Macrophage heterogeneity

It has long been recognized that macrophages isolated from different anatomical sites are heterogeneous with respect to many different properties including size, morphology, density, expression of cell surface proteins, and functional capabilities (reviewed in refs 12, 70). Because macrophage function is dependent, at least in part, on signals received from the immediate microenvironment, it has been suggested that macrophage heterogeneity may arise from the unique conditions within specific tissues. However, macrophages isolated from a given tissue also display heterogeneous functions, thus indicating that multiple mechanisms are probably involved in the generation of macrophage heterogeneity (12, 70). The variety of functions displayed by monocytes makes dissection of heterogeneity difficult, although subsets of monocytes that differ in size (71) or density (72 have been isolated. In particular, these cells show differential hydrogen peroxide production (73), phagocytosis (74), cytotoxicity (75), cytokine release (76), and markers for activation or differentiation (77). A potential problem in understanding the significance of this heterogeneity is that the isolation procedure may select for certain subpopulations of cells, making extrapolation to the in vivo situation uncertain (73, 75, 78, 79). Isolation of monocytes/ macrophages has been shown to have effects upon the levels of expression of certain cell surface molecules (64, 80). Thus, some perturbation of monocyte/ macrophage function might be the result of the purification technique used. Size fractionated monocytes isolated by elutriation show heterogeneous phagocytic capacity (81), cytokine production (77), prostaglandin secretion (77), and activation by GM-CSF for tumour killing and IL-la production (81). The large monocyte subpopulation has been shown to be more phagocytic than the small one (72, 74), more responsive to chemotactic stimuli (73), and exhibited enhanced cytotoxicity against certain target cells (73). In contrast, studies performed by velocity sedimentation separation show that large monocytes release more IL-1(3, prostaglandin E2, and TNFa in response to bacterial lipopolysaccharide (LPS) than do small monocytes (79). Large monocytes also produce more H202 than small monocytes (73). Monocyte subpopulations separated on the basis of their density through discontinuous gradients also differ from one another with respect to various biochemical characteristics. In fact, high density cells express high 5'-nucleotidase activity but low acid phosphatase and peroxidase activity, while the low density cells express low 5'-nucleotidase activity and high acid phosphatase and peroxidase activity (72, 77). Density defined monocyte subpopulations also differ in their expression of various functional activities. In fact, high density monocytes are less active than low density cells in antibody-dependent cell-mediated cytotoxicity reactions (75, 77) and less effective accessory cells in the generation of mitogen-proliferation responses (77). Monocytes are also heterogeneous with respect to their expression of a large number of cell surface proteins including Fc (FcR) and complement (CR) receptors, the structures recognized by some lectins, and various molecules defined by monoclonal antibodies (12, 70). In this regard, it is of interest that phenotypic and functional characterization of Fc-y receptor I (CD64)-negative monocytes defines a minor human monocyte subpopulation with high accessory and antiviral activities (82). Furthermore, it has been reported that CD14low/CD16+ small monocytes, representing around 10% of the monocytes in the human peripheral blood mononuclear cells, produce less TNFa, IL-1, and IL-6 than CD14high monocytes (83, 84). However, the CD14l0W/CD16+ monocyte subset has a higher expression of major histocompatibility complex (MHC) class II proteins (83).

7.2 Comparison of the efficacy of different purification techniques with respect to the source of macrophages

Mouse peritoneal macrophages represent an extensively used model for studies on macrophage functions. In fact, the peritoneal cavity provides an accessible site for harvesting fairly high numbers of resident macrophages, as they represent the major cell population present in the peritoneum (approximately 70%). However, the number of macrophages obtained from the peritoneum in noninflammatory conditions is generally insufficient for large scale studies and differs among various species (85). To increase the yield of macrophages, a sterile peritoneal exudate can be induced by injection of an eliciting agent. Notably, macrophages obtained from a stimulated animal differ from those found in a resident population in that many of the cells are recently immigrated from the circulation and are likely to be morphologically, biochemically, and functionally different. In this regard, differences have been reported in the adherence properties of peritoneal macrophages isolated from elicited mice. In fact, these cells attach less to microexudate-coated plates as compared to resident macrophages (19). Furthermore, differences in function can also be associated with differences in the eliciting agent used (86).

Satisfactory purification of rodent peripheral blood monocytes in suspension has not been achieved so far, since in rat and mice, these cells occur as a minor population of the peripheral blood leukocytes overlapping with lymphocytes in size and density. This problem appears to be restricted to rodent blood. Recently, a two-step procedure for the isolation of monocytes from rat blood, characterized by a high yield and purity, has been described (87). In the human system, the peripheral blood represents the major source of macrophages. Although these cells are present in the pool of mononuclear cells at low abundance (10-15%), their purification can be easily achieved by using physical methods (isopycnic centrifugation or elutriation) which allow a consistent enrichment of the monocyte fraction.

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