摘要:

AbstractInterleukin‐8 (IL‐8) such as LUCT (lung giant cell carcinoma‐derived chemotactic protein), NAP (neutrophil activating protein) and MDNCF (monocyte‐ derived neutrophil chemotactic factor), and formylmethionyl‐ leucyl‐phenylalanine (fMLP) are well‐known chemoattractants for human polymorphonuclear leukocytes (PMNs) and are able to stimulate phosphorylation of 64‐kd protein (p64) in these leukocytes. To elucidate the molecular mechanism of PMN activation with chemoattractants, we investigated the phosphorylation process of p64 in an intact cell. 32P‐Labeled PMNs were stimulated with LUCT/IL‐8, fMLP, leukotriene B4, or C5a, and phosphorylated proteins were analyzed by two‐dimensional electrophoresis and autoradiography. A marked phosphorylation of p64 was observed after stimulation. A new spot of phosphorylated p64 (pp64) could be detected on the gel stained with Coomassie Brilliant Blue, indicating that the isoelectric point (pI) of p64 shifted from 5.3 to a more acidic pI by the phosphorylation forming pp64. The spot of pp64 was shown to be dephosphorylated to p64 by treatment with calf intestine alkaline phosphatase. Other proteins having molecular masses 82, 66, 58, 55 and 50 kd were also phosphorylated. The fMLP‐ stimulated phosphorylation was time‐dependent and saturated within 5 min. Maximum stimulation was achieved with 10 nM fMLP. Phosphoamino acid analysis revealed phosphorylation of serine residues in pp64. Staurosporine (100 nM) and W‐7 (100 μΜ) significantly inhibited the phosphorylation of p64, but H‐7 slightly inhibited it. H‐8 and herbimycin A did not effect phosphorylation. Phorbol myristate acetate was found to stimulate significantly. Protein kinase C did not stimulate the phosphorylation. These data suggest that protein kinase C and calmodulin‐like protein are indirectly involved in the phosphorylation of p64 during chemoattractant‐activation of PMN.

ISSN:0741-5400    

DOI:10.1002/jlb.54.1.1

出版商:Wiley    

年代:1993

数据来源: WILEY

摘要:

AbstractLung giant cell carcinoma‐derived chemotactic protein (LUCT)/IL‐8 and fMet‐Leu‐Phe stimulate phosphorylation of a 64‐kd protein (p64) in 32P‐labeled human polymorphonuclear leukocytes (PMNs). The p64 was purified from cytosol of human PMNs (1.8 × 109 cells) by DEAE‐Sepharose CL6B column chromatography, hydroxyapatite HPLC, and reverse‐phase HPLC. By hydroxyapatite HPLC, p64s were separated and produced two peaks containing both nonphosphorylated and phosphorylated p64. Amino acid composition of each p64 was determined. Each p64 was directly subjected to amino acid sequencing, but the amino acid residue in the amino‐terminal of the proteins could not be detected. From the results of amino acid composition and other characters of p64, it is suggested that p64 is identical to I‐plastin, which is a leukocyte‐specific protein.

ISSN:0741-5400    

DOI:10.1002/jlb.54.1.10

出版商:Wiley    

年代:1993

数据来源: WILEY

摘要:

AbstractAbstract: Alveolar macrophages acquire lα‐hydroxylase activity in inflammation, and thereby metabolize 25 hydroxyvitamin D3 (25 D3) to the active metabolite, la,25‐dihydroxyvitamin D3 (1,25 D3, calcitriol). Cal‐ citriol is a potent differentiation agent that modulates mononuclear phagocyte activation and effector functions. The mediators that induce macrophage lα‐hydroxylase activity are not well delineated. Furthermore, it is unclear whether calcitriol is a product only of terminally differentiated macrophages or whether less mature mononuclear phagocytes can produce it as well. The ability of newly recruited monocytes to produce calcitriol as an autocrine differentiation agent is particularly important in inflammation, as it may substantially expand the functional repertoire of these cells. To assess the effects of cytokines on lα‐hydroxylase activity, blood monocytes were cultured in the presence and absence of human recombinant tumor necrosis factor a (TNF‐α), interferon‐7 (IFN‐γ), and interleukins 1 and 2 and then incubated with 25 D3 substrate. The conditioned media were assayed for calcitriol by high‐performance liquid chromatography and competitive receptor binding assay. No detectable calcitriol was produced by unstimulated monocytes. However, all the cytokines markedly increased monocyte calcitriol production (range 133‐151 pg/mg protein; in all cases P<.001). We then determined whether calcitriol production was suppressed by preincubation with either dexamethasone or the putative uremia toxin guanidinosuccinic acid (GSA). Dexamethasone pretreatment significantly inhibited subsequent cytokine‐induced calcitriol production by monocytes, as did GSA (average 69 and 63% of control, respectively).

ISSN:0741-5400    

DOI:10.1002/jlb.54.1.17

出版商:Wiley    

年代:1993

数据来源: WILEY

摘要:

AbstractTumor necrosis factor a (TNF‐α) has been shown to be an important mediator of the lethal effects of endotoxin in several experimental models of septic shock. However, studies with a recombinant human interleukin‐1 (IL‐1) receptor antagonist protein (IL‐lra) suggest a role for IL‐1 as a mediator of septic shock as well. In the present study, we show that mice treated in vivo with Corynebacterium parvum are primed for the production of interferon‐γ (IFN‐γ) and exhibit an enhanced capacity to produce serum IL‐Ια, TNF‐a, and IL‐6 when challenged intravenously with lipopolysaccharide (LPS). The majority of C. parvum ‐treated mice die within 24 h of an LPS challenge. Pretreatment with a rat antimouse TNF‐α monoclonal antibody (mAb) protected 90% of the animals against the lethal endotoxin challenge, while an anti‐IFN‐γ mAb gave approximately 75% protection. The anti‐IFN‐7fgamma; mAb also caused a reduction in LPS‐ induced serum TNF‐α and IL‐Ια. Anti‐IL‐la, anti‐ IL‐1β, and anti‐IL‐6 neutralizing mAb did not protect against lethality when administered to mice prior to the LPS challenge. These results indicate that TNF‐α and IFN‐γ are major mediators of endotoxin shock in C. parvum‐treated mice. The results further suggest that the IFN‐γ produced by C. parvum‐primed mice in response to an LPS challenge serves as a stimulus for enhanced production of TNF‐α and IL‐Ια. These findings are consistent with an increasing body of evidence suggesting a major role for IFN‐γ in lethal endotoxemia.

ISSN:0741-5400    

DOI:10.1002/jlb.54.1.23

出版商:Wiley    

年代:1993

数据来源: WILEY

摘要:

AbstractFlow cytometry, immunohistology, and SDS‐ PAGE Western blot analysis were used to characterize two novel anti‐mouse mononuclear phagocyte differentiation antigens defined by monoclonal antibodies. One antibody, Monts‐4, recognized an 80‐100 kd cell‐surface protein expressed on resident macrophages in the peritoneum and stained macrophages in the splenic white pulp and marginal zone, liver, lymph node paracortical regions, Peyers patches, and cortex of the thymus. Monts‐4 did not stain blood monocytes or monocyte‐derived inflammatory macrophages in the peritoneal cavity. Macrophages that were Monts‐4 positive became Monts‐4 negative within 24‐72 h after culturing in vitro. Monoclonal antibody SK39 recognized a 180 kd cell‐surface molecule expressed on inflamed peritoneal monocytes/macro‐ phages and stained macrophages in the splenic red pulp, cortex of the thymus, subcapsule and medullary regions of lymph nodes, and in sites of acute and chronic inflammation. No differences were seen in the expression of these antigens in the immunodeficient SCID versus normal BALB/c mouse. A comparison of the distribution and molecular weights of the Monts‐4 and SK39 antigens with other described mononuclear phagocyte‐specific antigens, including F4/80 and those defined by the Ml/70, ERTR9, MOMA1, MOMA2, and SER4 monoclonal antibodies, shows these to be novel mononuclear phagocyte‐specific differentiation antigens.

ISSN:0741-5400    

DOI:10.1002/jlb.54.1.30

出版商:Wiley    

年代:1993

数据来源: WILEY

摘要:

AbstractWe examined leukemic cells, HL‐60, an acute promyelocytic leukemia cell line, after differentiation induced by 1,25‐dihydroxyvitamin D3 (D3) and retinoic acid (A) for infection of Legionella pneumophila, the etiologic agent of Legionnaires' disease. We investigated the effect of interferon γ (IFN‐γ) on the differentiated cells and on the intracellular growth of the bacteria. An examination of morphological and antigenic changes in the cells was also included in the study. After 4‐day incubation with 10‐6M D3 or A, the HL‐60 cells differentiated into monocyte‐like (D3‐HL‐60) or mature granulocyte‐like (A‐HL‐60) cells, respectively. They were then infected with L. pneumophila. Intracellular multiplication of the bacteria was evident in D3‐HL‐60 cells but not in HL‐60 or A‐HL‐60 cells. D3‐HL‐60 cells required a 24‐h infection time for the intracellular growth of L. pneumophila. D3‐HL‐60 cells activated with human recombinant IFN‐7 for 1‐24 h (γ‐IFN‐D3‐HL‐60 cells) before infection markedly inhibited L. pneumophila multiplication, the effect of IFN‐γ being dose dependent. Surface marker analysis was carried out in HL‐60, D3‐HL‐60, and 7‐IFN‐D3‐HL‐60 cells. On D3‐HL‐60 cells, CDllb, CDllc, CD14, and CD35 antigen increased, whereas CD71 and HLA‐DR antigen decreased. This finding suggested that HL‐60 cells differentiated into monocyte‐like cells; the acquisition of the complement receptors, CDllb(CR3) and CD35(CR1), seemed to be important for phagocytosis and for the subsequent intracellular multiplication of L. pneumophila. The γ‐IFN‐ D3‐HL‐6O cells showed an increase of CD16, CD36, CD71, and HLA‐DR antigen, suggesting that they were in an activated state. Our study indicated, first, that D3 can induce human leukemic cells to differentiate into functional monocyte‐macrophage‐like cells that can support the intracellular multiplication of L. pneumophila and, second, that these differentiated leukemic cells can be activated by IFN‐γ to markedly inhibit bacterial growth.

ISSN:0741-5400    

DOI:10.1002/jlb.54.1.40

出版商:Wiley    

年代:1993

数据来源: WILEY

摘要:

AbstractIndividual steps of granulocyte maturation, such as lineage commitment, proliferation, maturation, and migration from the marrow to the peripheral blood, may be influenced by distinct interactions with the bone marrow stroma. To identify candidates of membrane components involved in maturational stage‐specific interactions, we studied changes in the expression of cell adhesion molecules along the granulocyte maturational pathway. Three‐color flow cytometric measurements were used to measure levels of cell adhesion molecules along this pathway. The α chains of VLA‐4 (CD49d) and VLA‐5, the inte‐ grin 01 chain (CD29), and CD31 (PECAM‐1) were expressed in high density on all early myeloid cells but down‐modulated during postproliferative maturation. CD44 and L‐selectin were expressed on CD34+ myeloid progenitor cells and mature granulocytes but down‐ modulated during the intermediate stages of maturation. The granulocyte receptor for endothelial selectins, sLex, was specifically expressed by myeloid progenitor cells. sLex was down‐modulated during the intermediate stages of granulocyte maturation but up‐regulated again during terminal maturation. In contrast, CD67, a putative granulocyte adhesion molecule, was negative on progenitors, transiently up‐regulated during the intermediate stages of maturation, and almost absent from the surface of mature granulocytes. These results show that each stage of granulocyte maturation is associated with the expression of a unique combination of cell adhesion molecules. L‐selectin, CD44, and 01 integrins were regulated as previously described for immature lymphopoietic cells and may therefore play general roles in the compartmen‐ talization and development of leukocytes. In contrast, sLex and CD67 were specifically expressed by myeloid cells and could be specifically important for compartmen‐ talization of distinct phases of granulocyte maturation.

ISSN:0741-5400    

DOI:10.1002/jlb.54.1.47

出版商:Wiley    

年代:1993

数据来源: WILEY

摘要:

AbstractThe decline of plasma fibronectin after surgery, trauma, and burn, as well as during severe sepsis after injury, appears to limit hepatic Kupffer cell phagocytic activity. Intravenous infusion of gelatin‐coated particles to simulate blood‐borne particulate collagenous tissue debris in the circulation after injury also depletes plasma fibronectin. We used soluble gelatin conjugated with 1251‐labeled dilactitol tyramine (DLT‐gelatin) as a model of soluble collagenous tissue debris. We studied its blood clearance as well as organ localization in normal and postburn rats. Fibronectin‐deficient plasma harvested early after burn exhibited limited ability to support in vitro phagocytic uptake of the gelatinized microparticles by Kupffer cells in liver tissue from normal rats. However, Kupffer cells in liver tissue from normal and postburn rats phagocytized the test particles at a normal rate when incubated in normal plasma. The DLT‐gelatin ligand bound to fibronectin in a dose‐ dependent manner as verified by its capture with anti‐ fibronectin coated plastic wells when coincubated with purified fibronectin. By gel filtration chromatography, the binding of fibronectin with the DLT‐gelatin ligand was readily detected, resulting in the formation of a high‐ molecular‐weight complex. In normal animals the plasma clearance and liver localization of 125I‐DLT‐gelatin was competitively inhibited by infusion of excess nonradioactive gelatin. The blood clearance and liver localization of the soluble gelatin ligand were also impaired after burn injury during periods of fibronectin deficiency similarly to the pattern observed with gelatin‐coated microparticles. By autoradiography, the cellular site for the uptake of the 1251‐DLT‐gelatin was primarily but not exclusively hepatic Kupffer cells; 125I‐DLT‐asialofetuin and 125I‐ DLT‐ovalbumin were removed by hepatocytes and sinusoidal endothelial cells, respectively. Thus, gelatin conjugated with 125I‐DLT can be used to simulate blood‐ borne soluble collagenous tissue debris after burn. It rapidly binds to plasma fibronectin before its hepatic Kupffer cell removal, and its blood clearance is markedly delayed after burn injury during periods of plasma fibronectin deficiency.

ISSN:0741-5400    

DOI:10.1002/jlb.54.1.56

出版商:Wiley    

年代:1993

数据来源: WILEY

摘要:

AbstractAbstract: Tumor necrosis factor a (TNF‐α) more than doubles tritiated thymidine ([3H]TdR) uptake in mouse macrophages stimulated by macrophage colony‐ stimulating factor (CSF‐1). However, nothing is known of how TNF‐α affects this increase or even whether it is manifested by increased cellular proliferation. Here we characterize the effects of TNF‐α on CSF‐l‐stimulated proliferation of both primary cells (bone marrow‐derived macrophages, BMMs) and a cloned growth factor‐dependent macrophage cell line (SI). We show that the TNF‐ α‐induced increase in [3H]TdR uptake of CSF‐l‐stimu‐ lated macrophages is directly proportional to an increase in the DNA content of the culture and that the effects of TNF‐α are direct and independent of cell number. TNF‐a decreases the population doubling time of log‐phase growing macrophages having quite different growth rates to the same (approximately 30%) extent: the doubling time of BMMs decreases from 24 to 17 h and that of SI cells from 17 to 13 h. TNF‐α exerts its effects on log‐phase growth by increasing to the same proportion CSF‐l‐stimulated proliferation at all concentrations of CSF‐1; that is, TNF‐α does not shift, but rather amplifies, the CSF‐1 dose‐response curve. Although TNF‐α alone does not stimulate macrophage proliferation, its presence in S1 cell cultures coming to quiescence after withdrawal of CSF‐1 greatly increases subsequent CSF‐l‐stimulated [3H]TdR uptake as the cells reenter the cycle. Finally, we show that both human and mouse TNF‐α increase CSF‐l‐stimulated log‐phase growth and reentry of quiescent cells into the cycle equally on a molar basis (halfmaximal stimulation of approximately 0.3 nM). The latter observation argues that the growth‐stimulatory effects of TNF‐α are mediated via the 55‐60‐kd TNF receptor. We conclude that TNF‐α acts directly on growth‐ competent macrophages to decrease significantly the population doubling time in a manner that enhances the mitogenic effects of CSF‐1.

ISSN:0741-5400    

DOI:10.1002/jlb.54.1.65

出版商:Wiley    

年代:1993

数据来源: WILEY

摘要:

AbstractThe mechanisms responsible for asbestos‐ induced pulmonary epithelial cell cytotoxicity, especially oxidant‐independent mechanisms, are not established. We determined whether human polymorphonuclear leukocyte (PMN) proteases contribute to asbestos‐induced damage to human pulmonary epithelial‐like cells (PECs) assessed using an in vitro chromium‐51 release assay. Serine antiproteases, phenylmethylsulfonyl fluoride and αι ‐antitrypsin, each ameliorated PEC injury induced by amosite asbestos and PMNs. A role for a specific proteinase, human neutrophil elastase (HNE), is supported by the facts that (1) asbestos increased HNE release assessed by an enzyme‐linked immunosorbent assay technique (1.7 ± 0.5 vs. 2.8 ± 0.5 μg/ml; P<.025), (2) purified HNE or porcine pancreatic elastase (PPE) each alone caused PEC detachment, (3) asbestos plus either HNE or PPE caused PEC lysis similar to that mediated by asbestos and PMNs, and (4) cationic agents released from PMNs were unlikely to be involved because polyanions did not ameliorate injury resulting from asbestos and PMNs. Compared to elastase, cathepsin G caused less PEC detachment and negligible augmentation in asbestos‐ induced PEC lysis. Asbestos increased the association of 125I‐labeled elastase with PECs nearly 50‐fold compared with PPE alone (14.4% vs. 0.3%, respectively; P<.01) and nearly 10‐fold compared with another particle, opsonized zymosan. We conclude that PMN‐derived proteases, especially elastase, may contribute to asbestos‐ induced lung damage by augmenting pulmonary epithelial cell injury.

ISSN:0741-5400    

DOI:10.1002/jlb.54.1.73

出版商:Wiley    

年代:1993

数据来源: WILEY