antigen expression (fibroblast interferon/immune interferon/lymphokine)
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1 Proc. Nati. Acad. Sci. USA Vol. 8, pp , April 1983 Immunology Macrophage activation: Dissociation of cytotoxic activity from Ia-A antigen expression (fibroblast interferon/immune interferon/lymphokine) ELLIOTT J. BLUMENTHAL, WALDEN K. ROBERTS, ADRIANA VASIL, AND DAVID W. TALMAGE Department of Microbiology and Immunology, University of Colorado Health Sciences Center, Denver, Colorado 8262 Contributed by David W. Talmage, December 2, 1982 ABSTRACT Peritoneal macrophages were obtained from DBA/2 mice that were untreated or after the injection of bacillus Cainette-Guerin (BCG), thioglycollate broth, proteose-peptone broth, or gamma-irradiated P-815 tumor cells. These macrophages were "activated" to become cytotoxic for a fibroblast cell line (L 929) by the addition of lymphokines (LKs), lipopolysaccharide (LPS), or fibroblast interferon (IFN-.3), and the expression of I region-associated antigens (Ia-Ad) on the macrophages was examined both before and after activation. Thioglycollateelicited macrophages became Ia-A' when activated by LKs, but they remained la-a- when activated by LPS or IFN-f3. Resident macrophages and proteose-peptone-elicited macrophages remained Ia-A- when activated with LKs. Macrophages from BCGinfected mice were both Ia-A' and cytotoxic for tumor cells without further treatment. In contrast, macrophages from mice injected with gamma-irradiated P-815 mastocytoma cells were Ia- A+ but not cytotoxic, and these macrophages could not be made cytotoxic by incubation with LKs. The cultured macrophage-like cell lines P388D1 and WEHI-3 became Ia-A+ after incubation with LKs, and this treatment amplified the cytotoxicity of both cell lines. We conclude that a number of factors are important in determining whether Ia-A expression accompanies macrophage activation and that Ia-A is irrelevant as a surface marker for macrophage activation. I region-associated antigens (la) have been detected on distinct subpopulations of macrophages. Most macrophages from the spleen, thymus, and liver are la', whereas la- macrophages predominate in the peritoneal cavity (1-3). It has been shown that only the Ia+ subset of macrophages is effective in presenting antigen to T cells (4, 5). In addition, Ia expression appears to be a prerequisite for macrophages to act as accessory cells for lymphokine (LK) production and lymphocyte proliferation (6, 7). Recently, it has been demonstrated that LK preparations can induce la- macrophages to become Ia+ in vitro (8-1). Similar preparations of LKs are known to contain macrophage-activating factor (MAF) which stimulates macrophages to become cytotoxic for tumor cells (11). This raises the questions of whether the Ia' macrophage is the cytotoxic macrophage and whether La antigens can be used as a surface marker for macrophage activation. In this report, we present evidence indicating that the cytotoxic macrophage can be either Ia-A+ or Ia-A-, depending upon the procedures used for elicitation and activation of it. Also, Ia-A was found not to be a useful marker for macrophage activation because Ia-A expression was neither necessary nor sufficient for macrophage cytotoxicity. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C solely to indicate this fact. 231 MATERIALS AND METHODS Mice. Our source of peritoneal macrophages was DBA/2 mice that were less than 3 months old (The Jackson Laboratory). Elicitation of Peritoneal Macrophages. Peritoneal exudate cells were collected by lavage with 5 ml of sterile Eagle minimal essential medium. Resident cells were removed from untreated mice. Cells were also obtained (a) 3-4 days after the injection of 1 ml of 3% thioglycollate in broth that had been "aged" for at least 6 months at 4C or (b) 3 days after intraperitoneal injection of 1 ml of 1% proteose-peptone broth. The bacillus Calmette-Guerin (BCG)-elicited macrophages were induced by injection of.1 ml of medium containing 2-3 X 17 BCG organisms into the tail vein. Four weeks later,.1 ml of purified protein derivative (2 mg/ml) was injected intraperitoneally; 5 days later the exudate cells were removed by lavage. The P-815-elicited macrophages were induced by intraperitoneal injection of 3 X 16 irradiated (5, R; 1 R = 2.58 x 1-4 C/kg); P-815 tumor cells 5-6 days later the exudate cells were removed by lavage. All of the cell preparations were then washed three times with minimal essential medium before counting and use. Macrophage Cell Lines. The two macrophage-like cell lines WEHI-3 and P388D1 were generously supplied by John Kappler and Philippa Marrack (National Jewish Hospital and Research Center, Denver, CO). These cells were grown in RPMI medium supplemented with.5 mm 2-mercaptoethanol, penicillin, streptomycin, and 1% fetal calf serum. Production of LKs. The LKs were prepared as described (12). Briefly, spleens were removed from CF1 mice that were infected or had been injected intravenously with 2 X 17 BCG organisms 18 days earlier. To the spleen cell suspension (usually 5 x 1' cells per ml) was added concanavalin A (Con A) (Pharmacia, Uppsala, Sweden) to 5,Ag/ml, and the cells were incubated for 2 hr at 37C in a chamber containing 95% air/ 5% CO2. The cells were then collected by centrifugation and resuspended in minimal essential medium supplemented as above but without Con A, and the incubation was continued for 24 hr. The cells were then removed by centrifugation and the culture supernatants were combined and used as a crude source of MAF and immune interferon (IFN-y). Purification of IFN-y. The spleen cell culture supernatant from the LK preparation was partially purified by ammonium sulfate precipitation and passed over a Con A-Sepharose column as described (12). Assay for Macrophage Activation. The cytotoxicity assay we used for LK activation of macrophages has been described Abbreviations: IFN-,B, fibroblast interferon (type I); IFN-y, immune interferon (type II); LPS, lipopolysaccharide; BCG, bacillus Calmette- Guerin; MAF, macrophage-activating factor; LK, lymphokine; Con A, concanavalin A.
2 232 Immunology: Blumenthal et al in detail elsewhere (12, 13). Briefly, mouse L 929 cells were labeled with [3H]thymidine, trypsinized, and added to 16-mm culture wells of a cluster plate (Costar, Cambridge, MA) at a concentration of 1 x 15 cells per well. After incubation for another 24 hr, a suspension containing 4-1 X 15 peritoneal macrophages per ml in minimal essential medium containing 1% fetal calf serum was added. Following a 2-hr incubation, the nonadherent cells were removed and the medium was replaced with.5 ml of LKs diluted in minimal essential medium/1% fetal calf serum. These cultures were then incubated for 48 hr at 37C in 95% air/5% C2, the plates were removed, 5 ul of a 1 mg/ml solution of pancreatic DNase (Worthington) was added to each well, and the plates were incubated an additional 2 min at 37TC. This terminal incubation with DNase facilitated the release of [3H]thymidine from dead cells. The culture supernatants from each well were then added to vials containing 5 ml of Biofluor (New England Nuclear) and the radioactivity was determined by liquid scintillation counting. Normally, <5% of the total radioactivity was released in the absence of LKs whereas >5% was released when LKs were present during the assay. Antibodies. The hybridoma MK-D6 (anti-ia-ad) cell line was generously provided by John Kappler and Philippa Marrack. The antibody produced by this cell line has been classified as belonging to the IgG2a class and has been mapped to Ia-Ad by its reactivity with BLO.D2 or D2.GD but not BLO.LG. A goat anti-mouse fluorescein-conjugated IgG (Meloy, Springfield, VA) was used as an indirect label to quantitate the Ia- Ad antigens on the macrophages. Quantitation of Ia-Ad Antigens. Peritoneal macrophages elicited by various methods were plated onto Lab-Tek tissue culture chamber slides (Lab-Tek, Naperville, IL) at a concentration of 2 x 15 cells per chamber. After a 3-hr period in minimal essential medium/1% fetal calf serum for the cells to become adherent, the nonadherent. cells were removed by washing twice with the same medium and the adherent cells were either stained (time ) or were incubated 1-3 days with various "activating" agents and subsequently examined for Ia- Ad antigen expression. Prior to the staining of the cells, the medium was aspirated and replaced with 1% paraformaldehyde solution for 15 min at room temperature. The cells were then washed twice with minimal essential medium/5% rabbit serum (Colorado Serum, Denver, CO) and allowed to equilibrate with minimal essential medium/5% rabbit serum for 2 min to allow the rabbit serum to bind to any available Fc receptors. After the 2-min incubation, the minimal essential medium/5% rabbit serum was removed and fresh minimal essential medium/5% rabbit serum containing a 1:8 dilution of the MK-D6 ascites fluid was added to each well. The slide was kept at 4 C for 4 min. After this period the medium was removed from each well, the cells were washed twice with cold minimal essential medium/5% rabbit serum, and then minimal essential medium/ 5% rabbit serum supplemented with a 1:3 dilution of the goat anti-mouse fluorescein-conjugated antibody was added and the cells were incubated for another 4 min at 4 C. The cells were then washed twice with minimal essential medium/ 5% rabbit serum to remove any unbound antibodies, the chamber was removed. from the slide, and a coverslip was placed over the cells and sealed. As controls, either normal mouse serum was used in the place of the MK-D6 antibody or the cells were stained only with the goat anti-mouse fluoresceinlabeled antibody. It was found that B cells stained positively in these controls, but these were readily distinguishable from macrophages by their morphology (small, rounded cells) and were not included with the macrophages that were counted. Proc. Natl. Acad. Sci. USA 8 (1983) A Zeiss microscope was used to observe the fluorescence of the cells, and photomicrographs were taken on Kodak Ektachrome film (16 ASA tungsten). The exposure time for the fluorescence microscopy under the blue light was 5 min. At least 2 cells were counted for each quantitation, and the positive cells were characterized by having good peripheral staining; the controls did not show any staining. RESULTS Effect of LKs on la-ad Expression by Macrophages. Fig. 1 demonstrates how different populations of macrophages stain with the anti-ia-ad antiserum followed by the fluorescein-labeled anti-mouse globulin antiserum. Cells that did not receive LKs during the incubation period were not stained (Fig. 1A); and LK-induced cells stained brightly (Fig. 1B). We chose the 3-day period for staining the cells because we found that la expression reaches its peak on this day and this correlates with the findings of others (8). When normal mouse serum was substituted for the anti-ia-ad antiserum in the procedure, LK-treated cells appeared similar to those in Fig. IA. In all cases there were a few stained B cells that reacted directly with the goat anti-mouse IgG-fluorescein and thus were positive in the normal serum controls. They were particularly plentiful in the resident cell populations (Fig. 1C), but they were easily distinguished from macrophages by their small size. There was no staining of either resident (Fig. 1C) or proteosepeptone-elicited macrophages (Fig. 1D) when cultured for 3 days in medium containing LKs. All of these macrophage populations had the same percentage of Ia-Ad-positive cells before the addition of LKs to the culture medium, the response after 3 days in culture was quite striking. In addition, peritoneal macrophages elicited with gamma-irradiated P-815 tumor cells had a high percentage of Ia-Adpositive cells when stained immediately after removal from the mice (Fig. 1E). These cells were very adherent, stained positively with a nonspecific esterase stain, and ingested latex beads, suggesting that they were macrophages (data not shown). Effect of LK Concentration on Macrophage Cytotoxicity and la-ad Expression. We next looked at the correlation between LK activation of macrophage cytotoxicity and the generation of Ia-Ad antigens on the macrophage cell surface. As the concentration of LK was decreased, both the cytotoxic potential and the Ia-Ad-inducing capability of the preparation were lost (Fig. 2). We have assayed more than 1 different LK preparations in this manner and, although both activities declined, Ia-Ad expression was always induced at LK dilutions that elicited no macrophage activation. In addition, 9% of both activities was lost after heating at 56 C for 1 hr or after treatment of the LK preparation with ph 2 buffer at 4 C for 12 hr (results not shown). Effects of LK on la-ad Expression and Cytotoxicity of Various Macrophage Preparations. The previous sets of data suggested that our LK preparation contained both Ia-Ad-inducing capability and macrophage activating factor(s) (MAF) and that these effects occurred together. We next wanted to examine different macrophage populations, or macrophages that had been elicited by different mechanisms, to determine whether they would respond in a similar manner. The potential of the macrophages to express Ia-Ad and to become cytotoxic was dependent upon how the macrophages were elicited (Table I). Resident, proteose-peptone-elicited, and thioglycollage-elicited macrophages all were capable of becoming cytotoxic for tumor cells, but only the thioglycollate-elicited macrophages expressed Ia-Ad in response to LKs. In addition, WEHI-3 and
3 Immunology: Blumenthal et al Proc. Nat Acad. Sci. USA 8 (1983) 233 FIG. 1. Effect of LKs on Ia-Ad expression by macrophages: thioglycollate-elicited macrophages cultured for 3 days in the absence (a, A) or presence (b, B) of LKs; resident macrophages cultured 3 days with LKs (c, C); proteose-peptone-elicited macrophages cultured 3 days with LKs (d, D); and gamma-irradiated P-815-elicited macrophages not cultured (e, E). Macrophages were photographed in white light (e-e) or in ultraviolet (blue) light (A-E) after staining with anti-ia-ad antiserum followed by fluorescein-labeled goat anti-mouse IgG. P388D1, the two macrophage-like cell lines were inducible for Ia-Ad expression as well as cytotoxicity. However, at the higher effector macrophage concentrations, both cell lines were cytotoxic for tumor cells without LK activation, although they remained negative for expression of Ia-Ad. Furthermore, macrophages elicited by intraperitoneal injection of gamma-irradiated P-815 mastocytoma cells were -positive initially for Ia- Ad antigen but were totally resistant to LK activation for tumor cell cytotoxicity. Finally, as reported by others (1416) we observed that BCG-elicited macrophages were both Ia-A positive and cytotoxic when assayed immediately after removal from the mouse, even in the absence. of LKs. When these macrophages were incubated in the absence of LKs they lost their Ia positivity.
4 234 Immunology: Blumenthal et al 1 CZ 8 ~~~~~~~~~~~~~.4 ~~~~~~// a 4 ", % LK in culture medium FIG. 2. Effect of LK concentration on peritoneal macrophage cytotoxicity and Ia-Ad expression. Increasing amounts of a LK preparation were added to macrophage cultures; cytotoxicity was measured by the percentage of total [3H]thjmidine released and expression was measured by percentage of Ia-A -positive cells. Each point represents the mean (SD, 2%) of triplicate determinations. Effects of IFN-y, Fibroblast Interferon (IFN-fi), and Lipopolysaccharide,(LPS) on Macrophage l-aad Expression and Cytotoxicity. Because we observed that the type of elicitation used to induce macrophage populations was important in determining whether Ia-Ad antigens were produced, we next Table 1. Effects of LKs on Ia-Ad expression and cytotoxicity of various macrophage preparations % [3H]thymidine % Ia-Ad positivet released* Macrophage On With With With With preparation* day no LK 2% LK no LK 2% LK Resident Proteosepeptone Thioglycollateelicited WEHI-3 (1 x 15) WEHI-3 (5 x 15) P388D1 (1 X 14) P388D1 (1 X 15) P-815-elicited BCG-elicitedl * All macrophage preparations except WEHI and P388D1 were assayed for Ia expression and [3H]thymidine release at a macrophage density of 2 x 15/cm2. For the exceptions, cell density (no./cm2) is shown in parentheses. tpercentages are based on counts of at least 2 cells in at least two different experiments; these values did not vary more than 15%. t Thymidine release was measured 2 days after the addition of macrophages to the labeled tumor cells. The mixtures were incubated with or without 2% LKs. Numbers are the mean values of triplicate determinations that did not vary more than 2% from this value. 11a expression was assayed 3 days after incubation of macrophages with or without 2% LKs. BCG-elicited macrophages were prepared, by the injection of 2-3 x 17 organisms in.1 ml into the tail vein of mice. Four weeks later,.1 ml of purified protein derivative (2 mg/ml) was injected intraperitoneally and 5 days later the cells were removed from the peritoneal cavity by lavage, washed, counted, and placed onto the chamber slides Os a- Cd d) *ax Pe Proc. Natl. Acad. Sci. USA 8 (1983) Table 2. Effects of IFN-y, IFN-13, and LPS on macrophage Ia expression and cytotoxicity % [3Hlthymidine released Activating No macro- With macrofactor added % Ia+ phages phages* None IFN-'y (1 units/ml) IFN-MP (1, units/ml) LPS (2,g/ml) *Tioglycollate-elicited peritoneal macrophages at a concentration of 2 x 15/cm2 were used for all assays. IFN-13 was obtained from Lee Biomolecular Research Labs (San Diego, CA) and was induced from virally infected (Newcastle disease virus) fibroblast cells. wanted to determine whether different macrophage-activating agents would show the same variability. To this end, we added three different macrophage-activating agents-ifn-y, IFN-, and LPS-to thioglycollate-elicited macrophages. All three activating agents were able to generate a cytotoxic response to tumor cells, but only IFN-y was able to induce the macrophages to express Ia-Ad (Table 2). The IFN-y used in this experiment had been purified by us to a specific activity of 1 X 16 interferon units/mg of protein by fractionation of a LK preparation through selective ammonium sulfate precipitation and Con A-Sepharose column chromatography (12). Our results correspond with those of Steeg and Oppenheim (17) who also observed that purified IFN-y induced the expression of la antigens on macrophages. The concentrations shown in Table 2 were found to be optimal for cytotoxicity, but even at concentrations up to 6, units of IFN-,B per ml there was no evidence of la expression. DISCUSSION In accord with results reported by others (18), we found that resident peritoneal macrophages or macrophages elicited with thioglycollate or proteose-peptone did not express Ia, whereas macrophages that appeared in response to the injection of BCG or P-815 tumor cells were Ia'. Treatment of the Ia- macrophages with LKs activated all of these populations to become cytotoxic for tumor cells, but the concomitant appearance of surface Ta depended upon the method used for the original elicitation (Table 1). Ia expression was invariably seen after- LK activation of thioglycollate-elicited macrophages, whereas no Ia could be detected on the surface of activated resident or proteose-peptone-elicited macrophage populations. Significant differences between these two populations have been reported with respect to 2 production (19), membrane-associated enzyme activity (2), and bactericidal capacity (21). We can speculate that only the recently recruited macrophages become Ia' (1), possibly because they are less mature as, suggested by Hogg and Parish (22), and that we are able to obtain these macrophages only with thioglycollate broth. Other investigators have used LKs to induce Ia expression on peritoneal macrophages and have found that resident macrophages respond only weakly (1), whereas both thioglycol-. late- (9) and proteose-peptone-elicited macrophages became Ia+ after in vitro incubation with LKs. It is unclear whether the difference in Ia induction between our proteose-peptoneelicited macrophages and those of Steinman et al(8) was due to a difference in the health of the mice used, in the proteosepeptone broth used, in the length of culture with LK (23), or in the Ia detection assay. However, we think that the important points to be made are that the method of elicitation can affect Ia expression, possibly due to differences in pros-
5 Immunology: Blumenthal et at taglandin synthesis by the macrophage populations (24), and that macrophage cytotoxicity and Ia expression can be dissociated. Thioglycollate-elicited macrophages that are activated by using IFN-p or LPS instead of LK remain la- (Table 2). This is additional evidence that the activated macrophage need not be Ia+ to be cytotoxic. LK treatment induced Ia expression in both the WEHI-3 and P388D1 cell lines and promoted cytotoxicity, although at relatively high cell concentrations LK was not necessary for target cell killing (Table 1). However, at lower cell concentrations, LK activation was required for cytotoxicity, suggesting that with these cell lines LK treatment amplifies an inherent low level of cytotoxicity. As expected from earlier work (14, 15), BCG infection induced macrophages that were both Ia+ and cytotoxic without further treatment, probably as a consequence of the infection stimulating LK production in vivo. In contrast, macrophages that were elicited by using irradiated P-815 cells were Ia' upon removal from the mice but were not cytotoxic, and these macrophages could not be made cytotoxic by subsequent LK treatment. This presents another interesting example of how tumor cells may escape immune surveillance-i.e., by paralyzing elicited macrophages so that they are unable to give a cytotoxic response to LK signals. This also shows that Ia expression is not sufficient for macrophage cytotoxicity. In previous work, Roberts and Vasil (12) were unable to separate MAF and IFN-y activities by using techniques of protein purification, protein inactivation, and differential LK induction. Using many of these same techniques, we also have been unable to resolve Ia-inducing factor(s) from MAF/IFNy activities. In addition, we have investigated the LKs produced by 64 T cell hybridoma clones and 29 subclones after stimulation with Con A (*) and found high qualitative and quantitative correlations among the production of MAF, IFNy, and Ia-inducing factor. Thus, we think that the possibility should be considered that the induction of la antigens, cytotoxicity, and the antiviral state represent different activities of a common factor. * Zlotnik, A., Roberts, W., Blumenthal, E., Marrack, P. & Kappler, J., T Cell Hybridomas as Sources of Lymphokines, Third International Lymphokine Workshop, August 1982, Philadelphia. Proc. Natl. Acad. Sci. USA 8 (1983) 235 This work was supported by National Institute of Allergy and Infectious Diseases Research Grant AI-347 and American Cancer Society Grant IM Cowing, C., Schwartz, B. D. & Dickler, H. B. (1978)J. Immunol 12, Beller, D. I. & Unanue, E. R. (198)J. Immunol 124, Richman, L. K., Klingenstein, R. J., Richman, J. A., Strober, W. & Berzofsky, J. A. (1979) J. Immunol 123, Yamashita, U. & Shevach, E. M. (1977) J. Immunol 119, Cowing, C., Pincus, S. H., Sachs, D. H. & Dickler, H. B. (1978) J. Immunol 121, Farr, A. G., Wechter, W. J., Keily, J. M. & Unanue, E. R. (1979) J. Immunol 122, Habu, S., Hayakawa, K. & Okumura, K. (1979) Cell Immunol 47, Steinman, R. M., Nogueira, N., Witmer, M. D., Tydings, J. D. & Mellman, I. S. (198)J. Exp. Med. 152, Steeg, P. S., Moore, R. N. & Oppenheim, J. J. (198)J. Exp. Med. 152, Scher, M. G., Unanue, E. R. & Beller, D. I. (1982) J. Immunol. 128, Fidler, I. J. & Roz, A. (1981) in Lymphokines, ed. Pick, E. (Academic, New York), Vol. 3, pp Roberts, W. K. & Vasil, A. (1982) J. Interferon Res. 2, Roberts, W. K. & Vasil, A. (1982) J. Immunol Methods 54, Marino, P. A., Whisnant, C. C. & Adams, D.. (1981)J. Exp. Med. 154, McCarthy, M. E. & Zwilling, B. S. (1981) Cell. Immunol 6, Nathan, C. F., Silverstein, S. C., Brukner, L. H. & Cohn, Z. A. (1979)J. Exp. Med. 149, Steeg, P. S. & Oppenheim, J. J. (1982) Fed. Proc. Fed. Am. Soc. Exp. Biol 41, 84 (abstr.). 18. Scher, M. G., Beller, D. I. & Unanue, E. R. (198)J. Exp. Med. 152, Soberman, R. J. & Karnovsky, M. L. (1981) in Lymphokines, ed. Pick, E. (Academic, New York), Vol. 3, pp Edelson, P. (1981) in Lymphokines, ed. Pick, E. (Academic, New York), Vol. 3, pp Miake, S., Takeya, K., Matsumoto, T., Yoshikai, Y. & Nomoto, K. (198)J. Reticuloend. Soc. 27, Hogg, N. & Parish, C. R. (198) Immunology 41, Beller, D. I. & Ho, K. (1982)J. Immunol 129, Snyder, D. S. (1982) Fed. Proc. Fed. Am. Soc. Exp. Biol. 41, 815, (abstr.).
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