摘要:

AbstractFibronectin (Fn) is a high molecular‐weight glycoprotein that can influence many aspects of monocyte function. The purpose of this study was to determine whether Fn could stimulate monocyte tumor necrosis factor (TNF) secretion. Monocytes were isolated from the peripheral blood of healthy volunteers by density gradient centrifugation and adherence to plastic (2 h). Plasma Fn was purified from the blood by gelatin‐Sepharose affinity chromatography. Monocytes were stimulated with Fn for 18 h and the supernatants were assayed for TNF activity using the L929 bioassay. Intact Fn stimulated the secretion of TNF in a dose‐dependent manner. Intact Fn–induced TNF secretion by monocytes was inhibited (50%) but not eliminated by the addition of the R‐G‐D–containing peptide GRGDSP. Limited proteolysis of the Fn molecule using insoluble chymotrypsin resulted in a fragment preparation that was dramatically more stimulatory than the intact Fn preparation. High‐performance liquid chromatographic (HPLC) purification of the fragments demonstrated that at least two fragments were capable of stimulating TNF secretion. Further purification by affinity chromatography and HPLC localized the stimulatory activity to the 120‐kd cell‐binding fragment. The possibility that the stimulatory activity was the result of endotoxin contamination was ruled out using macrophages from C3H/Hej mice. These results suggest that Fn fragments are potentially important molecules for activation of monocytes and may stimulate monocyte cytotoxic activity.

ISSN:0741-5400    

DOI:10.1002/jlb.51.1.59

出版商:Wiley    

年代:1992

数据来源: WILEY

摘要:

AbstractAntineutrophil cytoplasmic autoantibodies (ANCA) react with proteins found in the granules of neutrophils and the peroxidase‐positive lysosomes of monocytes, including myeloperoxidase (MPO), proteinase 3 (PR‐3), and elastase. ANGA‐associated diseases, such as Wegener's granulomatosis and polyarteritis nodosa, are characterized by necrotizing vascular inflammation. The inflammatory lesions typically contain both neutrophils and mononuclear phagocytes, with the latter sometimes predominating, for example, in the granulomatous lesions of Wegener's granulomatosis. We investigated the presence of the ANCA target antigens PR3, MPO, and elastase in mononuclear phagocyte cytoplasm during the course of differentiation in vitro and in alveolar and peritoneal macrophages. We observed that ANCA antigens were down‐regulated during mononuclear phagocyte differentiation, with the loss corresponding to that of peroxidase‐positive granules. This suggests that ANCA can directly interact only with monocytes and early exudative macrophages and not with mature macrophages.

ISSN:0741-5400    

DOI:10.1002/jlb.51.1.65

出版商:Wiley    

年代:1992

数据来源: WILEY

摘要:

AbstractMacrophages derived in vitro from bone marrow progenitors (bone marrow–derived macrophages, BMDMs) using either macrophage colony‐stimulating factor (CSF‐1) or granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) as the myelopoietic stimulus display differential functional, morphological, and mRNA phenotypes. The data presented here demonstrate further that CSF‐1– and GM‐CSF–derived BMDMs differ in immunologic capacity. GM‐CSF–derived BMDMs, when compared to CSF‐1–derived BMDMs, showed greater cytolytic activity against tumor necrosis factor α (TNF‐α)–resistant, but not TNF‐α–sensitive, tumor targets. In contrast, CSF‐1–derived BMDMs produced nitrite in response to lipopolysaccharide (LPS) alone, whereas GM‐CSF–derived BMDMs required interferon γ plus LPS treatment. The two BMDM populations also showed differential sensitivities to LPS for secretion of TNF‐α and nitrite, but the maximal inducible amounts of these factors and prostaglandin E2were similar between the BMDM populations. Lastly, GM‐CSF–derived but not CSF‐1–derived BMDMs showed an L‐arginine–dependent listeriacidal activity. These results show that the functional heterogeneity of CSF‐1– and GM‐CSF–derived macrophages is limited and appears to result largely from differences in the activational signals required by each BMDM population to elicit a given function.

ISSN:0741-5400    

DOI:10.1002/jlb.51.1.69

出版商:Wiley    

年代:1992

数据来源: WILEY

摘要:

AbstractThe visible excitation and emission wavelengths of the recently developed fluorescent Ca2+indicator fluo‐3 permit analysis of the intracellular Ca2+concentration, [Ca2+]i, in flow cytometry with a 488‐nm argon laser. The role of [Ca2+]iin human polymorphonuclear leukocyte heterogeneity was investigated in response to formyl‐methionyl‐leucyl‐phenylalanine (fMLP), C5a, and interleukin 8/neutrophil attractant/activation protein 1 (IL‐8/NAP‐1) by flow cytometry. The [Ca2+]ichanges in different subpopulations within a heterogeneous cell suspension were resolved upon stimulation with fMLP. Using an anti‐CD16 phycoerythrin‐conjugated antibody and fluo‐3 simultaneously, neutrophils affected and nonaffected in Ca2+mobilization were distinguished in two patients suffering from glycogen storage disease type lb. In normal neutrophils, a different time course of Ca2+mobilization of neutrophil subpopulations immediately after stimulation with fMLP was detected. In addition, after stimulation with a low concentration of IL‐8/NAP‐1 (10‐10M) two subsets of neutrophils appeared; one of them showed an increase in [Ca2+]i, while the other did not. These results indicate heterogeneity in the neutrophil signal transduction process involved in Ca2+mobilization. Therefore, flow cytometric analyses can resolve changes in single‐cell [Ca2+]idistribution patterns, which is important for the understanding of [Ca2+]iin neutrophil heterogeneous activation processes.

ISSN:0741-5400    

DOI:10.1002/jlb.51.1.77

出版商:Wiley    

年代:1992

数据来源: WILEY

摘要:

AbstractGranule‐poor human neutrophil cytoplasts, prepared without heat or cytochalasin B treatment so as to preserve both motile function and activatable respiratory burst oxidase, were investigated for their content of several isoforms of protein kinase C (PKC). Immunoblotting with isoform‐specific rabbit antibodies (Abs) to PKC revealed that both the α‐specific and the β(I and II)‐specific Abs recognized a protein band of 78 kd comigrating with PKC from rat brain cytosol. The γ‐specific antiserum did not detect any protein of this molecular mass. The cytoplast β‐PKC band was more readily detected than the cytoplast α‐PKC band. Antibodies to βI‐ or β‐II‐specific PKC sequences showed the βII subtype to be the predominant form of β‐PKC, although some βI was also found. The identity of the 78‐kd cytoplast bands as PKC was established by the fact that phorbol ester treatment of intact cytoplasts induced translocation of the bands from cytosol to membrane fractions. However, whereas PKC specific activity was similar in cytoplast lysates and brain cytosol, immunoreactivity of cytoplast α‐ and β‐PKC bands was considerably less than that of rat brain. Hydroxylapatite chromatography of partially purified cytoplast PKC revealed two major peaks of PKC activity precisely coeluting with brain a‐ and β‐PKC and displaying comparable enzymatic activities despite the relatively weak immunoreactivity of cytoplast α‐and β‐PKC. To our knowledge, this is the first demonstration that human neutrophil–derived cytoplasts contain α, βI, and βII forms of PKC and that each isoform translocates from cytosol to membrane upon exposure to phorbol ester at concentrations that induce superoxide production. In addition, our evidence raises the possibility that cytoplasts may also possess other isoforms of PKC that we are unable to detect with our α, β, and γ antibodies. Finally, the granule‐poor cytoplasts seem a particularly useful preparation in which to examine the role of individual PKC isoforms in neutrophil activation.

ISSN:0741-5400    

DOI:10.1002/jlb.51.1.84

出版商:Wiley    

年代:1992

数据来源: WILEY

摘要:

AbstractIn this brief definitive report, we show that over a 6‐h period and under serum‐free conditions, recombinant monocyte‐macrophage colony‐stimulating factor 1 (rCSF‐1) and lipopolysaccharide (LPS) synergize and induce macrophages to express higher levels of mRNA for interleukin 1α (IL‐1α), IL‐1β, tumor necrosis factor α (TNF‐α), and IL‐6 and to release more bioactivity than macrophages treated with LPS alone. This synergy was regulated by the amount of LPS in the culture medium. Paraformaldehyde‐fixed macrophages likewise showed augmentation of IL‐1 activity, but whereas all of the bioactivity associated with the fixed macrophages could be neutralized by anti‐IL‐1α antibody only ≍ 40% of the supernate activity could be attributed to IL‐1α. Preliminary data suggest that the augmenting effect induced by CSF‐1 cannot be explained solely on a quantitative basis because the addition of rIL‐1α to supernates of macrophages treated with LPS alone or with the combination of LPS and CSF‐1 resulted in an increase in thymocyte mitogenic activity to a level that could not be explained on an additive basis. Although the supernates contained TNF and IL‐6, antibody neutralization assays made it unlikely that these were directly responsible for the augmenting effect. These results suggest that CSF‐1 not only enhances basic genetic responses induced by LPS alone but also may induce a mechanism that amplifies cytokine bioactivity.

ISSN:0741-5400    

DOI:10.1002/jlb.51.1.93

出版商:Wiley    

年代:1992

数据来源: WILEY

ISSN:0741-5400    

DOI:10.1002/jlb.51.1.xii

出版商:Wiley    

年代:1992

数据来源: WILEY