BrIef CommunIcatIon: Tumor Suppression by a Lymphokine Released Into the Circulation of Mice With Delayed Hypersensitivity~'2 S. B. Salvin, J. S. Youngner, Jane Nishio, and Ruth Neta a SUMMARY-Lymphokines such as migration inhibitory factor and type 1/ interferon were released into the circulation of tuberculin-sensitive mice after i'v injection of old tuberculin. Daily administration of such sera into the tumor sites of mice given implants of sarcoma MC-36 markedly suppressed tumor growth.-J Natl Cancer Inst 55: 1233-1236, 1975.

Soluble mediators [lymphokines (LK's)] were released into the circulation of mice with delayed hypersensitivity after stimulation with specific antigen (1-3). High titers of migration inhibitory factor (MIF) and of a new type of mouse interferon (type II) occur in the sera of such mice. Bacteriostatic, mycostatic, and hematopoiesisinhibiting activities can also be demonstrated in these sera (4). Preliminary studies indicate that a cytotoxin may also be released into the circulation (Neta R, Salvin SB: Unpublished observations). In addition, tuberculinsensitive mice that release LK's into the circulation after stimulation with antigens from the tubercle bacillus show marked resistance to aerosol challenge with a virulent strain of Mycobacterium tuberculosis} H s7Rv (5). The production of these LK's is accomplished as follows: Mice are infected iv with 10 7 M. bouis, strain BCG, and 3 weeks later are inoculated with 50 mg old tuberculin (OT). Peak amounts of released LK's are found in sera of mice bled 3-4 hours after the inoculation of OT. MIF activity appears in the serum in such high concentrations that it is titratable; this is in contrast to the low concentrations of MIF produced in vitro by exposure of sensitized lymphocytes (peritoneal-exudate or spleen cells) to specific antigen (1) 2). The type II interferon released into the sera of mice with delayed hypersensitivity differs from conventional virus-induced interferon (type I) in stability at pH 2 and 56 0 C, and in host specificity. Also, highly purified antibody against type I interferon does not neutralize the activity of type II interferon (2). Since cell-mediated immunity (CMI) is believed to have a function in resistance to tumors, we tried to determine whether this resistance was mediated by the LK's associated with CMI (5). Our data indicate that passively administered LK-containing sera suppress the growth of tumor grafts in mice. MATERIALS AND METHODS

C3HeB/FeJ female mice weighing 20-25 g were purchased from The Jackson Laboratories, Bar Harbor, Maine. A transplantable, 3-methylcholanthrene-induced sarcoma (MC-36) was obtained from Dr. Bernard Fisher, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania. The tumor was serially passaged by sc implantation of tumor fragments into the abdominal area. Sera containing the soluble mediators MIF and type II interferon were obtained from Swiss Webster mice (Taconic Farms, Germantown, N.Y.). The mice were sensitized by iv infection with 5 X 106- 1 X 107 viable BCG cells. Three-4 weeks later, footpads of infected animals showed marked reactions to injection of 30 p.g

purified protein derivative (PPD). Three weeks after infection, the sensitized mice were challenged iv with 50 mg OT; they were exsanguinated 3-4 hours later. The separated sera were pooled, assayed for MIF and interferon activity as described previously (1), and stored at -70 0 C. The MIF titer of such sera was usually about 1 :64, and the type II interferon titer, about 1 :3,000. Control sera were obtained from normal, unsensitized mice challenged with 50 mg OT and bled 3 hours later, or from mice sensitized with BCG but challenged with saline or not challenged at all. Control sera containing type I interferon were obtained from mice inoculated iv with 2.5 X 109 plaque-forming units of Newcastle disease virus and bled 8 hours later. Within 24 hours after the solid tumor fragment (about 1.5-2.0 mm in diameter) was implanted sc in the abdominal area, 0.2 ml of either LK-containing or control serum was injected sc daily into the tumor implant area. When the tumors reached sufficient size, primarily in the control animals, the serum was inoculated directly into the tumor. The diameters of the tumors were measured frequently with calipers. If 1 U MIF is the amount of serum which will cause a 20% or greater inhibition of migration of normal mouse peritoneal exudate cells (1), then :::::10 MIF U /day were administered/mouse. This amount of serum also contained about 300 U type II interferon. Sera containing type I interferon were diluted so that the daily dose also contained 300 U activity. RESULTS

In six consecutive experiments, mice were treated daily for 20-36 days before final determinations of tumor size. In all experiments, tumor growth was markedly inhibited only in mice that had received LK-containing sera (table I). The cumulative results at the bottom of table I show that the average tumor volume was 916 mm- in mice given control sera, whereas in mice treated with LK-containing sera, tumor volume averaged only 24 mm" (see also figs. 1, 2). The inhibitory activity in the sera of BCG-infected and OT-challenged mice will be called tumor-inhibiting factor (TIF). Histologic examination revealed greater infiltration of mononuclear cells into the tumor sites in the mice treated with LK-containing sera than in those given control sera. It has not yet been determined whether this infiltration was due a) to the attraction and activation of macrophages by factors in the LK-containing sera, or b) to a toxic factor that suppressed or damaged the Received March 27, 1975; accepted July 3, 1975. Supported by the United States-Japan Cooperative Medical Science Program administered by the Public Health Service, National Institute of Allergy and Infectious Diseases (grant AI08528), by Public Health Service grant AIll945 from the National Institute of Allergy and Infectious Diseases, and by contract DADA-17-67-C7046 from the U.S. Army Medical Research and Development Command, Washington, D.C. 20314. S Department of Microbiology, School of Medicine, University of Pittsburgh, Pittsburgh, Pa. 15261. 1

2

JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 55, NO.5, NOVEMBER 1975

1233

1234

SALVIN, YOUNGNER, NISHIO, AND NETA

TABLE I.-Inhibition of sarcoma MC-36 by

LK-containing sera administered daily

Tumor mass (mmt) in mice given

Expt

1 2 3

b

4b 5

Duration (days) 30 31 28 32 31

Average volume of tumor mass

Control sera;

mean-ass

a

LK-containing sera; mean±SE a

Percent inhibition

(6) 998±159 (6) 780±450 (16)1,322±243 (15) 624±177 (12) 858±520

(8) (4) (14) (13)

18±38 22±63 52±54 24±62 (11 ) 6±58

99.8 97.2 97.0 96.3 99.3

(55)

(50) 24±55

98

916±310

o Number of mice in parentheses. In experiments 2 and 4, 18 additional mice were treated with sera containing 300 U type I interferon. The average tumor volume (712 mms) in these mice was not significantly different from the tumor volumes in mice given control sera. b

tumor cells and thereby attracted mononuclear phagecytes. DISCUSSION

Type I interferon, prepared by injection of viruses into mice, reportedly inhibited the multiplication of mouse leukemia L121O.cells in stationary suspension cultures (6). The degree of inhibition was related to the antiviral titer of the interferon preparations used. In addition, repeated daily ip injections of 20,000 U mouse brain interferon (type I) for 1-2 months increased the survival times of several strains of mice after ip challenge with Ehrlich ascites tumor cells, or with other ascites tumor cells such as RC-19 and Ll210 (7). The protective effect varied with the strain of mouse and the type of tumor cells. However, daily administration of type I interferon, as described here, did not protect mice against the solid tumor sarcoma MC-36, though LK-containing sera did show such protective activity. Sera containing TIF (corresponding to daily doses of 10 U MIF and 300 U type II interferon) administered daily for 3-4 weeks inhibited MC-36 tumor growth in mice by 97%. In contrast, 20,000 U type I interferon administered daily for 1-2 months were required to increase the survival times of the mice challenged with ascites cells (7). This marked activity of TIF-containing sera may be due to the high potency of TIF, to the synergistic effect of the several LK's known to be present in the sera, or to the high susceptibility of MC-36 under the conditions employed.

Inoculation of viable cells of BCG or components thereof has been reported to inhibit tumor growth in animals and man (8-11); the mechanism by which this tumor growth is restricted is not known. However, when lymphocytes from tuberculin-sensitive guinea pigs were exposed to specific antigen (PPD), injection of the supernatants into the tumor site produced a local accumulation of mononuclear cells and suppressed the growth of syngeneic tumor grafts in guinea pigs (12). Our data further emphasize the importance of LK's in suppression of tumor growth. Repeated injections of BCG into tumor sites may cause local accumulation of TIF and thereby restrict tumor growth. Thus our demonstration of TIF activity against a solid tumor in mice suggests an approach to tumor immunotherapy in animals and man. REFERENCES

(1) SALVIN SB, YOUNGNER JS, LEDERER WH: Migration inhibitory factor and interferon in the circulation of mice with delayed hypersensitivity. Infect Immun 7:68-75, 1973 (2) YOUNGNER JS, SALVIN SB: Production and properties of migration inhibitory factor and interferon in the circulation of mice with delayed hypersensitivity. J Immunol 111:19141922, 1973 (3) SALVIN SB, NISHIO J: In vitro cell reactions in delayed hypersensitivity. J Immunol 103:138-141, 1969 (4) SALVIN SB, NISHIO J, SHONNARD JT: Two new inhibitory activities in blood of mice with delayed hypersensitivity, after challenge with specific antigen. Infect Immun 9:631-635, 1974 (5) SALVIN SB, RIBI E, GRANGER DS, et al: Migration inhibitory factor and type II interferon in the circulation of mice sensitized WIth mycobacterial components. J Immunol 114:354-359, 1975 (6) GRESSER I, BROUTy-Boyt D, THOMAS M, et al: Interferon and cell division. I. Inhibition of the multiplication of mouse leukemia LI210 cells in vitro by interferon preparations. Proc Natl Acad Sci USA 66:1052-1058, 1970 (7) GRESSER I, BOURALl C: Antitumor effects of interferon preparations in mice. J Natl Cancer Inst 45:365-376, 1970 (8) ZBAR B, TANAKA T: Immunotherapy of cancer: Regression of tumors after intralesional injection of living Mycobacterium bovis. Science 172:271-273, 1971 (9) ZBAR B, BERNSTEIN ID, RAPp HJ: Suppression of tumor growth at the site of infection with living Bacillus Calmette-Guerin. J Nat) Cancer Inst 46:831-839, 1971 (10) BARTLETT GL, ZBAR B, RAPP HJ: Suppression of murine tumor growth by immune reaction to the Bacillus Calmette-Guerin strain of Mycobacterium bovis. J Natl Cancer Inst 48:245257, 1972 (11) ZBA~ B, RAPP HJ, RIBI EE: Tumor suppression by cell walls of Mycobacterium bovis attached to oil droplets. J Natl Cancer Inst 48:831-835, 1972 (12) BERNSTEIN D, THOR DE, ZBAR B, et al: Tumor immunity: Tumor suppression in vivo initiated by soluble products of specifically stimulated lymphocytes. Science 172:729-731, 1971

I.-Surface view of 2 mice inoculated sc 30 days previously with sarcoma MC-36. Control mouse (lett) was inoculated daily at tumor site with control serum; experimental mouse (right) was inoculated daily at tumor site with mouse serum containing 10 U TIF.

FIGURE

SALVIN, YOUNGNER, NISHIO, AND NETA

1235

2A 2.-Sections of 30-day-old tumors from mice. H & E. X 1,250. A: Control mice, showing cell composition of tumor; mice, showing increased infiltration of mononuclear cells and appearance of large vacuoles.

FIGURE

1236

B:

experimental

SALVIN, YOUNGNER, NISHIO, AND NETA

Tumor suppression by a lymphokine released into the circulation of mice with delayed hypersensitivity.

Lymphokines such as migration inhibitory factor and type II interferon were released into the circulation of tuberculin-sensitive mice after iv inject...
610KB Sizes 0 Downloads 0 Views