Quantitative analysis of total macrophage content in adult mouse tissues. Immunochemical studies with monoclonal antibody F4/80.

Abstract

Because of the widespread dispersion of mononuclear phagocytes throughout the body, there is little quantitative information on their total mass, relative numbers in different sites, or mobilization and redistribution in normal individuals or disease states. It is difficult to obtain such information by direct isolation of macrophages (M401 from all sites where they are known to occur and from tissues in which they are deeply embedded. The pool size of M4~ and their precursors in different compartments has been estimated by van Furth and his colleagues (1) by single-cell analysis after [SH]thymidine labeling, but this method favors enumeration of cells that turn over relatively rapidly, rather than more quiescent resident cells. In principle, antibodies that are specific for M4~ could be used to quantitate the content of antigen (Ag) in tissues directly, without cell separation. F4/80, a rat monoclonal antibody (Ab) directed against a plasma membrane glycoprotein of apparent Mr 160,000, is a specific and sensitive marker for mature mouse Mq~ after isolation (2) and in situ (3). In this study we have assayed F4/80 Ag content in various tissues of normal adult mice by adapting an absorption immunoassay developed by Williams and his colleagues (4) to measure lymphoid differentiation Ag in tissue lysates. We estimated M4~ number by calibrating F4/80 Ag content in a murine M4~ tumor-derived cell line, J774.2. Our results are in good agreement with immunohistochemical findings, and establish that relatively large amounts of F4/80 Ag are found not only in hemopoietic and lymphoid tissues, but also in other sites such as the gastrointestinal tract and normal kidney. This experimental approach provides a basis for further studies on the response of the mononuclear phagocyte system to inflammation and infection. Materials and MethodsAnimals. Tissues, organs, and peritoneal cells were obtained from 8-10-wk-old male mice of the C57BL/6 strain weighing 23 _ 2 g. Swiss mice, Pathology Oxford (PO), were used to prepare M~ target cells for binding assays. This work was supported in part by the Wellcome Trust and the Medical Research Council, UK. Address correspondence and reprint requests to S. Gordon. Preparation of M$ Target Cells. PO mice aged 8-10 wk were injected with 1.0 ml of thioglycollate broth intraperitoneally and peritoneal cells (TPC) harvested 4-6 d later. Cells were cultivated at 37°C in 5% CO~ in Dulbecco's modified Eagle's medium (DME) with 10% fetal calf serum (FCS) in 96-well plates (Sterilin Ltd., Middlesex, United Kingdom [UK]), at a density of 1 x 105 M4~ per well, for 2 d. They were then washed free of nonadherent cells, fixed with 0.125% vol/vol glutaraldehyde, and excess glutaraldehyde removed by washing and further incubation with 10% FCS in phosphatebuffered saline (PBS). Plates were stored in PBS with 10 mM sodium azide at 4°C for up to 12 wk. Preparation of Tissue and Cell Extracts.Organs: Animals were freshly killed by ether and organs removed into ice-cold extraction buffer (PBS containing 3 mM io...

Noninflammatory phagocytosis of monosodium urate monohydrate crystals by mouse macrophages: Implications for the control of joint inflammation in gout

“…Our alignment is based on expression of the macrophage markers F4/80 and BM 8 ( Figure 1A), and the ability of cell lines to phagocytose latex microparticles ( Figure 1B). The F4/80 antigen is recognized as the prototypical differentiation marker of mature macrophages in mice (16,17) and has been previously used to demonstrate the presence of mature macrophages in the synovial membrane of normal joint tissues (10). Consistent with previous phenotyping studies of mouse monocyte/macrophage cell lines (18), F4/80 and BM 8 markers closely correlated in expression.…”

Monocyte/macrophage expression of ABCA1 has minimal contribution to plasma HDL levels

“…It is therefore unlikely that a major part of cholesterol in HDL originates from the macrophages. However, macrophages may act as the initial donors of phospholipids and cholesterol to initiate the formation of mature HDL especially because the liver, where nascent HDL is formed from secreted apolipoproteins, contains the largest macrophage population in the body (approximately 20% of total body macrophages) (19). Once the mature HDL is formed, it can obtain cholesterol by other receptormediated pathways such as SR-BI (5) or from the lipolysis of remnant lipoproteins.…”

“…This study also suggests that steady state HDL levels represent the contribution from many tissues that express ABCA1 as opposed to only those with the greatest need for cholesterol efflux such as macrophages. A conservative estimate for the total number of macrophages in a mouse is approximately 10 8 cells (19). These cells comprise a very small portion of the total peripheral cells in the body.…”

Intestinal macrophages: differentiation and involvement in intestinal immunopathologies

“…Accordingly, intestinal macrophages are most abundant in the colon with slightly lower frequencies in the SI. Hence, the mucosa of the small and the large intestine represents the largest reservoir of tissue macrophages in humans [45] and mice [46]. Under homeostatic conditions, most of the intestinal macrophages are located in the subepithelial mucosa, i.e., at the preferential site of antigen entry (Fig.…”