Eur. J. Irnmunol. 1986.16: 375-380 Suppressed IFN-y production in carrageenan-treated mice 375

In summary the present study shows that no simple correlation exists between class I1 MHC antigen expression and antigen- presenting capacity, and that the differences observed are not due to effects on IL 1. It might be argued that this would not be found in a situation where class I1 antigen expression is constitutively very low as in the case of murine peritoneal macrophages and murine cell lines. However, recent work on the antigen-presenting capacity of the murine line P388Dl has shown that signals which down-regulate Ia expression have little effect on antigen presentation by the same cells [25]. It seems that T lymphocytes may require only low levels of class I1 MHC antigen expression on APC.

We are grateful to Dr. T. Prospero, Department of Pathology, Uitiver- sity of Cambridge, who performed the flow cytofluorometric analysis. We thank Barbara Pearce for technical assistance.

Received May 9, 1985; in revised form November 5, 1985.

5 References

1 Matis, L. A., Glimcher, L. H., Paul, W. E. and Schwartz, R. H.,

2 Mizel, S. B., Immunol. Rev. 1982. 63: 51. 3 Barnstable, C. J., Bodmer, W. F., Brown, G., GalfrC, G., Mil-

stein, C., Williams, A. F. and Ziegler, A., Cell 1978. 14: 9. 4 Brickell, P. M., McConnell, I., Milstein, C. and Wright, B.,

Immunology 1981. 43: 493. 5 Brickell, P. M., Richardson, N. E., McConnell, I. and Feinstein,

A., Clin. Exp. Immunol. 1983. 54: 117. 6 Coombs, R. R. A , , Wilson, A. B., Eremin, D., Gurner, B. W.,

Haegert, D. , Lawson, Y., Bright. S. and Munro, A., J . Immunol. Methods 1977. 18: 45.

Proc. Natl. Acad. Sci. U S A 1983. 80: 6019.

Fujio Suzuki", Hiroshi Maeda' and Richard B. Pollard'

Department of Microbiology, Kumamoto University Medical School, Kumamoto' and Division of Infectious Diseases, Department of Internal Medicine and Department of Microbiology, University of Texas Medical Branch and Virology Division, Shriners Burns Institute, Galveston'

7 Zamoyska, R., Waldmann, H. , Prospero, T. and Lennox, E.,

8 Salmon, J., Prostaglandins 1978. 15: 383. 9 Slocombe, P., Easton, A,, Boseley, P. and Burke, D. C., Proc.

Natl. Acad. Sci. USA 1982. 79: 5455. 10 Goeddel, D. V., Leung, D. W., Dull, T. J., Gross, M., Lawn, R.

M., McCandliss, R., Seeburg, P. A , , Ullrich, A., Yelverton, E. and Gray, P. W., Nature 1981. 290: 20.

11 Rhodes, J., Salmon, J. and Wood, J., Eur. J. Immunol. 1985. 15: 222.

12 Basham, T. Y. and Merigan, T. C., J . Im unol. 1983. 130: 1492.

Mol. Biol. Med. 1983. I : 137.

13 Kelley, V. E., Fiers, W. and Strom, T. B., 7 J . Immunol. 1984.132: 240.

14 Wong, G. W. H., Clark-Lewis, I., McKimrn-Breschkin, J. L., Harris, A. W. and Schrader, J. W., J . Immunol. 1983. 131: 788.

15 Rhodes, J., Jones, D. H. and Bleehen, N. M., Clin. Exp. Immunol. 1983. 53: 739.

16 Rosa, F., Hatat, D., Abadie, A., Wallach, D., Revel, M. and Fellous, M., EMBO J. 1983. 2: 1583.

17 Snyder, D. S. and Unanue, E. R., J . Immunol. 1982.129: 1803. 18 Hokland, M., Larsen, B., Heron, I . and Plesner, T., Clin. Exp.

19 Gerrard, T. L., Cupps, T. R., Jurgensen, C. H. and Fauci, A. S . ,

20 Beller, D. I., Kiely, J.-M. and Unanue, E. R., J. Immunol. 1980.

21 Raff, H., Picker, L. J. and Stobo, J. D., J. Exp. Med. 1980. 152:

22 Gonwa, T. A., Picker, L. J., Raff, H. V., Goyert, S. M., Silver, J.

23 Lombardi, G., Piccolella, E., Vismara, D., Colizzi, V. and Asher-

24 Arenzana-Siesdos, F. and Vierelizier, J. L., Eur. J . Immunol.

25 Zlotinik, A,, Shirnonkervitz, R., Kappler, J. and Marrack, P.,

Immunol. 1981. 44: 239.

Cell. Immunol. 1984. 85: 330.

124: 1426.

581.

and Stobo, J. D., J. Immunol. 1983. 130: 706.

son, G. L., Clin. Exp. Immunol. 1984. 58: 581.

1983. 13: 437.

Cell. Immunol. 1985. 90: 154.

Suppression of interferon gamma production in mice treated with carrageenan Effects of carrageenan (CAR) treatment on the response of interferon (IFN) produc- tion in vivo and in vitro after stimulation with an IFN-y inducer, staphylococcal enterotoxin A (SEA), was investigated. The IFN-y production in mice stimulated with SEA was impaired after i.v. administration of a 20 mglkg dose of CAR. Spleen cells (SC) from CAR-treated mice had decreased ability to produce IFN in vitro after stimulation with the same inducer. SC obtained from mice during the suppressive state inhibited IFN-y production when they were co-cultured with mononuclear cells prepared from spleens of untreated control mice. This suppressor cell activity could be removed from SC by an adherence technique to plastic surface. The SC with suppressor activity were not inactivated by treatments with monoclonal anti-Thy-1.2 antibody, anti-asialo GM1 antisera and anti-mouse immunoglobulin antisera followed by complement. The suppressive activity was detected in cell-free culture fluids of macrophage fractions containing suppressor cell activity. These results suggest that the decrease in IF"-y production in mice pretreated with CAR may associate with the presence of suppressor cells characterized to the monocytelmacrophage lineage.

[I 51171 1 Introduction ~

Correspondence: Fujio Suzuki, Department of Microbiology, Kumamoto University Medical School, Kumamoto 860, Japan

Abbreviations: IFN-y: Interferon gamma SEA: Staphylococcal enterotoxin A CAR: Carrageenan McP: Macrophages NK: Natural killer MNC: Mononuclear cells C: Complement PAC: Plastic-adherent cells CAR-mice: Mice treated with CAR N-mice: Normal control mice PACF Cell-free fluids from cultures of PAC SC: Spleen cells mAb: Monoclonal antibody(ies)

Carrageenans (CAR), high molecular weight substances extracted from seaweed [ 1, 21, interfere with humoral and cell- mediated immune responses in mice [3] and rats [4] through the modifications of macrophage (MQ) functions. It was reported [5] that a single injection of relatively small doses of CAR abrogated or weakened natural resistance of irradiated mice to foreign bone marrow transplants. I n vivo administra-

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1986 0014-298Ol8610404-0375$02.50/0

376 F. Suzuki, H. Maeda and R. B. Pollard Eur. J. Immunol. 1986.16: 375-380

tion of CAR causes decreased responsiveness under most experimental conditions [3, 4, 61, but it may also cause increased responsiveness, depending on the time of adminis- tration relative to that of antigen [l]. Interest in CAR as a tool to study cellular mechanisms was generated by the observation that the unfractionated form of CAR was taken up by MQ in vitro [8] and that it was directly toxic to these cells without impairing the viability and function of lymphocytes [9, 101. A suppressor cell activity in spleens of mice treated with CAR has also been demonstrated [ll-131. Hochman and his co- workers have described the in vivo (111 and in vitro [23] activa- tion of suppressor cells, which inhibit natural killer (NK) cell activity brought about by the exposure of cultured spleen cells (SC) to CAR. It was also reported [13] that the suppressor cells induced by CAR in mouse spleens inhibited the lytic functions of cytotoxic T lymphocytes. However, the effect of suppressor cells generated by CAR on the production of vari- ous interferons (IFN) has not been reported, even though IFN are important natural host proteins which exhibit antiviral, antitumor, anticellular as well as immunoregulatory activities [14]. In particular, IFN-y plays a pivotal role in the regulation or mediation of several important lymphocyte functions [15]. These include the regulation of antibody production [16], enhancement of NK cell activity against virus-infected cells and tumor cells [17], regulation of Ia antigen expression on MQ, [18], and priming of MQ, for tumor cell killing [19].

In the view of the potential importance of IFN-y in the immune responses, we designed a study to investigate the response of CAR-treated mice and SC from these mice to IFN-y inducers. In addition, effects of mouse splenic suppres- sor cells induced by CAR on the production of IFN-y and characterizations of the suppressor cells that affect an IFN-y induction were also investigated. The results described herein indicate that CAR-induced suppressor cells, which were char- acterized to monocyte/MQ lineage, can inhibit IFN-y produc- tion by normal mouse mononuclear cells (MNC) stimulated with an IFN-y inducer, staphylococcal enterotoxin A (SEA).

2 Materials and methods

2.1 Mice, virus and reagents

Six to 8-week-old inbred BALB/c mice were used in the exper- iments. The medium used for the cultivation of lymphocytes was RPMI 1640 (Gibco, Grand Island, NY) supplemented with 2 mM L-glutamine, 20 mM sodium hydrogen carbonate, 25 mM HEPES buffer and 10% heat-inactivated fetal calf serum (RPMI growth medium). For the cultivation of L929 fibroblast cells, Eagle's modified minimum essential medium (MEM) supplemented with 10% heat-inactivated newborn calf serum and 20 mM sodium hydrogen carbonate (MEM- growth medium) was used. RPMI 1640 medium supplemented with 25 mM HEPES buffer, 20 mM sodium hydrogen carbo- nate and 100 U/ml heparin (RPMI-plain medium) was used for the preparation of splenocytes after refrigeration by iced water. All media contained 100 U/ml penicillin and 100 pg/ml streptomycin. The Indiana strain of vesicular stomatitis virus was grown in mouse L929 cells and stored frozen at -80°C until use. The 50% tissue culture infectious dose (TCID50) of the virus was 107,5/ml in these cells. CAR (type 11, C-1138) consisting of the predominantly iota-type was obtained from Sigma, St. Louis, MO. SEA was employed as an IFN-y inducer [16,20-221. The SEA was supplied from the Microbial

Biochemistry Branch, Division of Microbiology, FDA, Cin- cinnati, OH. The reference standard murine IFN (G-002-904- 511) was obtained from the Antiviral Program of the National Institute of Allergy and Infectious Diseases, National Institute of Health, Bethesda, MD, and used to standardize the IFN assay. A monoclonal antibody (mAb) and antisera utilized included monoclonal anti-Thy-1.2 antibody (Accurate Chemi- cal and Scientific Corporation, Westbury, NY); goat anti- mouse immunoglobulin antiserum (anti-mouse Ig, anti- IgA + IgG + IgM antiserum; Cappel Laboratories, Inc., Cochraneville, PA); anti-asialo GM1 antiserum (Wako Pure Chemical Industries, Ltd., Osaka, Japan), and anti-mouse IFN-y antiserum (anti-IFN-y; Department of Microbiology, University of Texas Medical Branch, Galveston, TX). Low- tox-M rabbit complement (C) was obtained from Cedarlane Laboratories, Ltd., Hornby, Ontario, Canada. These reagents were used for the characterization experiments of suppressor cells appeared in spleens of CAR-mice.

2.2 Cells

Monolayer cultures of murine L929 cells were grown in 150- cm2 tissue culture plastic flasks (Corning Glass Work, Corn- ing, NY) in MEM-growth media and used as target cells for IFN titration. As described previously [22], whole spleens were aseptically removed and teased on a steel mesh immersed in RPMI-plain medium in a plastic dish. The cells passed through the mesh were then washed twice with RPMI-plain medium and suspended in RPMI-growth medium. Splenic cells obtained were cultured in a 24-well microtiter plate for IFN induction and/or assay of suppressor cell activities. As described previously [23], separation of MNC from SC was performed by Ficoll-Hypaque sedimentation (e = 1.074 kg/l density). A highly MQ-enriched population (plastic-adherent cells, PAC) and a lymphocyte population were prepared by an adherent technique using a plastic surface coated with an inac- tivated fetal calf serum [23]. Approximately 10% of the total number of SC adhered to the plastic surface. The nonadherent cell populations (non-PAC) obtained by this method con- tained less than 2% of MQ.

2.3 Carrageenan treatment

A single i.v. injection of 0.4 mg/mouse (20 mg/kg) of CAR dissolved in saline was given to mice [ll]. Splenocytes har- vested 5 days after the injection of CAR were used as a source of producer cells for IFN-y and as a source of inhibitor cells in assays of suppressor activity.

2.4 IFN induction

As described previously [22], SEA dissolved in saline was injected i.p. into mice at a dose of 6.25 mg/kg to induce IFN-y in the serum [22]. Sera were obtained at various times after the injection and assayed for antiviral activity. SC or splenic MNC prepared from CAR-mice were stimulated with 0.02 yglml of SEA to induce IFN in vitro [22]. In some experiments utilizing SEA as an inducer, splenic MNC from normal mice untreated with CAR (N-mice) were co-cultured with SC, PAC and non- PAC prepared from CAR-mice to detect suppressor cell activ- ity. Twelve to 72 (mainly 72) h after cultivation at 37"C, cul- ture fluids were harvested and clarified by centrifugation

Eur. J. Immunol. 1986.16: 375-380 Suppressed IFN-y production in carrageenan-treated mice 377

2 x lo7 cells/ml. Cultured media were then clarified by cen- trifugation (1250 x g, 30 rnin), and used in experiments.

(1250 x g for 30 min). In this system, 1 x lo6 celldm1 of splenic MNC from N-mice produced 150-200 U/ml of IFN in their culture fluids. The IFN induced by SEA in the sera of mice and the culture fluids of splenic MNC has been previously characterized as IFN-y [16, 20, 211. 3 Results

3.1 IFN-y production in SC cultures 2.5 IFN assay and IFN determination

Antiviral activity was determined by means of a plaque reduc- tion assay utilizing VSV on L929 cells [24]. The IFN titer was calculated as the reciprocal of the greatest dilution of the test sample that reduced virus plaques by 50% and compared to murine reference IFN. One unit of IFN as defined by murine IFN standard equaled approximately 0.62 units in this assay system. To determine whether IFN induced by SEA in vitro and in vivo had a nature of IFN-y, inactivation experiments of the antiviral activity with anti-IFN-y were performed, as described previously [22].

2.6 Treatment of SC with blockers for T cells, B cells and NK cells in vitro

Five groups of SC obtained from mice 5 days after CAR treat- ment were prepared at a concentration of 3 x lo7 cellslml in RPMI-growth medium. Three groups were treated with a 1 : 500 dilution of anti-Thy-1.2 mAb, a 1 : 10 dilution of goat anti-mouse Ig antiserum or a 1 : 50 dilution of anti-asialo GM1 antiserum. Following incubation at 4°C for 1 h, cells of these 3 treated groups and one other group were centrifuged (450 x g for 10 rnin), resuspended in 2 ml of a 1 : 10 dilution of Low- tox-M rabbit C in cytotoxicity medium (RPMI 1640 medium supplemented with 0.3% bovine serum albumin, 25 mM HEPES buffer, 2 mM L-glutamine and antibiotics) at a concen- tration of 3 X lo7 cells/ml, and incubated for 1 h in a 37°C water bath. All five groups were then washed three times (450 X g for 10 min), resuspended in RPMI-growth medium at various concentrations and co-cultured with MNC from N- mice in the presence of SEA.

2.7 Assay of suppressor cell activity

SC, PAC and non-PAC fractions, which were prepared from mice 5 days after CAR injection, were used as a source of putative suppressor cells. Various numbers of suppressor cells suspended in 0.25 ml of RPMI-growth medium were placed in 24-wel1, 16 mm diameter microtiter plates (Costar, Cam- bridge, MA). As the IFN producer cells, a fixed number of splenic MNC from N-mice in 0.25 ml of RPMI-growth medium was added to each well followed by 0.5 ml of RPMI- growth media containing 0.04 @ml of SEA. After incubation at 37°C for 72 h in 5% COz, culture fluids were removed, centrifuged (1250 x g, 30 min) and frozen immediately at -80 "C until determination of the IFN titers. Reduction of the IFN titers was compared with that of controls and was con- sidered as suppressive activity of the putative suppressor cells.

2.8 Preparation of PACF

Conditioning media from PAC cultures were used as PACF. SC obtained from mice 5 days after CAR treatment were cul- tured in RPMI-growth medium for 3 days at a cell density of

The production of IFN-y in the culture of SC obtained from mice 5 days after CAR treatment was studied. To induce IFN in vitro, 2 x lo6 cellsiml of SC prepared from CAR-mice were cultured with 0.02 pg/ml of SEA. As shown in Fig. 1, there was a significant decrease in the amount of IFN production detected in the cultures of SC from CAR-mice. When only 18 U/ml of IFN was detected in culture fluids harvested from SC cultures of CAR-mice 72 h after cultivation, the counter- part of N-mice produced 165 U/ml of IFN in their culture fluids. Similar results were obtained when various numbers of SC from CAR-mice were stimulated with SEA. As shown in Fig. 2, when 1.6 x lo7 cells/ml of N-mice SC produced 311 UI ml of IF" in their culture fluids, 88 U/ml of IFN activity was detected in the same fluids from CAR-mice. The reduction of

12 24 48 72 Hours After Cultimtion

Figure 1. Kinetics of IFN-y production in cultures of SC stimulated with SEA. SC (1 X lo6 cells/ml) obtained from N-mice (G) and mice 5 days after CAR treatment (-C) were incubated in vitro with 0.02 yglml of SEA and culture fluids harvested up to 72 h after cultiva- tion in the presence of SEA were assayed for IFN activity. The IFN titer was expressed as an average of the titers obtained from 5 cultures.

3% r

250 I

5 10 50 1w 5m No. of Spleen Cells in Cultures (105cells/ml)

Figure 2. IFN-inducing capability of SC from CAR- and N-mice in vitro. Various numbers of SC from CAR-mice (5 days post-CAR treatment, -C) and N-mice (-0.) were cultured at 37 "C for 72 h in 5% COz in the presence of SEA (0.02 bg/ml). The IFN titer was expressed as an average of the titers obtained from 5 samples.

378 F. Suzuki, H. Maeda and R. B. Pollard Eur. J. Immunol. 1986.16: 375-380

IFN production in this experiment was calculated as 72% (p < 0.001) at 1.6 x lo7 celldml, 77% (p < 0.001) at 4 x lo6 cells/ml and 80% (p < 0.001) at 1 x lo6 cellsiml of SC cultures when compared with controls. The antiviral activities induced by SEA in cultures of SC from N-mice and CAR-mice were completely neutralized by treatment of anti-IFN-y.

3.2 IFN-y production in mice

To determine circulating IFN, blood samples were obtained from mice after an i.p. dose of SEA 5 to 36 h after the injec- tion. In this experiment, mice 5 days after CAR treatment were used as CAR-mice. As shown in Fig. 3, when SEA was administered to N-mice, IFN in serum appeared within 10 h and increased to a peak level (1152 U/ml) at 21 h. The IFN levels disappeared gradually and were no longer detectable 36 h after the administration. However, only 68 Uiml of IFN was detected in serum obtained from CAR-mice 21 h after the stimulation with the inducer. The IFN production in CAR- mice was also examined on various days after the injection of CAR (data not shown). The suppression of IFN-y production

Figure 3. Kinetics of IFN-y induction by SEA in mice treated with or without CAR. Mice 5 days after CAR treatment (-@-) or N-mice (-3) were administered with SEA (6.25 mgikg, i.p.). At various intervals after the stimulation, mice were bled and serum specimens were assayed for IFN. The IFN titer was expressed as an average of those obtained from 5 serum specimens for 15 mice.

2 0 / / I ' I I A

1 10 p,

m Suppressor Effector

Figure 4. Effect of different numbers of SC obtained from mice 5 days after CAR treatment on the suppression of IFN-y production. Graded numbers of SC containing suppressor cell activity were mixed with 2 X lo6 cellsiml of MNC from spleens of N-mice in the presence of SEA at a final concentration of 0.02 pg/ml. Culture fluids harvested 72 h after cultivation were assayed for the antiviral activity. The decrease in IFN production was compared with the control and the percent suppression was calculated as described.

Table 1. Suppressor activities of SC from CAR-mice on IFN-y pro- duction by splenic MNC from N-mice

No. of SC from No. of MNC IFN titer Suppression p'" CAR-mice"' from N-miceh' (U/mI)" (% ) (celldml) (cellsiml)

0 1 x lob 170 0 3 x 106 0 20 3 x 106 1 x lo* 21 88 < O . ( M l 1 1 x 106 0 20 1 x 106 1 x 106 81 52 0.02

As putative suppressors, SC obtained from mice 5 days after CAR treatment were cultured with or without effector cells. As effectors, splenic MNC from N-mice were stimulated with SEA (0.02 Kgiml) in the presence or absence of putative suppressor cells. IFN titer was an average of the titers from 5 cultures. Culture fluids were harvested 72 h after cultivation. Student's t-test, compared with the control.

initially appeared on day 2 and persisted until day 9 after CAR treatment. However, IFN production in mice 13 days after CAR treatment did not differ from controls. The antivi- ral activities induced in sera of N- and CAR-mice 20 h after SEA administration were almost neutralized by treatment with anti-IFN-y.

3.3 Suppressor cell activity of SC from CAR-mice

To determine whether the decrease in IFN production in CAR-mice was associated with suppressor cells, co-cultivation experiments were performed. Splenic MNC prepared from N- mice were used as effectors, and SC obtained from CAR-mice were used as putative suppressors. Obtained results of the co- cultivation are shown in Table 1. Whereas 1 x lo6 cells/ml of effector cells stimulated with 0.02 ygiml of SEA produced 170 U/ml of IFN, only 21 U/ml (88% suppression, p < 0.001) of IFN were detected when the same number of effector cells was co-cultured with 3 x lo6 cellsiml of suppressor cells in the presence of SEA. When 1 X lo6 cells/ml of suppressor cells were co-cultured with the same number of effector cells, 81 U/ ml of IFN (52% suppression) was produced. The IFN activity detected in co-culture fluids between suppressor and effector stimulated with SEA was completely neutralized by treatment with anti-IFN-y.

Since suppressor cell activity was detected in SC from CAR- mice, the effect of varying concentration of these cells on IFN production by effector cells was examined. One ml of RPMI- growth media containing graded numbers of SC from CAR- mice was mixed with an equal amount of the medium with 2 X lo6 of effector cells/ml in the presence of 0.04 ygiml of SEA, and they were incubated at 37°C for 72 h (Fig. 4). In the experiment, the ratio of effector-to-suppressor cells ranged from 1 : 0.25 to 1 : 16. As compared with the IFN titer of the control (effector alone, 170 U/ml), significant levels of IFN were detected when SC containing suppressor cell activity were mixed with effector cells at ratios of 1 : 0.25 (IFN titer: 158 U/ml). SC from CAR-mice significantly inhibited IFN pro- duction when co-cultured with effector cells at a ratio of 1 : 4 or

Eur. J. Immunol. 1986.16: 375-380 Suppressed IFN-y production in carrageenan-treated mice 379

Table 4. Effect of PACF on IFN-y production in MNC cultures greater (more than 85% suppression). Suppression of approxi- mately 50% was observed when effector cells and suppressor cells were co-cultured at a ratio of 1 : 1.

3.4 Characterization of suppressor cells

To determine whether suppressor activity in SC from CAR- mice was associated with cells adherent to plastic surfaces (PAC) and non-PAC fractionated from spleens of CAR-mice were tested for their suppressive abilities (Table 2). Although SC from CAR-mice inhibited IFN production significantly

Table 2. Suppressor activities of fractionated SC from CAR-mice on IFN-y production by splenic MNC from N-mice

Effector cells co-cultured No. of IFN titer Sup- with”) putative (U/mI)h’ pres-

suppressors sion (cells x lo-’/ ( % )

ml)

SC from N-mice SC from CAR-micc SC from CAR-mice Non-PAC from N-mice Non-PAC from CAR-mice Non-PAC from CAR-mice PAC from N-micc PAC from CAR-mice PAC from CAR-mice

Control (effector alone)

10 40 10

10 40 10

10 40 10

0

180 48 97

I79 161 I66

172 26 77

1 65

0 71” 41

0 2 0

0 X4“ 53

0

a) Effector cells (splenic MNC from N-mice, 1 X lo6 cellsiml) were co-cultured with fractionated SC (4 or 1 X lo6 cellsiml) obtained from mice 5 days after CAR treatment in the presence of SEA (0.02 yglml).

b) Harvested culture fluids were assayed for IFN activity and the titers determined as the average values obtained from 5 cultures.

c) Student’s t-test, p < 0,001, compared with the control.

Table 3. Effect of treatment with a mAb and two antisera on suppres- sor activities of SC from CAR-mice

Effectors were co-cultured IFN titer Suppression p‘’ with PAC pretreated by”’ (U/m1)”] ( % )

Media 36 78 < 0.001 C 42 75 < 0.001 Anti-Thy-1.2 mAb + C 48 71 <0.001 Anti-mouse Ip + C 39 76 < 0.001 Anti-asialo GMI + C 33 80 < 0.(X)1 Control (effector alone) I65 0

After treatment with various agents, splenic PAC prepared from mice 5 days post-CAR treatment were co-cultured with splenic MNC from N-mice (effectors, 1 X lo6 cellslml) at 37°C for 72 h in the presence of SEA (0.02 ygiml). The procedures of the treat- ment with the blockers and C were described in the text. Culture fluids harvested 72 h after co-cultivation were assayed for IFN activity. The titer was expressed as the average obtained from 5 specimens. Student’s t-test, compared with the control.

Effector cells were culturcd IFN titer”’ Supprc\<ion p“ in” ( U m l ) (?)

Media alone (control) 178 0

Media supplemented with 2 5 4 PACF from N-mice 182 0 50% PACF from N-mice 1 KO 0 25% PACF from CAR-mice 66 63 0 01 50% PACF from CAR-mice 51 71 <OO01

In the presence of SEA (0.02 yglml), MNC from N-mice (1 x lo6 cellslml) were cultured in media supplemented with 2.5% or 50% of PACF. PACF were prepared from cultures of PAC derived from spleens of CAR- or N-mice, as described in Sect. 2.8. Culture fluids harvested 72 h after cultivation were assayed for IFN activity. The titer was expressed as the average obtained from 5 samples. Student’s t-test, compared with control.

when they were co-cultured with effector cells (1 x 106 cells/ ml) at a ratio of 1 : 4, the IFN production was not inhibited by non-PAC fractions even when added to effector cells at the same number of cells. The PAC fraction enriched for MQ reduced IFN production more than the unfractionated SC pre- pared from CAR-mice.

The fact that the suppressor cell activity was associated with the PAC fraction suggested that a population of MQ-like cells might be responsible for the suppression of IFN-y production. Although PAC containing MQ can be isolatedfrom SC prepa- rations by the adherent technique, this cell fraction may include other cell populations adherent to plastic surface such as adherent T cells and adherent B cells. Therefore, the sup- pressor activity of SC prepared from CAR-mice was examined after treatment with anti-Thy-1.2 mAb or anti-mouse Ig anti- serum or anti-asialo GM1 antiserum plus c. As shown in Table 3, anti-Thy-1.2 mAb plus C had no detectable effect on the suppressor activity of the PAC fraction from CAR-mice, indicating that the cells mediating the suppression of IFN pro- duction lacked detectable Thy-1.2 antigen. The PAC fraction that had been exposed to anti-mouse Ig or anti-asialo GM1 antisera plus C also did not produce further reduction of sup- pressor activity.

To study whether the suppression was mediated by the soluble factor(s) released from suppressor cells, the effect of PACF, which was a cell-free culture fluid of PAC obtained from CAR-mice, on the production of IFN-y by MNC from N-mice was examined. As shown in Table 4, PACF from CAR-mice inhibited the production of IFN-y by N-mice MNC stimulated with SEA, while the same preparations from N-mice showed no inhibitory effect.

4 Discussion

The purpose of the present study was to investigate the response of CAR-treated mice and SC from these mice to IFN-y inducers. In mice that were injected 5 days after CAR treatment, SEA did not induce a significant amount of IFN in the circulation (Fig. 3). The suppressive state for IFN-y pro-

380 F. Suzuki, H. Maeda and R. B. Pollard

duction was detected 2 days after CAR treatment, persisted almost 7 days and gradually disappeared by day 12. The IFN- producing ability of SC from CAR-mice was also reduced after the incubation with SEA as compared with the titers obtained from splenic MNC of N-mice (Figs. 1 and 2). Since the co- cultivation of SC from CAR-mice with splenic MNC of N-mice in the presence of SEA resulted in the marked suppression of IFN-y production, it suggested that a suppressor cell was gen- erated in spleens of mice after the treatment with CAR. The suppression of IFN-y production was dependent upon the number of suppressor cells in the cultures and 50% suppres- sion of IFN responsiveness was observed when equal numbers of splenic MNC of N-mice and SC from CAR-mice were co- cultured (Fig. 4). The splenic suppressor cells of CAR-mice had characteristics that aided in their identification. They were not inactivated by the treatment with anti-Thy-1.2 mAb plus C (Table 3), and therefore did not appear to be T lymphocytes. Because these suppressor cells were removed by the adherent technique to plastic surface (Table 2), and also conditioning media (PACF) from cultures of PAC containing suppressor cell activity inhibited IFN-y production by N-mice MNC stimulated with SEA (Table 4), it appeared that the cells of monocyte/McP series were involved. The failure to abrogate the suppressor activity by treatments with anti-mouse Ig or anti-asialo GM1 antisera plus C tended to eliminate the possi- bility that suppressor cells are B lymphocytes and/or NK cells (Table 3).

The presence of suppressor M@ that influence the immune response has been the subject of recent investigations, and several types of suppressor M@ have been described [25-271. Suppressor McP that inhibit the responsiveness of IFN-y pro- duction have also been reported by us previously [23, 281. When thermally injured mice were stimulated by various IFN inducers, only IFN-y production was decreased as compared with noninjured controls [22]. This decrease in IFN production in thermally injured mice was associated with presence of sup- pressor McP that were capable of inhibiting IFN production by normal splenic lymphocytes in v im [23].

Splenic suppressor cells from CAR-mice which declined the NK cell activity have been reported by Hochman and his co- workers in 1981 [ l l ] and suppressor cells induced by CAR were characterized by them as a monocyte/M@ lineage [ l l , 121. Yung and Cudkowicz [13] described the in v i m acti- vation of M@-like suppressor cells brought about by the expo- sure of cultured SC to CAR. This suppressor cell activity appeared within 5 days of culture and was capable of inhibit- ing the lytic function of CTL. The reason for the appearance of the suppressor cells in spleens of CAR-mice is not known. It is possible that low levels of suppressor McP activity similar to those of splenocytes obtained from CAR-mice may exist in the lymphoid organs of N-mice. These suppressor cells may well be a part of a versatile regulatory circuit since suppressor McP

Eur. J. Immunol. 1986.16: 375-380

induced by CAR inhibited the iytic abilities of NK cells and T lymphocytes [ 11, 121 as well as the production of IFN-y from T cells presented herein.

We thank Dr. J. C. Guckian, Department of Internal Medicine, Univer- sity of Texas, Medical Branch, Galveston, TX, for his interest. The excellent technical assistance of R. Brutkiewicz, S. Uchimura and the secretarial help of M. Fujii are acknowledged.

Received May 27, 1985; in revised form November 12, 1985.

5 References

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Anderson, W., Can. J. Pharmaceutical Sci. 1967. 2: 81. Di Rosa, M., J. Pharm. Pharmacol. 1972. 24: 89. Ishizuka, S . , Otani, S. and Morisawa, S., J. Immunol. 1972. 118: 1213. Everson, N. W., Stacey, R. L. and Bell, P. R. E., Transplantation 1977. 24: 393. Yung, Y. P. and Cudkowicz, G., J. Immunol. 1977. 119: 1310. Thomson, A. W. andFowler, E. F., Transplantation 1977.24: 397. Turner, E. V. and Higginbotham, R. D., J. Reticuloendothel. SOC. 1977. 22: 545. Allison, A. C., Harington, J. S. and Birbeck, M., J. Exp. Med. 1966. 124: 141. Catanzaro, P. J., Schwartz, H. J. and Graham, R. C., Am. J. Pathol. 1971. 64: 387. Thomson, A. W., Wilson, A. R., Cruickshank, W. J . and Jeffries, A. H., Experientia 1976. 32: 525. Hochman, P. S. , Cudkowicz, G. and Evans, P. D., Cell. Immunol. 1981. 61: 200. Cudkowicz, G. and Hochman, P. S. , Cell. Immunol. 1980.53: 395. Yung, Y. P. and Cudkowicz, G., J . Immunol. 1978. 121: 1990. Yabrov, A. A., Interferon and Nonspecific Resistance, Human Sci- ences Press, New York 1980. Baron, S. , Blalock, J. E., Dianzani, F., Fleischmann, W. R., Georgiades, J. A., Johnson, H. M. and Stanton, G. J., Ann. New York Acad. Sci. 1980. 350: 130. Johnson, H. M., Stanton, G. J. and Baron, S., Proc. SOC. Exp. Bid. Med. 1977. 154: 138. Herberman, R. B. (Ed.), Natural Cell-Mediated lmmunity Against Tumors, Academic Press, New York 1980. Steeg, P. S. , Moore, R. N., Johnson, H. M. and Oppenheim, J. J., J. Exp. Med. 1982. 156: 1780. Pace, J . L., Russell, S. W., Torres, B. A., Johnson, H. M. and Gray, P. W., J. Immunol. 1983. 130: 2011. Osborne, L. C., Georgiades, J. A. and Johnson, H. M., Infect. Immun. 1979. 23: 80. Osborne, L. C., Georgiades, J. A. and Johnson, H. M., IRCS J. Med. Sci. 1980. 8: 212. Suzuki, F. and Pollard, R. B., J. Immunol. 1982. 129: 1806. Suzuki, F. and Pollard, R. B., J. Immunol. 1982. 129: 1811. Campbell, J. B., Grunberger, T., Kochman, M. A. and White, S. L., Can. J . Microbiol. 1975. 21: 1247. Parkhouse, R. M. E., J. Immunol. 1966. 97: 663. Scott, M. T., Cell. Immunol. 1972. 5: 459. Folch, H., Yoshinaga, N. and Waksman, H., J. Immunol. 1973. 110: 835. Suzuki, F. and Pollard, R. B., J. Znterferon Res. 1984. 4: 223.