The role of activated macrophages in protection and immunopathology in tuberculosis

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THE ROLE OF ACTIVATED MACROPHAGES IN PROTECTION AND IMMUNOPATHOLOGY IN TUBERCULOSIS G . A . W . Rook

Dept. Medical Microbiology, School o f Pathology University College & Middlesex School o f Medicine Riding House St. London WIP 7PP

It is not at all clear that we have yet identified the mechanism of protective immunity against mycobacteria, though it is widely assumed to be the iymphokine-activatedmaerophage. The pathways so far defined may be more relevant to immunopathology than to protection, particularly in man. The literature is conflicting due to the technical difficulties of assessing g r o w t h of m y e o b a e t e r i a within macrophages during the prolonged periods of culture required. M. tuberculosis tends to kill macrophages which contain more than about 5 bacilli, so these detach and are lost if the wells are ril~sed out before the end of the experiment. Moreover, the released organisms grow in the medium at very variable rates, determined above all by the availabilityof iron. Such growth is rapid in foetal calf serum, but very slow in human serum until the organisms are numerous. The organisms also tend to clump, so colony counts can be m i s l e a d i n g unless s o n i c a t i o n or detergents are used. Finally, reports of mediators causing 50-90 % reductions in bacterial (or isotope incorporation) counts compared to controls, over culture periods of 4 days or more may not represent protective mechanisms. Assuming a normal replication time of about 24 h, after 4 days in culture, a deficit of 50 % represents a reduction of one generation in 4, while a deficit of 87.5 % still means only that the organisms are doubling in 4 days rather than in one.

In murine peritoneal macrophages a few mycobacteria may be killed at the moment of phagocytosis by the products of the oxidative burst if one has been triggered. However, total staffs (not kill) of M. tuberculosis can be induced by crude l~mphokines, recombinant gamma interferon (r!FNy) (Rook el al., 1986b) or class I1 MHC-restricted T-cell lines (Rook et al., 1985). The effect is abrogated by steroids (Rook et al., 1987) and probably does not involve the oxygen reduction products (Flesch and Kaufmann, 1988). In our hands, and using virulent M. tuberculosis, no other cytokine/lymphokine has a comparable effect, though very small decreases in counts may be seen with GM-CSF (granulocyte/macrophage colony-stimulating factor) if inflammatory macrophages are used. It is not dear whether this is a specific effect, or secondary to the beneficial effect which this mediator exerts on the health of macrophage cultures, because in a medium containing FCS (foetal calf serum), a mediator which results in decreased extracellular bacilli will mimic a true bacteriostatic effect. Mediators which, in our hands, have no effect at all on the growth of virulent M. tuberculosis inside m u r i n e p e r i t o n e a l macrophages include turnout necrosis factor (TNF), 1,25-(OH) 2 vitamin D3 (calcitriol), IL-2 and IL-4. We have no experience with M. avium strains used in Dr. Lowell's laboratory, or the immature bone-marrow macrophages used by Dr. Kanfmann. Our negative results

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with TNF and calcitriol deserve further comment. As discussed below, calcitriol is the most effective defined activator of antituberculosis activity in h u m a n monocytes (Rook et al., 1986b), and in man it is probably an integral part ,;f the macrophage activation pathv~ay. Thus, in conditions where there are chronic granulomata in man, there are changes in vitamin D metabolism attributable to the activation within the macrophages of a 1-hydroxylase which converts the inactive circulating form of vitamin D3 (25(OH) cholecalciferol) into calcitriol. In extreme cases, so much of the active metabolite is formed that it exerts not only local effects within the granulomata, but also spills out into the systemic circulation where it causes abnormalities of calcium metabolism. This enzyme activity increases in response to IFN~' in human but not in murine macrophages. This may imply that caicitriol is not a part of the macrophage activation pathway in the mouse, in which case our finding that it has no effect on the growth of M. tuberculosis in routine cells seems reasonable (reviewed in Rook, 1988). The failure of TNF in the murine peritoneal macrophage/M, tuberculosis system is also interesting. Experiments in vivo in mice with neutralizing antiTNF have shown a protective role for TNF both in the Listeria monocytogenes model, and during infection with BCG (Kindler et aL, 1989). "[his effect is only seeu during the very early stage of the infection, and it does not d e m o n s t r a t e a direct effect on macrophages. It seems probal.le that this early phase of control of proliferation of Listeria in the mouse (and by inference, perhaps of BCG as well) is mediated by a T-cell-independent release of IFN T. This is produced by NK cells in response to TNF released by macrophages and an unidentified component of the bacteria (Bancroft et al., 1989). Production of IFN-T is subsequently taken over by the T-celldependent pathway, and anti-TNF no longer has a detrimental effect. The fact that mycobacteria are potent triggers of TNF release is clearly

relevant to this argument. This release is largely due to the phosphatidyl inositol mannoside (lipoarabinomannan, or LAM) which is comparable in potency to LPS in this respect (Moreno et aL, 1989). LAM readily contaminates mycobacterial antigen preparations, and reports of other TNF-triggering components should be treated with caution. Nevertheless, other glycolipid and also peptidoglycan fragments are likely to share this property. Since M. tuberculosis is a potent trigger of TNF release, this cytokine is present in infected maerophage cultures. Thus one might not expect to see inhibition of mycobacterial growth following addition of more exogenous TNF, even if TNF does, in fact, have some direct ability to induce antimycobacterial mechanisms. It will be interesting to see whether addition of neutralizing antiTNF to such cultures will increase growth of the organisms. Our understanding of antimycobacterial m e c h a n i s m s in h u m a n macrophages is even more limited. It is generally agreed that IFNT has little or no effect, and may actually enhance the growth of M. tuberculosis inside human macrophages (Douvas et al., 1985; Rook et al., 1986a). In this respect also, man is different from the mouse, and neither the T-cell-independent nor the T-cell-dependent routes of production of IFNT which can cause stasis (even if not kill) of M. tuberculosis in the mouse, have been shown to be relevant to protection in man. We have also examined IL-1, IL-2, IFN-alpha and TNF in the human system without success. TNF has been reported to cause i n h i b i t i o n of M. avium in human macrophages (Bermudez and Lowell, 1988), but since TNF causes activation of H1V in human cell lines in vitro and increased circulating HIV antigen levels in patients, this mediator may be eontra-indicated in this context. Calcitriol, as mentioned earlier, has a reproducible effect, which is additive with the weak effect of IFN T seen with monocytes from some donors. Using monocytes, calcitriol is active at physiological concentrations (10-gM)

KILLING INTRACELLULAR (Rook et al., 1986b), but A.J. Crowle a n d colleagues have found that, with monocyte-derived macrophages matured in vitro, much higher levels are required. However, the maximal inhibition o f M. tuberculosis achieved with these mediators is still unconvincing as a protective pathway. O n the other hand, macrophages exposed to IFNy and calcitriol are greatly primed for LAM-induced release of TNF, which in our hands has no effect whatsoever on growth o f M. tuberculosis in h u m a n monocytes. We have therefore argued at length elsewhere that this pathway may be immunopathological rather than protective (Rook et al., 1989). The fever, weight loss, and necrosis which characterize tuberculosis are readily attributable to known effects of TNF, and injection o f TNF into murine skin sites prepared by an injection of mycobacterial antigen 24 hours earlier will cause violent necrosis (Rook et al., 1989). We are unable to detect free TNF

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in the serum o f tuberculosis patients, but on the other hand, their serum contains high levels of an inhibitor of this mediator (Rook et al., 1989). Perhaps the mechanisms so far studied by immunologists serve to "wall o f f " the lesion in those in whom the organism has become astabfished, or perhaps they are purely immunopathological. W h a t then protects the truly resistant individuals in whom M. tuberculosis fails to become established? It may be that, as often discussed by Kaufmann, the organisms are " h i d i n g " in macrophages, a n d that these cells must be killed so that the organisms are subjected to repeated cycles o f rephagocytosis by freshly arriving macrophages, or to some other novel antibacterial mechanism. This would be terribly difficult to mimic in vitro. A n d what should we deduce from the fact that tuberculosis is such an early complication o f H I V infection?

References,

BANCROFT,G.J., SH~.HAN,K.C., SCUFmm~R,R.D. & UNA~,a~E,E.R. (1989), Tumor necrosis factor is involved in the T-cell-independent pathway of macrophage activation in scid mice. J. lmmunol., 143, 127-130. BE~'auo~z, L.E. & YOUNO,L.S. (1988), Tumor necrosis factor, alone or in combination with IL-2, but not IFN-gamma, is associated with macrophage killing of Mycobacterium avium complex. J. Immunot., 140, 3006-3013. DouvAs, G.S., LOOXER,D.L., VAT'rER,A.E. & CROWL~,A.J. (1985), Gamma interferon activate~ human macrophages to become tumoricidal and leishmanicidal but enhances replication of macrophage-associated mycobaeteria. Infect. Immun., 50, 1-8. FLeSCU,I.E. & KAUFMAWN,,.q.H. (1988), Attempts to characterize the mechanisms involved in mycobacterial growth inhibition by gamma-interferon-activated bone marrow macrophages. Infect. lmmun., 56, 1464-1469. KINVLER,V., SAVP~NO,A.P., GnAU, G.E., PIOUeT, P.F. & VASSALU,P. 0989), The inducing role of tumor necrosis factor in the development of bactericidal granulomas during BCG infection. Cell, S6, 731-740. MORENO, C., TAVERNE, J., MEHLERT, A., BATE, C.A., BREALEY,R.3., MEAGER, A., RooK, (3.A.W. & PLAYFAm, J.H.L. (1989), Lipoarabinomannan from Mycobacterium tuberculosis induces the production of turnout necrosis factor from human and routine macrophages. Clin. exp. lmmunol., 76, 240.245. RooK, G.A.W. (1988), The role of vitamin D in tuberculosis. Amer. Rev. Resp. Dis., 138, 768-77,.. RooK, G.A.W., AL ArnVAn, R. (1989), Fhe role of cytokines in the immunopathohigy of tuberculosis and the ;egulation of agalactosyl lgG. Lymphokine Res., 8, 323-328. ROOK, G.A.W., CHA~aPION,B.R., STEELE, J., VAPmV,A.M. & S'rAm~ED, J.L. (1985), I-A restricted activation by T-ceU lines of anti-tuberculosis activity in murine mecrophages. Clin. exp. Immunol., 59, 414-420. ROOK, G.A.W., S'r~L~, J., AINSWOaT~I,M. & CH^~E~ON, B.R. (1986), Activation of macrophages to inhibit proliferation of Mycobacterium tuberculosis: comparison of the effects of recombinant gamma-interferon on human monoeytes and murine peritoneal macrophages. Immunology, 59, 333-338.

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ROOK,G.A.W., STEELE,J., AINSWOR~,M. & LEV~TON,C. (1987) A direct effect of glucocorticoid hormones on the ability of human and routine macrophages to control the growth of Mycobacterium tuberculosis. Europ. J. Resp. Dis., 71, 286-291. RooK, G.A.W., STEELE,J., FP.AHER,L., BARKER,S,, KARMALI,R., O-RIORDAN,J. ~¢ STANFORD,J. (1986), Vitamin D3, gamma interferon, and control of proliferation of Mycobacterium tuberculosis by human monocytes. Immunology, 57, 159-163. RooK, G.A.W., TAVE~e, J., LevFro~, C. & S~eL~, J. (1987), The role of gamma-interferon, vitamin D3 metabolites and tumour necrosis factor in the pathogeuesis of tuberculosis. Immunology, 62, 229-234.

The following manuscript arrived too late for distribution to, and commentary by, the other participants in the present Forum: MYCOBACTERIA AND MACROPHAGE ACTIVATION R. van Furth

Dept. of Infectious Diseases, University Hospital, Leiden (The Netherlands)

Introduction. The phenomenon of cell-mediated immunity has been known for more than a hundred years and is best elucidated by the experiments of Robert Koch (Koch, 1882). When normal guinea pigs are injected subcutaneously with a larger number of live tubercle bacilli, the wound first closes and seemingly heals, but after about 10 to 14 days exteasive ulceration develops and persists until the animal dies. However, when a small number of tubercle bacilli are injected, the animal's defences can cope with these microorganisms and the guinea pig will survive. The immunological status of this animal has been changed and when such a guinea pig is re-infected with a large number of tubercle bacilli, it will recover. The increased resistance against the infection with tubercle bacilli is based on an altered cell-mediated immunity. About fifty years ago, Mcrril Chase was the first to demonstrate that cellmediated immunity can be transferred by lymphocytes but not by serum

(Chase, 1945). At present, we know that cell-mediated immunity is the result of cooperation between two types of cells, lymphocytes and macropha~es. The specificity of the immunological reaction lies in the interaction between CD4 + lymphocytes and the antigen; the macrophages are only secondarily affected by lymphokines secreted by these lymphocytes. Cell-mediated immunity for tubercle bacilli can be demonstrated with the tuberculin test. When a sm~ll amount of an extract of tubercle bacilli (tuberculin) is applied to the skin, a local inflammatory reaction occurs. Histologically, this reaction is characterized by an accumulation of lymphocytes and exudate macrophages. The reaction is called a delayed hypersensitivity,reaction, because it reaches a mayamum after 2 to 3 days. Although the delayed hypersensitivity reaction and cellmediated immunity have the same mechanism in common, the arbitrary interchange of these terms is confusing (van Furth, 1972). The mechanism of cell-mediated immunity has been studied extensively in