To study cytokine regulation of the 5-lipoxygenase (5-LO)/leukotriene (LT) synthase pathway we have developed mouse bone marrow-derived mast cells (BMMC) that minimally express each protein of the pathway by using a novel culture system, lacking interleukin (IL)-3. When mouse bone marrow cells were cultured for 5 weeks with 100 ng/ml c-kit ligand (KL) and 10 units/ml IL-10, a population of >95% mast cells was obtained.

To examine the freeze-fracture appearance of membrane alterations accompanying the preparation of sperm membranes for fusions-the first preparatory stage occurring before physiological release of the acrosomal content, the second afterward-we induced the acrosome reaction in capacitated guinea pig spermatozoa by adding calcium to the mixture. The most common features observed before fusion of the acrosomal and plasma membranes were the deletion of fibrillar intramembranous particles from the E-fracture faces of both membranes, and the clearance of globular particles from the P face of the plasma membrane-events taking place near the terminus of the equatorial segment. Large particles, >12 nm, remained not far from the cleared E-face patches. The P face of the outer acrosomal membrane is virtually clear from the outset. In addition, when fusion was completed, occasional double lines of large particles transiently embossed the P face of the plasma membrane (postacrosomal) side of the fusion zone. Behind the line of fusion, another series of particle-cleared loci emerged. We interpreted these postfusion membrane clearances as a second adaptation for sperm-egg interaction. Induction of the acrosome reaction in media containing phosphatidylcholine liposomes resulted in their apparent attachment, incorporation, or exchange in both the originally and secondarily cleared regions. Our observations support the concepts that membranes become receptive to union at particledeficient interfaces, and that the physiologically created barren areas in freezefracture replicas may herald incipient membrane fusion.After epididymal maturation, four stages of the spermatozoon's life are critical for successful interaction with the egg (4,6,8,38). One step is activation, the change in the rate and form of the tail movement from that of a slow-driven windshield wiper to a faster, undulating whip. Another is capacitation, an ill-defined phenomenon normally occurring in the female genital tract while the sperm membranes are undergoing modification to facilitate fusion between the acrosomal and plasma membranes. The third phase is this fusion itself, the acrosome reaction. The last crucial episode is the preparation of the plasma membrane for fusion with the egg.The intramembranous events associated with these important stages have barely been examined

Leukotriene (LT) C4 synthase is an integral membrane protein that catalyzes the conjugation of LTA4 to reduced glutathione to form LTC4. LTC4 synthase has been cloned and characterized from transformed cell lines, but the protein has not been defined from a tissue source. LTC4 synthase was purified to homogeneity from human lung tissue, utilizing S-hexyl glutathione chromatography followed by LTC4 affinity chromatography. A greater than 100,000-fold purification with a yield of 8 to 25% (n = 4) was achieved. The purified LTC4 synthase migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as an 18-kD protein, and its 19 N-terminal amino acid sequence is identical to that of purified LTC4 synthase from KG-1 myeloid cells or from expression cloning of a KG-1 library in COS cells. Using a rabbit polyclonal IgG raised against purified LTC4 synthase, SDS-PAGE immunoblotting of LTC4 synthase from human lung tissue, eosinophils, KG-1 cells, and platelets showed an 18-kD protein. Immunofluorescence staining of alveolar macrophages in human lung sections with the anti-LTC4 synthase IgG revealed LTC4 synthase to be largely perinuclear in distribution. Thus, LTC4 synthase, the biosynthetic enzyme responsible for the formation of cysteinyl LTs, is present in lung tissue in a form apparently identical to that of hematopoietic cells.

SummaryGranulocytes with the hybrid characteristics of eosinophils and basophils have been identified in the bone marrow and peripheral blood of humans with myeloid leukemias. We now describe a technique by which such hybrid granulocytes can be developed in vitro from normal cord blood precursors cultured in the presence of recombinant human interleukin (rhlL) 3 (350 pM) and rhlL-5 (200 pM) in a plastic vessel coated with Matrigel TM. After 14 d in culture, 90 +_ 3% (mean _+ standard error of the mean) of the nonadherent cells cultured in the Matrigel-coated flasks contained both eosinophil and basophil granules, as indicated by staining with Wright's and Giemsa stains. Of the nonadherent cells, 93 +_ 1% contained cyanide-resistant peroxidase, and 88 _+ 2% were toluidine blue-positive, characteristic of eosinophil and basophil granules, respectively. Transmission electron micrographs showed hybrid cells containing ultrastructurally distinct eosinophil granules with developing crystalline cores and basophil granules with reticular structures. These 14-d cord blood-derived cell cultures showed strong hybridization signals for eosinophil-derived neurotoxin by RNA blot analysis and contained 78 ng histamine per 106 cells. When the granulocytes were removed from cytokine-containing medium and suspended without Matrigel in RPMI 1640 medium containing 10% fetal calf serum (FCS), more than 80% of the granulocytes excluded trypan blue for as long as 5 d, and 93% had developed into eosinophils at 6 d. Conditioned medium prepared over 48 h from the 14-d cell cultures (hybrid granulocytes) sustained the 4-d viability in vitro of 78% of peripheral blood eosinophils from atopic donors. In comparison, 13 % survived in RPMI 1640 containing 10% FCS alone. This viability-sustaining activity was nearly completely neutralized by an anti-granulocyte/macrophage colony-stimulating factor (GM-CSF) antibody and was only minimally reduced by anti-IL-3 or -IL-5. Thus, cells possessing both eosinophil and basophil granules by both histochemical and ultrastructural analysis can be developed from normal progenitors in vitro in response to eosinophilopoietic cytokines and Matrigel. Their subsequent spontaneous development into mature eosinophils suggests that hybrid granulocytes are part of a normal developmental sequence during eosinophilopoiesis, Furthermore, these hybrid granulocytes are capable of autoregulation through elaboration of GM-CSF, which sustains their viability.