Experimental variables for induction of activated cytotoxic macrophages

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[5] CELADA,A., GRAY,P.W., RINDERKNECHT,E. & SCHREIDER,R.D., Evidence for a gamma-interferon receptor that regulates macrophage tumoricidal activity. J. exp. Med., 1984, 160, 55. [6] CELADA,A., ALLEN,R., ESPARZA,I., GRAY,P.W. & SCHREIBER,R.D., Demonstration and partial characterization of the interferon-gamma receptor on human mononuclear phagocytes. J. clin. Invest. 1985, 76, 2196. [7] BUCHMEIER,N.A. & SCHREIBER,R.D., Requirement o f endogenous interferongamma production for resolution of Lister& monocytogenes infection. Proc. nat. Acad. Sci. (Wash.), 1985, 82, 7404.

EXPERIMENTAL

V A R I A B L E S F O R INDUCTION

OF ACTIVATED CYTOTOXIC MACROPHAGES b y M.S. Meltzer, D.L. Hoover, M.J. Gilbreath, R . D . S c h r e i b e r a n d C . A . Nacy

Dpt of Immunology, Walter Reed Army Medical Center, Washington, DC 20307 (USA) A preceding forum on macrophage activation concluded that the unmodified term <>was much too vague to be useful : activated macrophages should always be defined for the particular effector function at issue [1-3]. Within these limits, however, a working definition o f macrophage activation would include induction of specific cytotoxic effector functions not present in either resident tissue macrophages or in cells that accumulate at sites o f sterile inflammation (inflammatory macrophages). Analysis of this process must consider at least four interrelated variables : the cell that responds, the activation signal, the susceptible target and the cytotoxic assay. Changes in any one o f these variables markedly influences what one interprets as macrophage activation.

Responsive macrophages. Macrophage response to a single activation signal for induction o f cytotoxicity changes with cell maturation a n d / o r differentiation [4]. Although this concept seems eminently reaso-

nable, the ultimate macrophage response may not be predictable (fig. 1). For example, the level of tumoricidal a c t i v i t y i n d u c e d by i n t e r f e r o n gamma(IFN)-treated inflammatory macrophages is more than 10-fold greater than that induced by an equal number of resident peritoneal cells given identical treatment. Interestingly, differences in macrophage response are not related to number of IFN receptors: the number o f IFN receptors/cell for inflammatory and resident macrophages are comparable. In the face of large differences in cell response for induction of tumoricidal activity, an entirely different phenomenon occurs with IFNinduced macrophage microbicidal activity [5]. The number of macrophages infected with Rickettsia tsutsugamushi in resident and inflammatory cell populations are equal. Small amounts of IFN (~< 10 IU/ml) added to infected cell cultures activates macrophages to kill the intracellular bacteria. In contrast to tumoricidal activity, no difference in IFN dose-response was detected between resident and inflammatory cell populations for induction of microbicidal activity. Similarly, macrophages

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infected with amastigotes of Leishmania major also respond to IFN and develop microbicidal activity. In this case, microbicidal activity induced in the resident macrophage population is more than 10-fold greater than that induced in inflammatory macrophages. Thus we easily document changes in macrophage response to activation stimuli with cell differentiation. We also document a striking system specificity: for any particular target, resident macrophages may be more, less, or equally responsive to an activation signal than equal numbers of inflammatory cells. Furthermore, superimposed upon differences in cell maturation are even greater changes in cell response that may occur with any of several other intrinsic or extrinsic factors: variation in macrophage activation among certain strains of mice (A/J, C 3 H / H e J , P / J strains) or during certain viral and parasitic infections are examples that easily come to mind [6].

Activation signals. Recent reports that IFN is a macrophage activation factor in both human and murine effector systems raised expectations that macrophage activation would now be simplified. Indeed, recombinant IFN alone activates macrophages to kill tumour and microbial targets. However, macrophage physiology is not that simple : several investigators now document macrophage activation factors that are physicochemically and antigenically distinct from IFN. For example, a subclone of EL-4, a murine T-cell line, secretes a 25-Kd factor that activates macrophages to kill tumour cells; the same factor activates macrophages to kill skin-stage schistosomula of Schistosoma mansoni [7, 8]. Both cytotoxic activities are induced by IFN, yet the EL-4 factor is both physicochemically (heat-labile, acid-resistant, 25 Kd molecular weight) and antigenically distinct from IFN. Even more inte-

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resting are observations that, while IFN and the EL-4 lymphokine (LK) share some activities, this overlap is not complete: EL-4 LK does not induce fibroblast antiviral activity or increase the number of Fc receptors or Ia expression on macrophages; EL-4 LK also does not induce macrophage microbicidal activity against L. major. Thus, although the EL-4 LK is a macrophage activation factor, its range of activity is limited. Macrophages treated with LK from antigen/mitogen-stimulated murine spleen cell cultures develop microbicidal activity against R. tsutsugamushi or amastigotes of L. major. Both are obligate intracellular parasites. Microbicidal activity in each case is also induced by recombinant IFN. However, for both microorganisms, IFN represents about half the total activity in LK : LK depleted of IFN by anti-IFN immunoaffinity chromatography activate macrophages to kill intracellular rickettsia and leishmania [9]. As seen with EL-4 LK, non-IFN macrophage activation factors in spleen cell-derived LK share some but not all properties of IFN. These spleencell-derived factors, for example, do not activate macrophages for tumoricidal activity. The precise physicochemical characterization of these non-IFN macrophage activation factors is now under investigation. The recent discovery of similar factors in man has made this analysis both relevant and exigent IlO, 11]. Other macrophage activation factors in LK induce changes in macrophage function that are not induced by IFN. For example, the relative susceptibility of a macrophage population to infection by obligate intracellular parasites is affected by prior treatment with LK : the number of macrophages infected with L. major is reduced 3- to 5-fold by 4-h exposure to LK before infection [12]. LK treatment does not affect parasite viability or the uptake of inert particles such as latex beads or red blood cells. We term this LK-induced macrophage activity, resistance to infection. Similar LK-induced resistance to infection has been documented with rickettsia, legionella, and mycobacteria.

Activity in LK that induces resistance to infection is not affected by monoclonal or polyclonal anti-IFN in fluid or solid phase; m a c r o p h a g e s treated with recombinant murine IFN do not develop resistance to infection. The preceding clearly documents multiple macrophage activation factors in LK. IFN appears to be the most universal. With the exception of resistance to infection, all cytotoxic effector functions present in activated macrophages from Mycobacterium bovis strain BCGinfected mice can be induced in vitro with IFN. BCG remains the prototypic macrophage activation agent in vivo: IFN fulfills a similar role in vitro. Other macrophage activation factors described here have a more limited range of action. At this point in time, which if any of these LK are relevant in vivo during host resistance to neoplastic or infectious disease remains to be determined. In addition to primary macrophage activation factors, there are also secondary factors that modulate macrophage effector function. These accessory factors are second signals that by themselves have little or no effect on macrophage cytotoxicity, yet markedly influence macrophage response to a primary activation stimulus. Such accessory factors can either amplify or suppress macrophage activation. For example, we recently discovered a 25-Kd LK from the EL-4 cell line that dramatically amplifies IFN-induced macrophage microbicidal activity against L. major. This accessory amplification factor has no activity by itself, but synergistically increases IFN-induced macrophage leishmanicidal activity 5- to 10-fold. The EL-4 amplification factor is unaffected by polymyxin B and is physicochemically and antigenically (activity unaffected by anti-IFN immunoafflnity chromatography) unrelated to IFN. O f course, there are also a number of more familiar exogenous second signals that amplify macrophage activation. These are often derived from bacteria or other infectious pathogens and include heat-killed listeria, bacterial endotoxic lipopolysaccharides, lipid A, muramyl dipeptide and certain lectins.

MACROPHAGE Suppressive accessory factors also derive from endogenous and exogenous sources. Factors released from the EL-4 cell line suppress macrophage activation [13]. IFN or LK-induced macrophage microbicidal activity against L. major is markedly but selectively inhibited by EL-4 LK" LK-induced macrophage tumoricidal activity or resistance to infection are unaffected. Other endogenous factors that suppress some, but not all effector responses of LK-activated macrophages include prostaglandins, alpha foetoprotein and certain neuroendocrine mediators. Exogenous factors also selectively inhibit induction of macrophage cytotoxicity and include bacterial endotoxins, immune complexes and liposomes o f a particular phospholipid composition [14]. At first glance (or even hundreds of glances later), regulation of macrophage activation seems hopelessly complex. Induction of macrophage cytotoxic reactions by any of several different primary activation signals is regulated (amplified or suppressed) in turn by a host of seemingly unrelated second signals : one set o f signals turns on the radio and selects the station, another regulates the volume. Is this regulatory network of heterogeneous signals any more complex than the complement or coagulation cascade or the endocrine control of ovulation ?

Susceptible targets. Conceptually, this is the most straightforward o f the variables and perhaps the best documented. For example, one mechanism of macrophage-mediated cytotoxicity is through release of reactive oxygen species inclu-

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ding H202. Susceptibility to toxic effects of reagent H202 is certainly not uniform among either neoplastic or microbial targets: tumour cell susceptibility to lethal toxic effects o f reagent H202 varies over a > 10-fold concentration range; susceptibility o f two leishmania species, L. major and L. donovani varies over a 10-fold range [15]. Similar target cell variation has been documented for other macrophage-derived toxic effector molecules such as prostaglandins and tumour necrosis factor. Cytotoxicity assay. Small changes in the actual mechanics of the cytotoxicity assay keeping macrophages, activation signal and target otherwise constant can profoundly affect induction of macrophage cytotoxic responses. The ability of macrophages to respond to activation signals and kill neoplastic or microbial targets changes with time in culture (fig. 2). Macrophages placed in culture for various times before addition of LK or IFN progressively lose the ability to respond and develop cytotoxicity against tumour cell, rickettsia, leishmania or schistosomula targets. In each system, loss of macrophage response is irreversible but not secondary to changes in cell viability. In fact, in several instances, these same macrophages can kill the identical target by other mechanisms (antibody or phorbol-myristic-acetatemediated cytotoxicity). The ability of macrophages to respond to activation signals and develop cytotoxic activity differs between cells cultured as an adherent monolayer or as a cell pellet (table I). Macrophages

TABLE I. - - Macrophage mierobicidal activity (in parentheses: % infected cells) against L. major: cell pellet versus adherent monolayer culture.

Cultures treated with Macrophage culture Cell pellet Adherent monolayer

Medium 0 % (62 _+ 4) 0 ~ (58 + 3)

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210

11 e F O R U M D ' I M M U N O L O G I E not clear with any of these in vitro model systems which combinations of experimental conditions most closely resemble the counterpart in vivo reaction. Indeed, it could certainly be argued that no in vitro assay accurately reflects the normal physiologic reaction. An ultimate aim for characterization of reactions that induce activated cytotoxic macrophages is therapeutic intervention in neoplastic and infectious disease. Each of the preceding reactions has that potential.

cultured as a cell pellet respond to both LK and recombinant IFN to kill amastigotes of L. major. In contrast, macrophages c u l t u r e d as an a d h e r e n t monolayer, with or without the nonadherent peritoneal cells, respond less well to IFN and not at all to LK. Differences in macrophage response between adherent monolayer and cell pellet or suspension cultures are also documented for adenosine and lysine transport, glucose oxidation, procoagulant activity and release o f superoxide anion. It is

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References.

[1] NORTH, R.J., The concept o f the activated macrophage. J. Immunol., 1978, 121, 806-808.

[2] KARNOVSKY,M.L. & LAZDINS,J.K., Biochemical criteria for activated macrophages. J. Immunol., 1978, 121, 809-812. [3] COHN, Z.A., The activation of mononuclear phagocytes: fact, fancy and future. J. Immunol., 1978, 121, 813-816.

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[4] MELTZER, M.S., Tumor cytotoxicity by lymphokine-activated macrophages: [5] [6] [7]

[8]

[9]

[10] [11]

[12] [13] [14]

[15]

development of macrophage tumoricidal activity requires a sequence of reactions. Lymphokines, 1981, 3, 319-343. NACY, C.A., OSTER, C.N., JAMES, S.L. & MELTZER,M.S., Microbicidal effector reactions of activated macrophages against intracellular and extracellular parasites. Cont. Topics ImmunobioL, 1984, 14, 147-170. MELTZER, M.S., OCCHIONERO,M. ~r RUCO, L.P., Macrophage activation for tumor cytotoxicity: regulatory mechanisms for induction and control of cytotoxic activity. Fed. Proc., 1982, 41, 120-127. MELTZER,M.S., BENJAMIN,W.R. & FARRAR, J.J., Macrophage activation for tumor cytotoxicity: induction of macrophage tumoridical activity by lymphokines from EL-4, a continuous T-cell line. J. Immunol., 1982, 129, 2802-2807. NACY, C.A., JAMES, S.L., BENJAMIN,W.R., FARRAR, J.J., HOCKMEYER,W.T. & MELTZER,M.S., Activation of macrophages for microbicidal and tumoricidal effector functions by soluble factors from EL-4, a continuous T-cell line. Infect. Immun., 1983, 40, 820-824. NACY,C.A., FORTIER,A.H., MELTZER,M.S., BUCHMEIER,N.A. & SCHREIBER, R.D., Macrophage activation to kill Leishmania major: activation of macrophages for intracellular destruction of amastigotes can be induced by both recombinant interferon-gamma and non-interferon lymphokines. J. Immunol., 1985, 135, 3505-3511. HOOVER,D.L., NACY, C.A. & MELTZER,M.S., Human monocyte activation for cytotoxicity against Leishmania donovani: induction of microbicidal activity by interferon-gamma. Cell. Immunol., 1985, 99, 500-511. HOOVER,D.L., FINBLOOM,D.S., CRAWFORD,R.M., NACY, C.A., GILBREATH,M. MELTZER,M.S., A lymphokine distinct from interferon-gamma that activates human monocytes to kill Leishmania donovani. J. Immunol., 1986, 136, 1-5. NACV,C.A., MELTZER,M.S., LEONARD,E.J. & WVLER, D.J., Intracellular replication and lymphokine-induced destruction of Leishmania tropica in C3H/HeN mouse macrophages. J. Immunol., 1981, 127, 2381-2386. Ygcv, C.A., Macrophage activation to kill Leishmania tropica: characterization of a T-cell-derived factor that suppresses lymphokine-induced intracellular destruction of amastigotes. J. lmmunol., 1984, 133, 448-453. GILBREATH,M.J., NACY, C.A., HOOVER, D.L., ALVlNG,C.R., SWARTZ,G.M.Jr & MELTZER,M.S., Macrophage activation for microbicidal activity against Leishmania major: inhibition of lymphokine activation by phosphotidylcholine-phosphotidylserineliposomes. J. Immunol., 1985, 134, 3420-3424. NATHAN,C.F., Mechanisms of macrophage antimicrobial activity. Trans. roy. Soc. trop. Med. Hyg., 1983, 77, 620-630.