Vol. 17, No.3 Printed i U.S.A.
INFEcTiON AND ImmuNrTy, Sept 1977, p. 639-63
Copyright© 1977 American Society for Microbiology
In Vivo Studies on the Cellular Source of Migration Inhibitory Factor in Mice with Delayed Hypersensitivity S. B. SALVIN,* G. SONNENFELD,t AND J. NISHIO Department ofMicrobiology, School ofMedicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
Received for publication 10 March 1977
Mice sensitized intravenously with 300 Lg of BCG cell walls in Drakeol-Tween 80 and challenged intravenously 3 weeks later with 50 mg of old tuberculin released migration inhibitory factor (MIF) into the circulating blood in quantities that could be detected in serum dilutions of 1:64 to 1:128. When thymus-derived lymphocytes (T-cells) were absent at the time of sensitization, as in neonatally thymectomized mice or in athymic nude mice, detectable amounts of MIF were not formed. Sensitized mice treated with either anti-@ serum or anti-bone marrow-derived lymphocyte (B-cell) serum before intravenous challenge with old tuberculin released reduced amounts of MIF into the circulation. Mice lethally irradiated, reconstituted with B-cells, sensitized with BCG cell walls, and then challenged intravenously 3 weeks later with old tuberculin did not release MIF into the circulation. When T-cells were injected at least 10 days before challenge, however, MIF appeared.
Lymphokines, such as migration inhibitory factor (MIF) and type II interferon, are released into the circulation of mice that have been sensitized intravenously (i.v.) with Mycobacterium bovis strain BCG and then challenged i.v. with old tuberculin (OT) (11, 14). The mice were appropriately sensitized with either living mycobacteria, dead mycobacteria, or cell wall fractions in oil droplets, and were challenged about 3 weeks later with either OT or living mycobacteria. Within 2 to 3 h after this i.v. challenge, MIF can be detected in the circulating blood, with serum dilutions of about 1:128 inhibiting the migration of normal macrophages by at least 20%. The MIF released into the circulation has been partly purified (11) and has been found to be acid unstable, heat stable, and highly specific (14). Of importance in the production of MIF may be the type or types of cells exposed to the antigen and the mode of interaction of these cells. Accordingly, experiments were initiated to determine (i) the source and types of cells involved in production of MIF and (ii) the experimental environment in vivo for maxum release of MIF. In this paper, experiments are described indicating that (i) thymus-derived lymphocytes (T-cells) must be present for 10 days before the time of challenge for MIF to be formed, and (ii) cells of both thymic and bone marrow (B-cells) origin are involved in the release of MIF in vivo into the circulation. t Present address: Division of Infectious Diseases, Stanford University Medical School, Stanford, CA 94305.
639
MATERIAIS AND METHODS Animals. Female Swiss Webster mice weighing 25 to 30 g were purchased from Taconic Farms, Germantown, N.Y. Offspring were thymectomized within 12 h of birth. The athymic nude mice backcrossed to BALB/c mice were purchased from G. L. Bomholtgard, Ry, Denmark. The C3H/He and AKR mice were purchased from Jackson Laboratories, Bar Harbor, Me; the C3H/He Dub mice were purchased from Flow Laboratories, Dublin, Va. Sensitization. The sensitizing antigen consisted of the cell walls of M. bovis strain BCG, obtained through the courtesy of E. Ribi, Rocky Mountain Laboratory, National Institute of Allergy and Infectious Diseases, Hamilton, Mont. In the preparation of the inoculum, 24.75 mg of the cell walls and 0.12 ml of Drakeol 6/VR (Pennsylvania Refining Co., Butler, Pa.) were ground into a paste. A 0.85% sodium chloride solution (16.7 ml) with 0.2% Tween 80 was added, and the grinding was resumed until a stable emulsion was obtained (10). Footpad tests. Mice were inoculated in one hind footpad with 30 jg of purified protein derivative of OT (PPD) in 0.05 ml of physiological saline (Parke, Davis & Co., Detroit, Mich.) and in the other bind footpad with 0.05 ml of physiological saline. Twenty-four hours later, with the aid of a calipers-micrometer (SchnelltAster, Systems-Kroplin, Schtchtern, West Germany), the thickness of the footpads inoculated with PPD was compared with the thickness of footpads inoculated with saline. A delayed reaction was indicated when an increase of greater than 10% in thickness was observed. Migration inhibition. Mice were inoculated intraperitoneally (i.p.) with 3 ml of light mineral oil 5 to 10 days before cell harvest. The peritoneal cavity was rinsed out with Hanks solution, and the peritoneal
640
SALVIN, SONNENFELD, AND NISHIO
INFECT. IMMUN.
exudate cells therefrom were washed three times by centrifugation in Hanks solution. The cells were concentrated, and a tiny drop was planted on the surface of 3 ml of agar substrate in a Falcon tissue culture flask. The substrate contained medium 199, 0.5% Ionagar, and 20% (by volume) mouse serum, plus penicillin (100 U/ml) and streptomycin (100 ,ug/ml). The flasks were incubated at 370C for 48 h, at which time the radius of migration was measured under lowpower magnification with the aid of an ocular micrometer (9). Preparation of anti-0 serum. Thymus glands were excised from 3-week-old C3H/He mice, teased gently, passed through glass wool filters, and then washed twice in Hanks balanced salt solution (6). Viable cells (107), with viability determined by cell exclusion of trypan blue, were inoculated either i.p. in Hanks solution or subcutaneously in incomplete Freund adjuvant. Then the mice were inoculated i.p. once a week for 8 to 11 weeks with 1 x 107 to 2 x 107 washed, viable lymphocytes. Six days after the last injection, the mice were bled. A pool of control serum was simultaneously obtained from normal adult AKR
T-cells that the resulting sera were active on B- and spleen cells but not on mouse T-cells (Table 1). All sera were frozen and stored at -70°C. X-irradiation. Two different sources of irradiation were used: (i) a "37cesium unit, 0.662 meV, producing 57 rads/min at a distance of 35 mm from the source; and (ii) a Picker X-ray unit, 200 kV, 15 mA, 20 cm (428 rads/min) or 45 cm (100 rads/min) from the target. The dose rate was calculated with a Victoreen R meter and Frichi (ferrous sulfate) dosimetry.
mice.
The sera from both the anti-U and control mice first inactivated at 56°C for 30 min and then absorbed for 1 h at room temperature with normal mouse erythrocytes (10% of cell volume) and with Bcells (10% of cell volume). The resulting antisera were active on T-cells, but not on B-cells (Table 1). Preparation of anti-B-cell sera. Spleens from 5to 6-week-old neonatally thymectomized mice were excised, teased, filtered through glass wool, and then washed twice in Hanks solution (2, 5). Each of three rabbits was inoculated i.v. with 2 x 10i to 3 x 108 viable spleen cells initially and then reinoculated i.v. on days 8, 16, and 22 with 5 x 10i to 6.5 x 10i viable spleen cells. Seven days later, the animals were exsanguinated. Both experimental and normal rabbit sera were absorbed at room temperature 8 to 10 times with normal mouse liver cells, mouse erythrocytes, and were
mouse
so
RESULTS Influence of neonatal thymectomy on the in vivo release of MWF. Experiments were initiated to determine whether the presence of Tcells at the time of sensitization was essential for the release of MIF into the circulating blood. Mice were thymectomized within 18 h of birth, sensitized 4 to 5 weeks later with 300 jig of BCG cell walls in oil droplets, challenged 3 weeks later with 50 mg of OT, and exsanguinated 3 h thereafter. Nonnal mice were similarly sensitized by i.v. inoculation with 300 ,ug of BCG cell walls in oil droplets, challenged i.v. with 50 mg of OT, and then exsanguinated. The sera from nonthymectomized, adult, sensitized mice (controls) caused at least 20% inhibition of the migration of normal macrophages in dilutions of 1:64, 1:64, and 1:128, in three separate experiments. Each pool consisted of seven to ten mice. In contrast, in two of three separate experiments where pooled sera were assayed, the sera of neonatally thymectomized mice did not have detectable amounts of MIF. In the third experiment, MIF was detected in the low titer of 1:4. Each pool of the experimental mice consisted of the bleedings from seven to nine mice. Thus, removal of the thymus at birth eliminated the
TABLE 1. Influence of anti- #and anti-B-cell antiserum on lymphoid cells in vitro and in vivo In vitro mortalitya
Type of serum
Anti-0
In vivo mortality"
Control + Mortality (%) at given dilution of antiserum Source of target cells comple- Direct ro- Cell repliettes 1:5 1:10 cationd 1:20 (%)1 1:40 senun) 0 Thymus 10ie 96 98 99 97 0.08 3,562
Bonemarrow 8 4 6 3 Thymus 6 3 2.2 7,843 Bone marrow 8 1 Anti-B-cell 1 Thymus 3 3 4.1 14,812 Bone marrow 36 35 30 25 14 9 Normal rabbit Thymus 14 9 4.1 Bone marrow 9 8 a As measured by dye (trypan blue) exclusion after addition of antiserum and complement. I Splenic cells used. Adherent cells removed. Rosettes with chicken erythrocytes (7 x 10' spleen cells + 0.2% erythrocytes). 103 cells counted in each category (3). d Response of 10' spleen cells to concanavalin A (4). eAt least 100 cells counted to obtain each percentage.
Normal mouse
c
VOL. 17, 1977
CELL SOURCE OF MIF
ability of the mice to release MIF into the circulation on subsequent in vivo stimulation with BCG cell walls and challenge with OT. Release of MW into the circulation of athymic nude mice. The need for the presence of T-cells at the time of sensitization for MIF to be produced was confirmed in studies on athymic nude mice. Twenty such mice were sensitized i.v. with BCG in oil droplets, challenged with OT, and exsanguinated. Where the sera of control, sensitized mice produced MIF in titers of 1:64 to 1:128, the sera from the athymic nude mice did not have detectable quantities of MIF, i.e., titers of
INFEcTiON AND ImmuNrTy, Sept 1977, p. 639-63
Copyright© 1977 American Society for Microbiology
In Vivo Studies on the Cellular Source of Migration Inhibitory Factor in Mice with Delayed Hypersensitivity S. B. SALVIN,* G. SONNENFELD,t AND J. NISHIO Department ofMicrobiology, School ofMedicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
Received for publication 10 March 1977
Mice sensitized intravenously with 300 Lg of BCG cell walls in Drakeol-Tween 80 and challenged intravenously 3 weeks later with 50 mg of old tuberculin released migration inhibitory factor (MIF) into the circulating blood in quantities that could be detected in serum dilutions of 1:64 to 1:128. When thymus-derived lymphocytes (T-cells) were absent at the time of sensitization, as in neonatally thymectomized mice or in athymic nude mice, detectable amounts of MIF were not formed. Sensitized mice treated with either anti-@ serum or anti-bone marrow-derived lymphocyte (B-cell) serum before intravenous challenge with old tuberculin released reduced amounts of MIF into the circulation. Mice lethally irradiated, reconstituted with B-cells, sensitized with BCG cell walls, and then challenged intravenously 3 weeks later with old tuberculin did not release MIF into the circulation. When T-cells were injected at least 10 days before challenge, however, MIF appeared.
Lymphokines, such as migration inhibitory factor (MIF) and type II interferon, are released into the circulation of mice that have been sensitized intravenously (i.v.) with Mycobacterium bovis strain BCG and then challenged i.v. with old tuberculin (OT) (11, 14). The mice were appropriately sensitized with either living mycobacteria, dead mycobacteria, or cell wall fractions in oil droplets, and were challenged about 3 weeks later with either OT or living mycobacteria. Within 2 to 3 h after this i.v. challenge, MIF can be detected in the circulating blood, with serum dilutions of about 1:128 inhibiting the migration of normal macrophages by at least 20%. The MIF released into the circulation has been partly purified (11) and has been found to be acid unstable, heat stable, and highly specific (14). Of importance in the production of MIF may be the type or types of cells exposed to the antigen and the mode of interaction of these cells. Accordingly, experiments were initiated to determine (i) the source and types of cells involved in production of MIF and (ii) the experimental environment in vivo for maxum release of MIF. In this paper, experiments are described indicating that (i) thymus-derived lymphocytes (T-cells) must be present for 10 days before the time of challenge for MIF to be formed, and (ii) cells of both thymic and bone marrow (B-cells) origin are involved in the release of MIF in vivo into the circulation. t Present address: Division of Infectious Diseases, Stanford University Medical School, Stanford, CA 94305.
639
MATERIAIS AND METHODS Animals. Female Swiss Webster mice weighing 25 to 30 g were purchased from Taconic Farms, Germantown, N.Y. Offspring were thymectomized within 12 h of birth. The athymic nude mice backcrossed to BALB/c mice were purchased from G. L. Bomholtgard, Ry, Denmark. The C3H/He and AKR mice were purchased from Jackson Laboratories, Bar Harbor, Me; the C3H/He Dub mice were purchased from Flow Laboratories, Dublin, Va. Sensitization. The sensitizing antigen consisted of the cell walls of M. bovis strain BCG, obtained through the courtesy of E. Ribi, Rocky Mountain Laboratory, National Institute of Allergy and Infectious Diseases, Hamilton, Mont. In the preparation of the inoculum, 24.75 mg of the cell walls and 0.12 ml of Drakeol 6/VR (Pennsylvania Refining Co., Butler, Pa.) were ground into a paste. A 0.85% sodium chloride solution (16.7 ml) with 0.2% Tween 80 was added, and the grinding was resumed until a stable emulsion was obtained (10). Footpad tests. Mice were inoculated in one hind footpad with 30 jg of purified protein derivative of OT (PPD) in 0.05 ml of physiological saline (Parke, Davis & Co., Detroit, Mich.) and in the other bind footpad with 0.05 ml of physiological saline. Twenty-four hours later, with the aid of a calipers-micrometer (SchnelltAster, Systems-Kroplin, Schtchtern, West Germany), the thickness of the footpads inoculated with PPD was compared with the thickness of footpads inoculated with saline. A delayed reaction was indicated when an increase of greater than 10% in thickness was observed. Migration inhibition. Mice were inoculated intraperitoneally (i.p.) with 3 ml of light mineral oil 5 to 10 days before cell harvest. The peritoneal cavity was rinsed out with Hanks solution, and the peritoneal
640
SALVIN, SONNENFELD, AND NISHIO
INFECT. IMMUN.
exudate cells therefrom were washed three times by centrifugation in Hanks solution. The cells were concentrated, and a tiny drop was planted on the surface of 3 ml of agar substrate in a Falcon tissue culture flask. The substrate contained medium 199, 0.5% Ionagar, and 20% (by volume) mouse serum, plus penicillin (100 U/ml) and streptomycin (100 ,ug/ml). The flasks were incubated at 370C for 48 h, at which time the radius of migration was measured under lowpower magnification with the aid of an ocular micrometer (9). Preparation of anti-0 serum. Thymus glands were excised from 3-week-old C3H/He mice, teased gently, passed through glass wool filters, and then washed twice in Hanks balanced salt solution (6). Viable cells (107), with viability determined by cell exclusion of trypan blue, were inoculated either i.p. in Hanks solution or subcutaneously in incomplete Freund adjuvant. Then the mice were inoculated i.p. once a week for 8 to 11 weeks with 1 x 107 to 2 x 107 washed, viable lymphocytes. Six days after the last injection, the mice were bled. A pool of control serum was simultaneously obtained from normal adult AKR
T-cells that the resulting sera were active on B- and spleen cells but not on mouse T-cells (Table 1). All sera were frozen and stored at -70°C. X-irradiation. Two different sources of irradiation were used: (i) a "37cesium unit, 0.662 meV, producing 57 rads/min at a distance of 35 mm from the source; and (ii) a Picker X-ray unit, 200 kV, 15 mA, 20 cm (428 rads/min) or 45 cm (100 rads/min) from the target. The dose rate was calculated with a Victoreen R meter and Frichi (ferrous sulfate) dosimetry.
mice.
The sera from both the anti-U and control mice first inactivated at 56°C for 30 min and then absorbed for 1 h at room temperature with normal mouse erythrocytes (10% of cell volume) and with Bcells (10% of cell volume). The resulting antisera were active on T-cells, but not on B-cells (Table 1). Preparation of anti-B-cell sera. Spleens from 5to 6-week-old neonatally thymectomized mice were excised, teased, filtered through glass wool, and then washed twice in Hanks solution (2, 5). Each of three rabbits was inoculated i.v. with 2 x 10i to 3 x 108 viable spleen cells initially and then reinoculated i.v. on days 8, 16, and 22 with 5 x 10i to 6.5 x 10i viable spleen cells. Seven days later, the animals were exsanguinated. Both experimental and normal rabbit sera were absorbed at room temperature 8 to 10 times with normal mouse liver cells, mouse erythrocytes, and were
mouse
so
RESULTS Influence of neonatal thymectomy on the in vivo release of MWF. Experiments were initiated to determine whether the presence of Tcells at the time of sensitization was essential for the release of MIF into the circulating blood. Mice were thymectomized within 18 h of birth, sensitized 4 to 5 weeks later with 300 jig of BCG cell walls in oil droplets, challenged 3 weeks later with 50 mg of OT, and exsanguinated 3 h thereafter. Nonnal mice were similarly sensitized by i.v. inoculation with 300 ,ug of BCG cell walls in oil droplets, challenged i.v. with 50 mg of OT, and then exsanguinated. The sera from nonthymectomized, adult, sensitized mice (controls) caused at least 20% inhibition of the migration of normal macrophages in dilutions of 1:64, 1:64, and 1:128, in three separate experiments. Each pool consisted of seven to ten mice. In contrast, in two of three separate experiments where pooled sera were assayed, the sera of neonatally thymectomized mice did not have detectable amounts of MIF. In the third experiment, MIF was detected in the low titer of 1:4. Each pool of the experimental mice consisted of the bleedings from seven to nine mice. Thus, removal of the thymus at birth eliminated the
TABLE 1. Influence of anti- #and anti-B-cell antiserum on lymphoid cells in vitro and in vivo In vitro mortalitya
Type of serum
Anti-0
In vivo mortality"
Control + Mortality (%) at given dilution of antiserum Source of target cells comple- Direct ro- Cell repliettes 1:5 1:10 cationd 1:20 (%)1 1:40 senun) 0 Thymus 10ie 96 98 99 97 0.08 3,562
Bonemarrow 8 4 6 3 Thymus 6 3 2.2 7,843 Bone marrow 8 1 Anti-B-cell 1 Thymus 3 3 4.1 14,812 Bone marrow 36 35 30 25 14 9 Normal rabbit Thymus 14 9 4.1 Bone marrow 9 8 a As measured by dye (trypan blue) exclusion after addition of antiserum and complement. I Splenic cells used. Adherent cells removed. Rosettes with chicken erythrocytes (7 x 10' spleen cells + 0.2% erythrocytes). 103 cells counted in each category (3). d Response of 10' spleen cells to concanavalin A (4). eAt least 100 cells counted to obtain each percentage.
Normal mouse
c
VOL. 17, 1977
CELL SOURCE OF MIF
ability of the mice to release MIF into the circulation on subsequent in vivo stimulation with BCG cell walls and challenge with OT. Release of MW into the circulation of athymic nude mice. The need for the presence of T-cells at the time of sensitization for MIF to be produced was confirmed in studies on athymic nude mice. Twenty such mice were sensitized i.v. with BCG in oil droplets, challenged with OT, and exsanguinated. Where the sera of control, sensitized mice produced MIF in titers of 1:64 to 1:128, the sera from the athymic nude mice did not have detectable quantities of MIF, i.e., titers of
