摘要:

AbstractAbstract The subcellular localization of a heparan sulfate degrading endoglycosidase, heparanase, was studied in human neutrophils. Unstimulated cells were disrupted by nitrogen cavitation and fractionated on a Percoll density gradient into three components, separating the plasma membranes, specific granules, and azurophilic granules. Heparanase activity was measured by gel filtration analysis of35S‐labeled degradation fragments released from subendothelial extracellular matrix (ECM) or produced during incubation with soluble, ECM‐derived, heparane sulfate proteoglycans. Heparanase activity was found mainly in fractions containing the specific granules; this activity was inhibited by heparin. Freezing and thawing was not needed for recovery of the enzyme from the subcellular fraction, confirming previous data about its ready release. The mechanism of the ready release of heparanase from the specific granules requires further investigation.

ISSN:0741-5400    

DOI:10.1002/jlb.51.6.519

出版商:Wiley    

年代:1992

数据来源: WILEY

摘要:

AbstractAn in vitro system allowing the culture of ovine bone marrow–derived macrophages (BMMs) is described. Bone marrow (BM) cells from the sternum of 4‐to 9‐month‐old sheep were cultured in liquid suspension in hydrophobic bags with medium containing 20% autologous serum and 20% fetal calf serum (FCS). Cells with macrophage characteristics were positively selected and increased four‐ to fivefold between day (d) 0 and d18. Granulocytes and cells of lymphoid appearance including progenitor cells were negatively selected and were diminished 50‐fold during this 18‐d culture. The addition of macrophage colony‐stimulating factor (M‐CSF)–containing supernatants to liquid cultures did not significantly improve the yield of BMM in 18‐d cultures. In contrast, cell survival at d6 and macrophage cell yield at d18 depended on the concentration and source of serum in the culture medium. FCS and 1:1 mixtures of FCS and autologous serum were superior to autologous serum alone. Analysis of growth requirements of ovine BMMs suggested that they are under more complex growth control than their murine counterparts. In an [3H]thymidine incorporation assay with BM cells collected at different times of culture, d3 or d4 BM cells responded to human recombinant M‐CSF, human recombinant granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), bovine GM‐CSF, murine M‐CSF or murine M‐CSF‐containing supernatants, and bovine interleukin 1β (IL‐1β) in decreasing order of magnitude. Likewise, pure murine BMM populations harvested at d6 responded to homologous GM‐CSF, IL‐3, and human or murine M‐CSF. FCS did not stimulate the proliferation of murine BMMs (d6) and of ovine BM cells (d3 or d4). In contrast, ovine BM cells harvested at d12 responded to FCS by proliferation in a dose‐dependent manner but failed to proliferate in the presence of human or murine M‐CSF or M‐CSF‐containing supernatants of mouse and sheep fibroblasts containing mouse macrophage growth‐promoting activity. Likewise, various cytokine‐containing supernatants and recombinant cytokines (murine IL‐3, murine and human GM‐CSF, murine and bovine IL‐1β) did not promote proliferation of ovine d12 BM cells to an extent greater than that achieved with 15% FCS alone. Thus, ovine BMM proliferation is under the control of at least two factors acting in sequence, M‐CSF and an unidentified factor contained in FCS. The ovine BMM culture system may provide a model for the analysis of myelomonocytopoiesis in vitro.

ISSN:0741-5400    

DOI:10.1002/jlb.51.6.525

出版商:Wiley    

年代:1992

数据来源: WILEY

摘要:

AbstractBy selection and cloning in the presence of colony‐stimulating factor 1 (CSF‐1), we have obtained from separate explants of individual 12‐ or 13‐day mouse placentas four clonal cell lines dependent on CSF‐1 for survival and growth in culture. All four cell lines show characteristics consistent with their derivation from placental macrophages. We describe the effects of CSF‐1 on growth, morphology, and CSF‐1 receptor phosphorylation. One cell line, JPL2A, which shows relatively complete growth arrest in the absence of CSF‐1, was characterized in detail with respect to the effects of CSF‐1 on DNA synthesis and protein turnover and its response to the duration of CSF‐1 stimulation. In contrast to previous studies, our results suggest that extended stimulation leads to continuous recruitment of cells competent to reenter the cell cycle. These findings are discussed in terms of growth factor effects on the cell cycle and the usefulness of these cell lines for further study of growth factor signal transduction in macrophages.

ISSN:0741-5400    

DOI:10.1002/jlb.51.6.535

出版商:Wiley    

年代:1992

数据来源: WILEY

摘要:

AbstractThe human C1q receptor (C1q‐R) is a 65–70‐kd, highly acidic, hydrophobic glycoprotein that is expressed on a wide variety of cell surfaces. Although the C1q‐R itself appears to bind preferentially to C1q, the region of the ligand to which C1q‐R binds is the primary binding site for several other molecules, including fibronectin, laminin, and C1q inhibitor (chondroitin 4‐sulfate proteoglycan) as well as the complement C1r2C1s2tetramer. In order to further characterize the C1q‐R molecule with regard to its structure and function, highly purified C1q‐R was obtained from Raji cells using DEAE‐Sephacel and C1q‐Sepharose CL‐4B chromatography. Studies performed with125I‐labeled C1q‐R demonstrated that whereas the C1q‐R molecule binds poorly to a variety of human collagens including types II, III, and V, markedly enhanced binding is observed with type IV collagen and moderately enhanced binding with type I collagen. Amino acid composition studies show that the C1q‐R molecule contains approximately 44% hydrophobic and 12.6% hydrophilic residues with a ratio of negatively charged to positively charged residues of about 2:1. Treatment of125I‐labeled C1q‐R with endoglycosidase F lowers the apparent molecular size from 70 to 58 kd, whereas endoglycosidase H lowered the size to 64 kd. Treatment with neuraminidase, on the other hand, shifted the size of C1q‐R to 60 kd. These results suggest the presence of several highly sialylated complex‐type or high mannose–type N‐linked oligosaccharide side chains. Because purified C1q‐R has a blocked amino terminus, amino acid sequences representing internal fragments of the molecule were generated by electroblotting and in situ enzymatic digestion. When these short sequences were searched against the National Biomedical Research Foundation computer data base, a seven‐amino‐acid sequence, VSWQGQI, showed significant homology (100% and 80% in a five‐amino‐acid overlap, respectively) with the α chains of the human fibronectin (α5β1) and vitronectin (αvβ3) receptors, and to a lesser degree with epidermal growth factor receptor and T cell receptor. A second sequence, ISEDNIR, showed homology with mouse collagen type IV (86% in a six‐amino‐acid overlap), calmodulin (60% in a seven‐amino‐acid overlap), and aLeishmania majorsurface antigen, gp63. These observations seem to predict that C1q‐R has pockets of conserved sequences that are similar to those not only present in its ligand(s) but also in other cell surface receptors that may, in part, fulfill similar functions.

ISSN:0741-5400    

DOI:10.1002/jlb.51.6.546

出版商:Wiley    

年代:1992

数据来源: WILEY

摘要:

AbstractHuman mast cells developed in vitro when cord blood mononuclear cells were cocultured for 3 months with 3T3 embryonic mouse skin fibroblasts. The metachromatic cells that arose in these cultures contained histamine, a functional Fcεreceptor and granule proteases (tryptase, chymase), and they were definitively identified by the ultrastructural demonstration of crystal granules. We present a detailed ultrastructural analysis of this newly available system for the reliable development of human mast cells in vitro and provide criteria for definitive identification of the mast cell and basophil lineages in humans.

ISSN:0741-5400    

DOI:10.1002/jlb.51.6.557

出版商:Wiley    

年代:1992

数据来源: WILEY

摘要:

AbstractInterleukin 1 (IL‐1) production by A/J (A) and C57BL/6J (B6) mouse peritoneal macrophages stimulated with lipopolysaccharide (LPS) was determined. Strain A macrophages produced low levels of soluble IL‐1 bioactivity compared with B6 macrophages. This defect was not reversed by indomethacin, interferon‐γ, phorbol myristate acetate, or calcium ionophore A23187. In contrast, cytosolic IL‐1 bioactivity was similar in LPS‐stimulated A and B6 macrophages. Western blotting revealed that A macrophage supernatants contained lower levels of both 17‐kd IL‐1α and 17‐kd IL‐1β but similar levels of tumor necrosis factor α compared with B6 macrophages. Cytosolic levels of 31‐kd Pro‐IL‐1α and also 31‐kd pro‐IL‐1β were similar in A and 6 macrophages. Oligonucleotide probing indicated that A and B6 macrophages contained similar levels of IL‐1α and also IL‐1β mRNA. These findings indicate that LPS‐stimulated A macrophage culture supernatants contain low levels of both IL‐1α and IL‐1β compared with B6 macrophages and that these defects in IL‐1 production are posttranscriptionally regulated.

ISSN:0741-5400    

DOI:10.1002/jlb.51.6.570

出版商:Wiley    

年代:1992

数据来源: WILEY

摘要:

AbstractAlveolar macrophages (AMs) are important in the host response to aerogenous pulmonary bacterial infections, such asPasteurella haemolytica–inducedpneumonia in cattle. Previous work has shown that AMs enhanceP. haemolytica–mediated pulmonary endothelial cell (EC) damage in vitro. The purpose of this study was to determine the mechanism of AM‐enhanced EC damage using an in vitro AM‐EC coculture system consisting of AMs cultured on culture plate insert membranes and ECs in the underlying chamber. The addition of lipopolysaccharide (LPS) to the culture plate insert chamber resulted in EC damage indicated by51Cr release, which was enhanced in the presence of AMs. To determine the role of AM‐secreted cytokines, recombinant human interleukin 1 α (IL‐1) or tumor necrosis factor α (TNF) was added to ECs simultaneously with varying concentrations of LPS. Although TNF and IL‐1 alone had only marginal toxic effects on ECs, the simultaneous treatment of TNF or IL‐1 with LPS greatly increased the LPS cytotoxic effect on ECs. In addition, IL‐1 receptor antagonist eliminated the IL‐1 enhancement of LPS‐mediated EC toxicity. These results suggest that macrophage‐secreted cytokines synergistically enhance LPS‐mediated pulmonary EC damage.

ISSN:0741-5400    

DOI:10.1002/jlb.51.6.579

出版商:Wiley    

年代:1992

数据来源: WILEY

摘要:

AbstractInterleukin 1 (IL‐1) has been shown to modulate various functional activities of neutrophils, presumably by first binding to cell surface IL‐1 receptors. In this study, we characterized and identified IL‐1 receptors on bovine neutrophils using direct and competitive receptor binding studies and affinity cross‐linking analysis. The results of direct binding studies demonstrated that bovine neutrophils bound125I‐labeled bovine IL‐1 by a single affinity binding site (Kd= 4.6 nM, 5600 binding sites per cell). Competitive receptor binding studies demonstrated that unlabeled recombinant bovine IL‐1β, murine IL‐1α, and human IL‐1β competitively blocked neutrophil binding of125I‐labeled bovine IL‐1β. In contrast, the IL‐1 receptor antagonist (IL‐1ra) demonstrated no detectable binding to bovine neutrophils as judged by direct and competitive receptor binding assays. Affinity cross‐linking of125I‐labeled bovine IL‐1β to neutrophils was used to identify cell surface IL‐1 receptors. Two specific cross‐linked products were observed with molecular sizes of 89 kd (a deduced receptor size of 71.5 kd) and more than 200 kd. The latter may indicate a complex of IL‐1 receptor‐associated signal‐transducing components, IL‐1 receptors, and IL‐1. The presence of 100 nM unlabeled bovine, murine, or human IL‐1 during the receptor binding and cross‐linking reactions prevented the formation of cross‐linked complexes of125I‐labeled bovine IL‐1β and its receptor. In contrast, the IL‐1ra failed to inhibit the formation of cross‐linked complexes of125I‐labeled bovine IL‐1β and its receptor.

ISSN:0741-5400    

DOI:10.1002/jlb.51.6.586

出版商:Wiley    

年代:1992

数据来源: WILEY

摘要:

AbstractIn this study, superoxide formation was not immediately detected when polymorphonuclear leukocytes were treated with linoleyl alcohol–coated, carboxy‐modified latex beads. However, all other measures of neutrophil activation were present. Superoxide was not detected until 30 min after the initial exposure to beads. However, an O−2‐producing, NADPH‐dependent oxidase is active 15 min after exposure. When polymorphonuclear leukocytes are pretreated with cytochalasin B, superoxide production is detected immediately after exposure to the beads. Superoxide secretion after treatment with linoleyl alcohol–coated latex beads is compared with the response to other latex beads. The results imply that neutrophils form a phagolysosome around linoleyl alcohol–coated latex beads that is tightly sealed and does not allow superoxide to escape into the medium where it could be detected by the reduction of ferricytochrome c.

ISSN:0741-5400    

DOI:10.1002/jlb.51.6.591

出版商:Wiley    

年代:1992

数据来源: WILEY

摘要:

AbstractRecent studies have shown that bilateral decentralization (sympathectomy) of the superior cervical ganglia (SCG) of rats sensitized to the parasiteNippostrongylus brasiliensisattenuated the development of pulmonary inflammation following allergen challenge. Sympathectomy inhibited total leukocyte infiltration into lung lavage fluids, particularly neutrophil infiltration. To define the effects of decentralization of the SCG on neutrophil responses, peripheral blood neutrophils of rats were isolated and tested in in vitro chemotaxis and phagocytosis assays. Neutrophils from rats that were sympathectomized 7 days previously displayed a marked reduction in chemotaxis toN‐formyl‐methionyl‐leucyl‐phenylalanine and leukotriene B4compared to neutrophils from sham‐operated or unoperated groups. Although the degree of chemotaxis was greater in blood neutrophils from parasite‐infected rats than from uninfected rats, sympathectomy markedly reduced the chemotactic responses of both groups. In addition, neutrophils of sympathectomized rats were unresponsive to lipopolysaccharide‐induced metabolic activation as assessed by in vitro phagocytosis and oxidative reduction of nitroblue tetrazolium. Thus, decentralization of the SCG of rats affects the chemotactic responses and functions of neutrophils. Understanding the role of the sympathetic nervous system in modulating the behavior of neutrophils will shed light on the interactions between the nervous and immune systems.

ISSN:0741-5400    

DOI:10.1002/jlb.51.6.597

出版商:Wiley    

年代:1992

数据来源: WILEY