J. Exp. Med.
Volume 186, Number 1, July 7, 1997 39–45
The R ockefeller University Press • 0022-1007/97/07/39/07 $2.00
Interleukin 12 (IL-12) Is Crucial to the Development of
Protective Immunity in Mice Intravenously Infected
By Andrea M. Cooper,
and Ian M. Orme
Nutley, New Jersey 07110
Departm ent of Microbiology, Colorado State University, Fort Collins , Colorado 80523;
Departm ent of Inflam m ation and Autoim m une Diseases , Hoffm an-La Roche Incorporated,
CD4 T cells that secrete cytokines, resulting in activation of macrophages and the recruitment
of monocytes to initiate granuloma formation. The cytokine-mediating macrophage activation
), which is largely dependent on interleukin-12 (IL-12) for its induction.
To address the role of IL-12 in immunity to tuberculosis, IL-12 p40
M. tuberculosis and their capacity to control bacterial growth and other characteristics of
their immune response were determined. The IL-12 p40
terial growth and this appeared to be linked to the absence of both innate and acquired sources
. T cell activation as measured by delayed type hypersensitivity and lymphocyte accu-
mulation at the site of infection were both markedly reduced in the IL-12 p40
Therefore, IL-12 is essential to the generation of a protective immune response to
, with its main functions being the induction of the expression of IFN-
antigen-specific lymphocytes capable of creating a protective granuloma.
infection is associated with the emergence of protective
mice were infected
mice were unable to control bac-
and the activation of
ulation of protective CD4 T cells that secrete cytokines, re-
sulting in local activation of macrophages and the recruit-
ment of monocytes to initiate granuloma formation (1, 2).
The kinetics of this protective immunity, which leads to the
control and containment of the infection, and the onset of
bacterial clearance, is closely associated with the kinetics of
emergence and loss of CD4 T cells that secrete large amounts
of the cytokine IFN-
(2–4). This cytokine has been shown
to be pivotal in protective immunity, as illustrated by the
severe, disseminated form of tuberculosis seen in IFN-
gene-disrupted mice (5, 6).
In vivo and in vitro infection with
in the secretion of numerous cytokines (2). As has been
demonstrated previously (7), this response includes the se-
cretion of IL-12, a cytokine that appears to have powerful
immunopotentiating effects. Among these are the ability to
initiate the development of Th1 phenotype in naive T cells
(8, 9) and the ability to potentiate IFN-
antigen-activated Th1 cells (10). Hence, the role of this cy-
tokine in the protective immune response to
which is dependent upon the Th1-type pathway, is of in-
Previous studies have relied upon the treatment of in-
fected mice with exogenous IL-12, which led to increased
cquired cellular immunity to
infection is characterized by the emergence of a pop-
survival (11) and lowered bacterial numbers (11, 12). The
effect was not dramatic, suggesting that endogenous IL-12
was already present at sufficient levels. Depletion of IL-12
by antibodies did diminish the level of protection (12), but
was not sufficient to halt immunity. This may have been
due to incomplete neutralization of IL-12 activity by the
antibody. Therefore, we assessed the level of protection in
mice lacking endogenous IL-12. These mice having re-
cently been generated using homologous recombination to
disrupt the IL-12 p40 gene (13).
Using this mouse in a systemic model of infection, we
demonstrate that IL-12 is crucial for the development of
protective immunity as measured by IFN-
development of activated T cells, and the control of bacte-
rial growth. Of particular interest was the observation that
mice were unable to generate a delayed type
type of reaction, or for that matter
recruit organized mantles of lymphocytes into their gran-
ulomas, indicating that the presence of IL-12 is crucial to
cellular accumulation in these processes.
embryonic stem; HPR T, hypoxanthine phosphoribosyl transferase; PPD,
purified protein derivative.
Abbreviations used in this paper:
DTH, delayed type hypersensitivity; ES,
IL-12 in Immunity to Tuberculosis
Materials and Methods
Mice.C57BL/6J control mice were purchased from the Jack-
son Laboratories (Bar Harbor, Maine). Male and female gene-dis-
rupted mice were generated using homologous recombination in
embryonic stem (ES) cells as previously described (13). In brief, chi-
meric animals derived from the targeted 129/Sv ES cells that were
heterozygous for the IL-12 p40 mutation were mated to C57BL/6
mice. Progeny from this cross that were determined to be het-
erozygous for the IL-12 p40 (IL-12 p40
backcrossed to the C57BL/6 strain for a total of five backcrosses.
The backcrossed IL-12 p40
mice were then intercrossed and
screened to obtain mice homozygous for the IL-12 p40 mutation
). Homozyogotes were crossed to each other in order
to expand the colony and their progeny used in these experiments.
Experim ental Infections. A virulent laboratory strain of
(Erdman) was grown from a low passage seed lot in
Proskauer-Beck liquid media to midlog phase, aliquoted, and fro-
C. Mice were infected with 10
eral tail vein as described previously (1). The numbers of viable
bacteria in target organs was followed against time by plating se-
rial dilutions of whole organ homogenates on nutrient Middle-
brook 7H11 agar and counting bacterial colony formation after
20-d incubation at 37
C. The data are expressed as the log
of the mean number of bacteria recovered per organ (
Isolation of m RNA and Detection of Cytokine-specific Message by
Reverse Transcriptase Polym erase Chain Reaction.
sue was excised, placed in Ultraspec (Cinna/Biotecx, Friendswood,
TX ), homogenized, and R NA was extracted as described previ-
ously (12, 14). 1
g of total R NA was reverse transcribed, diluted,
and underwent PCR expansion of cytokine-specific cDNA. The
amount of cytokine-specific product was determined by the hy-
bridization of fluorescein-labeled cytokine-specific probe. The fluo-
rescein was detected by the enhanced chemiluminescence kit (ECL;
Amersham Corp., Arlington Heights, IL) and the resultant light
signal was detected on Hyper-film (Amersham). For the IL-12R
2 chain analysis, the R NA underwent a similar R T-PCR reac-
tion using the primers AGC CCT GAT TTA GCT GAA TCC AG
and GCT CTT CCT CTG GTG TTC GTG TTC. The amount
of specific product was determined by hybridization of the ampli-
con to a
2-specific oligonucleotide probe,
GGC AAG TGG TAC TCA ATC AAC TCA G. The hybrid-
ized filters were exposed and quantitated by integrating the volume
in individual amplicons using a phosphoimager (ImageQuant ver-
sion 3.3; Molecular Dynamics, Sunnyvale, CA). The amplicon
also underwent HPR T-specific hybridization as a control. The
linearity of the PCR reaction was determined empirically (15).
Data is expressed as the mean pixel value of four samples from four
separate mice. The significance of the difference between groups
was determined by an unpaired Student’s
of the signals from control versus infected tissue (
Secretion of IFN-
In Vitro.Spleens were harvested from con-
trol and IL-12 p40
mice both before and during infection. A
single cell suspension was prepared from pooled spleens, cells were
treated for 5 min with a 0.155 M ammonium chloride/0.010 M
potassium bicarbonate solution in order to lyse the red blood
cells, washed, and resuspended in complete DMEM (10% FCS,
buffered with Hepes). Cell suspensions were plated at 5
cells per well in 96 well plates and incubated for 5 d at 37
. The cells were stimulated with either medium alone, culture
filtrate protein antigens of
(strain Erdman). Culture filtrate proteins were received
from Dr. J.T. Belisle under the NIH contract AI25147. The con-
) mutation were further
bacteria via the lat-
Infected liver tis-
test comparing the means
C in 5%
, or whole live
centrations of IFN-
two-site sandwich ELISA using antibodies R 4.6A2 and X MG1.2
as previously described (2). The concentration of other cytokines
were determined using the same procedure but the antibody pairs
were as follows: TNF-
, MP6-XT3, and a polyclonal rabbit anti–
, 18352D; IL-10, JES5-2A5 and SXC-1; IL-4, BVD4-
1D11, and BVD6-24G2; IL-6, MP5-20F3, and MP5-32C11. All
ELISAs used gave detectable signals with concanavilin A–treated
splenocyte cultures. All tissue culture reagents were obtained from
Sigma Chem. Co. (St. Louis, MO) and all ELISA reagents were
obtained from PharMingen (San Diego, CA).
Determ ination of DTH.Infected mice were challenged in one
footpad with 10
g of purified protein derivative (PPD) of
(Connaught Labs, Canada) and in the other with vehicle
control. The swelling in each footpad was measured using vernier
calipers and the difference taken as the amount of antigen-specific
swelling. The PPD preparation did not induce swelling in nonin-
Histological Analysis. The lower right lobe of each mouse was
inflated with 10% formal saline and blocked with the lobes from
the other mice within the experimental group. Blocks were sec-
tioned to allow the maximum area of each lobe to be seen and sec-
tions were stained with hematoxylin and eosin. Slides were ex-
amined blind and analyzed for differences in the size of granuloma
formation and the characteristics of cells within the granulomas.
in cell supernatants was determined by a
IL-12 Is Cruc ial fo r the Co ntro l o f Bac te rial Re plic atio n.
for IL-12 in the expression of protective immunity to
infection has recently been demonstrated (2, 11,
14, 15). However, the advent of the mouse lacking the
gene for the IL-12 p40 subunit has allowed a more com-
plete determination of the role of this molecule. Therefore,
the IL-12 p40
mice were infected with virulent
and the growth of bacteria over time followed. As
can be seen in Fig. 1, the absence of bioactive IL-12 results
in the inability of the mice to control bacterial growth in
the three major organs. In a second experiment, the mice
were also unable to control growth and succumbed to in-
fection between days 40 and 45 of infection (intact mice
control bacteria and survive to old age before dying of re-
crudescent disease) (16).
Induction in Target Organs of Infected Mice Is Reduced
in the Absence of IL-12 p40.
To determine the reason for
the loss of control of bacterial growth the induction of
mR NA for protective molecules in the livers of infected
mice was examined. Fig. 2 demonstrates that, the level of
mR NA was drastically reduced in the IL-12 p40
mice. In addition, the expression of mR NA for a second
protective cytokine, TNF-
, was delayed in the IL-12
mice compared with the C57BL/6 mice. The de-
lay in expression of both of these cytokines probably re-
sulted in the delay of the induction of macrophage activa-
tion as demonstrated in the reduced expression of mR NA
for the gene for inducible nitric oxide synthase (17) (Fig. 2
The ability of these mice to generate another IFN-
–inducing molecule, IL-18 was also of interest. Fig. 2
Cooper et al.
demonstrates the equivalent levels of mR NA for this mole-
cule in the C57BL/6 mice and the IL-12 p40
the absence of IL-12, and therefore IFN-
that a strong Th2 type response could develop. Therefore,
the expression of mR NA for IL-4 and IL-13, cytokines
linked to this type of cellular response (18), was deter-
mined. In contrast with this reasoning, a lower induction of
both IL-4 and IL-13 was observed in the IL-12 p40
, it was possible
Absence of IL-12 Affects Both Innate and Acquired IFN-
Protein Production.The role of IFN-
tective Th1 response is well documented and the require-
ment for this molecule in early T cell activation is estab-
lished. To determine the levels of IFN-
in the initial stages of infection the ability of naive spleno-
cytes to release IFN-
in response to live
analyzed. IL-12 p40
splenocytes had a much reduced
ability to secrete IFN-
compared with the C57BL/6 sple-
in initiating a pro-
tuberculosis infection. Control (solid circles) and IL-12
p40?/? (open circles) mice were infected via the lat-
eral tail vein with 105 M. tuberculosis bacteria and
the number of viable bacteria present in the target
organs was determined over time. The data points
represent the mean and standard error of the bacte-
rial number in the organs of four individual mice.
This is a representative figure from one of two sim-
IL-12 p40?/? mice cannot control M.
cles) and IL-12 p40?/? (open circles) which were infected as in Fig. 1. RNA was extracted from the tissue and subjected to RT-PCR with primers specific for
the molecules noted in the graphics. The HPRT graph demonstrates that equivalent readable R NA was extracted for each sample. a demonstrates the
amount of IFN-?, TNF-?, and inducible nitric oxide synthase-specific mRNA in the livers of infected mice, whereas b demonstrates the amount of IL-18, IL-4,
and IL-13–specific mR NA in the same tissues. The data points represent the mean pixel values resulting from the analysis of tissue from four individual
mice. This data is representative of two separate experiments.
IL-12 p40?/? mice have altered mR NA induction in response to infection with M. tuberculosis. Liver tissue was taken from control (solid cir-
IL-12 in Immunity to Tuberculosis
nocytes (Fig. 3 a). The addition of neutralizing antibodies
to TNF-? and IL-12 demonstrates the role of both these
molecules in driving this response in the C57BL/6 mice
(Fig. 3 a).
The amount of antigen-specific IFN-? was determined
by culturing splenocytes from infected animals with M. tu-
berculosis culture filtrate proteins. Fig. 3 b demonstrates that
the lack of IFN-? mR NA in the liver is reflected by an ab-
sence of IFN-? protein in the spleens of infected animals.
Some IFN-? mR NA was seen in the livers of the IL-12
p40?/? mice at day 30 (see Fig. 2 a) although there is no
antigen-specific protein at this time point (Fig. 3 b). It is
possible that the IFN-? mR NA is a response to the high
levels of IL-18 mR NA seen in both C57BL/6 and IL-12
p40?/? mice throughout infection (19, 20) (Fig. 2 b).
To determine whether the absence of IFN-? might in-
fluence the induction and control of other cytokines, the
levels of several cytokines in the splenocyte cultures was
determined. Table 1 shows that antigen-specific release of
TNF-?, IL-10, and IL-6 was similar for both types of ani-
mal and that neither produced detectable IL-4 on day 30.
The Absence of IL-12 Affects the Developm ent of Antigen-spe-
c ific Ce llular Re c ruitm e nt. Using the footpad rechallenge model
the role of IL-12 in generating a recall response to specific
antigen was examined. Mice lacking IL-12 were unable to
mount a DTH in response to mycobacterial antigen even
after 30 d of infection (Fig. 4).
The ability of T cells to differentiate into IFN-?–producing
cells depends upon their ability to respond to IL-12 and
this in turn depends upon the expression of a receptor for
IL-12. To determine whether the absence of IL-12 could
affect the expression of the IL-12 receptor (IL-12R ) we
measured the amount of mR NA specific for the IL-12R
?2 chain in the infected liver (it was not possible to mea-
sure this molecule on the surface of the CD4 cells as there
is not yet an antibody to it). At the peak of immunity on
day 15 the mean ratios of IL-12R ?2 mR NA compared
with HPR T were 0.23 ? 0.02 for the C57BL6 mice and
0.07 ? 0.02 for the IL-12 p40?/? mice (P ?0.001).
cannot make IFN-? protein in
response to M. tuberculosis infec-
tion. Splenocytes from naive (a)
or infected (b) mice were cul-
tured, in vitro, at a concentration
of 2 ? 105 cells per well in 96-
well plates. In a, the cells from
naive control (solid bars) or IL-12
p40?/? (striped bars) mice were
exposed to live bacilli in the
presence or absence of anti-cyto-
kine antibodies (ND, not deter-
m ined). In b, the cells from in-
fected control (so lid c irc le s) or IL-12
p40?/? (open circles) mice were
exposed to purified culture fil-
trate proteins derived from M.
tuberculosis. The cells were cul-
tured for 4 d and the superna-
tants were then analyzed by
sandwich ELISA for IFN-?.
IL-12 p40?/? mice
response. Control and IL-12 p40?/? mice that were infected as in Fig. 1
were challenged in the left hind footpad with saline and the right hind
footpad with 10 ?g of purified protein derivative of M. tuberculosis on day
15 (solid bars) or day 30 (striped bars) of infection. The data represents the
mean and standard error of the difference in footpad swelling between the
saline and PPD challenged footpad for each of four mice. * P ?0.05 by
the Student’s t test.
IL-12 p40?/? mice fail to generate an antigen-specific recall
T able 1.
from Infected Mice*
Antigen-specific Cytokine Release by Splenocytes
10186 ? 10 59 ? 0 207 ? 8
129 ? 11 35 ? 0 272 ? 1
1455 ? 195
796 ? 470
*Cytokine release was measured by sandwich ELISA of culture super-
nantant from cells incubated in the presence of antigens derived from
‡Values are the mean and standard error (in pg/ml) of triplicate tests on
pooled cells from four animals.
Cooper et al.
The absence of a strong DTH in the IL-12 p40?/? mice
was mirrored in the lower number of lymphocytes in the
lung granulomas of IL-12–deficient animals (Fig. 5). Many
macrophages in the IL-12 p40?/? tissues appeared vacu-
olated due to the presence of large numbers of bacteria,
whereas bacteria are very sparse in the tissues of C57BL/6
We demonstrate here that the absence of the bioactive
IL-12 molecule results in unrestrained growth of M. tuber-
culosis bacteria in all target organs after a systemic infection.
This growth correlates with a much reduced expression of
mR NA for both IFN-? and TNF-? and the subsequent
absence of macrophage activation as evidenced by reduced
mR NA for inducible nitric oxide synthase. The reduction
in mR NA for IFN-? was mirrored by a dramatic reduction
of both nonspecific and antigen-specific IFN-? protein pro-
duction. Attendant to the absence of IFN-? there was a
marked delay in the expression of antigen-specific cellular
accumulation both at sites of infection and at sites of anti-
gen rechallenge. This was linked to a delayed T cell activa-
tion as manifested by the reduced induction in expression
of the IL-12R ?2 chain. Taken together, the lack of im-
munity seen in the IL-12 p40?/? mice is crucially linked to
the absence of IFN-? and to the inability of the mice to
generate antigen-specific activated T cells.
This report confirms our previous conclusion, based on
antibody depletion studies, that IL-12 is important in the
protective IFN-? response in tuberculosis (11, 14, 15). Pre-
viously, however, we had noted that expression of mR NA
for IFN-? preceded that for IL-12 (15), thus making it un-
clear which of these two molecules is the initiator of the
response. The ability of IL-12–deficient mice to produce a
small amount of IFN-? has been reported both here, in re-
sponse to M. tuberculosis, and previously in response to LPS
(13). However, this innate reponse to both stimuli was much
reduced in comparison to control mice and apparently is
unable to activate macrophages to an anti-microbial state.
It was also possible that this limited IFN-? production
may have been due to the activity of the IFN-?–inducing
factor IL-18 (19, 20). This molecule was first described as a
product from the livers of Proprionobacteria acnes–treated
mice and it was thus plausible that it would be induced in
M. tuberculosis infections (19). We show here that mR NA
for IL-18 was present in the livers of infected animals but
that it was not strongly modulated during infection. The
recently reported observation that this molecule needs to
be activated by the IL-1? converting enzyme (21) may ex-
plain the poor expression of IFN-? in the IL-12 p40?/?
mice even though IL-18 mR NA was detected. Therefore,
this clearly indicates that IL-18 is not able to compensate
for the lack of IL-12 production in mice infected with M.
R ecent developments suggest that IL-12 receptor ex-
pression is crucial to the maturation of the Th1 IFN-?–pro-
ducing phenotype among T cells. The ability to respond to
this recently described dimeric molecule (22, 23) is as im-
portant in the development of Th1 cells (24) as the pres-
ence of IFN-? (25). Conversely, an inability to respond to
IL-12 is essential to the development of a fixed Th2 phe-
notype (26). This inability of cells to respond to IL-12 is
due to the absence of IL-12R ?2 mR NA expression and
the expression of this molecule is dependent upon IFN-
?–mediated signaling (27). To address the role of this mol-
ecule in the response to M. tuberculosis, we quantitated the
amount of IL-12R ?2 chain induced in the liver as a result
of M. tub e rc ulo sis infection. Here, we report for the first time
that IL-12R ?2 receptor is induced in response to infection
and that this induction does not occur in the absence of IL-12
p40. That this increase occurs before the maximal antigen-
specific IFN-? production and killing of bacteria (1) sug-
gests that it is an important correlate of protection.
line, sectioned, and stained with hematoxylin and eosin. At sites of mononuclear accumulation in the lung tissue there are more lymphocytes in the con-
trol (A) tissue compared with IL-12 p40?/? (B) tissue. Original magnification ?200.
IL-12 p40?/? mice have defective granuloma formation. Tissue from infected control (A) and IL-12 p40?/? (B) mice was fixed in formal sa-
IL-12 in Immunity to Tuberculosis
We have previously suggested that IL-12 plays an impor-
tant role in the generation of a successful granuloma. This
was based on the observations that exogenous IL-12 im-
proves granuloma formation in old mice (14) and that anti-
IL-12 disrupts granuloma formation in young mice (12).
That there is very little detectable antigen-specific T cell
response in terms of IFN-? production, DTH, or lympho-
cyte accumulation at the site of infection suggests that the
defective granuloma formation in IL-12 p40?/? mice could
be linked to the importance of IFN-?–producing Th1 cells
in this mechanism. The importance of Th1 cells in the
generation of DTH has been suggested previously and the
absence of DTH in both this model and in the previous de-
scription of the IL-12 p40?/? mouse (13) fully supports this
contention. The reason for this importance may reside in
the recently described expression by Th1 cells of the recep-
tors for both E- and P-selectin (28); molecules that have
also recently been shown to be crucial to recruitment of
these Th1 cells into inflamed tissue (29).
However, IL-12 may affect cellular recruitment by re-
ducing the levels of IFN-?–dependent chemokines such as
RANTES, which is specific for activated or memory T cells
and which plays a role in DTH (30). There is also evidence
for IFN-?–independent mechanisms as in the absence of
IFN-?, IL-12 can cause profound inflammation in the
lungs (31) and directly induce the expression of cutaneous
lymphocyte-associated antigen on activated CD4 cells (32).
In addition, the lack of IL-12 in old mice may explain the
high incidence of recrudescent tuberculosis in this animal
model (16), and is in keeping with the clinical observation
that tuberculosis in elderly humans often presents in a mil-
iary form (33). Whatever the mechanism, it is clear the data
reported here supports the contention that a function of
IL-12 in the protective immune response to M. tuberculosis
may be the control of dissemination by maintaining the in-
tegrity of the granulomatous response.
It is clear that IL-12 is required both for the initiation,
maturation, and maintenance of protective immunity to
tuberculosis. Moreover, in contrast with the original de-
scription (13) of the immune capacity of these knockout
mice, we did not in the current study observe induction of
Th2 type cytokines such as IL-4. This cytokine usually oc-
curs later in the immune response to M. tuberculosis and is
not considered protective but instead probably contributes
to the clearance of mycobacterial antigens released after
bacteria are killed by activated macrophages (2). The ab-
sence of any IL-4 in these experiments might simply reflect
the time points examined, but it is nevertheless interesting
to observe even in the complete absence of a strong Th1
response there is no Th2 type response.
In conclusion, it appears that IL-12 is central to the gen-
eration of antigen-specific lymphocytes that are able to pro-
duce IFN-?. The data reported above suggest a straightfor-
ward explanation for this dependence, i.e, in the initial
interaction with the host the bacteria induces IFN-?, the
maximal expression of which is dependent upon IL-12 and
TNF-?; this large induction of IFN-? is required to induce
the expression of the IL-12R ?2 chain on naive T cells; the
expression of this receptor then allows the T cells to re-
spond to IL-12 and, therefore, to become antigen-specific
This work was supported by National Institutes of Health grant AI-40488.
Address correspondence to Andrea M. Cooper, Department of Microbiology, Colorado State University,
Fort Collins, Colorado 80524. Phone: 970-491-2833; FAX : 970-491-1815; E-mail: acooper@vines.
Received for publication 24 February 1997 and in revised form 4 April 1997.
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