lmmunochemistry, 1977. Vol. 14. pp. 143-147. Pergamon Press. Printed in Great Britain

R E G U L A T O R Y SUBSTANCES P R O D U C E D BY LYMPHOCYTES---IV FURTHER

CHARACTERIZATION OF THE O F D N A S Y N T H E S I S (IDS)*

INHIBITOR

Y U Z I R O N A M B A ¢ and BYRON H. W A K S M A N Department of Pathology, Yale University Medical School, 310 Cedar Street, New Haven, CT 06510, U.S.A.

(Received 30 July 1976) Abstract--Inhibitor of DNA synthesis (IDS)~, a soluble mediator which inhibits DNA synthesis of PHA-stimulated rat lymph node cells, was partially purified by conventional protein fractionation procedures from the culture supernatants of DA rat lymph node cells stimulated with concanavalin A (Con A) and shown to resemble IDS obtained by antigenic stimulation of specifically sensitized cells in its physicochemical properties. Antiserum was prepared by immunizing rabbits with this partially purified IDS preparation. IDS produced by Con A stimulation appeared to be antigenieally similar to that produced by antigenic stimulation, since both were absorbed in a parallel manner on a Sepharose 4B column coupled with this antibody. Neither Sepharose-conjugated rabbit anti-rat thymocyte IgG nor IgG from Lewis anti-DA allo-antiserum absorbed IDS. Conversely, DA rat thymocytes were unable to absorb the activity of rabbit anti-IDS. These findings suggest that IDS is not a major cell surface component.

INTRODUCTION

There are many immunologic phenomena in which thymus-derived cells or a 'supressor' subpopulation of these may act to inhibit the functional expression of T or B cells (reviewed in Gershon, 1974; M611er, 1975). As in other cell to cell interactions which affect immunologic responses, suppressor cells may act via humoral mediator(s). In preceding papers of this series, we have characterized a substance which appears to play such a role, the inhibitor of D N A synthesis (IDS) produced by sensitized lymphocytes of DA rats exposed to antigen (Namba & Waksman, 1975a, b, c, 1976a). IDS was shown to be distinct from lymphotoxin, proliferation inhibitory factor (PIF), and migration inhibitory factor (MIF). Since it is an attractive hypothesis that many cell components which participate in communication between cells may have originated from ancestral recognition molecules (Waksman & Namba, 1976), we were interested in determining the relationship between this 'suppressor' factor and components expressed on the surface of T-lymphocytes such as antigens of the major histocompatibility complex, Ia, beta-2 microglobulin and the T-cell receptor which binds antigen. In the present * This work was supported by NIH research grants AI-06112 and AI-06455 and NCI contract CB-43926. t On leave of absence from the Department of Pathology, Institute for Virus Research, Kyoto University, Kyoto, Japan. :~ Abbreviations used in this paper: BSA, bovine serum albumin; Con A, concanavalin A; DEAE, diethylaminoethyl; IDS, inhibitor of DNA synthesis; LNC, lymph node cells; MIF, migration inhibitory factor; OA, ovalbumin; PBS, phosphate-buffered saline; PHA, phytohemagglutinin; PIF, proliferation inhibitory factor; TCA, trichloroacetic acid.

study, we approached this problem by the use of an antiserum prepared against purified IDS. The experimental results indicate that this lymphokine is not a component expressed in abundance on the cell surface.

MATERIALS A N D M E T H O D S

Animals Inbred DA rats 4-6 months of age, bred in the Yale animal facility were used throughout.

Production of IDS IDS was produced from lymph node cells (LNC) stimulated in 2 different ways. One is the method described in preceding papers (Namba & Waksman, 1975a, b). Briefly, DA rats were immunized with 100 #g OA (ovalbumin, 5 x crystalline, Nutritional Biochemicals Co., Cleveland, OH) in Freund's complete adjuvant, given in the 4 footpads. LNC collected 9 days after immunization were cultured in 250ml tissue culture flasks (Falcon Plastics, Oxnard, CA) at a cell density of 5 x 106cells/ml in serum-free RPMI-1640 medium (Associated Biomedic Systems, Inc., Buffalo, NY) containing OA 10 #g/ml. After 48-hr incubation, culture fluids were harvested and centrifuged at 1000 g for 30 rain at 4°C, and the supernatant fluids stored at -10°C until used. For the preparation of pure IDS, LNC from normal rats were cultured under the same conditions with Con A (Calbiochem, La Jolla, CA) 2/~g/ml and OA 10 ~g/ml. The culture medium was changed every 2 days by low speed centrifugation, and the supernatants were stored at -10cC. Purification of IDS One liter of culture supernatant was concentrated with Diaflo ultrafiltration membrane PM-10 (Amicon Corp. Lexington, MA) to 50 ml and dialyzed against phosphatebuffered saline (PBS). The dialyzed material was applied to a Sephadex G-100 column Ibed volume approx 11.) 143

144

YUZIRO NAMBA and BYRON H. W A K S M A N

equilibrated with PBS. Ten milliliter fractions were collected and assayed for IDS activity, using the L N C - P H A assay system described below. Active fractions (6 × 104-9 × 1 0 4 daltons) were pooled and dialyzed against phosphate buffer (0.01 M sodium phosphate buffer, pH6.8) and applied to a 1.2 × 10cm DEAE-cellulose column (Cellex-D, 0.92m-equiv/g, Bio-Rad Laboratory, Richmond, CA) equilibrated with the same buffer. After washing the column with 20 ml of buffer, elution was carried out by increasing salt concentration stepwise up to 0.03 M sodium phosphate plus 0.06 M NaCI. The active fraction from DEAE-cellulose chromatography was dialyzed against 0.01 M sodium phosphate buffer, pH 6.8, and applied to a 1.0 × 3.0 cm hydroxyl apatite column (Sigma Chemical Co., St. Louis, MO) equilibrated with the same buffer. After stepwise elution with 6 ml volumes of buffer of increasing molarity, each fraction was dialyzed against PBS and stored at -10~C until used in experiments.

Analysis of partially purified IDS by aerylamide #el electrophoresis Disc electrophoresis was carried out according to the method of Ornstein and Davis (1964). Aliquots from active hydroxylapapite fractions were run on acrylamide gel electrophoresis (7.50, pH 8.6). After electrophoresis the gel was cut into 4 mm pieces, then each piece was extracted with 2 ml of PBS supplemented with 1 mg/ml BSA. After 48 hr of extraction at 4'C, an aliquot from the extract was assayed for IDS activity.

Assay (f IDS Assay of IDS using PHA-stimulated LNC was carried out exactly as in our previous papers (Namba & Waksman, 1975a, b). Briefly, wells in microtest culture plates (Linbro Chemical Co., New Haven, CT) were each seeded with 3 × 105 normal rat lymph node cells in 0.1 ml RPMI-1640 supplemented with 20% fetal calf serum. The cells were stimulated with PHA (PHA-P, Difco Labs., Detroit, MI), which was diluted with RPMI-1640 and added to the culture in 0.1 ml (final concentration of PHA was adjusted to 1.0/~l/mt). In addition, 0.1 ml of the fluid to be assayed (adequately diluted with RPMI-1640) was added to each culture. DNA synthesis was measured by adding 1/~Ci 3H-thymidine (5 Ci/mM) (New England Nuclear Co., Boston, MA) in 0.1 ml RPMI-1640 at 44 hr followed by harvesting and processing of the cells 4 hr later. One-tenth milliliter of the final cell suspension was soaked into glass filter paper (Whatman, CF/A), heat dried, and washed with cold TCA (10%) followed by ethanol and toluene washing before counting. Thymidine uptake was expressed as the difference between counts/min in test lymphocyte cultures and counts/min in cultures not stimulated with PHA. Antiserum

New Zealand rabbits were immunized subcutaneously with 100#g of partially purified IDS (the active fraction from the hydroxylapatite column) mixed with Freund's complete adjuvant. After one month, a booster injection of 100/~g of the same material was given i.v., followed by bleeding on the 10th day. The antiserum was heat inactivated at 60°C for 30min. Rabbit anti-rat thymus serum was prepared in this laboratory by 2 intravenous injections, one week apart, of 2 × 109 washed DA rat thymocytes, followed by bleeding one week later. Lewis anti-DA serum was obtained by 3 intraperitoneal injections of 2.5-5 x 108 pooled, washed DA rat spleen and LN cells at 2 week intervals, with bleeding 6 days after the last injection. Pooled sera in each case, were inactivated 30 min at 56 C.

Absorption of antiserum In some experiments, one ml of rabbit anti-IDS antiserum was absorbed twice with 1 × 108 normal DA rat thymocytes at 4°C for 1 hr.

Preparation of Sepharose-co~jugated antisera Conjugation of antisera to Sepharose 4B particles was carried out by the method of Axdn et al. (1967). Briefly, immunoglobulin was purified from each antiserum by ammonium sulfate precipitation and Sephadex G-200 fractionation and coupled with Sepharose particles which were activated by cyanogen bromide. Conjugation was carried out in 0.1 M carbonate buffer, pH 9.0. for 18 hr at 4 C .

Cytotoxicity test The cytotoxic activity of various antisera was measured by a 2-step method. Target cells were put into microculture wells in 0.2 ml PBS together with serially diluted antiserum in 0.1 ml. After incubation for 30min at 4°C, 0.1 ml of guinea-pig complement was added and the microplate was incubated at 37'~C for an additional 30 min. Trypan blue solution (0.5°0 in saline, 0.05 ml) was now added to each well and unstained cells were counted in a hemocytometer. RESULTS

Purification of IDS The first step of purification was fractionation on Sephadex G-100. This p r o c e d u r e had the additional p u r p o s e of eliminating C o n A to permit assay of fractions for IDS activity. C o n A b i n d s . d e x t r a n and is effectively r e m o v e d by this means. T h e I D S activity was found to be rather widely distributed (Fig. 1), a n d fractions of a p p r o x 6 9 x 104 d a l t o n s were p o o l e d and processed in the next purification step. SEPHADEX

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Characterization of IDS

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Fig. 2. DEAE-cellulose chromatography of the pool of active material obtained by Sephadex gel filtration. An aliquot of each fraction was added to the PHA-LNC system, after dialysis against PBS, in a final concentration of 10~. Fraction-4 is the active fraction and was processed in the next purification s t e p . . During DEAE-cellulose chromatography, IDS activity was eluted mainly in Fraction-4, but some activity was also found in Fraction-5 (Fig. 2). Four batches of Fraction-4 from DEAE-cellulose chromatography (equivalent to 41. of culture supernatant) were chromatographed on hydroxylapatite. The active material was eluted at 0.04 M sodium phosphate buffer (Fig. 3). The recovery of IDS activity was estimated as approx 10% of that in the original culture supernatant, while its specific activity was increased approx 100-fold.

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Absorption of IDS by Sepharose-conjugated antibody As shown in Table 1, rabbit antibody against IDS produced by Con A stimulation could bind IDS produced by the same stimulus. Absorption of the antiserum with DA rat thymocytes did not affect its ability to react with IDS, suggesting that IDS is not

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Analysis of the purified material by disc electrophoresis The material purified by hydroxylapatite column chromatography was subjected to acrylamide gel electrophoresis. As shown in Fig. 4, 1DS activity was detected in fractions corresponding to Band-3, one of the denser bands found in the gel. However, at least 4 other bands were also present, indicating that the purified material was contaminated by additional proteins.

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Characteristics of the antiserum against partially puri.fled IDS Rabbit antiserum against IDS, prepared as described in Materials and Methods, showed negligible hemagglutinating activity against DA rat red blood cells (negative at 1:10), in marked contrast with the rabbit anti-DA thymocyte serum (positive at 1:320) used in the absorption experiment described below. The cytotoxic activity of the anti-IDS serum for DA rat thymocytes was also low (91'~0 killed at 1:10 and 63~o at 1:20), while the anti-thymocyte serum killed 97~o thymocytes at 1:320 and 65°~ at 1:640. Lewis anti-DA antiserum also showed high cytotoxic activity against DA thymocytes (90~o killing at 160-fold dilution), as well as against peripheral DA lymphoIMM 14/2

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Fig. 4. Polyacrylamide disc gel electrophoresis of Fraction-3 obtained by hydroxylapatite chromatography. In the upper part of this figure, the pattern of the gel stained with Amido black 10B is presented. Since the gel shrinks during fixation and staining, the distance of each band from the starting point was corrected. The material extracted from each slice of the gel was dialyzed against PBS and added to the PHA-LNC system in a final concentration of 10~o.

146

YUZIRO NAMBA and BYRON H. WAKSMAN Table 1. Absorption of IDS by Sepharose-conjugated antibody" DNA synthesis (counts/min x 103)

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19.6 82.0 76.3 79.6

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87.6

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28.0 84.0

RPMI-1640 medium Rabbit anti-IDS

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4.5 control

Sepharose-conjugated antisera

aAntisera were conjugated with Sepharose beads as described in Materials and Methods. A Sepharose column of approx 10 ml was prepared for each antiserum, and 5 ml of IDS (fractionated on Sephadex and DEAE-cellulose and dialyzed against PBS in advance) was charged. After incubation for 30 min at room temperature, each column was eluted with 20 ml PBS. The eluted material was concentrated with polyethylene glycol No. 20,000 to 5 ml, dialyzed against RPMI-1640, then added to PHA LNC assay system in a final concentration of 20%. a component expressed in abundance on the thymocyte surface. This result agreed with the finding that xeno- and alloantibodies against rat thymus (rabbit anti-rat thymus and anti-DA), which were directed mainly to surface components of thymocytes and to the major histocompatibility complex, did not absorb IDS. Table I also shows that antibody against IDS produced by Con A stimulation reacts with IDS produced by antigenic stimulation virtually as well as with the homologous material. DISCUSSION The present study demonstrates that IDS produced by Con A stimulation is both antigenically and physicochemically similar to, if not identical with, that produced by antigenic stimulation. A similar relationship has also been reported for lymphotoxins produced with mitogen and with antigen by Kolb and Granger (1968) and for MIF produced by the two types of stimulation by Remold et al. (1972). Since it is much more convenient to use Con A than to use antigen for analysis of the IDS producing mechanism at cellular and subcellular levels, we will make this the basis of further studies of IDS producing lymphocyte subpopulations in subsequent papers of the present series. In this study, IDS produced by Con A-stimulated DA rat LNC was partially purified by conventional protein purification methods. An antiserum was prepared against it which proved to bind IDS activity effectively. DA thymocytes which are efficient sources of IDS (Namba & Waksman, unpublished data), were unable to absorb the binding activity from the antiserum and, conversely, the IDS-specific serum showed very low cytotoxic activity against thymocytes. Xenoand alloantibodies, strongly reactive with rat thymocytes and with elements of the major histocompatibility complex, failed to bind IDS. These findings argue against the possibility that IDS might be a cell surface component released by antigen or lectin.

The IDS molecule therefore appears to differ sharply from other immunoregulatory lymphokines which enhance lymphocytic responses (cooperation) or inhibit them (suppression). Thus allogeneic effect factor (Armerding et al., 1975), antigen-specific helper factor (Taussig et al., 1975), and antigen-specific suppressor factors (Takemori & Tada, 1975; Taniguchi et al., 1976; Kapp et al., 1976) all are described as specific gene products of the I region and are readily absorbed with anti-Ia antiserum. In some instances these appear to be firmly attached to the cell membrane (Takemori & Tada, 1975). The possibility that specific suppressor factors might actually be complexes of Ia with a non-specific factor such as IDS appears incompatible with the reported molecular weights of 45-50,000 for the specific factors and 75-80,000 for IDS (Namba & Waksman, 1975a, b). Similarly the difference in molecular weights appears to rule out the possibility that IDS might simply be a pool of specific factors of varying specificities. We are left with the alternative that there are really distinct antigen-specific and non-specific factors and that these are different in character as well as in function.

REFERENCES

Armerding D., Kubo R. T., Grey H. M. & Katz D. H. (1975) Proc. natn. Acad. Sci. U.S.A. 72, 4577. Ax~n R., Porath J. & Ernback S. (1967) Nature 214, 1302. Gershon R. K. (1974) Contemp. Top. lmmunobiol. 3, 1. Kapp J. A., Pierce C. W., De la Croix F. & Benacerraf B. (1976) J. lmmun. 116, 305. Kolb W. P. & Granger (3. A. (19681 Proc. natn. Acad. Sci. U.S.A. 61, 1250. Mtiller (3., Ed. (1975) Suppressor T Lymphocytes. Transplantn Rev. 26, 1. Namba Y. & Waksman B. H. (1975a) Inflammation 1, 5. Namba Y. & Waksman B. H. (1975b) J. lmmun. 115, 1018. Namba Y. & Waksman B. H. (1975c) Expl cell Res. 94, 23. Namba Y. & Waksman B. H. (1976a) J. Immun. 116, 1140. Namba Y. & Waksman B. H. (1976b) Unpublished data.

Characterization of IDS Ornstein L. & Davis B. J. (1964) Ann. N.Y. Acad. Sci. 121, 321. Remold H. G., David R. A. & David J. R. (1972) J. Immun. 109, 578. Takemori T. & Tada T. (1975) J. exp. Med. 142, 1241.

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Taniguchi M., Hayakawa K. & Tada T. (1976) J. Immun. 116, 542. Taussig M. J., Munro A. J., Campbell R., David C. S. & Staines N. A. (1975) J. exp. Med. 142, 694. Waksman B. H. & Namba Y. (1976) Cell. lmmunol. 21, 161.