Abstract
Mac-2, a 30-35-kDa galactose-binding protein, is synthesized at similar levels in murine peritoneal exudate macrophages whether recruited in response to an intraperitoneal pathogen Mycobacterium microti, to sterile inflammatory stimuli such as thioglycollate broth, or to concanavalin A. In elicited or activated macrophages up to 30% of Mac-2 is constitutively secreted, and secretion is stimulated markedly by calcium ionophore A23187. Only thioglycollate-elicited macrophages express cell surface Mac-2, and binding is mostly (> 80%) a result of affinity for cell surface carbohydrate structures. Mac-2 surface expression is markedly reduced upon further activation of thioglycollate-elicited macrophages with bacterial lipopolysaccharide in vitro. Polylactosamine structures are present on all macrophage populations examined as determined by binding of Lycopersicon esculentum lectin, whereas alpha-galactosyl residues detected by Griffonia simplicifolia isolectin B4 are expressed only on the thioglycollate-elicited macrophages, indicating that these residues are the major determinants responsible for Mac-2 surface expression. Chemical cross-linking experiments have identified binding of endogenous cell-surface Mac-2 to three glycoproteins of molecular masses of 92, 125, and 180 kDa containing alpha-galactosyl and polylactosamine structures on thioglycollate-elicited macrophages. The restricted cell surface distribution of Mac-2 on thioglycollate-elicited peritoneal macrophages, a population of recently recruited monocytes, suggests a role(s) in early events of macrophage infiltration and tissue fixation such as extravasion and cell-matrix interactions.
Highlights
A-galactosyl and polylactosamine structureosn thiogly- Suggestive evidence for extracellular roles comes from the findcollate-elicited macrophages
Detailed studies haveshown that Mac-WCBP30 secretion ing andsequencing studies haveshown that Mac-2 homologues from BHK cells and its targetedsecretion from apical domains are present in other rodent and human inflammatorymacro- of polarized Madin Darby canine kidney cells are independent phages (Cherayil et al, 1989, 1990) and further thatMac-2 is of the normalsecretory pathway traveledby most secretedand identical or closely similar to galactoside-binding proteins of plasma membraneproteins (Sat0 et al, 1993)
K CBP30hlac-2 namely L. esculentum lectin, G. simplicifolia isolectin B4, li: conophowas from BHK cells, and specific polyclonal rabbit antibodies were as rum agglutinin, or CBP30 all at 5 pg/ml final concentration
Summary
Min, and thencleared by centrifugation at 12,000x g for 10 minat 2 "C. Samples (0.4 ml) of each fraction were precleared with normal rabbit. Isolation of Lectin-Ligand Conjugates-Adherent cells labeled with [36Slmethioninefor 3 h a t 37 "C as described above were washed with plicifolia isolectin B4, biotinylated Lycopersicon esculentum (tomato) cold PBS and lysed in lysis buffer B, 1%Triton X-100, 20 mM Tris, pH lectin, and water-solublebiotin (longarm) NHS were fromVector Labo- 7.2,0.15 M NaCI, 1m~ phenylmethylsulfonyl fluoride, unitdml aproratories, Peterborough, U.K. Tetracarpidium conophorum agglutinin tinin (0.5 ml) a t 2 "C. K CBP30hlac-2 namely L. esculentum lectin, G. simplicifolia isolectin B4, li: conophowas from BHK cells, and specific polyclonal rabbit antibodies were as rum agglutinin, or CBP30 all at 5 pg/ml final concentration The latter described (Sato and Hughes, 1992). Harvested peritoneal exudate (1:lv/v) in lysis buffer A, the beads werewashed, solubilized, and anacells (5 x lo5) were washed in cold PBS; resuspended in PBS, 0.1% lyzed by SDS-polyacrylamide gelelectrophoresis as before. M e r incubation a t 37 "C for 3 h, cell monolayers were washed
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