Macrophage Activation: Dissociation of Cytotoxic Activity from Ia-A Antigen Expression (Fibroblast Interferon/Immune Interferon/Lymphokine) ELLIOTT J
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Proc. Nati. Acad. Sci. USA Vol. 80, pp. 2031-2035, 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 80262 Contributed by David W. Talmage, December 20, 1982 ABSTRACT Peritoneal macrophages were obtained from MATERIALS AND METHODS DBA/2 mice that were untreated or after the injection of bacillus Cainette-Guerin (BCG), thioglycollate broth, proteose-peptone Mice. Our source of peritoneal macrophages was DBA/2 broth, or gamma-irradiated P-815 tumor cells. These macro- mice that were less than 3 months old (The Jackson Labo- phages were "activated" to become cytotoxic for a fibroblast cell ratory). line (L 929) by the addition of lymphokines (LKs), lipopolysac- Elicitation of Peritoneal Macrophages. Peritoneal exudate charide (LPS), or fibroblast interferon (IFN-.3), and the expres- cells were collected by lavage with 5 ml of sterile Eagle min- sion of I region-associated antigens (Ia-Ad) on the macrophages imal essential medium. Resident cells were removed from un- was examined both before and after activation. Thioglycollate- treated mice. Cells were also obtained (a) 3-4 days after the elicited macrophages became Ia-A' when activated by LKs, but injection of 1 ml of 3% thioglycollate in broth that had been they remained la-A- when activated by LPS or IFN-f3. Resident "aged" for at least 6 months at 40C or (b) 3 days after intra- macrophages and proteose-peptone-elicited macrophages re- peritoneal injection of 1 ml of 1% proteose-peptone broth. mained Ia-A- when activated with LKs. Macrophages from BCG- The bacillus Calmette-Guerin (BCG)-elicited macrophages were infected mice were both Ia-A' and cytotoxic for tumor cells with- induced by injection of 0.1 ml of medium containing 2-3 X out further treatment. In contrast, macrophages from mice in- 107 BCG organisms into the tail vein. Four weeks later, 0.1 jected with gamma-irradiated P-815 mastocytoma cells were Ia- ml of purified protein derivative (2 mg/ml) was injected in- A+ but not cytotoxic, and these macrophages could not be made traperitoneally; 5 days later the exudate cells were removed cytotoxic by incubation with LKs. The cultured macrophage-like by lavage. The P-815-elicited macrophages were induced by cell lines P388D1 and WEHI-3 became Ia-A+ after incubation with intraperitoneal injection of 3 X 106 irradiated (5,000 R; 1 R LKs, and this treatment amplified the cytotoxicity of both cell lines. = 2.58 x 10-4 C/kg); P-815 tumor cells 5-6 days later the We conclude that a number of factors are important in deter- exudate cells were removed by lavage. All of the cell prep- mining whether Ia-A expression accompanies macrophage acti- arations were then washed three times with minimal essential vation and that Ia-A is irrelevant as a surface marker for mac- medium before counting and use. rophage activation. Macrophage Cell Lines. The two macrophage-like cell lines WEHI-3 and P388D1 were generously supplied by John Kap- I region-associated antigens (la) have been detected on distinct pler and Philippa Marrack (National Jewish Hospital and Re- subpopulations of macrophages. Most macrophages from the search Center, Denver, CO). These cells were grown in RPMI spleen, thymus, and liver are la', whereas la- macrophages medium supplemented with 0.5 mM 2-mercaptoethanol, pen- predominate in the peritoneal cavity (1-3). It has been shown icillin, streptomycin, and 10% fetal calf serum. that only the Ia+ subset of macrophages is effective in pre- Production of LKs. The LKs were prepared as described senting antigen to T cells (4, 5). In addition, Ia expression ap- (12). Briefly, spleens were removed from CF1 mice that were pears to be a prerequisite for macrophages to act as accessory infected or had been injected intravenously with 2 X 107 BCG cells for lymphokine (LK) production and lymphocyte prolif- organisms 18 days earlier. To the spleen cell suspension (usu- eration (6, 7). ally 5 x 10' cells per ml) was added concanavalin A (Con A) Recently, it has been demonstrated that LK preparations can (Pharmacia, Uppsala, Sweden) to 5 ,Ag/ml, and the cells were induce la- macrophages to become Ia+ in vitro (8-10). Similar incubated for 2 hr at 370C in a chamber containing 95% air/ of LKs are known to contain 5% CO2. The cells were then collected by centrifugation and preparations macrophage-acti- resuspended in minimal essential medium supplemented as vating factor (MAF) which stimulates macrophages to become above but without Con A, and the incubation was continued cytotoxic for tumor cells (11). This raises the questions of whether for 24 hr. The cells were then removed by centrifugation and the Ia' macrophage is the cytotoxic macrophage and whether the culture supernatants were combined and used as a crude La antigens can be used as a surface marker for macrophage ac- source of MAF and immune interferon (IFN-y). tivation. Purification of IFN-y. The spleen cell culture supernatant In this report, we present evidence indicating that the cy- from the LK preparation was partially purified by ammonium totoxic macrophage can be either Ia-A+ or Ia-A-, depending sulfate precipitation and passed over a Con A-Sepharose col- upon the procedures used for elicitation and activation of it. umn as described (12). Also, Ia-A was found not to be a useful marker for macrophage Assay for Macrophage Activation. The cytotoxicity assay activation because Ia-A expression was neither necessary nor we used for LK activation of macrophages has been described sufficient for macrophage cytotoxicity. Abbreviations: IFN-,B, fibroblast interferon (type I); IFN-y, immune The publication costs of this article were defrayed in part by page charge interferon (type II); LPS, lipopolysaccharide; BCG, bacillus Calmette- payment. This article must therefore be hereby marked "advertise- Guerin; MAF, macrophage-activating factor; LK, lymphokine; Con A, ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. concanavalin A. 2031 Downloaded by guest on September 24, 2021 2032 Immunology: Blumenthal et aL Proc. Natl. Acad. Sci. USA 80 (1983) in detail elsewhere (12, 13). Briefly, mouse L 929 cells were A Zeiss microscope was used to observe the fluorescence labeled with [3H]thymidine, trypsinized, and added to 16-mm of the cells, and photomicrographs were taken on Kodak culture wells of a cluster plate (Costar, Cambridge, MA) at a Ektachrome film (160 ASA tungsten). The exposure time for concentration of 1 x 105 cells per well. After incubation for the fluorescence microscopy under the blue light was 5 min. another 24 hr, a suspension containing 4-10 X 105 peritoneal At least 200 cells were counted for each quantitation, and the macrophages per ml in minimal essential medium containing positive cells were characterized by having good peripheral 10% fetal calf serum was added. Following a 2-hr incubation, staining; the controls did not show any staining. the nonadherent cells were removed and the medium was re- placed with 0.5 ml of LKs diluted in minimal essential me- dium/10% fetal calf serum. These cultures were then incu- RESULTS bated for 48 hr at 370C in 95% air/5% C02, the plates were Effect of LKs on la-Ad Expression by Macrophages. Fig. removed, 5 ul of a 1 mg/ml solution of pancreatic DNase 1 demonstrates how different populations of macrophages stain (Worthington) was added to each well, and the plates were with the anti-Ia-Ad antiserum followed by the fluorescein-la- incubated an additional 20 min at 37TC. This terminal incu- beled anti-mouse globulin antiserum. Cells that did not re- bation with DNase facilitated the release of [3H]thymidine from ceive LKs during the incubation period were not stained (Fig. dead cells. The culture supernatants from each well were then 1A); and LK-induced cells stained brightly (Fig. 1B). We chose added to vials containing 5 ml of Biofluor (New England Nu- the 3-day period for staining the cells because we found that clear) and the radioactivity was determined by liquid scintil- la expression reaches its peak on this day and this correlates lation counting. Normally, <5% of the total radioactivity was with the findings of others (8). When normal mouse serum released in the absence of LKs whereas >50% was released was substituted for the anti-Ia-Ad antiserum in the procedure, when LKs were present during the assay. LK-treated cells appeared similar to those in Fig. IA. In all Antibodies. The hybridoma MK-D6 (anti-Ia-Ad) cell line was cases there were a few stained B cells that reacted directly generously provided by John Kappler and Philippa Marrack. with the goat anti-mouse IgG-fluorescein and thus were pos- The antibody produced by this cell line has been classified as itive in the normal serum controls. They were particularly belonging to the IgG2a class and has been mapped to Ia-Ad plentiful in the resident cell populations (Fig. 1C), but they by its reactivity with BLO.D2 or D2.GD but not BLO.LG. A were easily distinguished from macrophages by their small size. goat anti-mouse fluorescein-conjugated IgG (Meloy, Spring- There was no staining of either resident (Fig. 1C) or proteose- field, VA) was used as an indirect label to quantitate the Ia- peptone-elicited macrophages (Fig. 1D) when cultured for 3 Ad antigens on the macrophages. days in medium containing LKs. All of these macrophage Quantitation of Ia-Ad Antigens.