After earlier accounts of macrophage research, Ilya Metchnikoff, a Russian zoologist known to be the father of cellular immunity, established the idea of a phagocyte system in the last decade of the nineteenth century (Gordon, 2008). Already after a few years, several different names had been given to these phagocytosing cells. The “reticulo-endothelial system” (RES) was coined by Aschoff to include a group of cells capable to take up intravenously injected diluted lithium carmine (Aschoff, 1924, Guilliams et al., 2014). RES was then replaced by the so-called “mononuclear phagocyte system” (MPS), as proposed in the late 1960s by Van Furth and colleagues to indicate a network of cells including monocytes and macrophages (resident and infiltrating), with the premise that all macrophages are derived from blood monocytes (van Furth et al., 1972). In the meantime, Wiseman (1934) introduced the term “histiocyte” to designate large cells with voluminous granulated cytoplasm, sometimes containing engulfed particles, usually found in lymph nodes and spleen; then this term was used as a synonym for the fully differentiated end cells of the MPS, derived from a CD34+ common precursor (Moore et al., 2014). Currently, but still in evolution over time, histocytes are considered cells of both the monocyte/macrophage and Langerhans/dendritic lineages, although dendritic cells (DCs) were initially excluded from MPS and their inclusion is still controversial (Jenkins and Hume, 2014, Moore, 2014). Also, the initial concept that “all macrophages are derived from blood monocytes” was definitely challenged and it is now clear that macrophages can self-renew in tissues and monocytes arise in three different waves: yolk sac, fetal liver, and then, after birth, from the bone marrow hematopoietic stem cell (Hoeffel and Ginhoux, 2018). However, although too simplistic as initially formulated, the MPS concept remains and can be considered as valid today as when it was originally conceived (Hume et al., 2019).

The last review on macrophage-dendritic cell lineage diseases in human medicine was published in 2016 (Emile et al., 2016) while the more updated one in the veterinary field was available in 2014 (Moore, 2014). As shown in both the cited reviews, the cellular populations of different histiocytic disorders/diseases (HD) display different immunophenotypes and thus distinct differentiation lineages within the MPS. Understanding this heterogeneity is relevant to define different therapeutic approaches according to the underlying pathogenesis. Overall, when a predominant histiocytic infiltrate is present, two main mechanisms are hypothesized for its recruitment: a) infectious disease, macrophages are primarily involved with phagocytosis and digestion of foreign antigens (Gross et al., 2005b); b) an immunomodulatory dysfunction, dendritic cells are poorly phagocytic and are specialized in processing and presenting antigens (Gross et al., 2005a, Gross et al., 2005c). In the first option, macrophages are among the first-line defense cells in granulomatous diseases, in the second option dendritic cells proliferate and accumulate. The therapeutic options can thus range from antibiotics to immunomodulatory agents.

In this scenario, it would be of great interest to evaluate the proportion of macrophages and dendritic cells in a tissue and/or in a given inflammatory population. With the use of the following antibodies, we aimed to identify macrophages within histiocytic cells: 1) an anti-Ionized Calcium Binding Adapter Molecule 1 (Iba-1), as a marker of all histiocytes, not able to differentiate between macrophages and dendritic antigen-presenting cells; 2) an Anti-S100A9 + Calprotectin (S100A8/A9 complex) antibody, MAC387 clone known as Macrophage + Monocytes antibody; 3) a macrophage marker Antibody Panel (CD11b, CD68, CD163, CD14, CD16). To independently distinguish macrophages from other histiocytic cell populations we used normal skin and skin from dogs infected with Leishmania infantum, since amastigotes live and multiply within macrophages.

A combination of these mentioned staining allowed to differentiate macrophages within the whole histiocytic infiltrate.