T Lymphocyte-Macrophage Interactions in Cellular Antibacterial Immunity

Immunobiol., vol. 161, pp. 361-368 (1982) Institut fur Medizinische Mikrobiologie, Freie Universitat Berlin, Berlin, Germany

T Lymphocyte-Macrophage Interactions in Cellular Antibacterial Immunity H.HAHNandS.H.E.KAUFMANN

Abstract Acquired resistance to facultative intracellular bacteria depends on a bicellular mechanism whereby specific T lymphocytes activate macrophages for enhanced bacteriocidal capaciry. In vivo, protection is paralleled by delayed-rype hypersensitiviry. In vitro correlates are specific T lymphocyte proliferation and interleukin induction. Macrophage activation results from complex cell interactions involving both T lymphocytes and macrophages. Although such interactions are not yet fully understood, it appears likely that interleukin-facilitated collab­ oration between Lyt 1 and Lyt 123 T lymphocytes is required. Most probably, H2-restricted interactions between antigen-presenting mononuclear phagocytes and Lyt 1 T lymphocytes induce secretion of interleukins which further recruit additional Lyt 1 T Lymphocytes from the Lyt 123 T lymphocyte set. In this way, the pool of Lyt 1 T lymphocytes capable of attracting and activating macrophages at the site of bacterial implantation via lymphokines (macrophage activating factor, migration inhibition factor) could be markedly enhanced.

Introduction A medically important group of bacterial pathogens is capable of intracel­ lular survival once they have been taken up by phagocytes (Table 1). The term, facultative intracellular bacteria, has been coined to account for this fact. Most, if not all, facultative intracellular bacteria cause infectious diseases which are distinguished by the formation of granulomatous lesions at the site of bacterial deposition and by delayed-type hypersensitivity (DTH) reactions to antigens of the invading bacteria. Antibodies, although produced in large amounts, are of little importance in protection, and vaccination procedures which exclusively lead to the formation of anti­ bodies (e.g. dead vaccines of bacterial components or products) do not endow immunity. In order to be protective, antigens have to be introduced in the form of live bacteria (e.g. BeG, Brucella vaccine, and others) or have to be incorporated in complete Freund-type adjuvant, a procedure not applicable to humans. MACKANESS and associates (1) have analyzed the underlying mechanisms in immunity to facultative intracellular bacteria. They have established - an early example of cellular cooperation in immunology - that immunity to facultative intracellular bacteria rests upon a bicellular mechanism, in which specific lymphocytes, subsequently identified as T lymphocytes, act as inducers, and mononuclear phagocytes as expressors of immunity to facul-

362 . H. HAHN and S. H. E. KAUFMANN

tative intracellular bacteria. Because of its dependence on cells rather than specific antibodies, this fonn of immunity is called cell-mediated antibacte­ rial immunity (eMI) or cellular antibacterial immunity. Mononuclear phagocytes express eMI by essentially three mechanisms (2-4): 1. Augmentation of the number of mononuclear phagocytes at sites of bacterial implantation; 2. Granuloma formation around proliferating bacteria; 3. Activation of mononuclear phagocytes by lymphokines secreted by antigen stimulated T lymphocytes. These changes in the mononuclear phagocyte compartment are the consequences of specific immune reactions between antigen and T lympho­ cytes. However, not only do T lymphocyte-antigen interactions affect mononuclear phagocytes, but mononuclear phagocytes in turn influence T lymphocyte activities, in this way regulatory control loops being estab­ lished. Specific T lymphocytes as inducers of antibacterial eMI The role of specific T lymphocytes in the establishment of immunity to facultative intracellular bacteria has been established by cell transfer experi­ ments. Viable lymphocytes derived from spleens, thoracic ducts, or induced peritoneal exudates of mice or rats immune to a variety of facultative intracellular bacteria were transferred to immunologically naive recipient animals. Upon subsequent challenge with homologous bacteria, the cell recipients turned out to be protected against infections. On the other hand, controls which had received lymphocytes from animals immune to nonre­ lated micro-organisms succumbed to the infection (5). The cells transferring protection were sensitive to treatment with anti-Thy 1 antiserum and hence are T lymphocytes (6-8). Mononuclear phagocytes as antigen presenting cells In the immune response leading to the production of T lymphocytes involved in the delivery of activating signals (e.g. macrophage activation in eMI, help in antibody production or help in the formation of cytotoxic T lymphocytes) T lymphocytes are not stimulated by antigen directly. Rather, antigen is first taken up by mononuclear phagocytes, processed, and subsequently presented in close association with cell membrane struc­ tures which are encoded by the H-2I locus of the major histocompatibility complex (9). On the other hand, antigen presented on non-immunological cells and in association with H-2K,D encoded products will lead to the production of T lymphocytes with cytolytic activity, important in antiviral and antitumor immunity (10).

T Lymphocyte-Macrophage Interactions . 363

Subpopulations of mononuclear phagocytes expressing H-21 region­ coded products (Ia) on their surfaces have been identified and are consid­ ered the main cell class involved in antigen presentation (11). It has been shown that in Listeria-immune animals la-rich mononuclear phagocytes are induced by reactions between specific T lymphocytes and antigen and that these cells are exclusively capable of interacting with T lymphocytes (12). A protein factor derived from antigen stimulated T lymphocytes was iden­ tified which was responsible for induction of la-expression. In this way, a control loop may exist whereby initial interactions of T lymphocytes with la-bearing macrophages cause bone marrow-derived cells to differentiate into la-bearing mononuclear cells.

H-2 restriction of T lymphocyte-macrophage interactions The association of antigen with cell membrane products encoded by the MHC entails an important control function of the MHC. As shown by ZINKERNAGEL and associates (13), adoptive protection to Listeria mono­ cytogenes by T lymphocytes is restricted at the recognition level by the H-21 locus, i.e., antigen presenting cells and reacting T lymphocytes have to share the H-21 haplotype. This requirement for histocompatibility at the H-21 locus is limited to a short initial period only during which antigen recognition by T lymphocytes on histocompatible mononuclear phagocytes takes place. It is followed by the release of lymphokines which in an antigen-nonspecific way, and not H-2-restricted, attract and activate mono­ nuclear phagocytes for enhanced bacteriocidal activity (Figure 1). Restric­ tion at the H-21 locus of the MHC was also found for DTH to soluble antigens, antigen-specific cell proliferation in vitro, help in antibody pro­ duction, and help in the generation of cytotoxic T lymphocytes (9).

T lymphocyte subsets and mononuclear phagocytes The fact that T lymphocytes are capable of recognizing antigen in association with H-21 in one, and H-2K,D coded products in another, instance suggests the existence of T lymphocyte subsets individually pro­ grammed to recognize antigens presented in differing ways and to emanate different signals - activation signals on the one hand, and cytolytic ones on the other. The use of antisera specific for differentiation antigens on T lymphocytes (Lyt antigens) has indeed allowed dissection of the T lymphocyte system into distinct subpopulations programmed for distinct biological functions (14). Although exceptions exist, it generally holds that it is the Lyt 1+ T lymphocytes which are restricted by the H-21 locus of the MHC and are programmed to deliver activation signals. Accordingly, Lyt 1+ T lympho-

364 . H. HAHN

and S.

H.

E.

KAUFMANN

cytes are involved in help in antibody formation, help in the generation of cytotoxic T lymphocytes, and in the mediation of DTH. Lyt 23+ T lymphocytes are controlled by the H-2K,D loci of the MHC. In part, they are able to exert cytotoxic effector functions, whereas another group of Lyt 23 + T lymphocytes constitutes suppressors of humoral and cellular immune responses. A third subpopulation, Lyt 1 + ,23 + T lymphocytes, is less well defined. It is likely that Lyt 1 +, 23 + T lymphocytes serve as precursors of Lyt 1 + T lymphocytes and/or Lyt 23 + T lymphocytes and fulfill regulatory functions. There is evidence that Lyt T lymphocyte subsets do not act in an isolated fashion, but rather interact with each other and with macrophages within regulatory circuits, direct cell contact as well as production of mediators (interleukins) being required for signal delivery.

Macrophages as suppressor cells Macrophages act also as suppressors of cell-mediated immune responses and thus are in yet another way involved in the regulation of CMI (15). Inhibitory effects of macrophages are mediated by soluble products, no­ tably prostaglandins (16). 2)

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Fig. 1. T cell-macrophage interactions in cell-mediated antibacterial immunity. Note that only steps 5 to 7 have been analyzed, whereas steps 2 to 4 lack direct experimental proof. From 28.

T Lymphocyte-Macrophage Interactions . 365

T lymphocyte subsets in antibacterial CMI Our own studies (17) have established that transfer of protection and DTH can be achieved with Lyt 1+,23+ T lymphocytes. However, partici­ pation of other T lymphocyte subsets in the adoptive mediation of protec­ tion and DTH must be inferred as well. Thus, using in vitro assays, we could show that Lyt 1+ T lymphocytes proliferate in response to mac­ rophage bound antigen and primarily cause macrophages to secrete inter­ leukins (see below) (18). Furthermore, we have recently succeeded in propagating in vitro Listeria-specific T lymphocyte lines cloned by the soft agar method (KAUFMANN et al., manuscript in preparation). Listeria­ specific T lymphocyte lines were not only active in vitro, showing antigen­ specific proliferation and induction of interleukin secretion, but in addition protection to live Listeriae as well as DTH to listerial antigen could be conferred upon naive recipient mice by Listeria-specific T lymphocyte lines. These findings strongly suggest that both biological activities are mediated by a single T lymphocyte population. From these combined in vitro and in vivo data, we suggest that after infection with the facultative intracellular bacterium, Listeria mono­ cytogenes, both Lyt 1+ and Lyt 1+, 23+ T lymphocytes are needed for optimum immune responses. These cell sets most probably are intercon­ nected within regulatory circuits whereby Lyt 1+, 23+ T lymphocytes either act as precursors or as positive modulators of Lyt 1 + T lymphocytes. Intercellular mediators in antibacterial CMI

Lymphokines There is no doubt that lymphokines released by antigen stimulated T lymphocytes are messenger molecules for the mediation of signals between the former and mononuclear phagocytes. More than 50 lymphokines have been described on the basis of phenomenology; of these, the ones most relevant to the expression of CMI are: Macrophage inhibitory factor (MIF), macrophage activating factor (MAF), and chemotactic factor (CF) (19). Evidence that lymphokines are involved in antibacterial CMI comes from experiments in which non-activated mononuclear phagocytes were incu­ bated in the presence of antigen and specific T lymphocytes. Subsequently, mononuclear phagocytes turned out to be activated for greater bacteriocidal capacity (20). Similar findings were obtained with mononuclear phagocytes incubated in supernatants from such reactions or in MIF-like purified preparations (21). Also, MIF has been shown to appear in immune animals in close temporal relationship with secondary antigen challenge (22); and injections of purified MIF into whole animals have reproduced symp­ tomatology of CMI (23). As to the cellular source of MIF, evidence has been produced that MIF is produced by immune Lyt 1+ T lymphocytes (24).

366 . H. HAHN and S. H. E. KAUFMANN

Interleukins

The term, interleukin, has been proposed for substances which are secreted by leukocytes and mediate signals between these cells (25, 26). So far, there has been agreement on the definition of two interleukins, inter­ leukin 1 (IL 1) and interleukin 2 (IL 2). IL 1 is identical with the monokine, lymphocyte activity factor (LAF). It is an antigen nonspecific factor with mitogenic activity for lymphocytes. IL 1 from mice has a molecular weight of 12,000-18,000 Daltons. Murine IL 2 has a molecular weight of 30,000-35,000 Daltons. It is secreted by activated T lymphocytes and has the capacity to promote long-term growth of T lymphocytes in vitro, hence its former name T cell growth factor. There is evidence that both IL 1 and IL 2 play a biological role in the differentiation and recruitment of cytotoxic T lymphocytes (26). Although direct proof is lacking, the demonstration of high interleukin activities in cultures consisting of Listeria-immune T lymphocytes, macrophages, and homologous antigen suggests that interleukins might also play a role in antibacterial CM!. Secretion of both IL 1 and IL 2 in cultures primarily depends on Listeria-immune Lyt 1 T lymphocytes (27 and KAUFMANN et al., manuscript in preparation). Thus, interactions between T lymphocyte subsets and macrophages in antibacterial CMI most likely take the following form: Listeria-specific Lyt 1+ T lymphocytes interact with la-associated antigen and activate mac­ rophages to secrete IL 1. In the presence of appropriately presented antigen (signal 1), IL 1 acts as signal 2, leading to activation of Listeria-specific Lyt 1 + T lymphocytes for the secretion of IL 2. Antigen (signal 1) and IL 2 (signal 2) are instrumental aids in the recruitment and/or differentiation of Lyt 1 + T lymphocytes from the specific Lyt 1 +, 23 + T lymphocyte pool. Also, proliferation of Lyt 1 + T lymphocytes takes place. These interactions result in the secretion of lymphokines, most probably MIF, MAF, and CF, which in turn attract macrophages and activate them for greater bacterioci­ dal activity. This schematic outline being hypothetical at this juncture Table 1. Facultative intracellular bacteria Mycobacterium tuberculosis Mycobacterium leprae Brucella spp. Listeria monocytogenes Erysipelothrix rhusiopathiae Yersinia spp. F rancisella Pasteurella multocida Salmonella typhi Salmonella paratyphi Treponema palIidum Legionella pneumophiia

T Lymphocyte-Macrophage Interactions . 367

certainly deserves further consolidation by experimental facts. In particular, it will be interesting to learn more about the identity of the T lymphocyte populations secreting IL 2 on the one, and macrophage activating factors on the other, hand as well as on the identity of the macrophage population(s) involved in antigen presentation and IL 1-secretion as compared to elimina­ tion of bacteria. Conclusion It is increasingly becoming apparent that in antibacterial eMI, like in other immunological systems, T lymphocyte subgroups and mononuclear phagocytes are interconnected to form regulatory loops, whereby one cell type influences the activity of the other. Especially the role of macrophages, not only as effectors, but also as antigen presenting cells in the induction of the immune response and as potent suppressors, is now being appreciated.

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368 . H. HAHN and S. H. E. KAUFMANN 14. CANTOR, H., and R. K. GERSHON. 1979. Immunological circuits: cellular composition. Fed. Proc. 38: 2058. 15. ALLISON, A. C. 1978. Mechanisms by which activated macrophages inhibit lymphocyte responses. Immunol. Rev. 40: 3. 16. STENSON, W. F., and C. W. PARKER. 1980. Prostaglandins, macrophages, and immunity. J. Immunol. 125: 1. 17. KAUFMANN, S. H. E., M. M. SIMON, and H. HAHN. 1979. Specific Lyt 123 T cells are involved in protection against Listeria monocytogenes and in delayed-type hypersensitiv­ ity to listerial antigens. J. expo Med. 150: 1033. 18. KAUFMANN, S. H. E., M. M. SIMON, and H. HAHN. 1981. Macrophage activation in immunity to facultative intracellular bacteria: which T cell subset(s) is (are) involved? In: O. Forster and M. Landy (eds.). Heterogeneity of mononuclear phagocytes. Academic Press, New York, 464. 19. BLOOM, B. R. 1980. The unspecificity of cellular reactions. J. Immunol. 124: 2527. 20. SIMON, H. B., and J. N. SHEAGREN. 1971. Cellular immunity in vitro. I. Immunologically mediated enhancement of macrophage bactericidal capacity. J. expo Med. 133: 1377. 21. FOWLES, R. E., I. M. FAJARDO, J. 1. LEIBOWITCH, and J. R. DAVID. 1973. The enhancement of macrophage bacteriostasis by products of activated lymphocytes. J. expo Med. 138: 952. 22. KAUFMANN, S., 1. WEBER, and H. HAHN. 1975. Macrophage inhibiting activity in serum and central lymph of Listeria-immune mice. Eur. J. Immunol. 5: 799. 23. COHEN, S. 1980. Lymphokines in delayed hypersensitivity. In: Immunology 80. M. Fougereau and J. Dausset (eds.). Academic Press, London, N ew York, Toronto, Sydney, San Francisco, 860. 24. KOHNER, A. 1., H. CANTOR, and J. R. DAVID. 1980. Ly phenotype of lymphocytes producing murine migration inhibitory factor (MIF). J. Immunol. 125: 1117. 25. AARDEN, 1. A. et al. 1979. Letter to the editor: Revised nomenclature for antigen­ nonspecific T cell proliferation and helper factors. J. Immunol. 123: 2928. 26. MOLLER, G. (ed.). 1980. T cell stimulating growth factors. Immunol. Rev. 51. 27. KAUFMANN, S. H. E., H. HAHN, and M. M. SIMON. 1982. T cell subsets induced in Listeria monocytogenes immune mice: Ly phenotypes of T cell interacting with mac­ rophages in vitro. Scand. J. Immunol., in press. 28. HAHN, H., and S. H. E. KAUFMANN. The role of cell-mediated immunity in bacterial infections. Rev. Inf. Dis. in press. Dr. H. HAHN, Institut fur Medizinische Mikrobiologie, Freie Universitat Berlin, 1000 Berlin 45, Germany