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The role of interferon-gamma in the induction of activated macrophages
MACROPHAGE ACTIVATION
203
[11] GRAY, P.W. & GOEDDEL, D.V., Cloning and expression of murine IFN-~ cDNA. Proc. nat. Acad. Sci. (Wash.), 1983, 80, 5842.
[12] SCHULTZ,R.M. & KLEINSCHMIDT,W.J., Functional identity between recombi[13] [14]
[15] [16] [17]
nant murine interferon-~ and macrophage activating factor. Nature (Lond.), 1983, 305, 239. Ruco, L.P. & MELTZER,M.S., Macrophage activation for tumor cytotoxicity: development of macrophage cytotoxic activity requires completion of a sequence of short-lived intermediary reactions. J. ImmunoL, 1978, 121, 2035. SCHULTZ,R.M., Synergistic activation of macrophages by lymphokine and lipopolysaccharide: evidence for lymphokine as the primer and interferon as the trigger. J. Interferon Res., 1982, 2, 459. SCHULTZ,R.M., PAVLIDIS,N.A., STYLOS,W.A. & CHIRIGOS,M.A., Regulation of macrophage tumoricidal function : a role for prostaglandins E. Science, 1978, 202, 320. SCHULTZ,R.M. & ALTOM, M.G., Potentiation of nonspecific immunotherapy of experimental lung metastases by indomethacin. J. Immunopharmacol., 1983, 5, 277. SCHULTZ,R.M., CmRIGOS, M.A., STOYCHKOV,J.N. & PAVLIDIS,N.A., Factors affecting macrophage cytotoxic activity with particular emphasis on corticosteroids and acute stress. J. reticuloend Soc., 1979, 26, 83.
T H E ROLE OF I N T E R F E R O N - G A M M A IN T H E I N D U C T I O N OF ACTIVATED M A C R O P H A G E S by R.D. Schreiber, A. Celada and N. Buchmeier
Department of Pathology, Washington University School of Medicine, St. Louis, MO (USA) Introduction. It is now well recognized that following in vitro culture with lymphokinecontaining supernatants, macrophages acquire or increase their capacity to kill a variety of neoplastic cells and intracellular or extracellular parasites. Macrophages that exhibit enhanced cytocidal activity have become known as ~ macrophages and the lymphokines that initiate this process as macrophage-activating factors (MAF). Whereas, in the past, these definitions have served an important descriptive function, they are today too imprecise to accurately characterize the functional state of a cell that is extremely pleotropic in the activities it can perform.
Thus, it has become necessary to define activation within the context of a particular cell function. More importantly, since induction of macrophage cytocidal activity is complex and can occur in a stepwise fashion, it has also become necessary to specify the pathway step being studied and the particular assay being used. In our laboratory, we define activated macrophages as those cells capable of lysing cytoplasmically labelled P815 mastocytoma cells in a short-term (24 h) assay. Moreover, we define MAF as the lymphokine that initiates this process by priming the macrophage so that it becomes more receptive to a second signal that triggers the actual tumoricidal response.
204
11 e F O R U M D ' I M M U N O L O G I E
IFN~f-dependent induction o f tumoricidal activity. Our initial goal was to define the molecular identify of MAF. Using supernatants from mitogen-stimulated cultures of either normal murine splenic cells or a T-cell hybridoma which produced large quantities of MAF and antiviral activities, we demonstrated that IFN~" was the major and possibly only lymphokine detected by our assay that was capable of initiating the macrophage cytocidal response (table I) [1-4]. These observations prompted us to focus our subsequent efforts on elucidating IFN~,'s mechanism of action at the molecular level and in attempting to validate its physiologic role in vivo. We felt that these studies were particularly warranted because of the general agreement among different laboratories that IFN~, could almost universally initiate macrophage activation in a variety of assay systems. Our studies have documented that macrophages carry on their plasma membrane a specific receptor for IFN-( and that ligand-receptor interaction is the event that initiates the macrophage activation response. The murine IFNy receptor was demonstrated by showing that elicited and bonemarrow-derived macrophages (1) absor-
bed either natural or recombinant IFN~, at 4~ or 37~ (2) formed specific rosettes with microspheres coated with recombinant IFN~f, and (3) bound radioidinated purified IFN~, in a saturable, reversible and specific manner. Macrophages expressed approximately 10,000 receptors/cell and at 4~ bound ligand with an affinity (Ka) of approximately 108M -1 [5]. We have also found a similar receptor on human mononuclear phagocytes [6]. These cells carried 5,000-10,000 receptors/cell and displayed a Ka for ligand of 109M- 1 at 4~ The IFN~" receptor on mononuclear phagocytes is not effected by states o f cellular d i f f e r e n t i a t i o n or activation and, in the human, appears to consist, at least in part, of a 70-Kd polypeptide chain. The biological relevance of the receptor has been validated by demonstrating a direct correlation between the degree of receptor occupancy and the extent of the cellular response induced. We have also investigated the structure-function relationships that exist within the IFN~molecule itself [4]. Using monoclonal antibodies specific for different epitopes on murine IFN-f, data was obtained to suggest that IFN~, displayed at least two topographically distinct functional domains. One domain was required for
TABLE I. - - Demonstration that IFN~. is the major MAF for inducing non-specific tumoricidal activity by murine macrophages. .
MAF and antiviral activities displayed: a) coordinate biosynthesis; b) identical heat and pH sensitivities; c) identical elution on polynucleotide and dye-ligand columns.
.
Hybridoma-derived MAF and IFN~, co-purified through sequential separation steps that resulted in a 100,000-fold purification and 40~ recovery of the two activities.
.
Recombinant murine IFN~,, procuced in E. coli and purified to greater than 97~ homogeneity quantitatively expressed MAF activity.
.
MAF activity produced by mitogen-stimulated normal T cells or the T-cell hybridoma was completely neutralized by two monoclonal antibodies specific for distinct epitopes on murine IFN~,.
MACROPHAGE ACTIVATION expression of the molecular site that binds to the receptor while the second domain appeared to be selectively required for expression of IFNT-dependent antiviral activity. These latter results thus raise the possibility that distinct regions of IFN T promote different cellular responses. Finally, we addressed the issue of whether IFNT plays a physiologic role in an in vivo situation [7]. For these studies, we used the model of Listeria monocytogenes infection in mice. By immunochemically quantitating IFN T production during the course of a sublethal infection, we were able to show a three-way correlation between (1) the production of IFN T by cells from infected mice, (2) the in vivo activation of macrophages, and (3) the clearance of bacteria from the peritoneal cavity and spleen and resolution of the infection. More importantly, we were able to demonstrate that immunochemical neutralization in vivo of endogenously produced IFN T inhibited the elaboration of activated macrophages and abrogated the capacity of the mice to resolve the infection. Thus, these results strongly suggest that IFN3, is an obligatory component in the physiologic pathway of macrophage activation. However, more data will be needed before IFN T can be assigned a direct macrophage activating role in vivo because of its ability to effect other pathway steps,
205
including the generation of T cells which secrete macrophage-activating lymphokines. Discussion.
The results presented herein represent one approach to studying IFN T as a prototypic macrophage-activating factor. At the present time, this particular lymphokine is the most easily studied, since it is available in purified recombinant form and monoclonal antibodies to both human and murine IFN T have been produced. The availability of these reagents thus allows for the conduct of detailed experiments which will ultimately elucidate the molecular interactions which underlie IFNT-dependent macrophage activation and define the physiologic importance of these in vitro observations. However, it must be stressed that these studies do not preclude the possibility that other MAF exist. In fact, the IFN T studies have already provided insights and reagents that are being used to look for other macrophage-activating lymphokines. However, it is now clear that such nonIFNT-MAF will have to ultimately be authenticated at the biochemical rather than the functional level, and their physiologic significance will have to be addressed.
References.
[1] SCHREIBER,R.D., ALTMAN,A. & KATZ, D.H., Identification of a T-cell hybridoma producing large quantities of macrophage-activating factor. J. exp. Med., 1982, 156, 677.
[2] SCHREIBER, R.D., PACE, J.L., RUSSELL, S.W., ALTMAN, A. & KATZ, D.H., Macrophage-activating factor produced by a T-cell hybridoma: physiochemical and biosynthetic resemblance to gamma-interferon. J. Immunol., 1983, 131, 826. [3] PACE, J.L., RUSSELL, S.W., SCHREIBER, R.D., ALTMAN, A. & KATZ, D.H., Macrophage activation: priming activity from a T-cell hybridoma is attributable to interferon-T. Proc. nat. Acad. Sci. (Wash.), 1983, 80, 3782. [4] SCHREIBER,R.D., HICKS, L.J., CELADA,A., BUCHMEIER,N.A. & GRAY, P.W., Monoclonal antibodies to murine gamma-interferon which differentially modulate macrophage activation and antiviral activity. J. Immunol., 1985, 134, 1609.
206
11 e F O R U M D ' I M M U N O L O G I E
[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
203
[11] GRAY, P.W. & GOEDDEL, D.V., Cloning and expression of murine IFN-~ cDNA. Proc. nat. Acad. Sci. (Wash.), 1983, 80, 5842.
[12] SCHULTZ,R.M. & KLEINSCHMIDT,W.J., Functional identity between recombi[13] [14]
[15] [16] [17]
nant murine interferon-~ and macrophage activating factor. Nature (Lond.), 1983, 305, 239. Ruco, L.P. & MELTZER,M.S., Macrophage activation for tumor cytotoxicity: development of macrophage cytotoxic activity requires completion of a sequence of short-lived intermediary reactions. J. ImmunoL, 1978, 121, 2035. SCHULTZ,R.M., Synergistic activation of macrophages by lymphokine and lipopolysaccharide: evidence for lymphokine as the primer and interferon as the trigger. J. Interferon Res., 1982, 2, 459. SCHULTZ,R.M., PAVLIDIS,N.A., STYLOS,W.A. & CHIRIGOS,M.A., Regulation of macrophage tumoricidal function : a role for prostaglandins E. Science, 1978, 202, 320. SCHULTZ,R.M. & ALTOM, M.G., Potentiation of nonspecific immunotherapy of experimental lung metastases by indomethacin. J. Immunopharmacol., 1983, 5, 277. SCHULTZ,R.M., CmRIGOS, M.A., STOYCHKOV,J.N. & PAVLIDIS,N.A., Factors affecting macrophage cytotoxic activity with particular emphasis on corticosteroids and acute stress. J. reticuloend Soc., 1979, 26, 83.
T H E ROLE OF I N T E R F E R O N - G A M M A IN T H E I N D U C T I O N OF ACTIVATED M A C R O P H A G E S by R.D. Schreiber, A. Celada and N. Buchmeier
Department of Pathology, Washington University School of Medicine, St. Louis, MO (USA) Introduction. It is now well recognized that following in vitro culture with lymphokinecontaining supernatants, macrophages acquire or increase their capacity to kill a variety of neoplastic cells and intracellular or extracellular parasites. Macrophages that exhibit enhanced cytocidal activity have become known as ~
Thus, it has become necessary to define activation within the context of a particular cell function. More importantly, since induction of macrophage cytocidal activity is complex and can occur in a stepwise fashion, it has also become necessary to specify the pathway step being studied and the particular assay being used. In our laboratory, we define activated macrophages as those cells capable of lysing cytoplasmically labelled P815 mastocytoma cells in a short-term (24 h) assay. Moreover, we define MAF as the lymphokine that initiates this process by priming the macrophage so that it becomes more receptive to a second signal that triggers the actual tumoricidal response.
204
11 e F O R U M D ' I M M U N O L O G I E
IFN~f-dependent induction o f tumoricidal activity. Our initial goal was to define the molecular identify of MAF. Using supernatants from mitogen-stimulated cultures of either normal murine splenic cells or a T-cell hybridoma which produced large quantities of MAF and antiviral activities, we demonstrated that IFN~" was the major and possibly only lymphokine detected by our assay that was capable of initiating the macrophage cytocidal response (table I) [1-4]. These observations prompted us to focus our subsequent efforts on elucidating IFN~,'s mechanism of action at the molecular level and in attempting to validate its physiologic role in vivo. We felt that these studies were particularly warranted because of the general agreement among different laboratories that IFN~, could almost universally initiate macrophage activation in a variety of assay systems. Our studies have documented that macrophages carry on their plasma membrane a specific receptor for IFN-( and that ligand-receptor interaction is the event that initiates the macrophage activation response. The murine IFNy receptor was demonstrated by showing that elicited and bonemarrow-derived macrophages (1) absor-
bed either natural or recombinant IFN~, at 4~ or 37~ (2) formed specific rosettes with microspheres coated with recombinant IFN~f, and (3) bound radioidinated purified IFN~, in a saturable, reversible and specific manner. Macrophages expressed approximately 10,000 receptors/cell and at 4~ bound ligand with an affinity (Ka) of approximately 108M -1 [5]. We have also found a similar receptor on human mononuclear phagocytes [6]. These cells carried 5,000-10,000 receptors/cell and displayed a Ka for ligand of 109M- 1 at 4~ The IFN~" receptor on mononuclear phagocytes is not effected by states o f cellular d i f f e r e n t i a t i o n or activation and, in the human, appears to consist, at least in part, of a 70-Kd polypeptide chain. The biological relevance of the receptor has been validated by demonstrating a direct correlation between the degree of receptor occupancy and the extent of the cellular response induced. We have also investigated the structure-function relationships that exist within the IFN~molecule itself [4]. Using monoclonal antibodies specific for different epitopes on murine IFN-f, data was obtained to suggest that IFN~, displayed at least two topographically distinct functional domains. One domain was required for
TABLE I. - - Demonstration that IFN~. is the major MAF for inducing non-specific tumoricidal activity by murine macrophages. .
MAF and antiviral activities displayed: a) coordinate biosynthesis; b) identical heat and pH sensitivities; c) identical elution on polynucleotide and dye-ligand columns.
.
Hybridoma-derived MAF and IFN~, co-purified through sequential separation steps that resulted in a 100,000-fold purification and 40~ recovery of the two activities.
.
Recombinant murine IFN~,, procuced in E. coli and purified to greater than 97~ homogeneity quantitatively expressed MAF activity.
.
MAF activity produced by mitogen-stimulated normal T cells or the T-cell hybridoma was completely neutralized by two monoclonal antibodies specific for distinct epitopes on murine IFN~,.
MACROPHAGE ACTIVATION expression of the molecular site that binds to the receptor while the second domain appeared to be selectively required for expression of IFNT-dependent antiviral activity. These latter results thus raise the possibility that distinct regions of IFN T promote different cellular responses. Finally, we addressed the issue of whether IFNT plays a physiologic role in an in vivo situation [7]. For these studies, we used the model of Listeria monocytogenes infection in mice. By immunochemically quantitating IFN T production during the course of a sublethal infection, we were able to show a three-way correlation between (1) the production of IFN T by cells from infected mice, (2) the in vivo activation of macrophages, and (3) the clearance of bacteria from the peritoneal cavity and spleen and resolution of the infection. More importantly, we were able to demonstrate that immunochemical neutralization in vivo of endogenously produced IFN T inhibited the elaboration of activated macrophages and abrogated the capacity of the mice to resolve the infection. Thus, these results strongly suggest that IFN3, is an obligatory component in the physiologic pathway of macrophage activation. However, more data will be needed before IFN T can be assigned a direct macrophage activating role in vivo because of its ability to effect other pathway steps,
205
including the generation of T cells which secrete macrophage-activating lymphokines. Discussion.
The results presented herein represent one approach to studying IFN T as a prototypic macrophage-activating factor. At the present time, this particular lymphokine is the most easily studied, since it is available in purified recombinant form and monoclonal antibodies to both human and murine IFN T have been produced. The availability of these reagents thus allows for the conduct of detailed experiments which will ultimately elucidate the molecular interactions which underlie IFNT-dependent macrophage activation and define the physiologic importance of these in vitro observations. However, it must be stressed that these studies do not preclude the possibility that other MAF exist. In fact, the IFN T studies have already provided insights and reagents that are being used to look for other macrophage-activating lymphokines. However, it is now clear that such nonIFNT-MAF will have to ultimately be authenticated at the biochemical rather than the functional level, and their physiologic significance will have to be addressed.
References.
[1] SCHREIBER,R.D., ALTMAN,A. & KATZ, D.H., Identification of a T-cell hybridoma producing large quantities of macrophage-activating factor. J. exp. Med., 1982, 156, 677.
[2] SCHREIBER, R.D., PACE, J.L., RUSSELL, S.W., ALTMAN, A. & KATZ, D.H., Macrophage-activating factor produced by a T-cell hybridoma: physiochemical and biosynthetic resemblance to gamma-interferon. J. Immunol., 1983, 131, 826. [3] PACE, J.L., RUSSELL, S.W., SCHREIBER, R.D., ALTMAN, A. & KATZ, D.H., Macrophage activation: priming activity from a T-cell hybridoma is attributable to interferon-T. Proc. nat. Acad. Sci. (Wash.), 1983, 80, 3782. [4] SCHREIBER,R.D., HICKS, L.J., CELADA,A., BUCHMEIER,N.A. & GRAY, P.W., Monoclonal antibodies to murine gamma-interferon which differentially modulate macrophage activation and antiviral activity. J. Immunol., 1985, 134, 1609.
206
11 e F O R U M D ' I M M U N O L O G I E
[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 <
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