Cell surface expression of mouse macrosialin and human CD68 and their role as macrophage receptors for oxidized low density lipoprotein

Abstract

We have previously identified a 94-to 97-kDa oxidized low density lipoprotein (LDL)-binding protein in mouse macrophages as macrosialin (MS), a member of the lamp family. Earlier immunostaining studies have shown that MS and its human homolog, CD68, are predominantly intracellular proteins. However, using sensitive techniques such as flow cytometry (FACS) and cell-surface-specific biotinylation, we now show that there is significant surface expression of these proteins. FACS analysis of intact cells using mAb FA͞11 showed small but definite surface expression of MS in resident mouse peritoneal macrophages but this was greatly enhanced with thioglycollate elicitation. Biotinylation of intact cells and detergent-solubilized cell preparations followed by immunoprecipitation revealed 10-15% of the total MS content of elicited macrophages on the plasma membrane. Similar results were obtained with untreated RAW 264.7 cells. FACS analysis of intact THP-1 monocytic cells showed minimal surface expression of CD68 on unactivated cells (4% of total cell content). Stimulation with phorbol 12-myristate 13-acetate increased both surface and total CD68 expression considerably. Furthermore, the specific binding at 4؇C and uptake at 37؇C of 125 I-labeled oxidized LDL by activated THP-1 cells was inhibited by 30-50% by CD68 mAbs KP-1 and EBM-11. Thus, although the surface expression of MS͞CD68 at steadystate represents only a small percentage of their total cellular content, these proteins can play a significant role in oxidized LDL uptake by activated macrophages in vitro and could contribute to foam cell formation in atherosclerotic lesions.

Vasculogenic mimicry by bone marrow macrophages in patients with multiple myeloma

“…Separation and culture of bone marrow macrophages and ECs Heparinized bone marrow aspirates were centrifuged on Ficoll gradient and part of the separated mononuclear cells was subjected to FACS (FACSCanto II, Becton Dickinson, San Jose, CA, USA) analysis using the monocyte/macrophage markers anti-CD14 phycocyanin5 (PC5)-labeled monoclonal antibody (mAb) and anti-CD68 fluorescein isothiocyanate (FITC)-labeled mAb (both from Dako, Glostrup, Denmark) after permeabilization with saponin (Ramprasad et al, 1996).…”

CD68/macrosialin: not just a histochemical marker

“…CD68 is able to bind modified LDL, phosphatidylserine, and apoptotic cells; 12,47,48 (iii) CD68 can rapidly shuttle between the plasma membrane and endosomes. 10 Indeed, the intrinsic ability of CD68 to bind and internalize oxLDL provided an option to consider its putative role in atherogenesis and intracellular lipid accumulation.…”

“…11 As was shown in cultured monocyte THP-1 cells, surface CD68 binds oxidized low-density lipoprotein (oxLDL) suggesting a putative role as a receptor for oxLDL. 12 This property of CD68 can be disrupted by anti-CD68 monoclonal antibodies. However, so far, the role of CD68 as a receptor for oxLDL has not been confirmed in vivo or in vitro with the purified protein.…”

Alteration of fracture stability influences chondrogenesis, osteogenesis and immigration of macrophages

“…On the other hand, ED2 remained demonstrable for longer when stability was less. Molecular biological studies with macrosialin mRNA, a specific marker for murine monocytes and macrophages [19,25], have shown a similar kinetics. On day 8 after the fracture a higher concentration was measured in the steel-nailed fractures than in the polypropylene-nailed fractures, but in contrast to the polypropylene-nailed fractures it dropped back again to the initial readings on day 12.…”