STUDY OF MONONUCLEAR PHAGOCYTES IN VIVO: OVERVIEW
D. S. Nelson
At a time when reductionism holds sway in biology, the study of macrophages or, indeed, any other cells, in vivo may seem quaintly old-fashioned. There are, however, good reasons, both general and specific, to complement the refined and power- ful tools of analysis in vitro with more unwieldy but very re- warding studies in vivo. Quite clearly, life in the body is a good deal more complicated than life in the tissue culture chamber: There is always a need to make sure that what is observed in vitro has some counterpart in vivo and is not an artifact. For some physiological and pathological processes, we either altogether lack models in vitro or have models that are incomplete. Among many examples are atherosclerosis and delayed-type hypersensitivity (DTH). In the former case, it appears to be very difficult to reproduce in vitro processes involving both a good deal of the thickness of the walls of arteries and the blood circulating within them (1). In the case of DTH, much is known or inferred from studies in vitro about the interaction between antigens and committed T cells leading to the production of lymphokines and the consequent
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attraction and immobilization of macrophages at the site of the reaction. In vivo, however, other important factors op- erate: The supply of monocytes from the bone marrow, their mobility in tissues, changes in vascular permeability, and the involvement of the clotting system (2), an involvement that is only now yielding to analysis in vitro (3). All who work in vitro acknowledge that tissue culture conditions fall a long way short of reproducing the internal environment, but sometimes there are still surprises. For example, the pres- ence of large amounts of ascorbic acid in the air spaces of the lung (4) might be expected to have some effect on its antimicrobial defences.
In many branches of biology, the importance, or otherwise, of some organ, tissue, or group of cells can readily be demon- strated by means of ablation experiments. In immunology, for example, it is relatively easy to produce a mouse devoid of both a thymus and thymus-derived lymphocytes, but otherwise intact. It is, however, difficult to produce an animal devoid of mononuclear phagocytes, but otherwise intact. Sublethal whole body irradiation or cyclophosphamide can temporarily abolish the supply of monocytes from the bone marrow, but these agents also affect many other types of cell, while leav- ing mature macrophages unaffected (5,6). Conversely agents that are toxic to macrophages in vitro, such as silica (7) or carrageenan (8), may either have only a partial and transient antimacrophage effect in vivo or may have more widespread ef- fects, for example, on the clotting and complement systems on other leukocytes and on unidentified components of DTH (9).
Some of the difficulties can be illustrated by reference to studies of three important activities of macrophages: The expression of acquired cellular resistance to infection; the expression of antitumor immunity; and participation in inflam- matory reactions leading to tissue destruction.
In experiments that can now be regarded as classics,
Mackaness and his colleagues showed that activated macrophages are responsible for the expression of acquired cellular resis- tance to infection and that they become activated under the influence of specifically committed T lymphocytes reacting with antigen (10-15). The evidence was derived very largely
from experiments carried out with intact animals. Analysis of the mechanisms of activation and of the antimicrobial ac- tivities of macrophages must depend equally largely on experi- ments in vitro. There are, however, problems to be overcome.
In the first place, the macrophages that are of the greatest importance appear to be those recently derived from blood-born monocytes and not those already resident in tissues (16). Ac- cordingly, experiments in vitro should begin with either blood monocytes or freshly induced exudates. While this is not par-
ticularly difficult in principle, large numbers of blood mono- cytes are not readily obtained from the usual laboratory ro- dents, while exudates induced, for example, in the peritoneal cavity differ in character according to the inducing stimulus and may not accurately reflect the character of the cell pop- ulations in infected animals. A second and more formidable problem is the discrepancy between the intensity of the anti- bacterial activities of macrophages in vivo and in vitro.
Organisms such as Listeria monocytogenes and Salmonella typhimurium can be phagocytosed and destroyed with quite for- midable speed by macrophages in the liver, spleen, or perito- neal cavity of immune mice (11,12). It has, however, proved extremely difficult to reproduce this degree of activity in vitro (11, unpublished experiments) even with apparently sensitive techniques (17). Similar considerations apply to other bacteria in the lung and in isolated pulmonary alveolar macrophages (18; D. Rowley, personal communication). Con- ceivably, this could be related to the high ascorbic acid content of the lung (4). Clearly, however, this discrepancy between the activities of macrophages in vivo and in vitro deserves investigation.
Activated macrophages exert powerful and selective but nonspecific cytotoxic effects on tumor cells in vitro (19-21).
Several questions arise about the relevance of this phenomenon to tumor immunity in intact animals. First, how can it be shown that macrophages play an essential role in resistance to a particular experimental tumor? We have discussed elsewhere the criteria for a formal demonstration for such a role, con- cluding that it is difficult to devise totally rigorous and realistic criteria but that the circumstantial evidence, in many cases, is quite strong (22). Second, how are activated macrophages brought to the site of deposition of tumor cells so that they can exert their cytotoxic effect? It seems like- ly that, at least for certain tumors studied in our laboratory, an immunological reaction very similar to that of DTH is in- volved (9,23,24). Among problems still deserving of investi- gation are
(1) whether such mechanisms are operative more generally in tumor immunity, including resistance to metastasis;
(2) the way in which antitumor effector mechanisms operate in the face of the powerful antiinflammatory factors produced, apparently, by all tumors (25); and
(3) the possible role of antibody or the integration of humoral and cellular mechanisms in tumor recognition (26).
The third question concerns the mechanisms of killing of tumor cells in vitro and in vivo. Activated macrophages may kill tumor cells in vitro in a variety of ways, for example, by
contact and the release or injection of lysosomes, by the production of cytotoxic factors, and by the release of argi- nase (22). In certain circumstances the release of arginase seems an adequate mechanism by itself (27,28). The signifi- cance of such a mechanism in vitro has, of course, been ques- tioned, but recently evidence has been produced that local de- pletion of arginine by macrophages can be sufficient to create an environment hostile to tumor cells in vivo (29). Finally, activated macrophages obtained from animals and maintained in vitro can lose their cytotoxic effect quite quickly and addi- tional stimuli may be required to restore it (30-33). The factors operating in vivo to preserve the cytotoxic effective- ness of macrophages also deserve attention.
Macrophages are very prominent in inflammatory reactions resulting in tissue damage. One of the most important human diseases in which such reactions occur is rheumatoid arthritis.
Based on observations of lesions in man and experimental ani- mals and on biochemical observations on cultured macrophages,
strong suggestions have been made that the inflammation and destruction of joint tissue are due in part to the activities of macrophages, notably the release of lysosomal enzymes, neutral proteases, and prostaglandins (34-36). Models for such a complex interaction of macrophages and organized mix- tures of target cells are difficult to devise in vitro. It seems reasonable to suggest that more ingenuity could usefully be applied to the study of macrophages in the available animal models in vivo.
Finally, man is a notoriously intractable subject for ex- periment, and the study of human macrophages in vivo seems likely to remain a very difficult one. Methods for measuring the phagocytic activity of the "reticuloendothelial system" in man have been available for some time but are only recently coming into fashion again, for example, in studies of defec- tive macrophage function in rheumatoid arthritis (37) and sys- temic lupus erythematosus (38). Almost literally at the inter- face between studies in vivo and studies in vitro, however, is the examination of cells obtained from the lung by broncho- alveolar lavage. This relatively benign investigation promises to yield useful knowledge of the roles of macrophages (and of other cells) in inflammatory processes in the human lung (39).
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