Identification of macrophage external membrane proteins and their possible role in cell adhesion.

Starch-activated mouse peritoneal macrophages (STpMAC) plated on plastic demonstrate the adhesive properties typical for activated pMAC: attaching as round cells and, within 15 min, spreading out with marginal membrane ruffles. These attached STpMAC were labeled by lactoperoxidase-catalysed 125I surface iodination, sodium dodecyl-sulfate-lysed, and the lysates electrophoresed on polyacrylamide gels which were examined by autoradiography. The STpMAC morphological phenotype correlates with the labeling of a particular protein (195,000, estimated mol wt). Normal pMAC (NpMAC), from unstimulated mice, do not spread and do not display the 195,000 band. Both pMAC band patterns, including the 195,000 band, are relatively resistant to trypsin digestion, as is pMAC adhesion itself trypsin-resistant. Neither class of pMAC exhibits fibronectin (Cell Adhesion Factor, LETS protein) which is a component in the adhesive matrix of cells forming trypsin-sensitive monolayers. When pMAC are tested against antifibronectin antibody, these cells do not give immunofluorescent staining. In summary, two functions in pMAC adhesion, enzyme resistance and the ability to spread, appear related to molecular properties distinctive for pMAC surface protein.

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Mouse macrophage elastase. Purification and characterization as a metalloproteinase

Biochemical Journal ◽

10.1042/bj1930589 ◽

1981 ◽

Vol 193 (2) ◽

pp. 589-605 ◽

Cited By ~ 167

Author(s):

M J Banda ◽

Z Werb

Keyword(s):

Culture Medium ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Serine Proteinase ◽

Peritoneal Macrophages ◽

Serine Proteinases ◽

Endogenous Inhibitor ◽

Purification And Characterization ◽

Predominant Form ◽

Mouse Peritoneal Macrophages

Macrophage elastase was purified from tissue-culture medium conditioned by inflammatory mouse peritoneal macrophages. Characterized as a secreted neutral metalloproteinase, this enzyme was shown to be catalytically and immunochemically distinct from the mouse pancreatic and mouse granulocyte elastases, both of which are serine proteinases. Inhibition profiles, production of nascent N-terminal leucine residues and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of degraded elastin indicated that macrophage elastase is an endopeptidase, with properties of a metalloproteinase, rather than a serine proteinase. Macrophage elastase was inhibited by alpha 2-macroglobulin, but not by alpha 1-proteinase inhibitor. Macrophage elastase was resolved into three chromatographically distinct forms. The predominant form had mol.wt. 22 000 and was purified 4100-fold. Purification of biosynthetically radiolabelled elastase indicated that this form represented less than 0.5% of the secreted protein of macrophages. Approx. 800% of the starting activity was recovered after purification. Evidence was obtained for an excess of an endogenous inhibitor masking more than 80% of the secreted activity.

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Effects of cytochalasin B on actin and myosin association with particle binding sites in mouse macrophages: implications with regard to the mechanism of action of the cytochalasins.

The Journal of Cell Biology ◽

10.1083/jcb.91.2.373 ◽

1981 ◽

Vol 91 (2) ◽

pp. 373-384 ◽

Cited By ~ 39

Author(s):

R G Painter ◽

J Whisenand ◽

A T McIntosh

Keyword(s):

Binding Sites ◽

Cytochalasin B ◽

Peritoneal Macrophages ◽

Immunofluorescent Staining ◽

Transmission Electron ◽

Mouse Macrophages ◽

Particle Binding ◽

Punctate Pattern ◽

Particle Ingestion ◽

Mouse Peritoneal Macrophages

The intracellular distribution of F-actin and myosin has been examined in mouse peritoneal macrophages by immunofluorescence microscopy. In resting, adherent cells, F-actin was distributed in a fine networklike pattern throughout the cytoplasm. Myosin, in contrast, was distributed in a punctate pattern. After treatment with cytochalasin B (CB), both proteins showed a coarse punctate pattern consistent with a condensation of protein around specific foci. After CB-pretreated cells were exposed to opsonized zymosan particles, immunofluorescent staining for F-actin and myosin showed an increased staining under particle binding sites. Transmission electron microscope (TEM) examination of whole-cell mounts of such preparations revealed a dense zone of filaments beneath the relatively electron-translucent zymosan particles. At sites where particles had detached during processing, these filament-rich areas were more clearly delineated. At such sites dense arrays of filaments that appeared more or less randomly oriented were apparent. The filaments could be decorated with heavy meromyosin, suggesting that they were composed, in part, of F-actin and were therefore identical to the structures giving rise to the immunofluorescence patterns. After viewing CB-treated preparations by whole-mount TEM, we examined the cells by scanning electron microscopy (SEM). Direct SEM comparison of the filament-rich zones seen by TEM showed that these structures resulted from the formation of short lamellipodial protrusions below the site of particle binding. Electron micrographs of thin-sectioned material established that these lamellipodial protrusions were densely packed with microfilaments that were in part associated with the cytoplasmic surface of the plasma membrane. The formation of particle-associated lamellipodia did not appear to represent merely a slower rate of ingestion in the presence of CB, because they formed within minutes of particle contact with the cell membrane and were not followed by particle ingestion even after a 1-h or longer incubation. Furthermore, their formation required cellular energy. These results suggest that cytochalasin B blocks phagocytosis of large particles by affecting the distances over which any putative actomyosin-mediated forces are generated.

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Iodinated fibroblast β-glucuronidase as a ligand for receptor-mediated endocytosis

Biochemical Journal ◽

10.1042/bj2290213 ◽

1985 ◽

Vol 229 (1) ◽

pp. 213-219 ◽

Cited By ~ 3

Author(s):

M F Dean ◽

S Diment ◽

C Ostlünd ◽

B M Jenne ◽

S Contractor

Keyword(s):

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Peritoneal Macrophages ◽

Affinity Column ◽

3T3 Cells ◽

Receptor Mediated Endocytosis ◽

3T3 Fibroblasts ◽

Different Types ◽

Mannose Receptors ◽

Mouse Peritoneal Macrophages

Antibodies raised to human placental beta-glucuronidase were shown to cross-react with the beta-glucuronidase secreted by mouse 3T3 fibroblasts, but did not react with other lysosomal enzymes. The beta-glucuronidase secreted by 3T3 cells was purified 15000-fold by chromatography on an affinity column made from this antibody and resolved into a single component, of Mr 68000, by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Iodinated samples of purified enzyme were taken up into mouse peritoneal macrophages by receptor-mediated endocytosis at a rate similar to that calculated previously for unlabelled enzyme, and uptake was competitively inhibited by yeast mannan. Binding of beta-glucuronidase to macrophages was saturable, with a Kd of 7 × 10(-9)l/mol, an affinity comparable with that calculated for the binding of mannosylated bovine serum albumin (Kd 1.3 × 10(-9)l/mol), a ligand specific for mannose receptors. Four times as many molecules of mannosylated albumin (12000) as of beta-glucuronidase (3000), however, bound to each cell. This purification and iodination procedure did not therefore have any adverse effect on the uptake properties of secreted beta-glucuronidase, and provides a ligand with which to investigate binding and specific endocytosis into a range of different types of cell.

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Structural evidence for distinct IgG subclass-specific Fc receptors on mouse peritoneal macrophages

Journal of Experimental Medicine ◽

10.1084/jem.152.5.1147 ◽

1980 ◽

Vol 152 (5) ◽

pp. 1147-1161 ◽

Cited By ~ 24

Author(s):

BC Lane ◽

J Kan-Mitchell ◽

MS Mitchell ◽

SM Cooper

Keyword(s):

Binding Proteins ◽

Sodium Dodecyl ◽

Fc Receptors ◽

Peritoneal Macrophages ◽

Fc Receptor ◽

Rabbit Igg ◽

Mouse Macrophages ◽

Specific Proteins ◽

Sepharose 4B ◽

Mouse Peritoneal Macrophages

Membrane proteins which selectively bind to the Fc portion of IgG were identified in the Nonidet P-40 extracts of radiolabeled thioglycollate- elicited mouse peritoneal macrophages. Affinity columns of various IgG preparations coupled to Sepharose 4B were used to absorb the Fc-binding proteins. Analysis of the acetic acid or sodium dodecyl sulfate (SDS) eluates from aggregated human IgG or antigen-complexed rabbit IgG columns revealed two Fc(gamma)/-specific proteins with apparent 67,000 and 52,000 mol wt. These proteins were not detected in acid or SDS eluates from F(ab')(2) columns or in eluates from IgG column, over which were passed lysates of Fc receptor-negative cells. With the use of affinity columns that contained aggregated mouse myeloma proteins of different IgG subclasses, we found that the 67,000-dahon protein selectively binds to IgG2a, whereas the 52,000-dalton protein binds to IgG1 and IgG2b. Neither protein was found in SDS eluates from IgG3 columns. Trypsin treatment of the macrophages before detergent lysis removed the 67,000-dalton protein, although it leaves intact the 52,000-dalton protein. These results provide structural confirmation for the existence of separate Fc receptors on mouse macrophages and indicate that the two Fc-binding proteins identified in this study represent all or part of the trypsin- sensitive Fc receptor which binds IgG2a and the trypsin-resistant Fc receptor which binds IgG2b and IgG1.

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