Mononuclear phagocytes, comprising circulating monocytes and tissue macrophages, are phylogenetically ancient cells deriving from the primordial amoeba that was enclosed into a complex pluricellular organism. During the course of evolution, macrophages retained some of the basic features of the amoeba, such as mobility and phagocytosis, and acquired new immunoregulatoiy properties as a result of the interaction with the emerging immune system. Engulfing bacteria and fighting with competitors are among the primordial functional activities of macrophages that pre-existed the development of the immune system and that are triggered by classical environmental stimuli such as bacteria, toxins, organic molecules, pH, osmotic changes, etc. The need to communicate with the immune system and with the neighbouring cells caused profound modifications in the number and nature of the signals recognized by macrophages, that learned to recognize distress signals derived from lymphocytes, such as lymphokines and antibodies, or from tissues, such as hormones, adhesion molecules, and matrix proteins.
Mononuclear phagocytes established themselves as ubiquitous cells to carry on an effective defence of the host. As monocytes, they circulate in the blood and, upon extravasation, they colonize virtually every tissue where they differentiate into macrophages. Because of the close association with tissue cells, it was important that macrophages remained in a resting state in which the offensive functions could be kept silent to avoid damage to the neighbouring cells. In fact, mononuclear phagocytes, as well as monocytes, require activation by tissue-, immune system-, or environment-derived signals, either alone or in combination, to express lytic properties. Activated macrophages can exert anti-cellular activities and be toxic for other eukaiyotic cells, as shown by the ability to kill tumour target cells as well as fungi (1,2).
Since the discovery of the important role played by macrophages in the defence against parasites and tumours, attempts were made to dissect the mechanisms responsible for the acquisition and the expression of effector functions and to develop experimental systems to study and measure the lytic activity against prokaryotic and eukaryotic targets. In vitro cytotoxicity assays have provided scientists with the tools to probe these functions. The association between response to stimulation and expression of toxicity against cellular targets led scientists to utilize these functions as a parameter for macrophage activation. Models were proposed in which the expression of cytolytic activity against tumour cells was the end-point of macrophage activation. The difficulty of utilizing a biological assay as reference was the fact that the assays existed in almost as many different versions as the number of laboratories utilizing them, and the general validity of the results was undermined. These difficulties were overcome in two ways. On one hand, major efforts were made to standardize the assays (3) as well as to understand how different experimental methods should measure distinct expression of macrophage activity. On the other hand, the mechanisms responsible for the lytic activity were elucidated and the phenomenological observations of lytic activity was associated to activation of biochemically defined pathways and/or secretion of toxic mediators. It is now recognized that:
(a) The lytic activity of macrophages is due to the concomitant expression of several toxic pathways/molecules.
(b) Each assay will be more sensitive to the expression of certain lytic mechanisms.
Therefore, it is no longer surprising that each assay may measure different levels of macrophage activation and various degrees of response to activating stimuli.
Monocytes/macrophages can be readily obtained from various anatomical locations in a large number of animal species, humans included; can be separated from other cell types with some degree of efficiency; and are available in sufficient numbers for several types of studies. While short-term maintenance of macrophages in culture is common, long-term cultivation of primary isolates has been rather unsuccessful both because of the relatively short life span and the inability of replication in vitro of mature, differentiated monocytes/ macrophages. Moreover, the results obtained from in vitro studies may not always completely reflect the in vivo activities of this cell type. In fact, the physical state of the macrophage donor, along with the nature of the material used to obtain the cells and the culture conditions employed, have a profound influence on macrophage functions in vitro. Therefore, it becomes sometimes convenient to utilize established macrophage cell lines as a source of functional monocytes/ macrophages.
This chapter introduces a series of procedures to assess monocyte/macrophage lytic functions in vitro, including various techniques for the analysis of cytolysis and cytostasis of tumour cells and methods, to evaluate killing of intracellular and extracellular micro-organisms by macrophages.
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