摘要:

AbstractWe report that pretreatment of human eosinophils with GM‐CSF, IL‐3, or IL‐5 enhanced the respiratory burst induced by opsonized particles. In order to gain more insight into the intracellular mechanism(s) involved in cytokine priming, the role of [Ca2+], and tyrosine kinases was studied. Optimal priming concentrations of GM‐CSF, IL‐3, and IL‐5 did not induce a rise in [Ca2+]i, and Ca2+‐depleted eosinophils ([Ca2+]i<20 nM) were still primed after preincubation with these cytokines. GM‐CSF, IL‐3, and IL‐5 induced phosphorylation of two proteins (102 and 122 kd) on tyrosine residues, as deduced from Western blot analysis with an antiphospho‐ tyrosine monoclonal antibody (4G10). This cytokine‐ stimulated tyrosine phosphorylation was not inhibited under Ca2+‐depleted conditions. In conclusion, this study demonstrates that GM‐CSF, IL‐3, and IL‐5 priming of the opsonized particle‐induced respiratory burst in human eosinophils is completely Ca2+independent. Moreover the tyrosine phosphorylation of a 102‐kd and a 122‐kd protein is Ca2+independent, suggesting that this event might be involved in cytokine priming.

ISSN:0741-5400    

DOI:10.1002/jlb.53.4.347

出版商:Wiley    

年代:1993

数据来源: WILEY

摘要:

AbstractWe studied the interactions between human neutrophils, as well as the purified human neutrophil serine proteases elastase (HNE) and cathepsin G (HNCG), and laminin. Our results show that intact laminin and two proteolytic fragments generated by HNE bind to neutrophils and stimulate cell migration. Domain‐ specific antilaminin monoclonal antibodies, rotary shadowing electron microscopy, and Western blotting mapped the two promigratory fragments on the laminin cross to the apical three‐armed region and long arm, respectively. In contrast, a fragment derived from the terminal ends of short arms neither bound to neutrophils nor stimulated migration. When neutrophils embedded in a reconstituted basement membrane gel were activated with phorbol myristate acetate, several stable, proteolytic laminin fragments were released into supernatants. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and Western blotting showed that these fragments appeared identical to those generated after digestion of soluble laminin with HNE and HNCG. Furthermore, release of laminin fragments by embedded neutrophils was inhibited by diisopropyl fluorophos‐ phate, and duplicated by incubating the basement membrane gel with purified HNE and HNCG. Our findings therefore suggest that neutrophils, through release of HNE and HNCG, are capable of digesting basement membrane laminin in vivo. In addition, the release of laminin fragments from damaged basement membranes may promote neutrophil migration and thereby accelerate inflammatory processes.

ISSN:0741-5400    

DOI:10.1002/jlb.53.4.354

出版商:Wiley    

年代:1993

数据来源: WILEY

摘要:

AbstractThe ability to activate peripheral blood lymphocytes (PBLs) in vitro with interleukin‐2 (IL‐2) is suppressed by the presence of autologous human pulmonary alveolar macrophages (AMs). AMs suppress both IL‐2‐induced proliferation and the induction of lympho‐ kine‐activated killer cell (LAK) activity in a dose‐ dependent manner (79 ±6% suppression of LAK activity at a 0.25:1 AM/PBL ratio). Increasing the IL‐2 concentration increased baseline LAK activity but did not prevent AM‐mediated suppression. At least two different mechanisms of suppression were observed, one diffusible in nature and the other contact dependent. Indomethacin prevented the component of inhibition that diffused across porous polycarbonate membranes, indicating prostaglandins as the diffusible inhibitor. In contrast, indomethacin had no effect when added alone into conventional AM‐ PBL cocultures, but a combination of indomethacin and anti‐transforming growth factorβ1(TGF‐β1)antibody did prevent inhibition. This result suggests thatTGF‐ß1acts as an additional contact‐dependent inhibitor. PBLs that were rendered unresponsive to IL‐2 completely recovered their responsiveness within4days after removing AMs from the coculture. These features suggest that pulmonary macrophages have multiple mechanisms for locally suppressing IL‐2 responses and lymphocyte activation.

ISSN:0741-5400    

DOI:10.1002/jlb.53.4.366

出版商:Wiley    

年代:1993

数据来源: WILEY

摘要:

AbstractAbstract: Studies of granule‐microtubule interactions in human neutrophils have suggested that mechanochemical ATPases such as kinesin or dynein may play a role in granule mobilization during neutrophil activation by inflammatory signals. In this study we show that proteins extracted from the surface of neutrophil granules, found previously to contain microtubule‐dependent ATPase activity, caused microtubules polymerized from phospho‐ cellulose‐purified rat brain tubulin to move across glass slides. Antibodies were generated against peptides based on two regions of the amino acid sequence ofDrosophilakinesin: the ATPase active site (amino acids 86‐99) in the head of the kinesin heavy chain and the tail of the heavy chain (residues 913‐933). These antibodies were found to recognize kinesin in rat brain extracts as well as kinesin‐ like polypeptides in extracts of human neutrophils. Furthermore, when used in immunoaflmity chromatography, these antibodies permitted the isolation of a protein from neutrophil granule extracts that was recognized byDrosophilakinesin antibodies. Subcellular localization by immunofluorescence microscopy showed this protein to be associated principally with the cytoplasmic granules of neutrophils.

ISSN:0741-5400    

DOI:10.1002/jlb.53.4.372

出版商:Wiley    

年代:1993

数据来源: WILEY

摘要:

AbstractActivated spleen T cells are invasive in hepatocyte and fibroblast cultures, and this property is dominantly expressed in T cell hybridomas. The invasive potential of the hybrids correlates with their capacity to disseminate in vivo. We have used this model to study the invasive and migratory properties of cytotoxic T lymphocytes (CTLs). TWo murine CTL clones were highly invasive, independent of their state of activation. CTL hybridomas, derived from one of the clones, were similarly invasive. In vivo, CTL hybridoma cells disseminated to extravascular sites in the liver, kidneys, lungs, ovaria, tubae, uterus, and lymphoid, mesenchymal, and fat tissues. Within 7 to 14 days, 106cells were lethal in 100% of mice. The adhesion molecules CD2, CD8, CD54, L‐selectin, and CD49d (VLA‐4 and LPAM‐1 α‐chain) were not expressed by all CTL hybridomas and therefore not indispensable for invasion in vitro and dissemination in vivo. In contrast, LFA‐1 (CDlla/CD18), CD44, and VLA‐6 (CD49f/CD29) were expressed on all hybrids. LFA‐1 antibodies inhibited CTL hybridoma invasion in vitro, but antibodies inhibiting CD44‐hyaluronate and VLA‐6‐laminin interaction had no effect. These results suggest that migration of cytotoxic T cells into non‐ inflamed tissues is independent of their activation state and does not require L‐selectin, LPAM‐1, CD2, and VLA‐4.

ISSN:0741-5400    

DOI:10.1002/jlb.53.4.381

出版商:Wiley    

年代:1993

数据来源: WILEY

摘要:

AbstractAbstract: We analyzed the phenotypic changes associated with monocyte activation and differentiation using a newly developed monoclonal antibody (B148.4). Among peripheral blood leukocytes, the antigen recognized by this antibody is expressed on monocytes and granulocytes at high and low density, respectively. Antigen expression is lost during in vitro differentiation of monocytes and is absent on tissue macrophages, indicating that expression of this antigen is related to monocyte differentiation. Only la, 25‐dihydroxyvitamin D3 and phorbol diesters, of several inducers tested, up‐regulate B48.4 antigen expression on cells (monocytes and certain myeloid cell lines) that constitutively bear the antigen, without, however, allowing its maintenance during monocytic differentiation or inducing it on negative cells. By im‐ munoprecipitation from B148.4* U937 cells, the antigen is a complex of a major 116‐kd and two minor 38‐ and 46‐kd molecules. Analysis of eight different tissues reveals that the antigen is shared with endothelial cells. Biochemical characteristics, cellular distribution, and expression pattern on monocytes, myeloid cell lines, and AML cells upon culture with different stimuli indicate that B148.4 is a novel monocyte differentiation antigen.

ISSN:0741-5400    

DOI:10.1002/jlb.53.4.390

出版商:Wiley    

年代:1993

数据来源: WILEY

摘要:

AbstractDevelopmental delays, which impair antibacterial host defense, are present in the neutrophil system of human preterm neonates. We hypothesized that diminished production of interleukin‐8 (IL‐8), a neutrophil chemotactic peptide, might in part be responsible for these defects. To test this, monocytes from the blood of preterm neonates, term neonates, and adults were isolated immunologically by “negative panning” and subsequently stimulated with interleukin‐1α (IL‐lα), tumor necrosis factor a (TNF‐α), or lipopolysaccharide (LPS), after which IL‐8 levels in the supernatants were measured by ELISA. Total cellular RNA was extracted and IL‐8 mRNA was assessed by Northern blotting and by competitive polymerase chain reaction (PCR) analyses. After stimulation with IL‐lα, IL‐8 accumulation was lowest in supernatants of monocytes from preterm neonates, intermediate in supernatants of monocytes from term neonates and greatest from monocytes of adults. Similarly, when stimulated with TNF‐α or LPS, monocytes from preterm neonates produced less IL‐8 than cells from term neonates and adults. These differences in IL‐8 concentrations paralleled differences in IL‐8 mRNA expression.

ISSN:0741-5400    

DOI:10.1002/jlb.53.4.399

出版商:Wiley    

年代:1993

数据来源: WILEY

摘要:

AbstractMedium‐chain triglyceride (MCT) and long‐ chain triglyceride (LCT) emulsions currently used in nutritional therapy were evaluated for their in vitro effect on neutrophil oxidative metabolism, phagocytosis, and bacterial killing activities. Neutrophils from healthy adult male volunteers were assessed after blood incubation with commercially available fat emulsions containing LCT, MCT, or a mixture of 50% MCT and 50% LCT at a final triglyceride concentration of 20 mg/ml. It was observed that MCT‐containing emulsions stimulated nitroblue tetrazolium (NBT) dye reduction by neutrophils as determined by a cytochemical NBT test performed directly on whole blood. This effect was dose dependent. However, after lipid removal by cell washing, the MCT‐treated neutrophils showed decreased production of hydrogen peroxide (H2O2) and NBT reduction in response to bacterial lipopolysaccharide or phorbol my‐ ristate acetate stimuli as well as impaired phagocytosis and killing ofStaphylococcus aureus.In contrast, the LCT emulsion did not alter any of the neutrophil functions evaluated. The present data suggest that MCTs elicit the oxidative metabolism of neutrophils, probably by phagocytosis of fat particles and, depending on the lipid concentration, this effect may not be reversible, leading to impairment of the cellular response to subsequent membrane stimuli.

ISSN:0741-5400    

DOI:10.1002/jlb.53.4.404

出版商:Wiley    

年代:1993

数据来源: WILEY

摘要:

AbstractThe inducible protein p71/73 marks the response of mouse macrophages to one of several stimuli (e.g., bacterial lipopolysaccharide or poly I:C) that trigger the expression of cytolytic activity when these cells have previously been primed for tumor cell killing by interferon‐γ (IFN‐γ). The results reported here identify this marker protein as the inducible prostaglandin endoperoxide synthase (PES), TIS10/PES‐2. Identification was based on four findings: (1) p71/73, like the TIS10/ PES‐2 protein, was associated with cellular membranes; (2) the sequence of amino acids in the NH2 terminus of both p71 and p73 was 96% identical to the predicted NH2‐terminal sequence of the TIS10/PES‐2 protein; (3) a polyclonal antiserum raised against the COOH‐terminal region of the TIS10/PES‐2 gene product recognized p71/73 in immunoblots; and (4) dexamethasone, which blocks induction of TIS10/PES‐2 expression, inhibited the induction of both p71/73 synthesis and tumoricidal activity in macrophage. Several regulatory roles for this protein in the activation process are possible.

ISSN:0741-5400    

DOI:10.1002/jlb.53.4.411

出版商:Wiley    

年代:1993

数据来源: WILEY

摘要:

AbstractFish oils are abundant in polyunsaturated fatty acids of the n‐3 series (in particular eicosapen‐ taenoic, 20:5 and docosahexaenoic acid, 22:6). Such fatty acids are generally considered to be beneficial in the prevention of cardiac disease and to have anti‐inflammatory properties. Neutrophil adherence is an essential early event in an acute inflammatory response, and we have demonstrated that both 20:5 and 22:6 stimulate adherence in vitro. Arachidonic acid (20:4, n‐6) was also stimulatory. Significant stimulation of adherence was seen from 5 to 80μM (nontoxic concentrations) 22:6, 20:5, or 20:4. At the lower fatty acid concentrations tested (≤40μM) 20:5 was less active than 22:6 or 20:4 at stimulating adherence. Above 40μM there was no difference in the ability of the three fatty acids to stimulate adherence. At the lower fatty acid concentrations tested (≤10μM) 22:6 was less active than 20:4, whereas above 10μM they were equally active. Immunofluorescent flow cytometric analysis of neutrophil integrin (adherence) receptors showed that the complement G3bi receptor (GDI lb) was up‐regulated by these fatty acids. There was no change in GDI la or GDllc. Saturated fatty acids of the same chain length were without effect on adherence or receptor expression. The findings suggest that these polyunsaturated fatty acids may, under certain conditions, be proinflammatory with respect to their acute effects on the interaction of neutrophils with microbes, endothelium, and other tissues.

ISSN:0741-5400    

DOI:10.1002/jlb.53.4.420

出版商:Wiley    

年代:1993

数据来源: WILEY