Proc. Nati. Acad. Sci. USAVol. 80, pp. 2031-2035, April 1983Immunology

Macrophage activation: Dissociation of cytotoxic activity from Ia-Aantigen expression

(fibroblast interferon/immune interferon/lymphokine)

ELLIOTT J. BLUMENTHAL, WALDEN K. ROBERTS, ADRIANA VASIL, AND DAVID W. TALMAGEDepartment 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 fromDBA/2 mice that were untreated or after the injection of bacillusCainette-Guerin (BCG), thioglycollate broth, proteose-peptonebroth, or gamma-irradiated P-815 tumor cells. These macro-phages were "activated" to become cytotoxic for a fibroblast cellline (L 929) by the addition of lymphokines (LKs), lipopolysac-charide (LPS), or fibroblast interferon (IFN-.3), and the expres-sion of I region-associated antigens (Ia-Ad) on the macrophageswas examined both before and after activation. Thioglycollate-elicited macrophages became Ia-A' when activated by LKs, butthey remained la-A- when activated by LPS or IFN-f3. Residentmacrophages and proteose-peptone-elicited macrophages re-mained Ia-A- when activated with LKs. Macrophages from BCG-infected mice were both Ia-A' and cytotoxic for tumor cells with-out further treatment. In contrast, macrophages from mice in-jected with gamma-irradiated P-815 mastocytoma cells were Ia-A+ but not cytotoxic, and these macrophages could not be madecytotoxic by incubation with LKs. The cultured macrophage-likecell lines P388D1 and WEHI-3 became Ia-A+ after incubation withLKs, and this treatment amplified the cytotoxicity of both cell lines.We conclude that a number of factors are important in deter-mining whether Ia-A expression accompanies macrophage acti-vation and that Ia-A is irrelevant as a surface marker for mac-rophage activation.

I region-associated antigens (la) have been detected on distinctsubpopulations of macrophages. Most macrophages from thespleen, thymus, and liver are la', whereas la- macrophagespredominate in the peritoneal cavity (1-3). It has been shownthat only the Ia+ subset of macrophages is effective in pre-senting antigen to T cells (4, 5). In addition, Ia expression ap-pears to be a prerequisite for macrophages to act as accessorycells for lymphokine (LK) production and lymphocyte prolif-eration (6, 7).

Recently, it has been demonstrated that LK preparations caninduce la- macrophages to become Ia+ in vitro (8-10). Similarpreparations of LKs are known to contain macrophage-acti-vating factor (MAF) which stimulates macrophages to becomecytotoxic for tumor cells (11). This raises the questions of whetherthe Ia' macrophage is the cytotoxic macrophage and whetherLa antigens can be used as a surface marker for macrophage ac-tivation.

In this report, we present evidence indicating that the cy-totoxic macrophage can be either Ia-A+ or Ia-A-, dependingupon the procedures used for elicitation and activation of it.Also, Ia-A was found not to be a useful marker for macrophageactivation because Ia-A expression was neither necessary norsufficient for macrophage cytotoxicity.

MATERIALS AND METHODS

Mice. Our source of peritoneal macrophages was DBA/2mice that were less than 3 months old (The Jackson Labo-ratory).

Elicitation of Peritoneal Macrophages. Peritoneal exudatecells were collected by lavage with 5 ml of sterile Eagle min-imal essential medium. Resident cells were removed from un-treated mice. Cells were also obtained (a) 3-4 days after theinjection of 1 ml of 3% thioglycollate in broth that had been"aged" for at least 6 months at 40C or (b) 3 days after intra-peritoneal injection of 1 ml of 1% proteose-peptone broth.The bacillus Calmette-Guerin (BCG)-elicited macrophages wereinduced by injection of 0.1 ml of medium containing 2-3 X107 BCG organisms into the tail vein. Four weeks later, 0.1ml of purified protein derivative (2 mg/ml) was injected in-traperitoneally; 5 days later the exudate cells were removedby lavage. The P-815-elicited macrophages were induced byintraperitoneal injection of 3 X 106 irradiated (5,000 R; 1 R= 2.58 x 10-4 C/kg); P-815 tumor cells 5-6 days later theexudate cells were removed by lavage. All of the cell prep-arations were then washed three times with minimal essentialmedium before counting and use.

Macrophage Cell Lines. The two macrophage-like cell linesWEHI-3 and P388D1 were generously supplied by John Kap-pler and Philippa Marrack (National Jewish Hospital and Re-search Center, Denver, CO). These cells were grown in RPMImedium supplemented with 0.5 mM 2-mercaptoethanol, pen-icillin, streptomycin, and 10% fetal calf serum.

Production of LKs. The LKs were prepared as described(12). Briefly, spleens were removed from CF1 mice that wereinfected or had been injected intravenously with 2 X 107 BCGorganisms 18 days earlier. To the spleen cell suspension (usu-ally 5 x 10' cells per ml) was added concanavalin A (Con A)(Pharmacia, Uppsala, Sweden) to 5 ,Ag/ml, and the cells wereincubated for 2 hr at 370C in a chamber containing 95% air/5% CO2. The cells were then collected by centrifugation andresuspended in minimal essential medium supplemented asabove but without Con A, and the incubation was continuedfor 24 hr. The cells were then removed by centrifugation andthe culture supernatants were combined and used as a crudesource of MAF and immune interferon (IFN-y).

Purification of IFN-y. The spleen cell culture supernatantfrom the LK preparation was partially purified by ammoniumsulfate precipitation and passed over a Con A-Sepharose col-umn as described (12).

Assay for Macrophage Activation. The cytotoxicity assaywe used for LK activation of macrophages has been described

Abbreviations: IFN-,B, fibroblast interferon (type I); IFN-y, immuneinterferon (type II); LPS, lipopolysaccharide; BCG, bacillus Calmette-Guerin; MAF, macrophage-activating factor; LK, lymphokine; Con A,concanavalin A.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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2032 Immunology: Blumenthal et aL

in detail elsewhere (12, 13). Briefly, mouse L 929 cells werelabeled with [3H]thymidine, trypsinized, and added to 16-mmculture wells of a cluster plate (Costar, Cambridge, MA) at aconcentration of 1 x 105 cells per well. After incubation foranother 24 hr, a suspension containing 4-10 X 105 peritonealmacrophages per ml in minimal essential medium containing10% fetal calf serum was added. Following a 2-hr incubation,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-bated for 48 hr at 370C in 95% air/5% C02, the plates wereremoved, 5 ul of a 1 mg/ml solution of pancreatic DNase(Worthington) was added to each well, and the plates wereincubated an additional 20 min at 37TC. This terminal incu-bation with DNase facilitated the release of [3H]thymidine fromdead cells. The culture supernatants from each well were thenadded to vials containing 5 ml of Biofluor (New England Nu-clear) and the radioactivity was determined by liquid scintil-lation counting. Normally, <5% of the total radioactivity wasreleased in the absence of LKs whereas >50% was releasedwhen LKs were present during the assay.

Antibodies. The hybridoma MK-D6 (anti-Ia-Ad) cell line wasgenerously provided by John Kappler and Philippa Marrack.The antibody produced by this cell line has been classified asbelonging to the IgG2a class and has been mapped to Ia-Adby its reactivity with BLO.D2 or D2.GD but not BLO.LG. Agoat anti-mouse fluorescein-conjugated IgG (Meloy, Spring-field, VA) was used as an indirect label to quantitate the Ia-Ad antigens on the macrophages.

Quantitation of Ia-Ad Antigens. Peritoneal macrophageselicited by various methods were plated onto Lab-Tek tissueculture chamber slides (Lab-Tek, Naperville, IL) at a con-centration of 2 x 105 cells per chamber. After a 3-hr periodin minimal essential medium/10% fetal calf serum for the cellsto become adherent, the nonadherent. cells were removed bywashing twice with the same medium and the adherent cellswere either stained (time 0) or were incubated 1-3 days withvarious "activating" agents and subsequently examined for Ia-Ad antigen expression.

Prior to the staining of the cells, the medium was aspiratedand replaced with 1% paraformaldehyde solution for 15 minat room temperature. The cells were then washed twice withminimal essential medium/5% rabbit serum (Colorado Serum,Denver, CO) and allowed to equilibrate with minimal essen-tial medium/5% rabbit serum for 20 min to allow the rabbitserum to bind to any available Fc receptors. After the 20-minincubation, the minimal essential medium/5% rabbit serumwas removed and fresh minimal essential medium/5% rabbitserum containing a 1:80 dilution of the MK-D6 ascites fluidwas added to each well. The slide was kept at 4°C for 40 min.After this period the medium was removed from each well,the cells were washed twice with cold minimal essential me-dium/5% rabbit serum, and then minimal essential medium/5% rabbit serum supplemented with a 1:30 dilution of thegoat anti-mouse fluorescein-conjugated antibody was addedand the cells were incubated for another 40 min at 4°C. Thecells were then washed twice with minimal essential medium/5% rabbit serum to remove any unbound antibodies, thechamber was removed.from the slide, and a coverslip was placedover the cells and sealed. As controls, either normal mouseserum was used in the place of the MK-D6 antibody or thecells were stained only with the goat anti-mouse fluorescein-labeled antibody. It was found that B cells stained positivelyin these controls, but these were readily distinguishable frommacrophages by their morphology (small, rounded cells) andwere not included with the macrophages that were counted.

A Zeiss microscope was used to observe the fluorescenceof the cells, and photomicrographs were taken on KodakEktachrome film (160 ASA tungsten). The exposure time forthe fluorescence microscopy under the blue light was 5 min.At least 200 cells were counted for each quantitation, and thepositive cells were characterized by having good peripheralstaining; 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 stainwith the anti-Ia-Ad antiserum followed by the fluorescein-la-beled anti-mouse globulin antiserum. Cells that did not re-ceive LKs during the incubation period were not stained (Fig.1A); and LK-induced cells stained brightly (Fig. 1B). We chosethe 3-day period for staining the cells because we found thatla expression reaches its peak on this day and this correlateswith the findings of others (8). When normal mouse serumwas substituted for the anti-Ia-Ad antiserum in the procedure,LK-treated cells appeared similar to those in Fig. IA. In allcases there were a few stained B cells that reacted directlywith the goat anti-mouse IgG-fluorescein and thus were pos-itive in the normal serum controls. They were particularlyplentiful in the resident cell populations (Fig. 1C), but theywere easily distinguished from macrophages by their small size.There was no staining of either resident (Fig. 1C) or proteose-peptone-elicited macrophages (Fig. 1D) when cultured for 3days in medium containing LKs. All of these macrophagepopulations had the same percentage of Ia-Ad-positive cellsbefore the addition of LKs to the culture medium, the re-sponse after 3 days in culture was quite striking. In addition,peritoneal macrophages elicited with gamma-irradiated P-815tumor cells had a high percentage of Ia-Adpositive cells whenstained immediately after removal from the mice (Fig. 1E).These cells were very adherent, stained positively with a non-specific esterase stain, and ingested latex beads, suggestingthat they were macrophages (data not shown).

Effect of LK Concentration on Macrophage Cytotoxicityand la-Ad Expression. We next looked at the correlation be-tween LK activation of macrophage cytotoxicity and the gen-eration of Ia-Ad antigens on the macrophage cell surface. As theconcentration of LK was decreased, both the cytotoxic poten-tial and the Ia-Ad-inducing capability of the preparation werelost (Fig. 2). We have assayed more than 10 different LK prep-arations in this manner and, although both activities declined,Ia-Ad expression was always induced at LK dilutions that elic-ited no macrophage activation. In addition, 90% of both activ-ities was lost after heating at 56°C for 1 hr or after treatmentof the LK preparation with pH 2 buffer at 4°C for 12 hr (resultsnot shown).

Effects of LK on la-Ad Expression and Cytotoxicity of Var-ious Macrophage Preparations. The previous sets of data sug-gested that our LK preparation contained both Ia-Ad-inducingcapability and macrophage activating factor(s) (MAF) and thatthese effects occurred together. We next wanted to examinedifferent macrophage populations, or macrophages that had beenelicited by different mechanisms, to determine whether theywould respond in a similar manner. The potential of the mac-rophages to express Ia-Ad and to become cytotoxic was depen-dent upon how the macrophages were elicited (Table I). Res-ident, proteose-peptone-elicited, and thioglycollage-elicitedmacrophages all were capable of becoming cytotoxic for tumorcells, but only the thioglycollate-elicited macrophages ex-pressed Ia-Ad in response to LKs. In addition, WEHI-3 and

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P388D1, the two macrophage-like cell lines were inducible forIa-Ad expression as well as cytotoxicity. However, at the highereffector macrophage concentrations, both cell lines were cy-totoxic for tumor cells without LK activation, although theyremained negative for expression of Ia-Ad. Furthermore, mac-rophages elicited by intraperitoneal injection of gamma-irra-diated P-815 mastocytoma cells were -positive initially for Ia-

FIG. 1. Effect of LKs on Ia-Ad expression by macrophages: thio-glycollate-elicited macrophages cultured for 3 days in the absence (a,A) or presence (b, B) of LKs; resident macrophages cultured 3 days withLKs (c, C); proteose-peptone-elicited macrophages cultured 3 days withLKs (d, D); and gamma-irradiated P-815-elicited macrophages not cul-tured (e, E). Macrophages were photographed in white light (e-e) orin ultraviolet (blue) light (A-E) after staining with anti-Ia-Ad anti-serum followed by fluorescein-labeled goat anti-mouse IgG.

Ad antigen but were totally resistant to LK activation for tu-mor cell cytotoxicity. Finally, as reported by others (1416)we observed that BCG-elicited macrophages were both Ia-Apositive and cytotoxic when assayed immediately after re-moval from the mouse, even in the absence. of LKs. Whenthese macrophages were incubated in the absence of LKs theylost their Ia positivity.

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2034 Immunology: Blumenthal et aL

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FIG. 2. Effect of LK concentration on peritoneal macrophage cy-

totoxicity and Ia-Ad expression. Increasing amounts of a LK prepa-

ration were added to macrophage cultures; cytotoxicity was measuredby the percentage of total [3H]thjmidine released and expression wasmeasured by percentage of Ia-A -positive cells. Each point representsthe mean (SD, 20%) of triplicate determinations.

Effects of IFN-y, Fibroblast Interferon (IFN-fi), and Li-popolysaccharide,(LPS) on Macrophage l-AAd Expression andCytotoxicity. Because we observed that the type of elicitationused to induce macrophage populations was important in de-termining whether Ia-Ad antigens were produced, we next

Table 1. Effects of LKs on Ia-Ad expression and cytotoxicity ofvarious macrophage preparations

% [3H]thymidine% Ia-Ad positivet released*

Macrophage On With With With Withpreparation* day 0 no LK 2% LK§ no LK 2% LK

Resident 8 4 2 2 53Proteose-peptone 5 5 8 4 67

Thioglycollate-elicited 6 5 98 6 84

WEHI-3(1 x 105) 1 1 100 7 48

WEHI-3(5 x 105) 1 1 100 19 46

P388D1(1 X 104) 1 1 100 2 42

P388D1(1 X 105) 1 1 100 24 55

P-815-elicited 58 8 15 5 4BCG-elicitedl 66 18 100 87 82

* All macrophage preparations exceptWEHI and P388D1 were assayedfor Ia expression and [3H]thymidine release at a macrophage densityof 2 x 105/cm2. For the exceptions, cell density (no./cm2) is shown inparentheses.

tPercentages are based on counts of at least 200 cells in at least twodifferent experiments; these values did not vary more than 15%.

t Thymidine release was measured 2 days after the addition of mac-

rophages to the labeled tumor cells. The mixtures were incubated withor without 2% LKs. Numbers are the mean values of triplicate de-terminations that did not vary more than 20% from this value.11a expression was assayed 3 days after incubation of macrophageswith or without 2% LKs.BCG-elicited macrophages were prepared, by the injection of 2-3 x107 organisms in 0.1 ml into the tail vein of mice. Four weeks later,0.1 ml of purified protein derivative (2 mg/ml) was injected intra-peritoneally and 5 days later the cells were removed from the peri-toneal cavity by lavage, washed, counted, and placed onto the cham-ber slides.

Table 2. Effects of IFN-y, IFN-13, and LPS on macrophage Iaexpression and cytotoxicity

% [3Hlthymidine releasedActivating No macro- With macro-factor added % Ia+ phages phages*

None 7 2 5IFN-'y (10 units/ml) 100 3 74IFN-MP (1,000 units/ml) 2 3 60LPS (20 ,g/ml) 3 4 32

*Tioglycollate-elicited peritoneal macrophages at a concentration of2 x 105/cm2 were used for all assays. IFN-13 was obtained from LeeBiomolecular Research Labs (San Diego, CA) and was induced fromvirally infected (Newcastle disease virus) fibroblast cells.

wanted to determine whether different macrophage-activatingagents would show the same variability. To this end, we addedthree different macrophage-activating agents-IFN-y, IFN-,and LPS-to thioglycollate-elicited macrophages. All three ac-tivating agents were able to generate a cytotoxic response totumor cells, but only IFN-y was able to induce the macro-phages to express Ia-Ad (Table 2). The IFN-y used in this ex-periment had been purified by us to a specific activity of 1X 106 interferon units/mg of protein by fractionation of a LKpreparation through selective ammonium sulfate precipitationand Con A-Sepharose column chromatography (12). Our re-sults correspond with those of Steeg and Oppenheim (17) whoalso observed that purified IFN-y induced the expression ofla antigens on macrophages. The concentrations shown in Ta-ble 2 were found to be optimal for cytotoxicity, but even atconcentrations up to 6,000 units of IFN-,B per ml there wasno evidence of la expression.

DISCUSSIONIn accord with results reported by others (18), we found thatresident peritoneal macrophages or macrophages elicited withthioglycollate or proteose-peptone did not express Ia, whereasmacrophages that appeared in response to the injection of BCGor P-815 tumor cells were Ia'. Treatment of the Ia- mac-rophages with LKs activated all of these populations to be-come cytotoxic for tumor cells, but the concomitant appear-ance of surface Ta depended upon the method used for theoriginal elicitation (Table 1). Ia expression was invariably seenafter- LK activation of thioglycollate-elicited macrophages,whereas no Ia could be detected on the surface of activatedresident or proteose-peptone-elicited macrophage popula-tions. Significant differences between these two populationshave been reported with respect to 02 production (19), mem-brane-associated enzyme activity (20), and bactericidal capac-ity (21). We can speculate that only the recently recruitedmacrophages become Ia' (10), possibly because they are lessmature as, suggested by Hogg and Parish (22), and that we areable to obtain these macrophages only with thioglycollate broth.

Other investigators have used LKs to induce Ia expressionon peritoneal macrophages and have found that resident mac-rophages respond only weakly (10), whereas both thioglycol-.late- (9) and proteose-peptone-elicited macrophages becameIa+ after in vitro incubation with LKs. It is unclear whetherthe difference in Ia induction between our proteose-peptone-elicited macrophages and those of Steinman et aL(8) was dueto a difference in the health of the mice used, in the proteose-peptone broth used, in the length of culture with LK (23), orin the Ia detection assay. However, we think that the im-portant points to be made are that the method of elicitationcan affect Ia expression, possibly due to differences in pros-

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taglandin synthesis by the macrophage populations (24), andthat macrophage cytotoxicity and Ia expression can be dis-sociated.

Thioglycollate-elicited macrophages that are activated byusing IFN-p or LPS instead of LK remain la- (Table 2). Thisis additional evidence that the activated macrophage need notbe Ia+ to be cytotoxic.LK treatment induced Ia expression in both the WEHI-3

and P388D1 cell lines and promoted cytotoxicity, although atrelatively high cell concentrations LK was not necessary fortarget cell killing (Table 1). However, at lower cell concen-trations, LK activation was required for cytotoxicity, suggest-ing that with these cell lines LK treatment amplifies an in-herent low level of cytotoxicity. As expected from earlier work(14, 15), BCG infection induced macrophages that were bothIa+ and cytotoxic without further treatment, probably as aconsequence of the infection stimulating LK production in vivo.In contrast, macrophages that were elicited by using irradi-ated P-815 cells were Ia' upon removal from the mice butwere not cytotoxic, and these macrophages could not be madecytotoxic by subsequent LK treatment. This presents anotherinteresting example of how tumor cells may escape immunesurveillance-i.e., by paralyzing elicited macrophages so thatthey are unable to give a cytotoxic response to LK signals.This also shows that Ia expression is not sufficient for mac-rophage cytotoxicity.

In previous work, Roberts and Vasil (12) were unable toseparate MAF and IFN-y activities by using techniques ofprotein purification, protein inactivation, and differential LKinduction. Using many of these same techniques, we also havebeen unable to resolve Ia-inducing factor(s) from MAF/IFN-y activities. In addition, we have investigated the LKs pro-duced by 64 T cell hybridoma clones and 29 subclones afterstimulation with Con A (*) and found high qualitative andquantitative correlations among the production of MAF, IFN-y, and Ia-inducing factor. Thus, we think that the possibilityshould be considered that the induction of la antigens, cy-totoxicity, and the antiviral state represent different activitiesof a common factor.

This work was supported by National Institute of Allergy and Infec-tious Diseases Research Grant AI-03047 and American Cancer SocietyGrant IM-204.

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