily patient symptom diary scores for each of three mastocytosis-related symptoms that had previously appeared to be alleviated by the use of this drug. Efficacy variables were compared for a 4-week baseline period, during which patients received open-labeled cromolyn sodium, and at 4-week intervals during a 16-week period of random assignment to cromolyn sodium or placebo. Overall disease severity and symptoms recorded in patient diaries were graded on a scale of 0 (absent) to 5 (incapacitating). The average physician assessment of disease severity and symptom scores of the patients in the placebo-treated group increased significantly during the randomization phase relative to patients in the cromolyn sodium-treated group, reflecting an exacerbation of symptoms with drug withdrawal (p less than 0.05 and less than 0.028, respectively). When the symptom scores were analyzed separately for gastrointestinal manifestations of disease (diarrhea, abdominal pain, nausea, and vomiting), cromolyn sodium treatment was significantly beneficial relative to placebo (p less than 0.02), whereas the benefit for nongastrointestinal manifestations did not reach statistical significance.
Cromolyn Sodium↗
Hypodense eosinophils and interleukin 5 activity in the blood of patients with the eosinophilia-myalgia syndrome.
The recent recognition of the eosinophilia-myalgia syndrome (EMS) associated with the ingestion of L-tryptophan prompted an analysis of the peripheral blood eosinophil phenotypes and of the serum eosinophil hematopoietins in this disorder. Five patients with an illness characterized by the abrupt onset of aching skeletal muscles, edema, thickening and induration of the skin, and marked blood eosinophilia associated with L-tryptophan ingestion provided eosinophils, serum, or both, for evaluation. Gradient sedimentation density analysis of the peripheral blood eosinophils from four of these patients revealed that 43 +/- 13% (mean +/- SEM) of the cells had converted to the abnormal (hypodense) sedimenting phenotype. When normodense eosinophils from the reference donors were cultured for 3 days in medium supplemented with increasing concentrations of serum from the patients with EMS, their viability increased in a dose-dependent manner to 45%, which was significantly augmented over the effect of normal serum. This eosinophil viability-sustaining activity was inhibited by 76 +/- 7% (mean +/- SEM; n = 3) by the addition of anti-interleukin 5 (IL-5) but not by neutralizing antibodies monospecific for either granulocyte/macrophage colony-stimulating factor (GM-CSF) or IL-3. IL-5, an eosinophilopoietic factor, converts normodense peripheral blood eosinophils in vitro to a hypodense sedimenting form with extended viability and augmented biologic responses to activating stimuli. Thus, the presence of IL-5 in the sera of patients with EMS may contribute to the development and maintenance of the eosinophilia and may regulate the conversion of the peripheral blood eosinophils to the hypodense phenotype with augmented pathobiologic potential.
Antibodies↗
Different mouse mast cell populations express various combinations of at least six distinct mast cell serine proteases.
Mouse serosal mast cells (SMCs) and Kirsten sarcoma virus-immortalized mast cells store large amounts of mast cell carboxypeptidase A and serine proteases in their secretory granules. Secretory granule proteins from 2.6 x 10(6) purified SMCs were separated by NaDodSO4/PAGE, trans-blotted to poly(vinylidine difluoride) membranes, and subjected to amino-terminal amino acid sequencing. Four distinct mast cell serine proteases were identified. With mast cell carboxypeptidase A, these serine proteases comprise the major proteins of mouse SMC secretory granules. Each of the four SMC serine proteases was distinct from the two serine proteases present in mucosal mast cells in the intestines of helminth-infected mice. The secretory granules of a Kirsten sarcoma virus-immortalized mast cell line contained three of the SMC-derived serine proteases and one of the mucosal mast cell-derived serine proteases. Thus, the family of mouse mast cell secretory granule serine proteases has at least six distinct members that can be expressed in different combinations in different mast cell populations.
Amino Acid Sequence↗
Human fibroblasts maintain the viability and augment the functional response of human neutrophils in culture.
When human neutrophils were co-cultured for 72 h with nontransformed human fibroblasts, 69 +/- 3% (n = 13) survived, as compared with survival levels of 2 +/- 1% (n = 15) and 26 +/- 6% (n = 7), respectively, for neutrophils cultured for the same time period in enriched medium alone or supplemented with 10 pM recombinant human granulocyte/macrophage colony-stimulating factor (rh GM-CSF). Conditioned medium from the human fibroblast cultures enhanced neutrophil survival in a dose-dependent fashion to the same level achieved with neutrophil/fibroblast co-cultures, and its soluble viability-sustaining activity was not inhibited by preincubation with neutralizing antiserum against rh GM-CSF. As compared with freshly isolated replicate samples, neutrophils co-cultured with human fibroblasts for 72 h exhibited augmented FMLP-stimulated superoxide production without spontaneous superoxide generation. This striking extension of survival and associated priming for a ligand response by neutrophils co-cultured with human fibroblasts suggests that fibroblasts may contribute to the proinflammatory properties of neutrophils in tissues.
Cell Survival↗
Manfred Martin Mayer: June 15, 1916-September 18, 1984.
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Allergy and Immunology↗
1990 Presidential Address: an alternative structure in departments of medicine for the career development of physician/scientists.
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Career Mobility↗
A morphometric study of normodense and hypodense human eosinophils that are derived in vivo and in vitro.
Hypodense eosinophils were obtained from two patients with the idiopathic hyperosinophilic syndrome (IHES), and hypodense eosinophils were derived by culturing normodense human eosinophils from control donors in the presence of endothelial cells alone, granulocyte/macrophage-colony-stimulating factor (GM-CSF) alone, or GM-CSF and fibroblasts. These eosinophils were examined ultrastructurally and stereologically for alterations in the volume density (Vv) of their electron-dense granules, the Vv of their lucent granules, the Vv of their lipid droplets, the numerical density of their granules with respect to cytoplasm (Nv), and the plasma membrane surface area-to-cell volume ratio (Sv) that might account for their decreased sedimentation density. The hypodense eosinophils that were obtained from the two patients with IHES exhibited a one-third reduction in granule Vv relative to normodense eosinophils from control donors, primarily because of a decrease in granule size. The culture-derived hypodense eosinophils exhibited 10% to 16% decreases in their granule Vv, significant increases in their lucent granules, and a approximately 7.5% decrease in their Sv. Calculation of the cell volume from cross-sectional area measurements showed that the eosinophils that had been cocultured with fibroblasts in the presence of GM-CSF increased their volume by approximately 15%. The eosinophils that had been cocultured with endothelial cells exocytosed some of their granules. In conclusion, a composite of factors including cell swelling, a decrease in the volume of the cytoplasm occupied by granules, and an increase in granule lucency contributes to the hypodense phenotype in vitro, but only cell swelling and hypogranulation are seen in cells from patients with IHES. The latter could reflect the response of 'primed' hypodense eosinophils in vivo to pertinent tissue ligands.
Adult↗
Characterization of cellular dermal infiltrates in human cutaneous mastocytosis.
Biopsies of lesional and nonlesional skin from 14 patients with localized cutaneous or associated systemic mastocytosis were examined by ultrastructural and immunohistochemical techniques. Mast cells within lesions of the dermis were highly variable between patients with regard to cell number and extent of degranulation, although lesional sites consistently contained more mast cells than did nonlesional sites. Two mast cell patterns were identified based upon granule morphology. In biopsies from 8 patients, the majority of granules contained electron-dense amorphous zones; crystalline lattices; and indistinct, incomplete solid scrolls forming parallel lamellae. In biopsies from 6 patients, in addition to these granules, there were also granules composed of electron-dense amorphous zones, reticulated matrices, and/or distinct scrolls with lucent cores interrupted by dense spheres. The granule morphology for the first group (N = 8) was identical with that seen in the preponderant type of skin mast cell of 6 normal control subjects, whereas the granule morphology of the second group (N = 6) displayed an abnormal ultrastructural phenotype for skin that included granule types normally found not only in skin but also in intestinal lamina propria and lung. For individual patients, the patterns of granule ultrastructure were consistent between clinically nonlesional and lesional skin. A minority of cells in both patient groups appeared primitive ultrastructurally, exhibiting rudimentary, Golgi-associated progranules; monocyte-like morphologic characteristics; and mitotic activity. Moreover, when mast cells in lesional skin were screened for a limited panel of surface antigens, they displayed common patterns of reactivity (M718+, HLA-DR/DQ+, CD4+), and in a selected case, immunoelectron microscopy confirmed the presence of these antigens on mast cell plasma membranes. Dermal mast cells from normal donors (N = 6) lack these epitopes. These observations suggest that infiltrates in cutaneous mastocytosis may exhibit phenotypic characteristics not only of cutaneous mast cells, but in some patients also of mucosal mast cells. In either circumstance, the mast cells may display antigenic determinants common to monocyte/macrophages. Concordance of granule phenotype between lesional and clinically uninvolved skin of individual patients furthers the notion that even localized mastocytosis reflects covertly defective systemic mast cell homeostasis.
Adult↗
Evidence for morphologic diversity of human mast cells. An ultrastructural study of mast cells from multiple body sites.
The ultrastructural features of 502 mast cells, including 34,187 granules, were studied from seven human tissue sites (lung, skin, colon, stomach, small bowel, breast parenchyma, and axillary lymph nodes). Granule areas and substructure were detailed according to the microenvironment (lung alveoli, lung bronchi, bowel mucosa, bowel submucosa, breast skin, breast parenchyma, and axillary lymph nodes). Although completely closed, discrete scrolls were present in some mast cell granules at all tissue locations, they were present more frequently in granules from bowel mucosa and lung (scroll-rich morphology). In contrast, most of the mast cell granules from skin, breast parenchyma, axillary lymph nodes, and bowel submucosa were rimmed by incomplete scrolls forming parallel lamellae; centrally, amorphous granular material and/or grating/lattice-like structures occurred (scroll-poor morphology). In breast, the latter granules had a mean granule area almost twice that of all other sites. Individual mast cells having granules with both scroll-rich and scroll-poor features were common in all tissue sites and microenvironments. In fact, 7.8% of mast cells had granules showing at least one completely closed, discrete scroll and grating/lattice-like structures, sometimes within the same granule. These observations indicate that mixed forms occur and that there is considerable morphologic diversity between the predominant mast cell found in mucosa and lung (scroll-rich morphology) and the predominant mast cell found in skin, breast parenchyma, axillary lymph nodes, and bowel submucosa (scroll-poor morphology).
Breast↗
Isolation and molecular cloning of mast cell carboxypeptidase A. A novel member of the carboxypeptidase gene family.
Mast cell carboxypeptidase A has been isolated from the secretory granules of mouse peritoneal connective tissue mast cells (CTMC) and from a mouse Kirsten sarcoma virus-immortalized mast cell line (KiSV-MC), and a cDNA that encodes this exopeptidase has been cloned from a KiSV-MC-derived cDNA library. KiSV-MC-derived mast cell carboxypeptidase A was purified with a potato-derived carboxypeptidase-inhibitor affinity column and was found by analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be a Mr 36,000 protein. Secretory granule proteins from KiSV-MC and from mouse peritoneal CTMC were then resolved by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblotted to polyvinylidine difluoride membranes. Identical aminoterminal amino acid sequences were obtained for the prominent Mr 36,000 protein present in the granules of both cell types. Based on the amino-terminal sequence, an oligonucleotide probe was synthesized and used to isolate a 1,470-base pair cDNA that encodes this mouse exopeptidase. The deduced amino acid sequence revealed that, after cleavage of a 15-amino acid hydrophobic signal peptide and a 94-amino acid activation peptide from a 417-amino acid preproenzyme, the mature mast cell carboxypeptidase A protein core has a predicted Mr of 35,780 and a high positive charge [Lys + Arg) - (Asp + Glu) = 17) at neutral pH. Although critical zinc-binding amino acids (His67, Glu70, His195), substrate-binding amino acids (Arg69, Asn142, Arg143, Tyr197, Asp255, Phe278), and cysteine residues that participate in intrachain disulfide bonds (Cys64-Cys77, Cys136-Cys159) of pancreatic carboxypeptidases were also present in mast cell carboxypeptidase A, the overall amino acid sequence identities for mouse mast cell carboxypeptidase A relative to rat pancreatic carboxypeptidases A1, A2, and B were only 43, 41, and 53%, respectively. RNA and DNA blot analyses revealed that mouse peritoneal CTMC, KiSV-MC, and bone marrow-derived mast cells all express a prominent 1.5-kilobase mast cell carboxypeptidase A mRNA which is transcribed from a single gene. We conclude that mouse mast cell carboxypeptidase A is a prominent secretory granule enzyme of mast cells of the CTMC subclass and represents a novel addition to the carboxypeptidase gene family.
Amino Acid Sequence↗
Cloning and characterization of the mouse gene that encodes the peptide core of secretory granule proteoglycans and expression of this gene in transfected rat-1 fibroblasts.
A mouse liver genomic library was probed with a 450-base pair AccI----3' gene-specific fragment of a mouse bone marrow-derived mast cell proteoglycan cDNA to isolate 15-18-kilobase (kb) genomic clones containing the gene that encodes the peptide core of mouse secretory granule proteoglycans. Based on the nucleotide sequences of its 2.0-3.5-kb subcloned fragments, this mouse gene consists of three exons. The first exon contains 41 base pairs of untranslated nucleotides that are present in the 5' region of the transcript and also encodes the hydrophobic 25-amino acid signal peptide. The second exon encodes a 48-amino acid sequence that would be predicted to be the N terminus of the peptide core after the signal peptide has been removed in the endoplasmic reticulum. The third exon encodes a 79-amino acid sequence that includes the 15 amino acids immediately preceding an alternating serine-glycine 21-amino acid sequence for glycosaminoglycan attachment, and the subsequent C-terminal 43 amino acids; this exon also contains the 424 untranslated nucleotides present in the 3' region of the transcript. Primer extension and S1 nuclease protection analyses were performed to determine the transcription initiation site of the mouse gene. Rat-1 fibroblasts were cotransfected with the selectable marker pSV2 neo and a lambda clone (lambda MG-PG1) to obtain two rat-1 fibroblast cell lines that had the mouse secretory granule proteoglycan gene integrated into their genomes. RNA blot analysis of both cell lines revealed the presence of the 1.0-kb secretory granule proteoglycan peptide core mRNA transcript, indicating that lambda MG-PG1 contained the entire mouse secretory granule proteoglycan peptide core gene including some of the regulatory elements in its promoter region. The gene that encodes the peptide core of mouse secretory granule proteoglycans is the first proteoglycan gene to have its complete exon/intron organization determined and to be transfected and expressed in another cell type.
Amino Acid Sequence↗
IL-5-dependent conversion of normodense human eosinophils to the hypodense phenotype uses 3T3 fibroblasts for enhanced viability, accelerated hypodensity, and sustained antibody-dependent cytotoxicity.
Human peripheral blood-derived eosinophils were assessed for their viability, density, and functional properties after 7 days of culture with purified mouse IL-5 and mouse 3T3 fibroblasts. Whereas none of the eosinophils remained viable after 7 days of culture in the absence of IL-5, 38 +/- 12% and 61 +/- 14% (n = 6, mean +/- SD) of the eosinophils survived in the presence of 1 pM IL-5 alone or 1 pM IL-5 in the presence of 3T3 fibroblasts, respectively (p less than 0.05). Direct contact between the fibroblasts and the eosinophils was not needed for this enhanced IL-5-dependent viability. After 7 days, 66 +/- 7% (n = 6) of the cocultured eosinophils were viable when the two cell types were separated by a 0.4-microns filter. As assessed by density-gradient centrifugation after 7 days of IL-5 exposure, all of the original normodense eosinophils became hypodense. The time course of this conversion was accelerated by the presence of 3T3 fibroblasts. Enhanced helminthic cytotoxicity was maintained by the 7-day cultured eosinophils only if they had been cocultured with fibroblasts. Eosinophils killed 10 +/- 11% (n = 5), 48 +/- 17%, and 31 +/- 15% of the larvae when they were cultured for 7 days in IL-5 alone, in IL-5 in direct contact with 3T3 fibroblasts, or in IL-5 with filter separation of the fibroblasts and the eosinophils, respectively. The ability of IL-5 to induce progenitor cells to differentiate selectively into eosinophils, and of 3T3 fibroblasts to facilitate the IL-5-mediated conversion of normodense eosinophils to hypodense eosinophils with increased viability and antibody-dependent cytotoxicity suggests a role for both hematopoietic and tissue factors in determining the presence and pathobiologic function of activated hypodense eosinophils in patients with hypereosinophilic conditions.
Animals↗