Products of activated lymphocytes. I. The use of radiolabeling techniques in the characterization and partial purification of the migration inhibition factor of the guinea pig.

General methods were developed and applied to the biosynthesis and purification of products of activated lymphocytes available in minute quantities. The activity studied here was the migration inhibitory factor (MIF) produced by purified protein derivative (PPD)- or concanavalin A (Con A)-stimulated lymphocytes obtained from one guinea pig or less. The methods selected yielded results in terms of two chemical parameters characteristic of the molecules involved, namely Kd on Sephadex G-75 and isoionic point, pI, on isoelectric focusing. When supernatants were fractionated on G-75 columns, there were several areas even in control supernatants which produced migration inhibition relative to medium controls. However, in PPD- and Con A-stimulated supernatants, at least one peak of MIF activity was found solely in the stimulated cultures, with a Kd of 0.15. A double-labeling technique was used to characterize the proteins of this peak. Control, unstimulated cultures were labeled with [14C]leucine and stimulated cultures were labeled with [3H]leucine. After mixing the supernatants and G-75 filtration, a major "ratiolabeled" broad peak. i.e. one with increased 3H/14C ratio, was found. When a narrow portion of this peak about Kd 0.15, containing most of the MIF activity, was subjected to analytical isoelectric focusing, all of the label was associated with proteins of lower net charge than albumin. A unique ratiolabeled peak was found in PPD- and Con A-stimulated fractions with a pI of approx. 5.3. A micropreparative isoelectric focusing technique was developed and yielded MIF activity in the same region as the major ratiolabeled peak. Further study will be required to ascertain whether the ratiolabeled protein is MIF. By following the Kd, pI, and 3H/14C labeling ratio, at least 14 products of activated lymphocytes, synthesized either de novo or in increased amounts, could be distinguished.

(Received for publication 21 September 1972) It is clear that the specific immunological information for delayed-type hypersensitivity reactions is carried by sensitized tymphocytes, probably thymus (T) 1 cells, yet the molecular mechanisms by which they mediate the various manifestations such as resistance to infection, tumor and allograft rejection, and ceil cooperation in vivo are unknown. Since their discovery, the products of activated lymphocytes (PALs) have held a special fascination by virtue of their possible role as biochemical mediators of the cell-mediated immune response in vivo, and more generally, as macromolecules which can produceprofound biological effects in vitro and in vivo in minute quantities. Because of the small amounts produced by antigen-stimulated lymphocytes, the factors have been extraordinarily difficult to purify and characterize by conventional biochemical techniques. However, as a result of the painstaking efforts of Remold et al. (1,2,16) and Dumonde et al. (3), using large pools of material, the migration inhibitory factor of the guinea pig (MIF) appears to be within a molecular weight range of 35-82,000, a density range of 1.352-1.452, of greater electrophoretic mobility in acrylamide gels than serum albumin and labile to neuraminidase, suggesting a glycoprotein nature. The difficulty is how to proceed from this work to the total isolation, purification, and characterization, in chemical terms, of macromolecules such as MIF, available in such limited quantities.
Our goal was to develop methods which would: (a) require fewer ]ymphocytes than previous ones, e.g. less than one guinea pig equivalent; (b) permit physical chemical characterization of molecules in terms of intrinsic molecular properties; (c) permit quantitative chemical assays ultimately to replace the rather variable bioassays presently in use; and (d) be sufficiently general to be applicable to a whole range of cell constituents and products. Our approach has been to adapt sensitive isotopic labeling methods to deal with this problem. The double-labeling technique, so useful for the study of phage and animal virus protein synthesis (4,5), has provided the basis for this approach. In this method, nonstimulated control lymphocytes are labeled, for example with [14C]leucine, and cultures of the same pool of sensitized cells stimulated with antigen or mitogen are labeled with [SH]leucine. Although many species of proteins may be labeled, by comparing the ratio of ~H/14C or absolute differences in labeling patterns, it is possible to identify and follow those proteins synthesized either de novo or in increased amounts by stimulated lymphocyte cultures. For convenience we shall designate these as "ratiolabeled peaks." Rosenberg and Levy (6) have recently used this technique to determine that the earliest increase in labeling ratio in phytohemagglutinin (PHA)-stimulated lymphocytes was associated with the nuclear fraction.
The present work will indicate that the radiolabeling techniques reveal significant increases in purified protein derivative (PPD)-or concanavalin A (Con A)-stimulated cultures of cell-associated and cell-released labeled proteins. The double-labeling profiles of supernatants on Sephadex G-75 columns and then on isoelectric focusing were analyzed. The characterization of MIF in these studies was completed by testing for biological activity after each fractionation step. With these techniques it has been possible to recover MIF activity associated with a single ratiolabeled peak from as few as 6 X 108 lymphocytes.

Materials and Methods
Animals.--Hartley guinea pigs were sensitized to tuberculin as described previously (7). The National Institutes of Health guidelines for handling of experimental animals were followed in all experiments.
Macrophage Migration Assay.--Supernatants and purified fractions derived from them were tested for their ability to inhibit the migration of normal peritoneal exudates as described in detail previously (7). The exudates were induced by intraperitoneal injection of 20 ml of light mineral oil (Marco152; Humble Oil & Refining Co., Brooklyn, N.Y.) and Drakeol 6VR (Pennsylvania Refining Co., Butler, Pa.) 1:1. Peritoneal cells were harvested 72 h after injection of the paraffin oil in Hanks' solution containing 0.5 U/ml heparin, washed twice with Eagle's minimal essential medium (MEM) (Grand Island Biological Company, Grand Island, N.Y.), and resuspended to a cell concentration of 30 X 106 viable cells/ml in MEM containing 15% normal guinea pig serum (NGPS) (North American Biologicals, Inc., Roekville, Md.), or in fractions reconstituted in the same medium. Migration areas were traced and measured after 24 and 48 h. For these experiments all migration percent inhibition was calculated relative to medium controls, i.e., the average migration of at least four replicate capillaries containing normal exudate cells migrated in fresh NGPS-MEM. This medium control is defined as 100% migration in these experiments.
Lymphocyte Cultures and Production of MIF.--Lymph node cells were obtained from tuberculin-sensitive guinea pigs 3-5 wk after sensitization and 7-10 days after skin testing with 10 /zg of tuberculin PPD (Ministry of Agriculture of Fisheries and Food, Central Veterinary Laboratory, Weybridge, England). The lymphocytes were teased in Hanks' solution containing 5% NGPS plus penicillin (100 U/ml) and streptomycin (100/zg/ml) and strained to remove tissue fragments as described previously (7). The normal guinea pig serum used for these experiments was heat inactivated at 56°C for 30 min. After washing twice in serum-free medium, the cells were resuspended at a concentration of 15 X 106 viable cells/ml in MEM supplemented with antibiotics and cultured in Falcon tissue culture flasks (catalog no. 3024; Falcon Plastics, Div. of B-D Laboratories, Inc., Los Angeles, Calif.) at a density of 8 X 106 viable cells/cm 2, using 40 ml of cell suspension per flask. For production of MIF, lymphocytes were stimulated either with PPD (25/~g/ml) or concanavalin A (Calbiochem, Los Angeles, Calif. ; purified by the method of Agrawal and Goldstein [8]) at a concentration of 1/zg/106 cells. The cultures were gassed and incubated in an atmosphere containing 10% CO2, 7% 02, and 83% N2. Mter incubation for 20 h, the cells were removed by centrifugation at 500 g for 15 rain.
For most studies, the cell-free supernatants were concentrated quickly to 5 ml with dry Sephadex G-25 (coarse grade; Pharmacia Fine Chemicals, Uppsala, Sweden) and desalted. An example of the procedure is as follows: to 40 ml of supernatants is added 13 g of dry Sephadex until all available fluid has been absorbed. The slurry is then transferred to a Boerner centrifuge filter (Arthur H. Thomas, Philadelphia, Pa.; catalog no. 4578-F25) and centrifuged at 1000 g for 5 min. In this step the solution is concentrated to approximately }~ of the initial volume. Mter an additional concentration step, the 5-ml concentrates were then immediately desalted on a G-25 (superfine) Sephadex column (2.5 X 25 cm; running buffer 0.01 M sodium phosphate, pH 7.5). The excluded peak was collected, frozen in dry ice methanol, and lyophilized.
When it became clear that the migration inhibitory activity was soluble in high salt concentrations while many other supernatant proteins were not, supernatants were directly lyophilized, dissolved in 5% original volume of 0.01 M HC1 as described previously (9), mixed with label marker, and centrifuged to remove salted-out proteins (1000 g, 15 min). The concentrated soluble material was then applied immediately to the Sephadex G-75 columns.
For fractionation of supernatants, desalted lyophilized supernatants, either control or MIF containing, were dissolved in 0.5 ml of sterile distilled water and mixed with 0.5 ml of a sample of a radioactive control supernatant as a marker. Occasionally, a small precipitate formed and was removed by centrifugation. The sample was then applied to the Sephadex G-75 column and fractions of 1 ml were collected at a flow rate of 5 ml/h. For establishing the labeled protein marker profile, 0.1 ml of each fraction was counted in a scintillation counter. For further fractionation in migration inhibition studies, every two tubes were pooled, dialyzed in sterilized dialysis bags (Union Carbide Corp., Chicago, Ill.) agianst 12 liters of distilled water overnight, and lyophilized.
For calibration of the columns, molecular weight standards employed were human gamma globulin, mol wt 140,000; human serum albumin, mol wt 67,000; ovalbumin, mol wt 45,000; chynmtrypsinogen A, mol wt 25,000; and cytochrome c, tool wt 12,400 (Schwarz/Mann Div., Becton Dickinson & Co., Orangeburg, N.Y.). Kd values were established using gamma globulin to indicate the exclusion volume and tritiated HzO to indicate the total column volume.
Incorporation of Labeled Amino Acid into Cell Associated and Cell-Rdeased Prol.ein.--For studying the incmporation of amino acid into total protein released by stimulated lymphocytes into the supernatants, lymph node cells from individual guinea pigs were cultured at a density of 10 X 106 cells/1 ml in Leighton tubes (Be]lco Glass, Inc., Vineland, N.J.). To one series of control cultures and another of PPD-stimulated cultures were added 1 /zCi of [14C]leucine (specific activity > 250 mCi/mM; New England Nuclear Corp., Boston, Mass.). At various times one tube of each set was cooled in ice, and the contents were transferred to centrifuge tubes and centrifuged at 250 g for 10 rain. The supernatants were precipitated with 4 ml of 5% trichloroacetic acid (TCA). The cell pellet was washed in MEM, centrifuged at 250 g, dis-solved in 0.5% sodium deoxycholate, and precipitated with 4 ml of 5% TCA. The precipitates were heated for 10 min on a steam bath, cooled, trapped in a fiber glass filter (Whatman GF/A, 25 ram), and washed with approximately 5 cc of 5% TCA. The filters were dried and counted in a scintillation mixture (1 vol toluene and 2 vol Triton X-100 [Rohm and Haas Co., Philadelphia, Pa.] containing 4 g/liter 2,5-diphenyloxazole [K & K Laboratories, Inc., Plainview, N.Y.]) at 75%.
The Double-Labeling Technique.--The basis of the technique is labeling of control cultures with leucine containing one isotope and the experimental with another, such that when the two are combined at a fixed ratio and fractionated, slight differences in synthesis of even small amounts of proteins can be detected as an increase in ratio in the stimulated to control samples. For these studies, lymph node cells from tuberculin-sensitive guinea pigs were washed and cultured in Joklik-modified MEM Spinner medium minus leucine at a density of 40 )< 106 cells/ml or 8 X 106 cells/cm 2. 5 ml of each suspension was cultured in a Falcon plastic culture flask (25 cm 2, no. 3012). Into one culture, for example the control culture, is added 100 #Ci [14C]leucine (> 250 mCi/mM) and into an experimental culture, i.e. a PPD-stimulated or Con A-stimulated culture, is added 500 #Ci [3H]leucine (30-50 Ci/mM). Experimental cultures were stimulated with PPD at 25 #g/ml or concanavalin A at 1 #g/106 cells. The flasks were gassed and cultured in a Dutton-type sealed plastic chamber (Norbo Machine Co., Huron, S. Dak.) in an atmosphere of 10% CO2, 7% oxygen, and 83% nitrogen (Matheson Co., Inc., East Rutheiford, N.J.). Mter 20 h of incubation, the cells were centrifuged at 500 g for 10 min. To the supernatants was added 0.2 ml of 40 mM cold leucine solution, after which they were ultracentrifuged at i00,000 g for 1 h in a Spinco ultracentrifuge (Spinco Div., Beckman Instruments, Inc., Palo Alto, Calif.). The labeled supernatants were desalted, and free labeled leucine was removed by passage through a G-25 Sephadex column (2.5 X 25 cm). The column was monitored by counting 50-#1 aliquots of each 3 ml fraction. The excluded protein peak was collected and lyophilized.
It was convenient to have an internal marker or labeled standard of unstimulated supernatant for further fractionation procedures. For this purpose, lymphocytes were cultured in medium as described above in the presence of 50 #Ci [14C]leucine, and the labeled marker supernatant was desalted and lyophilized as above.
Gel Filtration o/ Double-Labeled Supernatants.--To prepare the double-labeled mixture, lyophilized, desalted 14C-and 3H-labeled supernatants were dissolved in 0.5 ml of sterile distilled water and mixed together at a 3H/14C ratio of approximately 3. Mter clarification by centrifugation at 1000 g for 5 min, the solution was applied to the calibrated G-75 Sephadex column and eluted in 0.01 M phosphate buffer, pH 7.5, in 1-ml fractions. Radioactivity was counted in a Beckman scintillation counter (Beckman Instruments, Inc., Fullerton, Calif.) with the following characteristics: 3H efficiency, 38%; 14C efficiency, 60%. For establishing the double-labeling patterns, 0.2 ml of each fraction was mixed with 1 ml of water and 6 ml of scintillation fluid and counted under the following conditions: windows adjusted for 10% 14C spillover into the tritium channel, and less than 0.1% tritium spillover into the 14C channel. Under these conditions, the overall efficiency of tritium counting was 15% and that of 14C counting was 30%. The counts per minute were corrected for spillover, and either was plotted directly or converted to disintegrations per minute.
Discontinuous Aerylamide Gel Eleetrophoresis.--Narrow pools of Sephadex G-75 fractions were subjected to analytical acrylamide gel electrophoresis under conditions described by Remold et al. (1). Lyophilized Sephadex fractions in the region of K d = 0.1-0.2 (6-10 tubes) were dissolved in 1 ml of distilled water and dialyzed against 0.01 M glycine buffer, pH 6.8, for 5 h. A 200/zl aliquot was electrophoresed for 4 h on a 0.6 X 10 cm column of 7.5% acrylamide, pH 9.1, at 200 V. After separation, the gel was removed, frozen, and cut into 2-mm slices. Each slice was put into a scintillation vial and eluted in 1 ml of water overnight, 6 ml of scintillation fluid was added, and the vials were counted.
lsodeclric Focusing. -Analytical acrylamlde gels: Narrow pools (6 10 tubes) of Sq)hadex G-75 fractions in the region of MIF (Kd = 0.1-0.2) were dissolved in 2 ml of distilled water and dialyzed against distilled water for 5 h. To 2 ml of sample was mixed 1 ml of the following solution: 15 ml of acrylanride (30 g/100 ml q-bis-acrylamide (0.8 g/100 ml) ; 4 m] of letramethylethylenediamine (1:100), all from Eastman Kodak Co., Rochester, N.Y.; 1.5 ml of ampholine solution (40% LKB no. 8141 ampholine, pH 3 10, LKB Instruments, Inc., Rockville, Md.). The solution was degassed on a vacuum pump for 5 rain and 0.1 ml of a 2% ammonium persulfate solution (Fisher Scientific, Pittsburgh, Pa.) was added and the solution poured into a 0.6 )< 10.5 cm glass tube. After polymerization was complete (approximately 1 h at room temperature), 0.2(j'~: sulfuric acid was added to the upper reservoir and 0.4% triethanolamine to the bottom reservoir, and the gels were subjected to electrofocusing at constant voltage initially at 2 mA/gel. In each experiment, marker sperm whale myoglobin (Schwarz/Mann) was electrofocused in a parallel gel to indicate the completeness of the focusing process. The usual period for focusing was 34 h. The gels were frozen and cut into 2-ram slices, and counted. The indicator gel was cut out as well, extracted with 1.5 ml of boiled distilled water for 3 h, and the pH of each fraction measured. tube (OD 0.8 cm; ID 0.6 cm) in the descending end of which a platinum electrode was melted and afftxed. For highest recovery, the electrofocusing was carried out without a solid matrix, using a sucrose gradient to stabilize against convection and diffusion. For filling, this long side received 50% sucrose in 0.4% tfiethanolamine up to the mark. Then the right arm was stoppered in order to fill the left arm to the top with the same solution. The left arm was then stoppered with an ampule cap and the stopper on the right-hand arm was removed. The righthand arm was then filled with a 40-18% sucrose gradient containing ampholytes and 2 ml of sample. The sucrose gradient was prepared by mixing 1.65 ml of a denser solution containing 0.85 ml of 80% sucrose, 0.75 ml of sample, and 0.05 ml of ampholine solution, pH 3-10, with a lighter solution containing 1.25 ml of sample, 0.35 ml of 80e/e sucrose, and 0.05 ml of ampholine with constant stirring in a gradient mixer (see Fig. 1). The sample consisted of a narrow pool of lyophilized Sephadex G-75 fractions dissolved in distilled water and dialyzed for 5 h against distilled water. On top of the right arm, a reservoir made from an ampule stopper with a hole in the center was attached, filled with 0.2~ sulfuric acid, and the platinum anode was placed in contact with it. The left arm stopper was then removed and the sample was focused for approx. 16 h at 500 V constant voltage, 1 mA initial current. At the completion of the separation, the cathodal end was connected to a peristaltic pump infusing 50% sucrose at the bottom at a rate of 10 ml/h. A total of 20 fractions each containing 0.15 ml were collected from the top of the right-hand column in an LKB fraction collector. They were diluted with 1.5 ml of boiled distilled water and the pH was determined on each sample in a Radiometer pH meter (Radiometer Co., Copenhagen, Denmark). To remove the ampholytes the fractions were dialyzed extensively at 4°C, 24 h against 0.1 M glycine buffer pH 6.0, 24 h against 0.1 M NaC1, and 24 h against distilled water. In later experiments, gelatin (300 #g; Difco Laboratories, Inc., Detroit, Mich.) was added to each fraction to minimize adsorption of active material to the dialysis bag. The dialyzed samples were freeze dried, and later dissolved in MEM plus 15% NGPS for assay of MIF activity. Alternatively, each fraction was passed over a 0.5 X 20 cm Sephadex G-25 column in distilled water to remove ampholytes and sucrose, and then freeze dried directly.

Cumulative Protein Synthesis in PPD-Treated Normal and Tuberculin-Sensilive Lymph Node
Cells.--The first question to be faced in developing an isotopic-labeling technique for mediators of hypersensitivity is whether it is possible to see gross differences in incorporation of labeled amino acids into proteins in the supernatants of antigen-stimulated cultures. For such experiments lymph node cells from normal and tuberculin-sensitive guinea pigs were cultured in leucine-free medium in the presence or absence of PPD (25/~g/ml).
[14C]leucine was added at the beginning of culture and samples were removed at various times, cells and supernatants were separated by centrifugation, and cell-associated and supernatant proteins were separately precipitated by addition of 5 % cold TCA. The results of such an experiment are shown in Fig. 2. The amount of newly synthesized cellular protein generally exceeded that of released protein by 5-10-fold. Tuberculin PPD had neither nonspecific stimulatory nor toxic effects on protein synthesis or release from normal, nonsensitized guinea pig lymph node cells. In contrast, there was a clearly detectable stimulation in leucine incorporation into both cell-associated and released protein in the cultures of tuberculin-sensitive cells stimulated by tuberculin. The increase in synthesis in antigen-stimulated cells was first discernible at approx. 4 h and continued to increase such that by 20 h there was a reproducible difference of twofold or greater in leucine incorporation in both supernatant and cell-associated protein of stimulated cultures. These results follow closely the labeling kinetics described previously by Levy and Rosenberg (10), and correlate with the appearance of MIF activity at about 6 h after stimulation (11).

Relationship belween Labeling Profile on Sephadex G-75 Gel Filtration and
Migration Inhibitory Activity.--Because there was a significant increase in released labeled proteins, it was our expectation that we might see a unique labeled peak associated with MIF activity after gel filtration on Sephadex. In order even to be able to associate a labeled protein fraction with MIF activity, it was necessary to establish precisely on our calibrated columns the elution volume for MIF. Published results on the molecular size of MIF could not be relied upon because there were considerable differences in conditions for gel filtration and because large pools of fractions were invariably used. In order to provide an internal marker for this fractionation and testing of biological activity, a 14C-labeled nonstimulated marker supernatant was admixed with each concentrated supernatant (eq 40 ml) to be chromatographed on Sephadex G-75. From the breakthrough peak onward, 100 Sephadex fractions were collected from each column, and a sample from each tube was counted for radioactivity to establish the marker profile (Fig. 3 A) which was remarkably constant in all experiments. The marker profile indicated the breakthrough volume and a second constant peak at Ks 0.6. Three types of samples were fractionated: control supernatants, tuberculin-sensitive cells stimulated with PPD, and tuberculin-sensitive cells stimulated with concanavalin A. In these experiments, each 40 ml of supernatant fractionated represented 6 >( 108 lymph node cells, i.e., less than one guinea pig equivalent of lymphocytes. For determination of MIF activity ever)-two tubes were pooled, freeze dried, and reconstituted in MEM plus 15 % normal guinea pig serum for testing migration inhibitory activity on normal peritoneal exudate cells. While in previous work it has been common practice to calculate a migration index comparing the migration of cells in antigen-stimulated fractions relative to that in corresponding control fractions, for chemical fractionation purposes it is necessary to have a more absolute standard for 100% migration. It seemed important to be able to discriminate all migration inhibitory or toxic fractions of the column, and to determine later whether they were present only in antigenstimulated supernatants, rather than calculating only the relative percent inhibition.
The elution pattern on Sephadex G-75 columns of migration inhibitory activity is shown for control supernatant reconstituted with PPD ( Fig. 3 B), PPD supernatant (Fig. 3 C), and concanavalin A supernatant (Fig. 3 D). When migration inhibitory activity of every two fractions was analyzed, it became clear that there were many peaks of inhibitory activity associated with the supernatants. In four experiments using control supernatants, it was possible to define prominent inhibitory peaks at Ks 0.0, 0.09, and 0.26 which recurred in all supernatants, control or stimulated. We have chosen to present this particular experiment to indicate the maximal degree of nonspecific inhibitory activity which can be observed from lymphocyte culture supernatants. Nevertheless, in five experiments in which PPD-or con A-stimulated supernatants were examined in detail, it became clear that there was an additional new peak of migration inhibitory activity at Ks = 0.15, not present in controls, which was associated only with stimulated supernatants. Increased migration inhibitory activities at Ks 0.3-0.4 were seen in Con A supernatants. It was the peak at Ks 0.15 which was presumed to contain the antigen-or mitogen-induced MIF which was studied further.  To test a possible antigen dependence or specificity of the biological activity in these fractions, in some experiments every two fractions were pooled, half was reconstituted in medium alone and half in medium containing PPD (25 /ag/ml). Thus the effect of antigen on migration inhibitory activity was studied. In no case was the pattern of inhibitory activity altered by the addition of PPD, although the addition of PPD consistently enhanced already existing ~6"7" inhibitory activities by approximately 10-1~ Jo. This slight nonspecific enhancing property of PPD may account in some measure for earlier reports of antigen specificity of MIF.
Use of Double-Labdiug Technique for Analysis of Products of Activated Lymphocytes.--When elution profiles of PPD-stimulated and control supernatants labeled with [14C]leucine were compared, normalizing the curves to equal baseline counts, the patterns were essentially indistinguishable and resembled Fig. 3 A. Thus it was virtually impossible to distinguish any single peak increased in the PPD-stimulated cultures at the level of gross labeling. However, even under circumstances in which the bulk of total synthesized proteins produced may be similar, the double-labeling technique permits one to distinguish even small areas in which there is greater incorporation in the stimulated culture relative to the control. Accordingly, PPD-stimulated lymph node cells were cultured in the presence of [~4C]leucine and control supernatants in the presence of [aH]leucine. At 20 h the supernatants were processed, mixed together, and fractionated on the same Sephadex G-75 calibrated column. As can be seen in Fig. 4 B, a broad increased ratiolabeled peak was seen at Ka 0.05-0.2. The magnitude of the ratio difference of this peak was invariably small, generally 15% above the base-line ratio. It therefore did appear that there was an increase in some synthesized proteins in the PPD-stimulated culture, relative to control, which eluted in fractions known to contain MIF.
Because the mitogen concanavalin A has been found to induce MIF formation and stimulate lymphocytes to a greater degree than antigens (12,16), the effect of Con A on the double-labeling pattern was studied. The total labeling profile of a Con A and unstimulated control supernatant are shown in Fig. 4 A. After normalizing the raw data to equal base-line counts, a clear difference in gross counts between the stimulated and control cultures is seen. When Con A-stimulated cultures were labeled with [aH]leucine and the control with [~4C]leucine. the double-label profile indicated up to three ratiolabeled peaks, a major one maximal at Ke 0.1 and two smaller at Ka 0.3 and 0.5 (Fig. 4 C). The first peak eluted in the same position as that in which MIF and the ratiolabeled peak of PPD-stimulated supernatants were seen, although the increase in ratio with Con A was generally about twofold greater than with PPD. The broad area of this peak indicates that it must be comprised of several labeled proteins.
Reversal of labels in this experiment (Fig. 4 B, C) indicated that the increase in ratio is independent of the isotope used and related solely to whether the cultures were stinmlated or not. In general we chose to label the stimulated cultures with the weaker isotope (3H), so that differences would be underestimated if anything. In nine experiments with Con A-stimulated supernatants, the average ratio increase at the Ka 0.1 peak was 28.2 4-3.0%. Thus the ratiolabeling patterns for mitogen-and antigen-stimulated cells both showed an area of increased labeling in the region associated with biological activity. It cannot be emphasized too strongly that the degree of isotope incorporation and the reproducibility of the ratiolabeled patterns depends critically on the culture conditions, especially pH, cell density, concentration of stimulant, and amino acid-deficient medium.
Release of Prelabeled Protein.--Production of MIF has been found to be inhibited by agents which block RNA or protein synthesis (13,14,23), and it has been inferred that MIF represents a protein synthesized de novo. To ascertain whether any significant amount of preformed material was released from antigen-or mitogen-stimulated cells with a Ka similar to MIF, cultures were labeled either with [l~C]leucine or [3Hjleucine for 24 h in the absence of stimulation. After that prelabeling period, the cells were washed and incubated in the presence of cold leucine for a further 24 h, during which the [3Hlculture was stimulated with Con A. When the 24-48-h supernatants were filtered on Sephadex G-75 columns, although the total count profiles were similar to Fig. 4 A, a very different ratio pattern of prelabeled materials is seen (Fig. 4 D). It is clear that the large ratio peak of Ks 0.1 present in Fig. 4 C is absent, and that an increased ratio peak at Ks 0.5 is seen. This result suggests that the labeled peak associated with MIF is the result of newly synthesized species of proteins in the size range Ks 0.1, and is not significantly contributed to by preformed materials. The smaller size of prelabeled materials released after antigen stimulation may represent either different species of smaller molecules secreted by the cells, or degradation products of larger molecules. Similarly, the peak width or the "heterogeneity" of the principal peak at Ks 0.1 may indicate either that there are a number of proteins of slightly differing size in that region synthesized by stimulated cultures or that during the 20 h culture period there is partial degradation of a lesser number of synthesized molecules. Further analytical separation of the major ratiolabeled peak was necessary to clarify its degree of polydispersity or heterogeneity.
Analysis of Sephadex G-75 Ratiolabeled Peak at Ka 0.13-0.20 by Acrylamide Gel Electrophoresis.--It had previously been reported that MIF could be separated from a variety of proteins on acrylamide gel electrophoresis and that the peak migration inhibitory activity was obtained in fractions migrating anodally to albumin (1,16). Consequently, narrow pools of the ratiolabeled peak at Kd 0.13-2.0 from Sephadex G-25 columns were subjected to electrophoresis in 7.5% acrylalnide gels at pH 9.1 as described by Remold et al. (1). The total labeling pattern for the control and Con A gels is shown in Fig. 5 A, normalized to the same base line of counts, using the same supernatant pools shown in Fig. 4 A, C. Differences in labeling profiles between the stimulated and control samples were seen nlore clearly in the ratio curve shown in Fig. 5 B. Even with the narrow Sephadex pool of Kd 0.13-2.0, an increase in labeling of five to six distinct protein species was observed. Clearly the area of greatest increase in labeling in the stimulated fraction was found to migrate anodally to albumin, in the region described by Remold and David (16) to contain MIF.
The double-label technique permits one easily to study those fractions in which there is an increased ratio of components in stimulated relative to control fractions. However, it is also possible to express the same data as an absolute difference in the incorporation of leucine into the same proteins under standard conditions. The counts are corrected for efficiency into disintegrations per minute and the difference curve represents the absolute increase of labeled leucine incorporated (Fig. 4 C). By correcting further for specific activities the data could be expressed in micromoles of incorporated leucine. Thus different curves could prove useful for quantitating the absolute increase in specific proteins synthesized by activated lymphocytes.

Analysis of the Ratiolabeled Peak at Ka 0.13--0.20 by lsoelectric Focusing.--
The increase in labeling patterns of proteins migrating faster than albumin could reflect molecules of greater net negative charge than albumin or molecules of smaller size, since acrylamide gels are known also to separate by size (15). Therefore, a more absolute index of the charge characteristics of MIF was sought. Since the isoionic point, p/, is a characteristic of proteins and independent of the medium in which the isoelectric focusing is carried out, small pools of the ratiolabeled peak (Ka 0.13-0.2) were subjected to ampholine carrier electrophoresis in 7.5 % acrylamide gels over a pH gradient of 3-10.
The data in Fig. 6 represent results for isoelectric focusing of the same pools of material shown previously (Figs. 4 A, C, 5). A reproducible linear gradient was established from pH 3-8.6.
Two major labeled peaks can be recognized, one at pI 3.5-5, the other pI 5-6. However, the material at pI 3.5-5.0 shows no differences in the ratio of labeling (Fig. 6 B). The difference curve (Fig. 6 C) shows that the greatest difference in synthesis of protein in the Con A-stimulated culture relative to control is found in the area of pI 5-6 averaging pI 5.2 in five experiments. Thus, the ratiolabeled material has a higher pI than albumin and probably migrates faster than albumin on acrylamide charge gels by virtue of its smaller size.
The size fractionation and isoelectric focusing of the double-labeled supernatants indicated a markedly increased labeling pattern of materials with Kd value of approximately 0.15, and a pI value of 5.2. It remained to be demonstrated that there was biological activity associated with this labeled peak. Therefore, a simple micropreparative isoelectric focusing column was developed, shown in Fig. 1. In order to minimize the problems of adsorption to a solid matrix, the preparative ampholine gradient was stabilized with 18-40% sucrose gradient. Again, 40-ml supernatants of control, PPD-stimulated or Con A-stimulated cultures were processed, mixed with [1~C]marker supernatant, and filtered on Sephadex G-75. A pool of 10 tubes of Kd 0.12-0.18 was made and an aliquot of one-third was tested for migration inhibitory activity. An example of the inhibitory activity of this material relative to medium alone is given as follows: control 5%, PPD-stimulated 51%, Con A-stimulated 65% inhibition. The pooled fractions were focused in the microcolumn, and after reaching equilibrium, 20 fractions were collected (0.15 ml/fraction). After measuring the pH of each fraction, it was lyophilized and tested for MIF activity. The migration inhibitory patterns of two separate experiments are shown in Fig. 7. It immediately became apparent that the ampholines were not dialyzed away completely, and that some toxicity or spurious migration inhibition was produced, generally at a low level, even in control, non-MIF- containing Sephadex pools. Figs. 7 A-C were chosen to illustrate a high level of nonspecific inhibition encountered. Even after 3 days of dialysis, marked toxic peaks were present at pI 4.0 and 7.5-8.5. Even so, it was possible to see a markedly increased peak of migration inhibitory activity in the PPD (Fig.   7 B) and Con A (Fig. 7 C) fractions at pI 5-5.5. Addition of gelatin to the fractions diminished losses of activity. The nonspecific inhibitory activity of control supernatants (Fig. 7 E) is essentially accounted for entirely, simply by ampholine toxicity and is no greater than that produced by ampholine solution alone (7 D). In this experiment, there was again a marked increase in MIF 0.. 4-0.3 pH units, an activity activity associated with the fraction of pI " which is found essentially only in stimulated supernatants. Upon preparative isoelectric focusing, significant MIF activity from stimulated supernatants representing less than one ga~inea pig equivalent of lymph node cells was obtained in five of seven experiments. The average peak inhibition in that fraction in those experiments was 42.9 4-2.7 %.
Composite Picture of Synthesized Products of Activated L ymphocytes.--By use of techniques described above it is possible to characterize any ratiolabeled peak by three parameters: increase in label incorporation above control, as determined by the double-label difference curve; Kd on Sephadex G-75; and pI by isoelectric focusing. Individual double-labeled Sephadex fractions be-tween Ke 0.0 and Ke 0.2 were subjected to analytical isoe]ectric focusing in gels. The difference curves for the labeling pattern of each species of molecule with distinct pI was plotted against elution volume on Sephadex G-75. The composite picture so obtained (Fig. 8)  similar pI values. This result reinforces the importance of selecting narrow pools of the appropriate Kd fraction to achieve good resolution in isoelectric focusing. It indicates that the main broad ratiolabeling area seen on G-75 columns (Figs. 4B, C) is undoubtedly polydisperse. Of the fourteen ratiolabeled bands plotted, that with a Ke of 0.15 and pI of 5.2 is most closely associated with the biological MIF activity. DISCUSSION It is likely that some products of activated lymphocytes (PALs) will be biochemical mediators of delayed hypersensitivity reactions, and it is clear that they can exert profound biological effects in vivo and in vitro in minute quantities. For example, Remold et al. (16) and Dumonde et al. (3) found that significant inhibition of macrophage migration in the galinea pig could be pro-duced by as little as 400 ng of protein. While traditional methods of protein chemistry have been useful in providing us with our present information about the chemical nature of these substances, they have required large amounts of cells and given only small yields. More importantly, it is difficult to see how they can be pressed further into the fine resolntion required to characterize the various discrete factors. Further, the quantities of lymphocytes available from hypersensitive human donors are much too small, in general, to be amenable to analysis by these methods.
Our objectives were basically twofold. First, we wished to develop methods for following the PALs in small samples, our upper boundary condition being lymph node cells from one guinea pig. This necessitated the choice of isotopic labeling techniques for marking synthesized proteins, since it had been demonstrated previously that production of MIF was abolished by inhibitors of RNA and protein synthesis (13,14,23). Secondly, it was necessary to choose methods in which results could be expressed in terms which were intrinsic chemical parameters of the various molecules. Once these qualitative characteristics are established for each factor, more quantitative radioassays could be applied to their study, e.g., possibly for diagnostic assay. In this paper, we have dealt exclusively with the qualitative problem of developing isotopic labeling methods applicable to purification of MIF, and the relationship between isotopic labeling patterns and biological activity at each step.
The first studies were simply to indicate whether in antigen-or mitogenstimulated cells an increase in incorporation of labeled leucine into macromolecular protein could be established, and what the kinetics of this response were. The results (Fig. 1) confirm those previously of Levy and Rosenberg (10) on PHA-stimulated cells that a significant increase in total macromolecular protein incorporated into stimulated cultures was discernible as early as 4 h after stimulation. Further, the results showed that although the majority of the incorporation was into cell-associated protein, significant increases in synthesized proteins released into the medium occurred in stimulated cultures.
There has been much previous work on gel filtration patterns of MIF activitv using relatively large pools of eluate, and unfortunately many of the estimates for the size range of MIF are discordant (1,3,9,17,18). For detailed fractionation studies, it is obvious that the smaller the pool from Sephadex, the less the polydispersity and the higher the probability of good resolution by one more fractionation step based on charge. Therefore it was decided to test every two tubes for MIF activity past the void volume of our G-75 columns. When 40-cc supernatants were concentrated and used as sample, it soon became apparent that there were multiple peaks of migration inhibitory activity, even in control samples. The degree of inhibition varied with the preparative procedures and the macrophage populations tested, but the problem of distinguishing a MIF induced by stimulation from a variety of nonspecific inhibitor}" proteins became critical. Because of the nonspecific inhibitory activities, presentation of results in terms of migration inhibition by stimulated fractions relative to control was not really meaningful in chemical terms. For this reason, we chose to accept the medium control as the standard of 100% migration, which permitted us to define the Ka's for the nonspecific inhibitory proteins which may have confused the result. By testing 100 tubes, migration inhibitory activity could be localized within 10 tubes, and the elution volume for the peak activity associated with the unique migration inhibitory peak present only in stinmlated supernatants was approximately Ke = 0.15 (Fig. 3). The size marker used to calibrate these colunms closest to this peak was ovalbumin, with a Kd = 0.13 and tool wt of 45,000.
While there have been son'~e reports indicating that MIF activity in whole supernatants (11,19) or Sephadex fractions (20) may have antigen dependence or specificity for antigen, no evidence for specificity could be found in several recent and very thorough studies (1,3). In the present work we observed that the Ke 0.15 material did not lose migration inhibitory activity or radioactive counts by passage through PPD-immunoadsorbant columns, and thus no evidence for specificity of this MIF was seen here. PPD did, however, nonspecifically increase the degree of inhibition slightly in some experiments.
Having established the characteristics for elution of biological activity, attempts were made to introduce isotopic labels into proteins in that peak. The double-label technique, in which a stimulated sample is labeled with an amino acid of one isotope and the control with another, has proved very useful elsewhere for distinguishing small differences in synthesis in proteins over a background of a large amount of labeled and nonlabeled protein. While this study was in progress, Rosenberg and Levy (6) reported that this technique indicated increases in amino acid incorporation into cell-associated proteins in PHA-stimulated lymphocytes, and that the cell fraction showing the greatest increase early after stimulation was the nuclear fraction. In the present experiments when double-labeled supernatants, e.g. control labeled with [14C]leucine stimulated with [aH]leucine, were admixed and subjected to gel filtration on the same columns in which MIF activity was studied, at least one and often multiple broad peaks of increased labeling were associated with the stinmlated fraction, defined here as ratiolabeled peaks. The increase associated with the PPD-stinmlated supernatants was small, but the peak was always in the region of Kd approx. 0.1. Because the degree of stinmlation was low, we turned to Con A-stimulated culture supernatants. Con A has been shown to affect T cells exclusively (21) in the mouse, and to induce formation of MIF and skin reactive factor in the guinea pig (12). When Con A and control double-labeled supernatant mixtures were fractionated on G-75 columns, a significant broad peak emerged at Kd approx. 0.1, as well as smaller peaks at Kd 0.35 and 0.5 (Fig. 4). The magnitude of the increase of the Ke 0.1 peak in nine experiments was 28.2% -4-3~. For further fractionation a narrow pool was selected from this broad double-labeled peak in the region containing most MIF activity (ca. Kd 0.15).
Since it had been shown by Remold et al. (1,16) that MIF could be purified after Sephadex fractionation from other serum proteins by disc acrylamide gel electrophoresis and that it migrated anodally to albumin, the electrophoretic pattern of our narrow ratiolabeled pool was examined under their conditions. We were able to resolve five to six peaks, in which the majorit:y of label migrated anodally to albumin (Fig. 5). Thus, the ratiolabeled pattern indicated a heterogeneity of the Sephadex material, but confirmed that there was a significant amount of radioactivity in precisely the region identified by Remold et al. as containing MIF activity. However, this migration pattern could have been due either to a higher net charge, or to molecular sieving effects of polyacrylamide. Because relative migration is an ambiguous, parameter we chose isoelectric focusing as our method for charge separation, since at equilibrium proteins migrate to their isoelectric points, pI, which are intrinsic characteristics of the molecules. The analytical isoelectric focusing results indicated the narrow Sephadex Kd 0.15 pool contained two major labeled areas, one of which was the same in control supernatants, and the other was found to be significantly elevated only in stimulated supernatants (Fig. 6). This area centered at 5.3 indicating a lower net charge than guinea pig albumin (pI = 4.8). With a micropreparative isoelectric focusing procedure, it was possible to establish a unique migration inhibitory peak in the region of pI 5.2 -4-0.5 associated with the ratiolabeled area (Fig. 7). Because recovery of biological activity from acrylamide gels is low, we chose to stabilize the ampholine gradients with a sucrose gradient to obviate adsorption to a solid matrix. Our results indicate that this activity has a lower net negative charge than serum albumin and that the greater mobility on disc acrvlamide gels is probably due to sieving effects since MIF is smaller than albumin. They are also consistent with earlier observations using diethylaminoethyl fracfionation in which MIF activity was eluted in the alpha globulin region at lower ionic strength than albumin and prealbumin. (3,22,23). Analysis of the ratiolabeled profiles on isoelectric focusing gels of individual fractions eluting from G-75 columns between the void volume and Ke 0.2 disclosed the presence of at least 14 ratiolabeled peaks synthesized either de now or in increased amounts in the stimulated cultures. Several had similar Kd values but could be distinguished by pI values, and vice versa, and the need for using narrow Sephadex pools became readily apparent. It has thus been possible to characterize a variety of products of activated lymphocytes by the increase in isotope incorporation, by Kd on Sephadex, and by pI on isoelectric focusing.
This represents a first attempt using new methods and many additional experiments will be required to achieve full purification and characterization of MIF, and to establish whether the specifically ratiolabeled peak here associated with MIF activity is indeed the active component. We recognize limitations in the present approach. For example, even though one labeled peak may ultimately be isolated, it may be contanfinated with a number of nonlabeled proteins which will not be detectable by our method. Of course the methods need to be extended to other antigens than PPD, although PPD and Con A did induce the same ratiolabeled peak and MIF activity. The quantitative aspects of this method require further study. Dumonde et al. (3) have pointed out the importance of quantitation in terms of protein weight required to give certain percent migration inhibition in dilution experiments, e.g., MI30. However, there are difficulties in reporting specific activities on the basis of protein weight, since most of the protein in the supernatants is contributed by contaminating serum from dying cells, conditions that vary from experiment to experiment. The potential of the radiolabeling methods is ultimately to permit quantitation in absolute terms of the increase in isotope, or micromoles of leucine, incorporated into a specific protein in antigen-stimulated cultures. At this time we can only state that the net amount of label found in the ratiolabeled peak associated with MIF activity represents 0.1-0.5% of the total macromolecular counts, i.e. synthesized protein, in the supernatants. It is our expectation that the methods would simplify the prevailing complex situation by identifying the 15-20 biological activities already reported with a smaller number of chemical species.
We believe that the radioisotopic methods may prove helpful for permitting the isolation and characterization of many of the molecules in antigen-stimulated lymphocytes synthesized either de now or in increased amounts after antigen stimulation. The methods are sufficiently general in their application to be useful in studying synthesis of cellular components in the various lymphocyte subpopulations, and in macrophages affected by antigen. With further study they may be adapted to useful clinical assays in situations where the availability of l}mphocytes is limited. SUMMARY General methods were developed and applied to the biosynthesis and purification of products of activated lymphocytes available in minute quantities. The activity studied here was the migration inhibitory factor (MIF) produced by purified protein derivative (PPD)-or concanavalin A (Con A)-stimulated lymphocytes obtained from one guinea pig or less. The methods selected yielded results in terms of two chemical parameters characteristic of the molecules involved, namely Kd on Sephadex G-75 and isoionic point, pI, on isoelectric focusing.
When supernatants were fractionated on G-75 columns, there were several areas even in control supernatants which produced migration inhibition relative to medium controls. However, in PPD-and Con A-stimulated supernatants, at least one peak of MIF activity was found solely in the stimulated cultures, with a Kd of 0.15. A double-labeling technique was used to characterize the proteins of this peak. Control, unstimulated cultures were labeled with [14C]leucine and stimulated cultures were labeled with [3H]leucine. After mixing the supernatants and G-75 filtration, a major "ratiolabeled" broad peak, i.e. one with increased 3H/14C ratio, was found. When a narrow portion of this peak about Kd 0.15, containing most of the MIF activity, was subjected to analytical isoelectric focusing, all of the label was associated with proteins of lower net charge than albumin. A unique ratiolabeled peak was found in PPD-and Con A-stimulated fractions with a pI of approx. 5.3. A micropreparatire isoelectric focusing technique was developed and yielded MIF activity in the same region as the major ratiolabeled peak. Further study will be required to ascertain whether the ratiolabeled protein is MIF. By following the Kd, p[, and 3H/uC labeling ratio, at least 14 products of activated lymphocytes, synthesized either de now or in increased amounts, could be distinguished.