Macrophage-activating factor for cytotoxicity produced by a human T-cell hybridoma

CELLULAR

IMMUNOLOGY

87,626-636 (1984)

Macrophage-Activating Factor for Cytotoxicity Produced by a Human T-Cell Hybridoma MASAHIRO

HIGUCHI,

NORIKO

NAKAMURA,

SHU-ICHI

TSUCHIYA,

YOSHIROKOBAYASHI,AND TOSHIAIU OSAWA’ Division

of

Chemical Toxicology and Immunochemistry, Faculty of Pharmaceutical sciences, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan Received March 27, 1984; acceptedApril 14, 1984

Human MAF-C (macrophage-activation factor for cytotoxicity)-producing hybridoma H2-E35 was prepared by somatic cell fusion of PHA-activated peripheral blood lymphocytes with emetine/actinomycin D-treated cloned human acute lymphatic leukemia cells (CEM). The following activities were assayed (1) macrophage-migration-inhibitory factor (MIF), (2) macrophageactivation factor for glucose consumption (MAF-G), (3) macrophage-activation factor for 02formation (MAF-0), and (4) macrophage-activation factor for cytotoxicity (MAF-C). After anionexchangechromatography, MAF-C activity could be distinguishedfrom MIF and MAF-0 activities. It is shown that MAF-C is not the same as MAF-G from the culture supematants of CEM 1I, a parent cell line of H2-E3-5. Furthermore, MAF-C from H2-E3-5 culture supematants activated differentiated macrophages but not monocytes.

INTRODUCTION Antigens or mitogens stimulate production by T cells of lymphokines that affect macrophage functions. These lymphokines stimulate and increasemetabolic, secretory, and functional activities of macrophages. Whether most of these phenomena are induced by one molecule or not is an important unanswered question. The crude supernatant of cultured lymphocytes can not be used to solve this problem because it may contain many biologically active substances.We and others (l-3) have shown by the use of T-cell clone or T-cell hybridoma that more than one molecule is involved in the activation of macrophages. Ratliff et al. (1) reported a macrophage-activation factor (MAF)‘-producing hybridoma which did not produce interferon (IFN) and concluded that MAF and IFN are not the same, and Gemsa et al. (2) reported that the culture supematants from two different T-cell clones stimulate macrophages differently. We previously reported the establishment of a human T-cell hybridoma secreting lymphotoxin (LT), macrophage-migration-inhibitory factor (MIF), mac’ To whom correspondence should be addressed. 2Abbreviations used MAF, macrophage-activating factor; IFN, interferon; LT, lymphotoxin; MIF, migration-inhibitory factor; MAF-G, macrpohage-activating factor for glucose consumption; MAF-0, macrophage-activating factor for 02- formation; MAF-C, macrophage-activating factor for cytotoxicity; PHAP, phytohemagglutinin-P; PBS, 10 mM sodium phosphate buffer (pH 7.2) containing 0. I5 M NaCl; PBL, peripheral blood lymphocytes; PMA, phorbol myristic acetate; NBT, nitroblue tetraxolium; CSF, colonystimulating factor; MCF, macrophage chemotactic factor. 626 0008-8749184$3.00 Copyright Q 1984 by Academic Press,Inc. AU rights of reproduction in any form reserved.

MACROPHAGE-ACTIVATING

FACTOR

627

rophage-activation factor for glucose consumption (MAF-G), and macrophage-activation factor for 02- formation (MAF-0) (3-5), and showed that at least two different molecules, MAF-G and MAF-0, are involved in the activation of macrophages. In this study we developed a hybridoma H2-E3-5 which produces macrophageactivation factor for cytotoxicity (MAF-C) which lacked IFN activity. By anionexchange chromatography, we isolated MAF-C lacking MAF-0 activity from the culture supernatant of H2-E3-5 and isolated MAF-G lacking MAF-C activity from the culture supernatant of CEM 11, the parent cell line of the hybridomas. From these results, we concluded that MAF-C from the H2-E3-5 culture supematant does not possessMAF-0 activity and that it is also different from MAF-G derived from the CEM 11 culture supernatants. Furthermore, we demonstrated that MAF-C from the H2-E3-5 culture supematant could activate differentiated macrophages but not monocytes. MATERIALS

AND METHODS

Stimulation of human peripheral blood lymphocytes (PBL) with a T-cell mitogen. Human PBL were isolated from a healthy donor by Ficoll-Urografin density gradient centrifugation (8). Contaminating red cells were lysed in 0.87% T&buffered NH&l. The lymphocytes ( lo6 cells/ml) were cultured in RPM1 1640 containing 60 mg/liter of kanamycin, 2 mM glutamine, 5 X 1OM5M 2-mercaptoethanol, 10%fetal calf serum (enriched medium), and 5 pg/ml of phytohemagglutininP (PHA-P, Difco, Detroit, Mich.) at 37°C for 40 hr. Treatment of a clone of CEM (CEM 11) with emetine and actinomycin D. Emetinel actinomycin D-treated CEM cells were prepared essentially by the method described previously (4). Briefly, a clone of CEM (CEM 11) was suspended in RPM1 1640, containing 20 mM 4-(2-hydroxyethyl)-I-piperazineethanesulfonic acid (Hepes; pH 7.2), at a cell density of 2 X lO”/ml and then treated with 5 X 10m5M emetine hydrochloride (Nakarai Chemical Ltd., Kyoto, Japan) and 0.25 pg/ml of actinomycin D (Makor Chemicals Ltd., Jerusalem, Israel) at 37°C for 2 hr. These concentrations of emetine hydrochloride and actinomycin D completely inhibited proliferation of CEM 11 cells. The cells were washed four times with 10 mit4 sodium phosphate buffer (pH 7.2) containing 0.15 M NaCl (PBS) in order to remove free emetine and actinomycin D. Hybridization. PHA-P-activated PBL were centrifuged and incubated with 0.1 M N-acetyl-Dgalactosamine at 37°C for 20 min in order to remove cell-bound PHA-P. These PBL were fused with emetine/actinomycin D-treated CEM 11 cells, as described previously (6). Mitomycin C-treated CEM 11 cells were added to the fused cells. The mixture was subcultured in 0.2-ml culture wells in a 96-well microplate (Falcon, Oxnard, Calif.; No. 3042). Every day during the first week, 100 ~1 of the medium in each well was replaced by 100 ~1 of fresh enriched medium. The hybrid cell lines showed good growth in all of the wells within 2 weeks after fusion. However, in the control wells, to which emetine/actinomycin D-pretreated CEM 11 cells or mitomycin C-pretreated CEM 11 cells were added, no cell growth was observed. Detection of surface markers. Surface markers of fused cells were detected by the two-step binding assayusing monoclonal antibody OKT3, OKT4, OKT5, OKT8, or OKTl 1 (Ortho Pharmaceutical Co., Raritan, N.J.) and ‘251-labeledanti-mouse Ig according to the method described previously (7). Briefly, 20 ~1 of fused cells (1.5 to

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2.5 X 1O7cells/ml) in PBS containing 1% bovine serum albumin and 0.1% sodium azide was put into wells of a U-bottomed polyvinylchloride plate precoated with the above buffer overnight. To the cells was added 5 ~1of OKT3,OKT4,OKT5, OKTS, or OKTl 1 antibody. Each mixture was incubated at room temperature for 1 hr, centrifuged, and then washed three to five times with the above buffer. To the cell pellets was added 50 ~1 of ‘251-labeledanti-mouse Ig ( lo5 cpm/well), and then the mixture was incubated at 0°C for 1 hr and washed five times with the above buffer. The cell-bound radioactivity was measured with an autogamma counter. Preparation of supematants of PHA-P-activated PBL. PBL were cultured at a density of lo6 cells/ml in enriched medium containing 5 &ml of PHA-P at 37°C for 48 hr. After centrifugation, the supernatants were stored at -20°C. PHA-P itself does not show MIF, MAF-G, MAF-0, and MAF-C activities. Preparation of O-80% ammonium sulfate precipitates fraction of the culture supernatant of H2-E3-5. Ammonium sulfate was added to the culture supernatant of H2-E3-5 to 80% saturation. After centrifugation, the precipitates formed at O-SO% saturation were dissolved in PBS and dialyzed against PBS to remove ammonium sulfate. By this procedure 300 ml of the culture supernatant was concentrated to 25 ml. Assayfor MIF activity. We employed the agarose-droplet method using oil-induced peritoneal exudate cells of guinea pigs (Shizuoka Agricultural Cooperative Association for Laboratory Animals, Hamamatsu, Japan) (8). The migration-inhibitory activity can be expressed as the percentage of inhibition where migration distance in test sample x 100. migration distance in control sample Assayfor glucose consumption ofperitoneal exudate cells (MAF-G). MAF-G activity was determined as the glucose consumption of oil-induced peritoneal exudate cells of guinea pigs, according to the method of Onozaki et al. (9). The MAF-G activity can be expressed as the percentage of glucose consumption where % glucose remaining in test sample % consumption = 1 x 100. % glucose remaining in control sample 1 ( To quantify MAF-G activity, 100 units of MAF-G was defined as the amount of MAF-G in 1 ml of ammonium sulfate precipitate fraction of the H2-E3-5 culture supernatant. To determine the amount of MAF-G activity, serial dilutions were assayed. Assay for cellular Or- formation of U-937 cells by nitroblue tetrazolium (NBT) reduction (MAF-0). The MAF-0 assaywas performed by a modification of the method of Henry (10). After incubation of the U-937 cells with a test sample as described above, phorbol myristic acetate (PMA; Sigma Chemical Co., St. Louis, MO.) was added to a final concentration of 0.1 &ml, and the mixture cultured for 2 hr at 37°C. NBT was then added to a final concentration of 1 mg/ml, and the mixture cultured for 30 min at 37°C. The number of U-937 cells and NBT-positive cells were counted. The reduction of NBT to formazan is mostly 02- dependent (11). Cells were judged positive if stained blue-black by precipitated formazan (12). MAF-0 activity is expressed as the percentage of NBT-positive cells where % NBT-positive cells =

number of NBT-positive cells x 100. number of U-937 cells

MACROPHAGE-ACTIVATING

FACTOR

629

To quantify MAF-0 activity, 100 units of MAF-0 was defined as the amount of MAF-0 in 1 ml of ammonium sulfate precipitate fraction of the H2-E3-5 culture supernatant. To determine the amount of MAF-0 activity, serial dilutions were assayed. Assay for tumor cytotoxicity of monocyte-derived macrophages (MAF-C). MAF-C activity was evaluated as the cytotoxicity toward tumor cells of monocyte-derived macrophages, according to a modification of the method of Cameron et al. (13). Briefly, PBL freshly isolated from human blood were suspended at a concentration of 2.5 X lo6 cells/ml in minimum essential medium (MEM) with 10% human blood group type AB serum. Two-hundred-microliter aliquots of the cell suspension were added to microtiter plate wells (Falcon). After incubation for 1 hr at 37°C the nonadherent cells were removed by gently washing twice with warm (37°C) RPM1 1640. Two hundred microliters of RPM1 1640 containing 10% human blood group type AB serum was added to each well, and the monocyte preparations were allowed to develop into macrophages by incubation for 5 to 6 days at 37°C. After the 5- to 6-day incubation, the medium was removed and the macrophages were incubated for an additional 24 hr with test samples. Then the number of macrophages adhering to the wells were counted and washed. [3H]TdR-prelabeled K562 cells were added to each well (E/T = 10). After a 48hr incubation, release of incorporated [3H]TdR from K562 was determined by radioactivity counting. MAF-C activity is expressed as the percentage of tumor cytotoxicity where % tumor cytotoxicity =

release in test sample - spontaneous release total count - spontaneous release - release in control sample - spontaneous release x 100. total count - spontaneous release

To quantify MAF-C activity, 100 units of MAF-C was defined as the amount of MAF-C in 1 ml of ammonium sulfate precipitate fraction of the H2-E3-5 culture supematant. To determine the amount of MAF-C activity, serial dilutions were assayed. Assay for tumor cytotoxicity of monocytes. Instead of monocyte-derived macrophages, plastic-adherent monocytes were used for the MAF-C assay. Other details were as above. Assay for interferon (ZFN). Interferon activity was determined by a cytopathiceffect-inhibition assay with vesicular stomatitis virus in WISH (HeLa) cells. Assay for colony-stimulating factor (CSF). CSF activity was assayed by the soft agar method reported by Bradley et al. ( 14). Assay for macrophage chemotactic factor (MCF). MCF activity was assayedusing a modified Boyden chemotaxis chamber according to the method of Snyderman and Pike (15). Mono Q column anion-exchange chromatography. Samples were dialyzed against 0.05 M Tris-HCl (pH 8.0). A linear sodium chloride gradient system was prepared by combining the initial 0.05 M Tris-HCI (pH 8.0) buffer and the final 0.05 M TrisHCl buffer containing 1.0 M NaCl (pH 8.0). One milliliter of a sample was applied to a HR5 15 (50 X 5 mm, i.d.) Mono Q column, which was eluted with a linear sodium chloride concentration gradient of 0 to 0.5 M from fraction 3 to 26 and 0.5 to 1.0 M from fraction 27 to 28. Fraction volumes were 1 ml. All fractions were dialyzed against PBS.

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ET AL.

RESULTS Establishment of Hybridomas PHA-P-activated PBL were successfully fused with emetine/actinomycin D-treated CEM 11 cells, a clone of CEM cells, as described under Materials and Methods. Within 2 weeks after fusion, good growth of hybridomas was observed in all wells, while both emetine/actinomycin D-treated CEM 11 cells and mitomycin C-treated CEM 11 cells in control cultures completely lost their proliferating activity within a week, suggesting that all of the growing cells were hybrid cells. Surface Phenotypes of Hybrid Cells We tested the binding of monoclonal antibodies OKT3, OKT4, OKT5, OKT8, and OKTl 1 to hybrid clone H2-E3-5 and CEM 11 cells by the two-step binding assay, and the results obtained were compared by using Student’s t test (Table 1). H2-E3-5 expressed less OKT3- and more OKT4-, OKT5-, OKT8-, and OKTl lreactive antigens than did CEM 11, suggestingthat H2-E3-5 is a T-cell hybrid clone. MAPC Production by H2-E3-5 In order to obtain MAF-C-producing hybridoma cells, we assayedMAF-C activity in the culture supematant of all hybrid cell lines, and we obtained an MAF-Cproducing hybrid cell line, H2-E3. The cloning of H2-E3 was carried out by limited dilution (0.5 cells/well) using mitomycin C-treated CEM 11 cells as feeder cells. MAF-C activities in the culture supematants of 8 clones of H2-E3 were assayed(Fig. 1). Thus, a high MAF-C-producing hybrid clone, H2-E3-5, was obtained. Next, we compared the MAF-C activity of PHA-P-activated PBL culture supematant (PHA sup), CEM 11 culture supematant (CEM 11 sup), and H2-E3-5 culture supematant (H2-E3-5 sup). As shown in Fig, 2, H2-E3-5 produced more MAF-C than did PHAP-activated PBL, whereas CEM 11 produced much weaker MAF-C activity. At high concentrations of H2-E3-5 sup (lo-50%), an inhibitory effect was observed. Analysis of MIF, MAF-G, MAP0, and MAF-C Activities in the Culture Supernatant of Hybridomas In order to study the relationship of factors which affect macrophage functions we assayedMAF-C, MIF, MAF-G, and MAF-0 activities in the culture supematants of MAF-C-producing hybridomas H2-E3-5, H2-Dl l-1, and H2-Dl l- 11. In the culture TABLE 1 Surface Phenotypes of Cloned Sublines Two-step binding (cum/lo6 cells)” Sample CEM 11 H2-E3-5

+OKT3 12919 3231 (
+OKT4

+OKT5

+OKT8

18625 22465 (<0.2)

3997 498 1 (<0.2)

2828 10153 (
+OKT 11 2675 43 16 (<0.05)

0 All experiments were carried out in triplicate. The values are expressed as geometric means. bThe values in parenthesesare P values compared with the CEM 11 cell line by Student’s t test.

MACROPHAGE-ACTIVATING MAF-C

FACTOR

631

ACT IV I TYW

0

10

20

PHA-sup

H2-E3-1

H2-E3-2

HZ-E3-3

HZ-E3-4

H2-E3-5

H2-E3-6

HZ-E3-7

H2-E3-E

FIG. 1. MAF-C activity in the culture supematant of 8 clones of H2-E3. Each lot of cells was cultured at a cell density of IO6cells/ml for 48 hr, followed by assaying for MAF-C activity in the supematants as described under Materials and Methods. Each pair of horizontal bars represents the results obtained with two dilutions (1: 10, 1:100). PHA sup was obtained as described under Materials and Methods.

supernatant of H2-D 11- 1, MAF-C, MAF-G, low MAF-0, and very weak MIF activities were detected; in the culture supematant of H2-D 1I- 11, MAF-C, MIF, MAF-G, and low MAF-0 activities were detected; and in the culture supematant of H2-E3-5, MAF-C, MAF-G, relatively low MAF-0, and no MIF activities were detected (Table 2). These results strongly suggestthat MAF-C is a different molecular species from MIF and MAF-0. Fractionation of 80% Ammonium Surfate Precipitates of H2-E3-5 Sup by AnionExchange Chromatography Ammonium sulfate precipitates of H2-E3-5 sup (H2-E3-5 concentrated sup) were subjected to anion-exchange chromatography on Mono Q. The components were eluted from the column with a linear gradient of NaCl. Figure 3A shows NaCl con-

0

01

1

CONCENTRATION

IO

50 too)

FIG. 2. Dose-response curves of MAF-C activity of PHA sup (O), H2-E3-5 sup (O), and CEM 11 sup (m). PHA sup, H2-E3-5 sup, and CEM 11 sup were obtained and MAF-C activity assayed as described under Materials and Methods.

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TABLE 2 MAF-C, MIF, MAF-G, and MAF-0 Activities in Culture Supematants of Hybridomas’ Activity (2) Sample

MAF-C

MIF

MAF-G

0

0 18.1 f 0.1 15.0 -t 1.8 17.9 + 1.1

0

Control H2-Dl l-1 H2-Dl l-l 1 H2-E3-5

12.1 f 1.2

1.4 -t 1.5

19.1 + 1.0

15.2 + 0.9

26.6 + 2.2

k1.6

0

MAF-0 2.8 + 0.9 8.1 -+ 1.5

7.1 f 2.2 12.1 + 2.5

DCulture supematantsof hybridomas were obtained, diluted to a final concentration of 10%with appropriate culture medium, and all activities assayedas described under Materials and Methods.

centration and absorbance at 280 nm. Figures 3B-D show MAF-C, MAF-G, and MAF-0 activities, respectively, in each fraction eluted from the column. Both MAFC and MAF-G activities were present in fractions 13 to 20, with a peak at 15. From

0

1,

I 5

I

,

10 F IACTION

I

* 15

,I* 20

25

NO

FIG. 3. The elution pattern of ammonium sulfate precipitates of H2-E3-5 culture supematant. Anion exchanger, Mono Q, was packed in an HR 5/5 column (50 X 5 mm, id.). Elution buffer: 0.05 M TrisHCI, pH 8.0. Flow rate: 2.0 ml/min, 1.0 ml/fraction. Sample: 1 ml of H2-E3-5 concentrated sup. Linear sodium chloride concentration gradient from 0 to 0.5 M for fractions 3 to 26 and 0.5 to 1.0 M for fractions 27 to 28. Each fraction was dialyzed against PBS and assayedas described under Materials and Methods. (A) NaCl concentration ( - - - ) and ODrm nm(-). (B) MAF-C activity in each fraction. (C) MAF-G activity in each fraction. (D) MAF-0 activity in each fraction.

MACROPHAGE-ACTIVATING

633

FACTOR

the results shown in Fig. 3, we concluded that MAF-C could not be separated from MAF-G, whereas it could be separatedfrom MAF-0 by this column chromatography. The ammonium sulfate precipitates of CEM 11 sup were also subjected to the anion-exchange chromatography under the sameconditions. A peak of MAF-G activity was also detected in fraction 15 in this case. As shown in Table 3, however, only MAF-G activity was detected in fraction 15 from CEM 11, while MAF-C and MAFG activities were detected in fraction 15 from H2-E3-5. These results indicate that MAF-C and MAF-G activities are unlikely to be due to the same molecule. IFN, CSF, and MCF Activities in H2-E3-5 Sup Table 4 shows the anti-viral activity of PHA sup and H2-E3-5 concentrated sup. We did not detect anti-viral activity in H2-E3-5 concentrated sup. It was also found that IFN in PHA sup was mainly IFN-7, because anti-viral activity in PHA sup was mostly abolished by pH 2 treatment. Furthermore, we assayedCSF and MCF activities in PHA sup and H2-E3-5 concentrated sup. While PHA sup was found to contain both CSF and MCF activities, these activities were not detected in H2-E3-5 concentrated sup. Efect of H2-E3-5 ConcentratedSup on Monocyte-Derived Macrophages and Monocytes In order to clarify which stage of differentiation of macrophages is stimulated by MAF-C from H2-E3-5, we prepared not only monocyte-derived macrophages but also monocytes for use as effector cells. Table 5 shows tumoricidal activities of these effector cells activated with PHA sup, H2-E3-5 concentrated sup, or lipopolysaccharide (LPS). PHA sup activates both monocyte-derived macrophages and monocytes, whereas H2-E3-5 concentrated sup and LPS activate monocy-tederived macrophages but not monocytes. These results suggestthat different molecules are concerned in activation of macrophages and monocytes to kill tumor cells. DISCUSSION The data presented in this paper demonstrate that MAF-C in this study is a different molecular species from MIF, MAF-0, and MAF-G, and that there are at least two MAF-Cs, which differ in the maturation stage of the cells they affect. TABLE 3 MAF-C and MAF-G Activities in Fraction 15 from H2-E3-5 Culture Supematant and CEM 11 Culture Supematant” Activity (%x)~

Sample

PHA sup (50%)

PHA sup (10%)

H2-E3-5 cont. sup. (10%)

H2-E3-SFr. 15 (50%)

CEM Il-Fr. 15 (50%)

MAF-C Expt 1 Expt 2 MAF-G

36.2 -t 1.9 10.9 f 1.3 -

26.2 k 2.8 7.1 + 2.9 -

29.0 + 2.8 9.1 f 1.8 22.7 + 1.1

29.4 f 0.6 14.3 + 1.3

-0.7 +- 2.5 12.3 f 2.4

’ All samples were obtained and all activities assayedas described under Materials and Methods. ’ Values in parenthesesare final concentrations in culture medium.

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TABLE 4 IFN Activity in PHA Sup and H2-E3-5 Sup” Sample pH 2 treatment

PHA sup

H2-E3-5 cont. sup

-

4090 682

143 X43

+

’ All samples were obtained and all activities assayedas described under Materials and Methods.

Four assay systems, measuring MIF, MAF-G, MAF-0, and MAF-C, were used primarily. MIF activity can be defined by the migration inhibition of guinea pig peritoneal exudate cells. MAF-G activity can be defined by the stimulation of glucose consumption of guinea pig peritoneal exudate cells. MAF-0 activity can be defined by the stimulation of the 02- formation of U-937 cells triggered by PMA. MAF-C activity can be defined by the activation of human monocyte-derived macrophages to kill tumor cells. In our previous report (3), we concluded from a study of the culture supematant of a MIF-, MAF-G-, or MAF-O-producing human T-cell hybridoma that at least three molecules are concerned in the expressionof MIF, MAF-G, and MAF-0 activities. In the present study, we established an MAF-C-producing hybridoma, H2-E3-5. In the culture supematant of H2-E3-5 we detected MAF-C, MAF-G, little MAF-0, and no MIF activities. We then concentrated the H2-E3-5 sup by 80% ammonium sulfate precipitation and fractionated it by anion-exchange chromatography. MAFC could be separated from MAF-0 but could not be separated from MAF-G by this chromatography. Then we fractionated the culture supematant of the parent cell line, CEM 11, under the same conditions and found that MAF-G fractions that were eluted at the same positions as MAF-C fractions from H2-E3-5 do not show MAFTABLE 5 Tumor Cytotoxicity by Activated Monocytes and Monocyte-Derived Macrophages” Activity (%)b PHA sup (50%)

PHA sup (10%)

H2-E3-5 cont. sup (10%)

Monocyte Expt 1 Expt 2

17.8 + 1.3 8.7 + 1.2

10.2 + 0.4 -

2.7 f 0.7 -

1.0 + 0.9

Monocytederived macrophages Expt 1 Expt 2

36.2 f 1.9 15.0 + 1.9

26.2 + 1.9 -

29.0 -c 2.8 -

40.5 + 2.3

Effector cells

’ All samples were obtained and all activities assayedas described under Materials and Methods. b Values in parenthesesare final concentrations in culture medium.

MACROPHAGE-ACTIVATING

FACTOR

635

C activity. Thus, the results of anion-exchange chromatography show that, although MAF-C was eluted together with MAF-G, MAF-C activity and MAF-G activity are possibly not due to the same molecule. The heterogeneity of factors that affect macrophage functions has been studied by many investigators (3-5, 11, 18-23). Onozaki et al. (9) reported that MIF samples recovered from an immunoadsorbent column of an anti-MIF antibody, highly specific for MIF, had MAF-G activity. Therefore, MIF and MAF-G were thought to be identical. In our recent work (3), we observed that at least two molecules are involved in the expression of MIF and MAF-G activities, one MAF-G does not show MIF activity and the other MAF-G may or may not show MIF activity. Kniep et al. (16) reported that MAF-C was distinct from MIF. Their results are consistent with ours. Furthermore, Henry (10) demonstrated that mitogen-induced PBL sup showed MAF-C and MAF-0 activities. However, we demonstrated in this study that MAF-C derived from H2-E3-5 was clearly different from MAF-0. The relationship between MAF-C and IFNy has been discussed by several investigators. Pace et al. (17) reported that mouse IFN-y produced by recombinant DNA technology possessesMAF-C activity, and Nathan et al. (18) reported that human IFN-7 produced by recombinant DNA technology stimulates production by human monocyte-derived macrophages of H202. Schreiber et al. (19) reported that their MAF-C, derived from a mouse MAF-C-producing hybridoma, could not be separated from IFN-7. These results show that the IFN-7 molecule possessesMAF-C activity. However, these results did not solve the problem of whether all MAF-Cs have IFN activity or not. In the H2-E3-5 concentrated sup, we detected MAF-C activity but not IFN activity, so we concluded that this MAF-C molecule does not possessIFN activity. However, there still exists a possibility that some inhibitors of IFN activity are included in the culture supematant of H2-E3-5. There are several reports on human MAF-C assaysystems( 12,2 1,22). The authors used two kinds of effector cells. In one kind of assay system, plastic-adherent, microexudate, or serum-coated plastic-adherent monocytes were used. Fischer et al. (2 1) reported that lymphokine-rich supematant containing IFN-7 and partially purified IFN-7 activates monocytes to kill tumor cells. In the other assay systems, plasticadherent monocytes were cultured for some days and differentiated into macrophages before use. Cameron et al. ( 13) reported that lymphokines and LPS activate monocytederived macrophages to kill tumor cells. We established a different assaysystem using monocytes as effector cells in addition to our MAF-C assaysystem using monocytederived macrophages as effector cells. We assayedMAF-C activity of PHA sup, H2E3-5 concentrated sup, and LPS and found that, although it is not certain whether or not a single molecular speciesin the PHA sup is active toward two kinds of cells, PHA sup could activate both monocytes and monocyte-derived macrophages, while H2-E3-5 concentrated sup and LPS could activate only monocyte-derived macrophages.Thus, MAF-C derived from H2-E3-5 was found to activate only differentiated macrophagesto kill tumor cells. Hammerstrom et al. (22) reported that LPS activation of monocytes to kill tumor cells occurred at a very low level but gradually increased during an in vitro culture and suggestedthat the in vitro culture is a primary signal and LPS is a triggering signal. Our results suggestthat MAF-C from H2-E3-5 may function as a triggering signal.

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ET AL.

ACKNOWLEDGMENTS This investigation was supported by researchgrants from the Ministry of Education, Science,and Culture of Japan; the Suzuken Memorial Foundation; and the R. Naito Foundation for Medical Research.

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