1.3.4.1 Heterogeneity and activation of macrophages

Macrophage heterogeneity is a well-documented phenomenon, perhaps first observed by Metchnikoff, who described a progression of infiltrating cell types in inflammatory exudates. It has also long been recognized that macrophages isolated from different anatomical sites display a diversity of phenotypes and capabilities. Because macrophage function is dependent in part on signals received from the immediate microenvironment, it is suggested that macrophage heterogeneity may arise from unique conditions within specific tissues. Obviously, the sterile, anaerobic environment of the spleen or peritoneum will impart different constraints on resident macrophages than does the aerobic environment of the alveolar macrophage, which contains numerous external factors. Antibodies directed against specific membrane antigens have been used to compare macrophage from different tissues. For instance, human breast milk macrophages express an antigen not observed on monocytes, alveolar macrophages, or peritoneal cells. Furthermore, human alveolar macrophage express high levels of MHC class II antigen, whereas the opposite is found for peritoneal macrophages.

It has been just as quickly recognized that macrophages isolated from a given tissue display heterogeneous function. For example, only a portion of peritoneal macrophages express low levels of 5'-nucleotidase, and immune elicitation of peritoneal macrophages results in predominantly macrophages with low 5'-nucleotidase activity, presumably because of an influx of monocytes. Thus, functional heterogeneity results from the spectrum of maturational states in a given isolate because of the influx of monocytes and/or local proliferation.

Because macrophages are responsible for numerous inflammatory processes, it becomes important to distinguish between normal or steady-state haematopoiesis and induced haematopoiesis associated with immunological challenge. Production of the macrophage lineage from bone marrow progenitors is normally controlled by M-CSF, which is constitutively produced by many cell types. In response to invasive stimuli and inflammation, monocyte numbers increase dramatically, as do serum levels of M-CSF. In addition, GM-CSF appears in the serum. Although there appear to be a large overlap of macrophage progenitors able to respond to M-CSF or GM-CSF, the very different structures and signal transduction mechanisms of the receptors for M-CSF and GM-CSF suggest that the differentiation pathways they initiate, would be dissimilar.

M-CSF-derived macrophages are larger, have a higher phagocytic capacity, and are highly resistant to infection by vesicular stomatitis virus compared to GM-CSF-derived macrophages. Conversely, GM-CSF-derived macrophages are more cytotoxic against TNF--resistant tumour targets, express more MHC class II antigen, more efficiently kill Listeria monocytogenes, and constitutively secrete more PGE.

The production of functionally distinct macrophage populations gives the nonspecific immune system added flexibility to respond to immunological or inflammatory stimuli. It is probable that the nature of an immune response is dictated in large part by the functional phenotype(s) of the macrophages present within the lesion. The existence of distinct subsets of helper T lymphocytes (T cells) also suggest that the predominance of T1 (IFN- and IL-2 producing) or T2 (IL-4 and IL-10 producing) cells may, in turn, favor the production or activation of a particular macrophage subset.

In addition, the orchestration and regulation of cytokine production during inflammatory responses constitute a key determinant of both the resolution of challenge and the limitation of host tissue damage. Hence, the sequential appearance within inflammatory lesions may allow the most appropriate response at a given stage of an immune response. Analysis of temporal production of cytokines during immune responses suggests that different macrophage populations participate at various stages, or that the changing conditions within the lesion differentially affect the functions of distinct macrophage populations.

Several studies correlate the presence of certain macrophage populations with disease states. Human macrophages expressing an undefined antigen detected by the 27E10 monoclonal antibody are observed in acute inflammatory exudates in cases of contact dermatitis, gingivitis, and psoriasis, but are absent in chronic inflammation arising from osteoarthritis, tuberculoid leprosy, and rheumatoid arthritis. In human liver, heart, and kidney grafts, strong infiltration of 27E10-positive macrophages is associated with acute rejection, whereas the RM3/1-positive phenotype is associated with an uncomplicated clinical course. Accumulation of 25F9-positive macrophages correlates with tumour progression and poor prognosis. These studies suggest that phenotypic analysis of macrophage subsets might be of use diagnostically, even if the specific role of these macrophage populations is unclear.

It follows that the term macrophage refers to a heterogeneous population of cells that differ in their origin, development stage (differentiation), local adaptation and thus also in their function and purpose. The nomenclature of individual macrophage types (particularly inflammatory) is rather confusing and terms such as stimulated, activated, induced, elicited etc. are often used interchangeably. Basically, two main macrophage groups can be distinguished: resident (normal) and inflammatory (exudate) macrophages.

The term activated macrophages is reserved for macrophages possesing specifically increased functional activity. The process of differentiation is not to be confused with activation, the process trough which differentiated macrophages acquire an increase ability to perform specific functions. Characteristically, resident tissue macrophages are relatively quiescent immunologically, having low oxygen consumption, low levels of major histocompatibility complex (MHC) class II gene expression, and little or no cytokine secretion. Resident macrophages are, however, phagocytic and chemotaxic and retain some proliferative capacity.

There are two stages of macrophage activation, the first being a primed stage in which macrophages exhibit enhanced MHC class II expression, antigen presentation, and oxygen consumption, but reduced proliferative capacity. The agent that primes macrophages for activation is IFN-, a product of stimulated and cells. But many other factors, including IFN-, IFN-, IL-3, M-CSF, GM-CSF and TNF-, can also prime macrophages for selected functions.

Primed macrophages respond to secondary stimuli to become fully activated, a stage defined by their inability to proliferate, high oxygen consumption (through NADPH oxidase), killing of facultative and intracellular parasites, tumour cell lysis, and maximal secretion of mediators of inflammation, including TNF- , , IL-1, IL-6, reactive oxygen species, and nitric oxide produced by iNOS. Agents capable of providing secondary signals are diverse and include LPS, heat-killed gram-positive bacteria, yeast glucans, GM-CSF and phorbol esters. The distinction between primed and fully activated macrophages is usually arbitrary, depending in large part on the stimulus used and the functional assessed.

Macrophages stimulated for tumouricidal activity show decreased MHC class II gene transcription and are generally poor presenters of antigen, despite their secretion of IL-1. Moreover, the process of tumour cell lysis is a multistep event, determined by the sensitivity of the target cell itself. Thus, activation to kill one target does not necessarily include the ability to kill all targets.

When human (but not murine) macrophages are expossed to IFN- they express a 1-hydroxylase. This enables them to convert inactive circulating 25-hydroxycholecalciferol into the active metabolite, 1,25-dihydroxycholecalciferol (also known as vitamin D or calcitriol). Macrophages have receptors for this derivative, and it exerts additional activating effects on these cells, and perhaps some negative feedback on lymphocytes. This pathways is of some importance in man, since production of calcitriol can be so great that it leaks from the site of macrophage activation into the peripheral circulation, where it can exert its better known effects on calcium and phosphate balance. Detectable hypercalcaemia can result.

There is also evidence that activated macrophages can be deactivated. Prostaglandin E may have this effect, and some effector mechanisms (but not all) are steroid sensitive. Recently a macrophage deactivating factor (MDF) has been purified from a tumour cell supernatant. This cytokine block activation by IFN- of increased capacity for production of ROI and, to some extent, of nitric oxide. So too do IL-4, calcitonin gene related peptide (CGRP), and TGF-.