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In vivo release of lymphokines in different strains of mice
In Vivo Release
of Lymphokines
in Different
Strains
of Mice’
Attempts were made to induce the two lymphokines, migration inhibitory factor and interferon II. in rii’o with antigens from the tubercle bacillus. Of 11 strains of mice assayed, marked variations occurred in the capacity to release these lymphokines. This capacity to respond, however, was altered by the mode of presentation of the antigen. The migration inhibitory absent
factor and interferon in the nonresponding
II appeared strains.
together
in the responding
strains.
and
both
were
INTRODUCTION Lymphokines are soluble mediators associated with delayed hypersensitivity and/or cell-mediated immunity. They have been demonstrated primarily in l.itro by incubation of sensitized lymphoid cells with specific antigen or of nonsensitized lymphoid cells with mitogens, such as concanavalin A or phytohemagglutinin. Lymphokines have been released in rviro into the circulation of the intact mouse after intravenous sensitization with either 1 mg whole dead tubercle bacilli in Drakeol-Tween 80 emulsion or 300 pg cell walls of Mycdmcterium hovis strain BCG, also in Drakeol-Tween 80 emulsion. Three weeks later, the mice were challenged intravenously with 50 mg old tuberculin (OTY and 2 to 4 hr later exsanguinated (1). The two lymphokines studied most extensively were migration inhibitory factor (MIF) and interferon (IF). Interferon released by tuberculous animals challenged with specific antigen (OT) was different from the interferon induced either by viral challenge or by bacterial lipopolysaccharide (LPS), i.e., interferon II vs interferon I, respectively (2). In patients with immunologic diseases or with certain persistent disseminated infections, deficiencies in the release of one or more lymphokines, such as migration inhibory factor (MIF) or mitogenic factor (MF), may develop (3). Since different strains of mice vary in their capacity to develop cellular immunity (4). investigations were initiated to determine whether such variations in cellular immunity could be correlated with variations in production of and release of lymphokines ipr t,i\*o. In ’ This Diseases
work was supported (Grant AI-16064)
by the Public Health Service. (S.B.S.) and by the American
National Institute Lung Association
recipient of a National Institute of Health Research Fellowship ’ Abbreviations used: in BCG. :~~(,ohtrc.terilr)?l hoi,i.s strain CWIDr, cell walls of BCC in Drakeol-Tween 80 emulsion; interferon: LPS, bacterial lipopolysaccharide: MIF, migration
Award. BCG: CFA. IF 1. Type inhibitory
of .4llergy (R.N.). Dr.
and Infectious Ruth Neta is a
IF 32CA 06519. complete Freund’s adjuvant: I interferon: IF II. Type factor: OT. old tuberculin,
173 000%8749/8O/OSOl73-06%02.00/O Copyright in 1980 by Academic Pre\$. All rights of reproduction in any form
Inc. rex~wed.
II
174
SHORT
COMMUNICATIONS
this paper, the in viva release of MIF and type II interferon (IF II) will be shown to vary between strains of inbred mice. MATERIALS
AND METHODS
Mice. Outbred Swiss Webster mice were purchased from Taconic Farms, Germantown, New York. Outbred CFW mice were obtained from the Lobund Institute, Notre Dame, Indiana. The inbred strains, namely, C,,Bl/Ks, BALB/cBy, DBA/2, C,,Bl/6, CBA/Ca, AKR, DBA/l, A/J, and C,H/He strains, were purchased from Jackson Laboratories, Bar Harbor, Maine. NZB/W were obtained through the courtesy of Dr. V. Kelly, Montefiore Hospital, Pittsburgh, Pennsylvania. Tubercfe bacilli. Whole dead tubercle bacilli consisted of acetone-dried M. tuberculosis Jamaica Strain. Cell walls of M. bovis strain BCG were obtained through the courtesy of Dr. Edgar Ribi, Rocky Mountain Laboratory, Hamilton, Montana. Old tuberculin (OT) was purchased from Jensen-Salsbury, Kansas City, Missouri. Incomplete Freund’s adjuvant was purchased from Difco Laboratories, Detroit, Michigan. Migration inhibition of peritoneal-exudate cells. Five to ten days before harvest of peritoneal macrophages, donor mice were inoculated intraperitoneally with 3 ml of light mineral oil. The peritoneal cavity was subsequently washed out with Hank’s solution, and the resulting cells washed three times by centrifugation in Hanks’ solution for 10 min at 1000 rpm. The cells were then concentrated by centrifugation, drawn up in a Pasteur pipet, and a tiny drop placed on the surface of an agar substrate (5). The substrate consisted of medium 199,0.5% ionagar, 20% by volume of the mouse serum or plasma in 3-ml volumes dispensed in 30-ml Falcon tissue culture flasks. After incubation at 37°C for 48 hr, the cells were examined under low power microscope for their distance of migration in comparison with cells in flasks containing control mouse serum. An average inhibition of 20% was considered evidence for the presence of MIF, with dilutions of the serum producing this degree of inhibition presented as the titer. Interferon ussay. Interferon was determined by an assay using encephalomyocarditis virus (EMC) hemagglutination reduction yield (6). Briefly, L-929 cells were grown overnight in minimum essential medium (MEM) with 10% FCS in glass tissue culture tubes to form monolayers. The medium was removed and the cells were washed twice with phosphate-buffered saline. The experimental and control sera to be assayed for interferon were diluted IO-fold to obtain 10-l to 10e6 dilutions of each serum in MEM containing 2% fetal calf serum. Tubes were inoculated in triplicate with 1 ml of each of the serum dilutions. After an additional 24-hr incubation at 37”C, the medium was removed, cell monolayers were washed with phosphate-buffered saline, and 0.25 ml EMC virus (hemagglutination titer, 1: 16,384) in a buffer containing 0.5% gelatin and 0.2% lactoalbumin hydrolysate in Hanks’ solution was added to the cell monolayers. After 30 min of incubation, cells were washed twice with the phosphate buffer, and fresh MEM with 2% fetal calf serum (FCS) was added. The tubes with infected L-cell monolayers were returned to the incubator to allow for the growth of the virus for an additional 18 hr. The virus was harvested by repeated freezing and thawing. The titer of the virus was measured in a hemagglutination assay, employing human “0” red blood cells. The titer of interferon is expressed as a reciprocal of the dilution of the serum that produced a reduction of 0.5 log hemagglutination yield of EMC virus.
SHORT
COMMUNICATIONS
17s
RESULTS Release of MIF in Different
Strains
of Mice
Sensitization of Taconic Farms Swiss outbred mice (SW) with 300 pg BCG cell walls, challenge with 50 mg OT 3 weeks later, and exsanguination 2 to 4 hr later consistently yielded MIF in the resulting sera in titers from I:64 to 1:512. These sera usually consisted of pooled samples from 5 to 20 mice. Such high titers of MIF were accompanied by equally high titers of IF-II (I). Control mice, i.e., normal mice challenged with OT or sensitized mice not challenged, invariably failed to release detectable quantities of either of the two lymphokines into the circulation. The questions arose as to whether the capacity to release such lymphokines as MIF and IF II into the circulation in vi\,o varied from mouse strain to mouse strain and therefore what mechanisms regulated this release. Accordingly, different strains of inbred mice were sensitized intravenously, as above, with 300 Fg BCG cell walls in Drakeol-Tween 80 emulsion, challenged intravenously 3 weeks later with 50 mg OT, and 2-4 hr later exsanguinated. Appropriate controls were included (Table I). Twelve different strains were assayed for their capacity to release MIF in L’IVO. Although the experimental conditions were identical, the amounts of MIF released into the circulation at 4 hr postchallenge varied in the strains from a titer of 1:.512 to undetectable amounts. The outbred strain, CFW, obtained from the Lobund Institute. consistently did not yield detectable quantities of MIF in \sirw after appropriate sensitization and challenge. The three high responders in the inbred strains. namely. BALBlcByJ, C,,Bl/KsJ, and DBAI2, all belonged to the H-2” histocompatibility type. In contrast, the three H-2” strains, namely, AKR, CBAiCaJ, and C,H/He, were all non- or low responders. The DBAil and NZBiW mice were nonresponders, while the CiBl/6J was a moderate responder. The mice were also exsanguinated at 2, 6, and 24 hr after challenge in order to determine whether the maximum release of MIF and IF II into the circulation varied according to the strain of mouse and not according to the time of bleeding. The highest titers occurred at 4 hr postchallenge in the different mouse strains studied.
in separate experiments involving intravenous sensitization with 300 pg in Drakeol-Tween 80 emulsion and intravenous challenge with 50 mg OT 3 weeks later. CFW mice did not release detectable quantities of interferon in four consecutive experiments. In the ffth experiment (Table l), a low titer of interferon was detected, where control Swiss Webster mice had titers of 23,000. To establish whether different strains of mice vary in their ability to release interferon in ~‘i~lo, mice of various strains were similarly sensitized and challenged, and the sera assayed for interferon (Table 1). Most striking was the observation that interferon titers in the different strains varied from greater than 1 x 10” in C,,Bl/KsJ to less than 10 in DBA/ 1. Also striking was the parallelism in the amounts of MIF and interferon released into the blood of different strains of mice.
176
SHORT
COMMUNICATIONS TABLE
Titers Strain
of MIF
and IF II Released
1
into the Circulation
H-2 Type
of 11 Mouse
Strains
MIF
IF
C,,Bl/Ks
H-2”
512
BalblCBy
H-2”
512
> 10,000”
DBAIZ
H-2d
512
> 10,OOOh
C,,BlI6
H-2”
128
360,000
C3H/He
H-2”
o-4
AKR
H-2”
0
620 470
CBA/Ca
H-2k
0
500
NZBIW
H-zd’L
0
DBAil
H-2q
0
A/J
H-2”
SW
Outbred
512
CFW
Outbred
0
>256
320,000” 1,200,000
< 1,000
N.D.’
o Titers of pools of sera from separate experiments. included in each assay and this sample varied within b Maximum titer was not determined. c Not done.
A standard reference a twofold range.
interferon
sample
was
Subcutaneous treatment with CFA of Swiss Webster responder mice, before or after intravenous sensitization with BCG cell walls in Drakeol-Tween 80 emulsion, markedly affected the amount of interferon II released into the circulation (7). When responder A/J mice were treated subcutaneously with CFA 2 weeks before sensitization, the amount of interferon released into the circulation was increased. The question was then asked whether similar treatment of nonresponder mice would affect the capacity of the sensitized mice to release interferon on challenge. Nonresponder C,H/HeJ mice were treated subcutaneously with 1 mg dead tubercle bacilli in 0.5 ml CFA or with 300 pg BCG cell walls in Drakeol-Tween 80 emulsion 2 weeks before intravenous sensitization. Three weeks after the intravenous sensitization and 5 weeks after the subcutaneous treatment, these experimental mice, in addition to control mice that had been only intravenously sensitized, or sensitized with CFA, were challenged with 50 mg OT and exsanguinated 3 to 4 hr later. Examination of the sera for interferon and MIF (Table 2) revealed that subcutaneous treatment with CFA or with cells in Drakeol-Tween 80 emulsion
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before intravenous sensitization resulted in such mice responding to OT challenge by release of increased amounts of interferon and MIF into the circulation, similar to those released into the blood of A/J mice simultaneously intravenously sensitized and challenged. DISCUSSION Under the experimental conditions described in this paper, variations occur in the release of MIF and IF II in lvi\-o into the sera of different strains of sensitized mice. Thus, some strains, such as C,7Bl/KsJ, released both MIF and IF II in high titers (1:512 and 1: lo”-- IO”, respectively), whereas other strains, such as DBAIl, did not release either lymphokine into the circulation in detectable quantities. Variations in cell-mediated resistance to infection in different strains of mice have been described. In a study involving 22 strains of mice challenged with low doses of I14yc~ohucterium trrhrrc~rrlosi,s strain H,,,R, , differences in susceptibility to infection were noted (8). Similarly, four lines of outbred mice showed marked differences in their capacity to develop resistance against lethal tuberculous infection with H:>;R, after immunization with H:,,R;, or BCG (4). More recently. resistance to infection with Listrrin l~lo,loc’!‘togcrlcs has been shown to vary in different strains of mice. C,,Bl/6 mice were more resistant to intravenous inoculation than BALBic mice, where the resistance was measured by survival (LD,,,). microbial counts in liver and spleen, and adoptive transfer of immunity (9). Delayed-type hypersensitivity to BCG was markedly different in two inbred strains of mice, SWMiMs and C:,H/He, as measured by footpad reactions. spleen index, and macrophage disappearance reactions (10). Although variations in cell-mediated immunity and in delayed-type hypersensitivity to specific organisms have been indicated in different strains of mice, little has been reported on the occurrence of such differences in the production of lymphokines. A possible basis for such differences not being noted may be the fact that studies with lymphokines frequently involved induction in \‘itro by nonspecific mitogens. such as Con A, instead of specific antigen. One recent report, however, does demonstrate that splenic lymphocytes cultured in the presence of con A in a serum-free medium differ in their capacity to produce MIF, depending on the particular strain of mouse (11). TABLE Release
Strain
of mice
C,,H,HeJ C,H/HeJ C,,H/HeJ A/J” ” and ’ did
of Serum
Interferon
after
2
Pretreatment
of Nonresponder
Subcutaneous treatment
IF titer
CFA CWIDR None None
870.000 12.000 -: IO 950.000
Mice”
All mice were intravenously sensitized with 300 pg BCG cell walls in Drakeol-Tween 3 weeks later challenged intravenously with 50 mg old tuberculin. SW mice, sensitized subcutaneously with 1 mg CFA and challenged intravenously not release detectable quantities of MIF into the circulation.
MIF
titer 256 16 0 ,256 80 emulsion
with SO mg OT,
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SHORT
COMMUNICATIONS
Difficulty has been encountered in attempts to separate MIF from IF II by physicochemical methods (1, 2). This similarity between the two lymphokines is further exemplified by the observations that specific stimulation of BCG-sensitized mice of responder strains with specific antigen (OT) results typically in the in viva release of both MIF and IF II, whereas nonresponder strains release neither lymphokine under similar experimental conditions. In the studies reported in this paper, the differences in IF II titers between responder and nonresponder strains were most extensive (up to 105-fold). Differences were also noted in the amount of interferon I produced by different strains of mice in response to viral challenge (12). Several viruses were used as inducing agents in this study, and variations in the amounts of interferon I induced by each virus were present. These differences, however, were not as striking as those observed in the present study in the release of interferon II (12). The capacity of the strains to respond also varied when a particular virus was used as the inducing agent. The production of interferon I could be linked to the minor histocompatibility locus H-28, the linkage group of which is unknown (12). The relationship of the capacity to produce interferon II and other lymphokines to a particular histocompatibility locus in the mouse is in doubt. For example, the protective effect of BCG against infection with Schistosoma mansoni was shown not to be linked to the H-2 locus (13). In contrast, delayed-type hypersensitivity to synthetic polypeptide antigens was found to be linked to the H-2 locus (14). Thus, antigen structure and mode of antigen presentation may define the involvement of a gene or group of genes in the resulting immune response. The importance of the nature of the antigen and its mode of presentation to the animal in lymphokine production are further emphasized by the observation that mice nonresponsive after intravenous sensitization with BCG and intravenous challenge with OT did elicit a response when the animal was pretreated with CFA. ACKNOWLEDGMENT The author (R.N.) expresses cells for the assay of interferon.
her thanks
to Dr. Gary
Burleson
for supplying
the EMC
virus
and the L
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
Salvin, S. B., Youngner, J. S., and Lederer, W. H. Infect. Immun. 7, 68, 1973. Youngner, J. S., and Salvin, S. B. .I. Zmmunol. 111, 1914, 1973. Rocklin, R. E., Rosen, F., and David, J. R. N. Engl. J. Med. 282, 1340, 1970. Youmans, G. P., Youmans, A. S., and Kanai, K. Amer. .Z. Rrsp. Dis. 80, 753, 1959. Salvin, S. B., and Nishio, J. J. Zmmund. 103, 138, 1969. Jameson, P., Dixon, M. A., and Grossberg, S. E. Proc. Sue. E.xp. Eiol. Med.. 155, 173, 1977. Neta, R., and Salvin, S. B. Fed. Proc. 38, 1458, 1979. Pierce, C., Dubos, R. J., and Middlebrook, G., .I. Exp. Med. 86, 159, 1947. Cheers, C., and McKenzie, I. F. C. Infect. Zmmun. 19, 755, 1978. Nakamura, R. M., and Tokunaga, T. Infect. Zmmun. 22, 657, 1978. Adelman, N., Cohen, S., and Yoshida, T., J. Zmmunol. 121, 209, 1978. deMaeyer, E., and deMaeyer-Guignard, J. Znterfrron, in press, (1979). Civil, R. H., and Mahmoud, A. A. F. J. Zmmunol. 120, 1070, 1978. Davis, S., Shearer, G. M., Mosez, E., and Sela, M. .Z. Zmmund. 115, 1530, 1975.
of Lymphokines
in Different
Strains
of Mice’
Attempts were made to induce the two lymphokines, migration inhibitory factor and interferon II. in rii’o with antigens from the tubercle bacillus. Of 11 strains of mice assayed, marked variations occurred in the capacity to release these lymphokines. This capacity to respond, however, was altered by the mode of presentation of the antigen. The migration inhibitory absent
factor and interferon in the nonresponding
II appeared strains.
together
in the responding
strains.
and
both
were
INTRODUCTION Lymphokines are soluble mediators associated with delayed hypersensitivity and/or cell-mediated immunity. They have been demonstrated primarily in l.itro by incubation of sensitized lymphoid cells with specific antigen or of nonsensitized lymphoid cells with mitogens, such as concanavalin A or phytohemagglutinin. Lymphokines have been released in rviro into the circulation of the intact mouse after intravenous sensitization with either 1 mg whole dead tubercle bacilli in Drakeol-Tween 80 emulsion or 300 pg cell walls of Mycdmcterium hovis strain BCG, also in Drakeol-Tween 80 emulsion. Three weeks later, the mice were challenged intravenously with 50 mg old tuberculin (OTY and 2 to 4 hr later exsanguinated (1). The two lymphokines studied most extensively were migration inhibitory factor (MIF) and interferon (IF). Interferon released by tuberculous animals challenged with specific antigen (OT) was different from the interferon induced either by viral challenge or by bacterial lipopolysaccharide (LPS), i.e., interferon II vs interferon I, respectively (2). In patients with immunologic diseases or with certain persistent disseminated infections, deficiencies in the release of one or more lymphokines, such as migration inhibory factor (MIF) or mitogenic factor (MF), may develop (3). Since different strains of mice vary in their capacity to develop cellular immunity (4). investigations were initiated to determine whether such variations in cellular immunity could be correlated with variations in production of and release of lymphokines ipr t,i\*o. In ’ This Diseases
work was supported (Grant AI-16064)
by the Public Health Service. (S.B.S.) and by the American
National Institute Lung Association
recipient of a National Institute of Health Research Fellowship ’ Abbreviations used: in BCG. :~~(,ohtrc.terilr)?l hoi,i.s strain CWIDr, cell walls of BCC in Drakeol-Tween 80 emulsion; interferon: LPS, bacterial lipopolysaccharide: MIF, migration
Award. BCG: CFA. IF 1. Type inhibitory
of .4llergy (R.N.). Dr.
and Infectious Ruth Neta is a
IF 32CA 06519. complete Freund’s adjuvant: I interferon: IF II. Type factor: OT. old tuberculin,
173 000%8749/8O/OSOl73-06%02.00/O Copyright in 1980 by Academic Pre\$. All rights of reproduction in any form
Inc. rex~wed.
II
174
SHORT
COMMUNICATIONS
this paper, the in viva release of MIF and type II interferon (IF II) will be shown to vary between strains of inbred mice. MATERIALS
AND METHODS
Mice. Outbred Swiss Webster mice were purchased from Taconic Farms, Germantown, New York. Outbred CFW mice were obtained from the Lobund Institute, Notre Dame, Indiana. The inbred strains, namely, C,,Bl/Ks, BALB/cBy, DBA/2, C,,Bl/6, CBA/Ca, AKR, DBA/l, A/J, and C,H/He strains, were purchased from Jackson Laboratories, Bar Harbor, Maine. NZB/W were obtained through the courtesy of Dr. V. Kelly, Montefiore Hospital, Pittsburgh, Pennsylvania. Tubercfe bacilli. Whole dead tubercle bacilli consisted of acetone-dried M. tuberculosis Jamaica Strain. Cell walls of M. bovis strain BCG were obtained through the courtesy of Dr. Edgar Ribi, Rocky Mountain Laboratory, Hamilton, Montana. Old tuberculin (OT) was purchased from Jensen-Salsbury, Kansas City, Missouri. Incomplete Freund’s adjuvant was purchased from Difco Laboratories, Detroit, Michigan. Migration inhibition of peritoneal-exudate cells. Five to ten days before harvest of peritoneal macrophages, donor mice were inoculated intraperitoneally with 3 ml of light mineral oil. The peritoneal cavity was subsequently washed out with Hank’s solution, and the resulting cells washed three times by centrifugation in Hanks’ solution for 10 min at 1000 rpm. The cells were then concentrated by centrifugation, drawn up in a Pasteur pipet, and a tiny drop placed on the surface of an agar substrate (5). The substrate consisted of medium 199,0.5% ionagar, 20% by volume of the mouse serum or plasma in 3-ml volumes dispensed in 30-ml Falcon tissue culture flasks. After incubation at 37°C for 48 hr, the cells were examined under low power microscope for their distance of migration in comparison with cells in flasks containing control mouse serum. An average inhibition of 20% was considered evidence for the presence of MIF, with dilutions of the serum producing this degree of inhibition presented as the titer. Interferon ussay. Interferon was determined by an assay using encephalomyocarditis virus (EMC) hemagglutination reduction yield (6). Briefly, L-929 cells were grown overnight in minimum essential medium (MEM) with 10% FCS in glass tissue culture tubes to form monolayers. The medium was removed and the cells were washed twice with phosphate-buffered saline. The experimental and control sera to be assayed for interferon were diluted IO-fold to obtain 10-l to 10e6 dilutions of each serum in MEM containing 2% fetal calf serum. Tubes were inoculated in triplicate with 1 ml of each of the serum dilutions. After an additional 24-hr incubation at 37”C, the medium was removed, cell monolayers were washed with phosphate-buffered saline, and 0.25 ml EMC virus (hemagglutination titer, 1: 16,384) in a buffer containing 0.5% gelatin and 0.2% lactoalbumin hydrolysate in Hanks’ solution was added to the cell monolayers. After 30 min of incubation, cells were washed twice with the phosphate buffer, and fresh MEM with 2% fetal calf serum (FCS) was added. The tubes with infected L-cell monolayers were returned to the incubator to allow for the growth of the virus for an additional 18 hr. The virus was harvested by repeated freezing and thawing. The titer of the virus was measured in a hemagglutination assay, employing human “0” red blood cells. The titer of interferon is expressed as a reciprocal of the dilution of the serum that produced a reduction of 0.5 log hemagglutination yield of EMC virus.
SHORT
COMMUNICATIONS
17s
RESULTS Release of MIF in Different
Strains
of Mice
Sensitization of Taconic Farms Swiss outbred mice (SW) with 300 pg BCG cell walls, challenge with 50 mg OT 3 weeks later, and exsanguination 2 to 4 hr later consistently yielded MIF in the resulting sera in titers from I:64 to 1:512. These sera usually consisted of pooled samples from 5 to 20 mice. Such high titers of MIF were accompanied by equally high titers of IF-II (I). Control mice, i.e., normal mice challenged with OT or sensitized mice not challenged, invariably failed to release detectable quantities of either of the two lymphokines into the circulation. The questions arose as to whether the capacity to release such lymphokines as MIF and IF II into the circulation in vi\,o varied from mouse strain to mouse strain and therefore what mechanisms regulated this release. Accordingly, different strains of inbred mice were sensitized intravenously, as above, with 300 Fg BCG cell walls in Drakeol-Tween 80 emulsion, challenged intravenously 3 weeks later with 50 mg OT, and 2-4 hr later exsanguinated. Appropriate controls were included (Table I). Twelve different strains were assayed for their capacity to release MIF in L’IVO. Although the experimental conditions were identical, the amounts of MIF released into the circulation at 4 hr postchallenge varied in the strains from a titer of 1:.512 to undetectable amounts. The outbred strain, CFW, obtained from the Lobund Institute. consistently did not yield detectable quantities of MIF in \sirw after appropriate sensitization and challenge. The three high responders in the inbred strains. namely. BALBlcByJ, C,,Bl/KsJ, and DBAI2, all belonged to the H-2” histocompatibility type. In contrast, the three H-2” strains, namely, AKR, CBAiCaJ, and C,H/He, were all non- or low responders. The DBAil and NZBiW mice were nonresponders, while the CiBl/6J was a moderate responder. The mice were also exsanguinated at 2, 6, and 24 hr after challenge in order to determine whether the maximum release of MIF and IF II into the circulation varied according to the strain of mouse and not according to the time of bleeding. The highest titers occurred at 4 hr postchallenge in the different mouse strains studied.
in separate experiments involving intravenous sensitization with 300 pg in Drakeol-Tween 80 emulsion and intravenous challenge with 50 mg OT 3 weeks later. CFW mice did not release detectable quantities of interferon in four consecutive experiments. In the ffth experiment (Table l), a low titer of interferon was detected, where control Swiss Webster mice had titers of 23,000. To establish whether different strains of mice vary in their ability to release interferon in ~‘i~lo, mice of various strains were similarly sensitized and challenged, and the sera assayed for interferon (Table 1). Most striking was the observation that interferon titers in the different strains varied from greater than 1 x 10” in C,,Bl/KsJ to less than 10 in DBA/ 1. Also striking was the parallelism in the amounts of MIF and interferon released into the blood of different strains of mice.
176
SHORT
COMMUNICATIONS TABLE
Titers Strain
of MIF
and IF II Released
1
into the Circulation
H-2 Type
of 11 Mouse
Strains
MIF
IF
C,,Bl/Ks
H-2”
512
BalblCBy
H-2”
512
> 10,000”
DBAIZ
H-2d
512
> 10,OOOh
C,,BlI6
H-2”
128
360,000
C3H/He
H-2”
o-4
AKR
H-2”
0
620 470
CBA/Ca
H-2k
0
500
NZBIW
H-zd’L
0
DBAil
H-2q
0
A/J
H-2”
SW
Outbred
512
CFW
Outbred
0
>256
320,000” 1,200,000
< 1,000
N.D.’
o Titers of pools of sera from separate experiments. included in each assay and this sample varied within b Maximum titer was not determined. c Not done.
A standard reference a twofold range.
interferon
sample
was
Subcutaneous treatment with CFA of Swiss Webster responder mice, before or after intravenous sensitization with BCG cell walls in Drakeol-Tween 80 emulsion, markedly affected the amount of interferon II released into the circulation (7). When responder A/J mice were treated subcutaneously with CFA 2 weeks before sensitization, the amount of interferon released into the circulation was increased. The question was then asked whether similar treatment of nonresponder mice would affect the capacity of the sensitized mice to release interferon on challenge. Nonresponder C,H/HeJ mice were treated subcutaneously with 1 mg dead tubercle bacilli in 0.5 ml CFA or with 300 pg BCG cell walls in Drakeol-Tween 80 emulsion 2 weeks before intravenous sensitization. Three weeks after the intravenous sensitization and 5 weeks after the subcutaneous treatment, these experimental mice, in addition to control mice that had been only intravenously sensitized, or sensitized with CFA, were challenged with 50 mg OT and exsanguinated 3 to 4 hr later. Examination of the sera for interferon and MIF (Table 2) revealed that subcutaneous treatment with CFA or with cells in Drakeol-Tween 80 emulsion
SHORT
177
COMMUNICATIONS
before intravenous sensitization resulted in such mice responding to OT challenge by release of increased amounts of interferon and MIF into the circulation, similar to those released into the blood of A/J mice simultaneously intravenously sensitized and challenged. DISCUSSION Under the experimental conditions described in this paper, variations occur in the release of MIF and IF II in lvi\-o into the sera of different strains of sensitized mice. Thus, some strains, such as C,7Bl/KsJ, released both MIF and IF II in high titers (1:512 and 1: lo”-- IO”, respectively), whereas other strains, such as DBAIl, did not release either lymphokine into the circulation in detectable quantities. Variations in cell-mediated resistance to infection in different strains of mice have been described. In a study involving 22 strains of mice challenged with low doses of I14yc~ohucterium trrhrrc~rrlosi,s strain H,,,R, , differences in susceptibility to infection were noted (8). Similarly, four lines of outbred mice showed marked differences in their capacity to develop resistance against lethal tuberculous infection with H:>;R, after immunization with H:,,R;, or BCG (4). More recently. resistance to infection with Listrrin l~lo,loc’!‘togcrlcs has been shown to vary in different strains of mice. C,,Bl/6 mice were more resistant to intravenous inoculation than BALBic mice, where the resistance was measured by survival (LD,,,). microbial counts in liver and spleen, and adoptive transfer of immunity (9). Delayed-type hypersensitivity to BCG was markedly different in two inbred strains of mice, SWMiMs and C:,H/He, as measured by footpad reactions. spleen index, and macrophage disappearance reactions (10). Although variations in cell-mediated immunity and in delayed-type hypersensitivity to specific organisms have been indicated in different strains of mice, little has been reported on the occurrence of such differences in the production of lymphokines. A possible basis for such differences not being noted may be the fact that studies with lymphokines frequently involved induction in \‘itro by nonspecific mitogens. such as Con A, instead of specific antigen. One recent report, however, does demonstrate that splenic lymphocytes cultured in the presence of con A in a serum-free medium differ in their capacity to produce MIF, depending on the particular strain of mouse (11). TABLE Release
Strain
of mice
C,,H,HeJ C,H/HeJ C,,H/HeJ A/J” ” and ’ did
of Serum
Interferon
after
2
Pretreatment
of Nonresponder
Subcutaneous treatment
IF titer
CFA CWIDR None None
870.000 12.000 -: IO 950.000
Mice”
All mice were intravenously sensitized with 300 pg BCG cell walls in Drakeol-Tween 3 weeks later challenged intravenously with 50 mg old tuberculin. SW mice, sensitized subcutaneously with 1 mg CFA and challenged intravenously not release detectable quantities of MIF into the circulation.
MIF
titer 256 16 0 ,256 80 emulsion
with SO mg OT,
178
SHORT
COMMUNICATIONS
Difficulty has been encountered in attempts to separate MIF from IF II by physicochemical methods (1, 2). This similarity between the two lymphokines is further exemplified by the observations that specific stimulation of BCG-sensitized mice of responder strains with specific antigen (OT) results typically in the in viva release of both MIF and IF II, whereas nonresponder strains release neither lymphokine under similar experimental conditions. In the studies reported in this paper, the differences in IF II titers between responder and nonresponder strains were most extensive (up to 105-fold). Differences were also noted in the amount of interferon I produced by different strains of mice in response to viral challenge (12). Several viruses were used as inducing agents in this study, and variations in the amounts of interferon I induced by each virus were present. These differences, however, were not as striking as those observed in the present study in the release of interferon II (12). The capacity of the strains to respond also varied when a particular virus was used as the inducing agent. The production of interferon I could be linked to the minor histocompatibility locus H-28, the linkage group of which is unknown (12). The relationship of the capacity to produce interferon II and other lymphokines to a particular histocompatibility locus in the mouse is in doubt. For example, the protective effect of BCG against infection with Schistosoma mansoni was shown not to be linked to the H-2 locus (13). In contrast, delayed-type hypersensitivity to synthetic polypeptide antigens was found to be linked to the H-2 locus (14). Thus, antigen structure and mode of antigen presentation may define the involvement of a gene or group of genes in the resulting immune response. The importance of the nature of the antigen and its mode of presentation to the animal in lymphokine production are further emphasized by the observation that mice nonresponsive after intravenous sensitization with BCG and intravenous challenge with OT did elicit a response when the animal was pretreated with CFA. ACKNOWLEDGMENT The author (R.N.) expresses cells for the assay of interferon.
her thanks
to Dr. Gary
Burleson
for supplying
the EMC
virus
and the L
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
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