Although all of the protocols outlined above can be used successfully, it must be acknowledged that techniques based upon the ability of monocytes/macrophages to adhere to different surfaces may exhibit several disadvantages. They include poor cell viability, low yield, and contamination of cell preparations with other cell types. Based on the assumption that macrophages are not the only cell types capable of adhering to plastic or glass surfaces, adherence techniques alone cannot yield a 100% pure macrophage population. The recovered adherent cells generally contain cell types other than macrophages in small, but appreciable, numbers. In addition, macrophages are a heterogeneous group of cells that may differ in their adherence capacity. Thus, adherence-based techniques may not result in a recovery of all subpopulations in proportion comparable to that present in their starting cell sample. In the light of the well-known effect of adherence on macrophage activation, this method cannot be utilized for purification of macrophages to be used in a short-term assay, if the cells are to be considered 'non-activated'. Macrophages isolated by adherence-based methods should be allowed to revert to a non-activated status for at least 24 hours. Many of the activation parameters have returned to their basal levels at that time (26-28, 30, 32, 34). Alternatively, if activation functions of monocytes/macrophages are to be studied, it may be more advantageous to perform these studies on cells purified by physical methods. In fact, macrophages obtained by these procedures generally are considered to be in a 'non-activated' state. The use of physical methods of separation are discussed further in Section 6.
5.1 Effects of different surfaces on macrophage morphology and functions
The surface to which the monocytes/macrophages adhere can influence their morphology, survival, differentiation, and functional activities. In this regard, differences in cell phenotypes have been shown to be induced by cultivation on different substrates. In particular, it has been reported that in vitro differentiation of human peripheral blood monocytes to macrophages is dependent on the conditions of monocyte culture (39). Cultivation of monocytes on plastic, glass, or microexudate-coated glass gives rise to cells exhibiting epithelioid-like morphology. In comparison, cultivation of monocytes on collagen matrices results in cells resembling human resident tissue macrophages. The two cell populations differ in morphology, phagocytic activity (39, 58), receptors (39), and surface antigen expression (39). In addition, culture of monocytes on plastic or glass surfaces induces the expression of non-specific, extracellular cytotoxic activity against tumour target cells (45, 58). In contrast, monocytes cultured on collagen-coated surfaces do not develop a significant cytotoxic activity (45). It has also been reported that culture of monocytes on plastic surfaces, but not on FN-coated surfaces, results in some changes in the biosynthesis of polysaccharides
(59). The nature of the substrate has also been shown to modulate the expression of some calcium-binding proteins (MRP8 and MRP14), involved in the process of substrate adherence (60). In particular, adherence of monocytes to laminin, FN, or collagen results in an enhanced surface expression of MRP8 and MRP14, whereas adherence to untreated plastic surfaces is completely ineffective and resembles culture in suspension (60). Additional effects of adherence to different substrates have been described by Gudewicz and co-workers (61). These authors reported that adherence to collagen matrices primes monocytes to an increased response to phorbol ester stimulation of respiratory burst and arachidonic acid metabolism. In contrast, no such priming effect is observed in monocytes adhered to FN or laminin.
5,2 Effects of different detachment procedures on macrophage physiology
In early studies, results obtained after adherence of monocytes/macrophages to solid surfaces with subsequent removal of cells by a rubber policeman (62), were not completely satisfactory because of the variability in the number of viable cells recovered and of their poor responsiveness to activating stimuli (54, 63). However, membrane damage can be significantly reduced by incubating the adherent monocytes with ice-cold PBS prior to their removal by scraping (64). Subsequently, it was found that it is possible to coat Petri dishes with a microexudate from different cells before incubation of mononuclear cells and to remove adherent monocytes/macrophages by EDTA (51). This method, however, has the drawback of needing plastics conditioned by a previously resident adherent cell line. A modification of this method has been subsequently described based on pre-treatment of plastic surfaces with serum (52). Effective release of monocytes/macrophages has also been achieved by means of lignocaine solutions. Initial studies in which the lignocaine method was employed reported a variable release of monocytes/macrophages and, in particular, very low viability of the recovered cells (56). The finding that the pH of the lignocaine solution was a critical determinant of the recovery, viability, and reproducibility of the separation procedure permitted development of a modified lignocaine method that has been more successful (57 and Protocol 7). No consistent difference among the various detachment techniques is generally observed with regard to cell recovery, viability, adherence, morphology, neutral red uptake, phagocytosis of yeast cells, non-specific esterase staining, chemotaxis, and contamination by T and B lymphocytes. Methods such as EDTA treatment may be less damaging to the cell membrane than scraping. However, this compound has been reported to remove or alter the structure of adhesion molecules on the cell surface, rendering them unrecognizable to specific monoclonal antibodies (65). Moreover, the metabolism of macrophages released from glass or plastic by EDTA treatment may be depressed and cells should be allowed to recover from the separation procedure for one to several hours before physiological measurements are attempted (18).
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