Myeloid cells play a central role in inflammation. In the early acute stage, tissue resident macrophages (MΦ), granulocytes and mast cells, as well as the complement system and soluble inflammatory mediators, all contribute to the host response to injury. Later, MΦ recruited to the inflammatory lesion play a dominant role in the maintenance and resolution of inflammation 1. The ability to track leukocyte movements in various mouse models of inflammation has been invaluable in defining the roles of the selectins 2, integrins 3, Fc-receptors 4, complement 5 and chemokines 6–8 in the evolution of inflammatory cell recruitment and the subsequent propagation of inflammation.
Studies of murine inflammation often rely on laborious assessment of cellular infiltrates by differential cell counting or by indirect assay of cell recruitment [such as the presence of myeloperoxidase as a marker of neutrophils (Neu) recruitment], as there are currently no definitive markers available for the specific flow-cytometric identification of murine myeloid cells either in naive or immune challenged mice 5–9. Several markers for staining murine myeloid cells are readily available, which provide a limited ability to identify andcharacterize myeloid cells. CD11b is highly expressed on many myeloid cells, such as monocytes (Mo), Neu, eosinophils (Eos) and subsets of MΦ 10. However, CD11b is notably absent from the surface of many MΦ such as alveolar MΦ 11, several MΦ populations in the spleen and Kupffer cells in the liver 12, 13. Furthermore CD11b is expressed by NK cells 10, some dendritic cells (DC) 14 and subsets of lymphocytes. F4/80 is oneof the best characterized MΦ markers present on most tissue MΦ, but it is expressed at low levels on alveolar MΦ and Mo and not expressed by some subpopulations present in the splenic white pulp and the thymus 15. This marker, however, is also not unique to MΦ, its expression being detected on Eos16 and subsets of DC 17. The Gr-1 epitope, which is found on two receptors Ly-6G and Ly-6C has been useful in the initial identification of Neu, by their high expression 18, but it has now become apparent that many cell types (Neu, Mo, Eos, plasmacytoid pre-DC and subsets of T cells 18–21) express this epitope. An additional useful but uncharacterized Neu marker, the polymorphic 7/4 antigen, has been shown to be expressed on Neu22 and subsets of immunologically activated MΦ 23, 24, and has been successfully used to deplete myeloid committed cells from murine bone-marrow 25, 26. We have recently identified the murine β-glucan receptor (βGR) 27 and using a novel mAb (2A11) demonstrated that βGR was predominantly expressed on Mo, Neu and MΦ 28, 29. Expression of βGR was notably absent from Eos, but was detected on some DC and at lower levels on Gr-1+ T cells and plasmacytoid DC 29. We have also generated monoclonal antibodies against the murine MΦ mannose receptor (MR) 30, which is mainly expressed by MΦ and selected endothelial cells in situ31. Although surface expression seems to be low, compared to the intracellular pool, we have been able to assess surface expression by FACS on several isolated MΦ populations 29.
To characterize myeloid cells directly and to define new criteria for their rapid ex vivo identification we have utilized the above mentioned receptors and differentiation antigens in combination. Both 7/4 and βGR in conjunction with the more established myeloid markers CD11b, F4/80 and Gr-1 provide an efficient approach to the analysis of resting leukocyte populations from multiple tissues. Furthermore, the extended phenotypes that we have defined in this manuscript are retained during several models of local and systemic inflammation validating this approach for general myeloid cell analysis during immunological challenge. These studies highlight the significant heterogeneity of expression of these antigens within the Mo/MΦ-lineage, allow recently recruited Mo to be distinguished from other inflammatory and resident myeloid cells present in a lesion and form a basis for future characterization of myeloid cell subpopulations and the analysis of cell influx/efflux and death at the site of injury.