By
From the * Immunobiology Section, Laboratory of Parasitic Diseases, and Lymphocyte Biology
Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National
Institutes of Health, Bethesda, Maryland 20892-1892
The early induction of interleukin (IL)-12 is a critical event in determining the development of
both innate resistance and adaptive immunity to many intracellular pathogens. Previous in vitro
studies have suggested that the macrophage (M) is a major source of the initial IL-12 produced upon microbial stimulation and that this response promotes the differentiation of protective T helper cell 1 (Th1) CD4+ lymphocytes from precursors that are primed on antigen-bearing dendritic cells (DC). Here, we demonstrate by immunolocalization experiments and
flow cytometric analysis that, contrary to expectation, DC and not M
are the initial cells to
synthesize IL-12 in the spleens of mice exposed in vivo to an extract of Toxoplasma gondii or to
lipopolysaccharide, two well characterized microbial stimulants of the cytokine. Importantly,
this production of IL-12 occurs very rapidly and is independent of interferon
priming or of signals from T cells, such as CD40 ligand. IL-12 production by splenic DC is accompanied by
an increase in number of DCs, as well as a redistribution to the T cell areas and the acquisition of markers characteristic of interdigitating dendritic cells. The capacity of splenic DC but not
M
to synthesize de novo high levels of IL-12 within hours of exposure to microbial products in vivo, as well as the ability of the same stimuli to induce migration of DC to the T cell areas,
argues that DC function simultaneously as both antigen-presenting cells and IL-12 producing
accessory cells in the initiation of cell-mediated immunity to intracellular pathogens. This
model avoids the need to invoke a three-cell interaction for Th1 differentiation and points to
the DC as both a sentinel for innate recognition and the dictator of class selection in the subsequent adaptive response.
Interleukin 12 (IL-12) is a key cytokine in the induction
of cell-mediated immunity to intracellular pathogens. In
the innate response to these microbial agents, IL-12 triggers
the production of IFN- Macrophages (M In contrast to M The early synthesis of IL-12 is of crucial importance in
determining both innate and adaptive host resistance to the
intracellular protozoan, Toxoplasma gondii, and live replicative forms (tachyzoites) as well as parasite extracts have
been shown to be potent inducers of the cytokine from
peritoneal inflammatory M Experimental Animals.
Female C57BL/6, CBA, and C3H/
HeJ mice were obtained from the Division of Cancer Treatment,
National Cancer Institute (Frederick, MD). C57BL/6-SCID/SzJ
mice were purchased from The Jackson Laboratory (Bar Harbor,
ME) and bred at the National Institute of Allergy and Infectious
Diseases (NIAID) animal facility (Bethesda, MD). IFN- Microbial Stimuli.
Tachyzoites of the virulent RH T. gondii
strain were maintained by passage on human foreskin fibroblasts
and soluble tachyzoite antigen (STAg) was prepared as previously
described (10). Escherichia coli LPS was purchased from Sigma
Chemical Co. (St. Louis, MO).
In Vitro Cell Culture and Cytokine Production Assays.
Resident
and inflammatory M Flow Cytometry.
Low density spleen cells (LOD) were prepared from collagenase-digested spleens as previously described
(15), except that immediately after collagenase treatment, spleen
digests were washed in PBS containing 5 mM EDTA (PBS/
EDTA) and from this point onwards the cell suspension was always handled in buffers containing EDTA. This treatment allows
for release of interdigitating DC (IDC) from the inner periarteriolar lymphoid sheaths (PALS) that are normally lost in the presence of Ca2+ (16, 17).
and TNF from unsensitized NK
and T cells. At the same time, IL-12 selectively promotes
the differentiation of Th1 CD4+ cells, which produce the
same effector lymphokines upon restimulation with antigen. Thus, the induction of IL-12 early in infection initiates innate resistance to the pathogen while ensuring the induction of the correct class of adaptive host response (1).
)1 activated by microbial stimulation
produce high levels of IL-12, and it has been assumed that
these cells provide the major source of the cytokine in Th1
response initiation (2). Indeed, in vitro studies with TCR
transgenic CD4+ cells primed by antigen-pulsed cells showed
IL-12-producing M
to be highly effective in inducing selective Th1 cell differentiation (3). However, the model of
class selection suggested by these experiments requires that
both antigen-bearing dendritic cells (DC) and IL-12-producing M
travel from the site of infection to lymphoid
tissues where the responding T cells are found, and at
present, no evidence exists for such M
migration. Furthermore, a model in which IL-12 produced by M
acts in
a paracrine fashion to drive Th1 development of microbe-specific T cells would lead to Th1 differentiation of all recently activated precursors in the local microenvironment,
thus limiting the ability of the immune system to independently control Th1 responses to different antigens and potentially leading to inflammatory responses to self-antigens. A similar objection applies to models of type 1 response
initiation involving other "third-party" IL-12-producing
cells such as neutrophils (4).
, DC constitute a highly efficient system for capturing antigens in the periphery and delivering
them to the T cell areas of lymphoid tissues (5, 6). It is believed that this allows perusal of peripheral antigens by T
cells that recirculate between the blood and lymphoid compartments. In addition, DC possess many specializations that
allow them to function as efficient APCs, such as high levels of MHC products, adhesion and costimulatory molecules, extensive surface area, and high motility (5, 6). These
properties suggest that DC act as the priming APC for most
T cell responses and thus would be ideally placed to produce IL-12 at a site where it acts directly on those T cells
responding to DC-presented immunogenic MHC-peptide
complexes derived from infectious agents.
in vitro (7). Nevertheless,
inflammatory M
populations that are elicited by local injection of an irritant, or, for that matter, any population obtained after in vivo or in vitro manipulation, may not be
representative of the naive cells that initiate IL-12 responses.
Therefore, we have used immunolocalization techniques to
identify the cells first making IL-12 after in vivo stimulation
with T. gondii products or LPS. Our results clearly demonstrate that DC, not M
, are the cells involved in the IL-12
response to these microbial stimuli, which also simultaneously trigger DC recruitment to the T cell areas of the
spleen. Together, these findings support a two cell model
of in vivo T cell activation in which the DC serves as both the APC and the IL-12-producing initiator of the Th1 response.
gene-targeted (knockout; KO) mice (11) were originally provided by
D. Dalton and T. Stewart (Genentech, Inc., South San Francisco,
CA) and backcrossed for seven generations on the C57BL/6 background at Taconic Farms (Germantown, NY). IL-12 p40 KO
mice (12) (backcrossed for five generations on the same B6 background) were originally donated by Jeanne Magram (Hoffman-La
Roche, Nutley, NJ) and bred at the NIAID animal facility. CD40
ligand (CD40L) KO mice (13) maintained on a mixed B6 × 129/J background and control mice (B6 × 129)F2 were purchased from The Jackson Laboratory. All animals were housed in
specific pathogen-free conditions and were used at 6-9 wk of age.
were obtained from animals that had been
either untreated or inoculated intraperitoneally 4 d previously
with 1.5 ml of 3% thioglycollate (Sigma Chemical Co.). Cells
were plated in RPMI 1640 (GIBCO BRL, Gaithersburg, MD)
containing 2% FCS in 96-well plates for 2 h at 37°C. The medium and unbound cells were then removed and replaced with 200 µl of RPMI 1640 complete medium in the presence or absence
of STAg or RH tachyzoites. In some experiments, cultures were
supplemented with 100 U/ml of rMuIFN-
(provided by Genentech, Inc.). Bulk splenocytes were cultured under the same
conditions as the peritoneal cells. Supernatants were harvested at
6 h for TNF-
, at 18 h for IL-12 p40, and at 72 h for IFN-
determinations. These measurements were performed by two-site
ELISA, as previously described (14).
)2 anti-rat
IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).
N418 supernatant (ATCC HB 224 [American Type Culture
Collection, Rockville, MD]; reference 20) containing 25 µg/ml
rat IgG was subsequently added, followed by biotin-conjugated
goat F(ab
)2 anti-syrian hamster IgG cross-adsorbed against rat
and mouse serum proteins (Jackson ImmunoResearch Laboratories); TriColor-streptavidin (Caltag, San Francisco, CA) was
added, together with PE-conjugated 53-6.7 (anti-CD8
; PharMingen, San Diego, CA). Washes and reagent dilutions were in
WS except before the N418 step when all reagents and WS also included 0.1% saponin to allow antibody access to intracellular compartments.
Immunohistochemistry.
Spleen fragments were frozen in embedding medium (Cryoform; International Equipment Co., Needham, MA). 6-µm frozen sections were cut, air-dried, fixed in acetone, and rehydrated in Tris-buffered saline (TBS) containing
0.05% Tween 20. Endogenous peroxidase was blocked with 0.3%
H2O2. For IL-12 p40 staining, sections were incubated with
C17.15 (10-20 µg/ml) or an isotype-matched control, followed
by biotin-conjugated mouse F(ab)2 anti-rat IgG (Jackson ImmunoResearch Laboratories) and horseradish peroxidase (HRP)-streptavidin (DuPont, Boston, MA). HRP localization was revealed
using a metal-enhanced diaminobenzidine (DAB) substrate (Pierce,
Rockford, IL), resulting in brown/black staining. For double labeling with anti-T cell or anti-B cell markers, sections were first
stained as above; after developing the HRP, sections were re-blocked with 0.3% H2O2 and were further stained with either
FITC-H57-597 (anti-TCR-
; PharMingen) or FITC-RA3-6B2
(anti-B220; PharMingen), added in a solution containing 25 µg/ml
of rat IgG; this was followed by HRP-conjugated rabbit antifluorescein (BIODESIGN International, Kennebunkport, ME). For
double labeling with N418, sections were stained for IL-12 as
above or with NLDC-145 and HRP-mouse F(ab
)2 anti-rat IgG
(Jackson ImmunoResearch Laboratories); after developing HRP
with metal-enhanced DAB, sections were reblocked with 0.3% H2O2 and were further blocked with avidin followed by biotin
(both from Vector Laboratories, Burlingame, CA; used according
to manufacturer's instructions). N418 supernatant containing 25 µg/ml rat IgG was subsequently added, followed by biotin-conjugated goat F(ab
)2 anti-syrian hamster IgG cross-adsorbed against
rat and mouse serum proteins (Jackson ImmunoResearch Laboratories) and HRP-streptavidin.
M-derived IL-12 has traditionally
been considered a major factor driving Th1 responses, based
on the observation that M
produce high levels of IL-12
after exposure to microbial products or after microbial infection in vitro (1, 2). However, most experiments examining IL-12 synthesis by M
in response to microbial stimuli to date, including our own (10), have primarily made use of inflammatory cells such as those that can be isolated
from the peritoneal cavity of mice after elicitation with
thioglycollate. To study IL-12 production by resting, unactivated M
in response to microbial products, resident
peritoneal exudate cells (PEC) from LPS hyporesponsive
C3H/HeJ mice were compared to thioglycollate-elicited PEC
(thio-PEC) from the same mouse strain for the ability to
produce various monokines after in vitro infection with T. gondii or after exposure to a soluble T. gondii antigen extract
(STAg). As shown in Fig. 1, infection of freshly isolated
resident PEC with live T. gondii tachyzoites or incubation
with STAg did not result in production of detectable IL-12
p40 despite the fact that the same cells produced TNF. In
contrast, as previously reported (10), thio-PEC produced
substantial levels of both IL-12 p40 and TNF in response to
infection or exposure to STAg (Fig. 1). Addition of exogenous IFN-
to the cultures, which dramatically augments production of IL-12 p40 by inflammatory M
stimulated
with T. gondii (21), did not correct the selective defect in
IL-12 p40 production by resident cells despite the fact that
it increased their production of TNF by 10-fold (Fig. 1).
Resident M
were able to produce IL-12 p40 upon exposure to STAg or after live infection only when incubated
for 24 h before stimulation, especially if IFN-
was included in the preculture (data not shown). Furthermore, preincubation in the presence of IFN-
was required to
obtain substantial levels of IL-12 p40 production in response to STAg with bone marrow-derived M
populations and monocytic cell lines (data not shown). These results suggest that resting, unprimed M
cannot serve as a
significant source of IL-12 to drive Th1 responses to T. gondii. Thus, a model in which M
-derived IL-12 is a pivotal factor in driving Th1 responses to microbial infections requires that the infected M
be already "primed" for IL-12
production, in a manner analogous to thioglycollate elicitation or preculture in medium containing IFN-
.
Production of IL-12 by Spleen Cells in Response to T. gondii Antigens.
To identify a source of IL-12 not requiring
priming, we tested unfractionated spleen cells because previous studies had identified them as a source of T-independent IFN- production (22). Surprisingly, whole splenocytes produced significant levels of IL-12 p40 after STAg
exposure or T. gondii infection in vitro, but relatively little
TNF (Fig. 1). This raised the possibility that the spleen contains a population of cells that can produce IL-12 directly in response to microbial stimuli, without priming (Fig. 1).
When spleen cells were separated into adherent and nonadherent cells, STAg-induced IL-12 production was enriched
in the adherent fraction, composed primarily of M
and
DC, and was relatively depleted in the nonadherent lymphocyte fraction (data not shown). Adherent cells constitute only a small proportion of all splenocytes (<5%). Therefore, the high levels of IL-12 seen with unfractionated spleen cells, close to those elicited from the same
number of cells of homogeneous populations of thio-M
(Fig. 1), seem likely to reflect extremely high levels of production by a small subpopulation of cells.
To determine if IL-12 production by spleen cells in response to T. gondii also occurs in vivo, B6 mice were injected intravenously with STAg. Spleen cell suspensions
from mice injected with STAg, but not from uninjected
mice or from mice injected with PBS alone, spontaneously
produced IL-12 p40 during overnight culture (Fig. 2 A).
Maximal IL-12 p40 production could be seen as early as 3 h
after systemic administration of STAg and declined progressively thereafter (Fig. 2 A). The IL-12 produced in
vivo appeared to be bioactive because spleen cells from
STAg-injected mice produced increased levels of IFN-
upon in vitro restimulation of spleen cells with STAg or
LPS. This enhancement of IFN-
production was specifically dependent upon IL-12 induction in vivo because it was not seen in IL-12 p40-deficient mice or in wild type
mice treated with anti-IL-12 antibodies at the time of
STAg administration (Fig. 2 B). Together, these results
demonstrate that spleen cells can synthesize IL-12 p40 in
response to stimulation with T. gondii products in vitro or
in vivo, and that IL-12 production elicited by these molecules in vivo can prime an IFN-
response.
IL-12 Production by DC In Situ in Response to Systemic Administration of Microbial Products.
The ability of STAg to
induce IL-12 p40 production in the spleen in vivo offered
an opportunity to phenotype the IL-12-producing cells in
situ. Spleen sections from STAg-injected C57BL/6 mice showed numerous intensely stained IL-12 p40+ cells (Fig. 3 B)
that were not seen in sections from uninjected control mice
(Fig. 3 A). Induction of IL-12 p40 was specifically dependent on exposure to the parasite extract because it was not found in sections from animals injected with PBS, hen egg
lysozyme, or ovalbumin (OVA; Fig. 3 C and data not
shown). Intensely stained IL-12 p40+ cells could be detected as early as 3 h after STAg injection and the staining
peaked between 6 and 12 h, and then declined progressively. 24 h after injection, staining was barely visible and
the sections resembled those of control animals (data not shown). IL-12 p40+ cells had dendritic profiles and formed
abundant "nests" surrounding central arterioles (Fig. 4 D),
suggesting that they might represent IDC. Indeed, staining
of serial sections demonstrated that IL-12 p40+ cells localized exclusively in the T cell areas of the white pulp and
were excluded from the red pulp or the B cell areas (Fig. 4,
D-F). This was true except at 3 h after injection, when IL-12 p40+ cells showed a more diffuse location, with many cells
found at the edge of the T cell area and others interspersed
with B cells in the marginal zone (Fig. 4, A-C, and data
not shown). This picture is consistent with the IL-12-producing cells being in the process of migrating into the T
cell area (see below). LPS coinjected intravenously with OVA
also induced the appearance of IL-12+ nests of dendritic
profiles surrounding central arterioles in spleen sections (Fig.
3 D). These were not seen in control sections injected with
OVA alone (Fig. 3 C). Nevertheless, IL-12 p40 staining of
putative IDC in response to LPS injection was consistently weaker and involved fewer cells than in response to STAg
(Fig. 3 D). Importantly, IL-12 staining could not be detected in M in the red pulp or those in the marginal zone
(including MOMA-1+ [23] metallophils and marginal zone
M
), either at early times or up to 96 h after injection of
STAg (Figs. 3 and 4, and data not shown). IL-12 p40 staining by M
was also not detected in LPS-injected animals
(Fig. 3 D and data not shown).
|
To demonstrate that the IL-12 p40+ cells were indeed
DC, sections were double stained with anti-IL-12 p40 and
N418, a marker for mouse DC (20). As shown in Fig. 4 G,
IL-12+ cells were also positive for N418 (white arrow) although some N418+ cells did not appear to stain for IL-12,
particularly those N418+ cells at the edge or outside the
T cell area (Fig. 4 G, black arrow). Furthermore, staining of
serial sections with different antibodies demonstrated that
IL-12+ cells colocalized with cells positive for the NLDC-145 marker (18), also known as DEC-205 (24), which is
highly expressed by IDC in situ (data not shown). To confirm these results and to allow more accurate immunophenotyping of IL-12-producing cells, LOD suspensions were
prepared in Ca2+-free media from mice injected with STAg
or from controls injected with PBS, and then analyzed by
flow cytometry. A distinct subpopulation of LOD from
STAg-injected but not from PBS-injected mice could be
stained intracellularly with anti-IL-12 p40 antibodies (Fig. 5, A and B). All IL-12+ cells were also N418bright, confirming the observations from immunohistochemistry that
N418/dull splenic M
, which, like DC, are enriched in
LOD, do not produce significant levels of IL-12 after exposure to STAg in vivo (Fig. 5 B). Remarkably, most IL-12-producing N418+ cells were also positive for CD8
(Fig. 5, C and D), a marker that, like DEC-205, is expressed by IDC (16, 17). 67% of all the CD8
+ N418+
DC in STAg-injected animals stained for IL-12; in contrast, only a few (12%) of the CD8
N418+ DC were
positive for IL-12 and the intensity of IL-12 staining of
these cells was lower than that of the CD8+ DC (mean fluorescence 223 versus mean fluorescence 644; see Fig. 5 D).
We conclude that high levels of IL-12 production in mouse spleen can be detected shortly after systemic administration
of two different microbial products, STAg and LPS. This
IL-12 production almost exclusively involves a large proportion of CD8
+ DEC-205+ IDC, under conditions in
which production of the same cytokine by splenic M
cannot be detected.
IL-12 production
by DC in vivo in response to exposure to STAg was independent of the mouse strain used. It could be seen in both
C57BL/6 and BALB/c mice, two strains known to vary in their predisposition to Th2 responses (25), and was also
seen in (B6 × 129)F2 (data not shown and Fig. 3 E). To
determine whether STAg-induced IL-12 production by
DC in vivo is dependent on priming by IFN-, responses
to STAg were compared between B6 and IFN-
KO
mice. Spleen cells from both strains isolated after STAg injection spontaneously produced comparable amounts of
IL-12 p40 in culture (Fig. 2 A). Similarly, IL-12 staining in
the spleens of IFN-
KO mice intravenously injected with
STAg was indistinguishable from that in wild-type B6 controls, being restricted to dendritic profiles surrounding central arterioles (Fig. 3 G). Furthermore, the kinetics of IL-12
induction in vivo were identical between the two mouse
strains, with staining peaking at 6-12 h and disappearing by
24 h after injection (data not shown).
IL-12 production by both murine and human DC in
vitro has been previously reported (26). The major
mechanisms involved in IL-12 induction appear to be signaling through DC surface CD40 molecules after cross-linking by T cell-expressed CD40L, or direct signaling
through MHC class II molecules on DC, cross-linked by the TCR (30, 31, 33). Spleen cells from CD40L KO mice
secreted substantial levels of IL-12 p40 in response to live
parasite infection or STAg stimulation in vitro, although
these levels were somewhat lower than those produced by
cells from (B6 × 129)F2 control mice (Fig. 6). To examine
the CD40L dependence of IL-12 production by DC in response to STAg in vivo, CD40L KO mice were injected with STAg as before and spleens removed 6 h later. Staining of sections from CD40L KO mice was comparable to
that of sections from B6 mice or (B6 × 129)F2 controls,
suggesting that STAg-induced IL-12 production by IDC does
not require cross-linking of CD40 on DC by CD40L on
T cells (Fig. 3, E and F). To exclude the possibility that
other cognate T cell-DC interactions or T cell-derived cytokines might be responsible for the STAg effect, SCID
mice were similarly injected with STAg and analyzed in
parallel to the CD40L KO mice. Again, SCID spleen sections showed IL-12 p40 staining comparable to wild-type
controls (Fig. 3 H). SCID spleen cells also made high levels
of IL-12 p40 in response to STAg or live infection in vitro
(Fig. 6). Thus, microbe-induced IL-12 production by spleen cells in vitro or by DC in vivo does not require interactions with lymphocytes and is likely to reflect a direct effect on
the cells of one or more components of the microbial extract, or an indirect effect through induction of DC-activating inflammatory cytokines produced by nonlymphoid
cells.
Mobilization of DC to the Inner PALS after Systemic Administration of STAg.
Mouse spleen contains a subset of relatively immature DC outside or at the margin of the T cell
area that may act as precursors for IDC and that probably
represent the bulk of DC in conventionally prepared spleen
cell suspensions (20, 34). In vivo, LPS induces functional
maturation of these marginal zone DC and causes them to
move into the PALS area (35 and Reis e Sousa, C., unpublished data). The striking restriction of IL-12 p40 staining
to CD8+ DEC-205+ DC in the T cell areas, seen as early
as 6 h after STAg or LPS injection, could be due to stimulation of resident IDC by the microbial products and/or to
a rapid redistribution of IL-12-producing IDC precursors
to the inner PALS. Consistent with the latter, IL-12 p40+
DC could be seen at early times after STAg injection outside the T cell area (see above). Examination of spleen sections from STAg-injected animals demonstrated a striking
accumulation of double-stained N418+ NLDC-145+, as
well as single-stained N418+ cells, in nests surrounding the
central arterioles (Fig. 4 I), corresponding to the locations
where IL-12-producing cells were found. This contrasted
markedly with the more diffuse distribution of DC seen in
sections from control animals (Fig. 4 H). At earlier times after injection, the distribution of splenic DC was intermediate between that seen in control animals and the exclusive
localization in the T cell areas seen after 6 h, much like the
distribution of IL-12+ cells at 3 h (Fig. 4, A-C). The overall number of DC in spleen sections from STAg-injected
mice also appeared to be greater than in control animals
(compare Figs. 4, H and I). This was confirmed by counting different cell subpopulations in the low density fraction of spleen. 6 h after STAg administration, STAg-injected
mice had ~50% more N418+ cells in their spleens than
control PBS-injected animals, whereas no change was seen
in the relative number of B220+ cells between the two groups
(Table 1). The increase in splenic DC in STAg-injected mice
reflected an increase in the number of both NLDC-145+
and NLDC-145
cells (Table 1), consistent with the results
obtained by immunohistochemistry, which suggested that
many of these recruited cells were moving into the T cell
areas of spleen and becoming DEC-205+ IDC. The increase in DC numbers did not explain the 40% total increase in spleen cellularity seen after STAg injection, suggesting that other cell types were also being recruited to the
spleen (Table 1). Some of these cells appeared to be M
(Reis e Sousa, C., unpublished data) although, unlike DC,
they did not produce IL-12 (see Fig. 5). Thus, like LPS
(35), STAg increases immigration of DC into the spleen
and induces a redistribution of these immigrants and of resident splenic DC to the inner PALS.
Understanding the regulation of IL-12 production during the course of immune responses is the focus of much
research because of the importance of this cytokine in driving the development of Th1 cell responses, as well as in
regulating innate immunity (1). Two pathways for IL-12
production by M have been clearly identified, one involving stimulation by T cell-derived, membrane-bound, or
soluble CD40L during M
-T cell interactions, and another through direct stimulation of M
cells by microbial
products (1). In contrast, little is known about the ability of
DC to produce IL-12 in response to microbial stimuli.
Several reports have shown that interaction with T cells appears to be the main pathway for induction of IL-12 production by DC, and that M
activators such as LPS or bacteria have much less effect on these cells (26, 28, 30, 31). T
cell-dependent DC-derived IL-12 is induced during cognate interactions, through ligation of CD40 on DC by
CD40L expressed on the activated T cells, as well as
through direct signaling by MHC class II molecules cross-linked by the TCR (30, 31). This has suggested a model in
which the Th1 development often associated with microbial infections is attributed to two separate sources of IL-12.
Initial IL-12 production is presumed to occur in response
to microbial stimulation of M
and predispose for Th1 development (2, 3, 36). Later IL-12 production by antigen-presenting DC interacting with antigen-specific T cells is
thought to synergize with M
-derived IL-12 to promote
Th1 differentiation (37). In this model, M
act as a bridge
between the innate and the adaptive immune systems, sensing infection and helping to induce protective Th1 responses to microbes.
However, several lines of evidence suggest that maximal
IL-12 production by M in response to bacteria or microbial products requires prior activation of the cells (38).
This is particularly evident in the inability of freshly isolated, resting peritoneal cells, bone marrow-derived M
, or
M
cell lines to produce IL-12 in response to infection
with live Toxoplasma or to stimulation with Toxoplasma antigens, as reported in this study, despite being able to produce other cytokines such as TNF (Fig. 1 and data not shown). The selective inability of resting M
to produce
IL-12 even after infection with live parasites raises questions about a model in which M
-derived IL-12 is crucial
for inducing IFN-
-mediated protective responses to infection.
We have found an alternative source of IL-12 in response to microbes in the adherent fraction of mouse
spleen cell suspensions stimulated in vitro. This source was
independent of pretreatment of mice with inflammatory
stimuli and appeared to represent DC rather than splenic
M in preliminary fractionation studies (data not shown).
This is consistent with our observations that noninflammatory M
such as those found in the spleen do not produce
appreciable levels of IL-12 in direct response to microbial
stimuli (Fig. 1). Furthermore, we have found no evidence
for high levels of IL-12 production by splenic M
after
STAg injection in vivo, even using a sensitive flow cytometric technique (Fig. 5). In contrast, we clearly demonstrate that DC in the spleen can produce high levels of IL-12
p40 in response to two microbial products delivered systemically, in circumstances in which neither T cell help nor IFN-
is available. Production of IL-12 p40 is transient and
extremely rapid and involves a significant fraction of splenic
DC. This hitherto undiscovered abundant source of IL-12,
independent of IFN-
, could be responsible for the increase in serum levels of IL-12 found after systemic administration of LPS, which is seen in wild type as well as IFN-
-
deficient mice (19, 41).
IL-12 is composed of two subunits, p40 and p35, which
can form a bioactive heterodimer or a p40 homodimer that
acts as an IL-12 receptor antagonist (1). We have not been
able to stain spleen sections or cell suspensions for IL-12
p75, even under conditions in which IL-12 p40 was clearly
induced (data not shown). This is likely due to the fact that
the heterodimer is made at levels 10-50-fold lower than
those of the IL-12 p40 subunit (1). Staining for IL-12 p35
did not help resolve whether IDC produced the heterodimer since the p35 subunit was found to be constitutively expressed in spleen white pulp of unstimulated mice,
particularly in B cell areas, and expression did not change
upon injection of STAg (data not shown). This result is
consistent with a previous report that used in situ hybridization with probes for the p35 subunit of IL-12 and also
detected hybridization in the B cell area (42). Interestingly,
in the same report it was shown that probes for the IL-12
p40 subunit hybridized strongly to the T cell areas of spleen
after intraperitoneal injection of LPS (42), a result entirely
consistent with the observations made here that both LPS
and STAg induce production of high levels of IL-12 p40 on IDC. However, in spite of our inability to stain for IL-12 p75, IL-12 production in animals injected with STAg was
bioactive in that neutralization of the cytokine in vivo abrogated the enhancement of IFN- production during subsequent in vitro restimulation of spleen cells with STAg or
LPS (Fig. 2 A). Thus, we believe that the production of IL-12
p40 by DC is likely to translate into bioactive p75 cytokine
that can act on Th precursors (Thp) to drive Th1 development, and on NK cells to elicit IFN-
secretion.
Although the main DC population staining for IL-12
p40 in situ was found deep in the T cell areas (Fig. 4, D-F)
and expressed the IDC markers CD8 and DEC-205 (Fig.
5), occasional dendritic profiles scattered among marginal
zone B cells also stained for IL-12 in situ at early times (3 h)
after intravenous injection of STAg (Fig. 4, A-C). As these
profiles were not seen at later times after injection (
6 h),
it is probable that they represent marginal zone DC and/or
newly arrived immigrant DC that are in the process of migrating to the inner PALS where the bulk of the IL-12+
DC were found. This interpretation is consistent with the
redistribution of DC to the T cell areas observed after administration of STAg (Fig. 4, H and I) or LPS (35 and Reis
e Sousa, C., unpublished data). It is also consistent with the
increase in DC number in the spleen observed after STAg
injection (Table 1), which probably represents an influx of
blood DC released from nonlymphoid organs, as seen in
response to systemic LPS (43, 44). Together, these results
suggest that the effect of microbial products is, in part, on
IDC precursors, and that IL-12 production is another consequence of the general DC activation induced by STAg or
LPS that leads to maturation of nonlymphoid DC with recruitment to lymphoid organs and migration to the T cell
areas (6).
The redistribution of activated IDC precursors to the T
cell areas may explain why maximal in vitro IL-12 production by spleen cells isolated from STAg-treated animals is
seen earlier (3 h after injection; Fig. 2 B) than the maximal
IL-12 staining of IDC observed in situ (6-12 h after injection; Figs. 3 and 4): as DC move into spleen T cell areas to
become IDC, they become increasingly resistant to isolation by simple mechanical dissociation of spleens into a cell
suspension (17). However, the 40% increase in numbers of
splenic CD8+ DEC-205+ N418+ DC seen after STAg
injection (Table 1), presumed to be due to immigration/
maturation of IDC precursors, is not sufficient to explain
the fact that 67% of these cells produce IL-12 p40 by flow
cytometric analysis (Fig. 5). Thus, in addition to activating IDC precursors, it is likely that microbial products also act on small numbers of preexisting IDC in the T cell areas.
Production of IL-12 by DC in response to microbial
products such as STAg and LPS is likely to be important in
the development of cell-mediated immunity to infection.
IL-12 production by M at the site of infection (see below) is unlikely to influence the development of Th1 cells
taking place at a distance in draining lymph nodes. In contrast to M
, DC are specialized APC for transporting antigens from the periphery to lymphoid tissues (5, 6). IL-12
production by lymphoid DC derived from recent immigrants that brought microbial antigens from the site of infection would be more likely to influence Th1 development in the lymphoid microenvironment than would the
IL-12 produced by M
that remain at the peripheral site of
invasion. This is especially true because these DC would be
presenting microbial peptides in association with MHC molecules to the responding antigen-specific T cells, ensuring juxtaposition of the IL-12 source and the responding Thp. In
fact, even in cases in which there is abundant draining of
microbial products to lymphoid tissues, such as those mimicked here by intravenous injection of STAg or LPS, DC
rather than M
appear to be the main source of IL-12
(Figs. 3-5). Thus, we can propose a revised model for the role of M
- versus DC-derived IL-12 in immunity to microbial infections. At the site of infection, local signals trigger the migration of nonlymphoid DC bearing microbial
antigens to lymphoid tissues. These cells produce IL-12 due
to direct stimulation by microbial products and present antigen to microbe-specific T cells, triggering clonal expansion and predisposing the Thp to differentiate towards Th1
effectors. During the DC-T cell interaction, engagement of CD40 on the DC by CD40L upregulated on the T cell (30,
31), as well as direct signaling through MHC molecules (31),
serve to sustain IL-12 production by DC, further driving
Th1 development of the responding cells. DC-derived IL-12
might also activate NK cells in lymphoid tissues, which then
produce IFN-
locally, further predisposing Thp to differentiate towards Th1 effectors.
On the other hand, IL-12 produced by tissue M in response to the infectious organism could be important at the
site of infection. This might require IFN-
as a priming
signal that, during the innate phase of the immune response, could be provided by recruited NK cells that were
activated in lymphoid tissues by DC. Alternatively, inflammation itself could prime tissue M
to make IL-12 in response to microbial products, consistent with our observations that inflammatory thio-M
in vitro do produce IL-12
in response to Toxoplasma, even when derived from IFN-
KO mice (Fig. 1 and reference 14). In either case, M
-derived IL-12 could then further stimulate NK cells at the
site of infection. This would lead to increased secretion of
IFN-
by these cells, which, in turn, would potentiate the microbicidal activity of these M
, as well as increase their
ability to produce additional IL-12 (38, 39). This establishes
a positive feedback loop that would ensure maximal activation of M
effector cells at the site of infection. That loop
can eventually be broken by the known ability of M
to
also produce the antiinflammatory cytokine, IL-10, ensuring that full-blown activation does not lead to debilitating
immunopathology (1, 9). In addition, during the adaptive
phase of the immune response, IL-12 production by M
may help maintain the differentiated phenotype of Th1 effectors at the site of infection.
This model postulates that the priming APC produces
IL-12 directly and that trans-acting M-derived IL-12 is
not essential for Th1 development. Dispensing with the
need for trans-acting IL-12 ensures that IL-12 produced in
response to infection acts specifically on the T cells responding to microbial antigens rather than on bystander T
cells, and explains how Th1 and Th2 responses to different
antigens could occur simultaneously in the same microenvironment. Thus, in this model, DC and not M
provide
the bridge between adaptive and innate immunity.
Address correspondence to Dr. Caetano Reis e Sousa, LBS/LI/NIAID/DIR, Bldg 10, Rm 11N311, 10 Center Dr, MSC 1892, National Institutes of Health, Bethesda, MD 20892-1892. Phone: 301-496-4047; FAX: 301-480-7352; E-mail: caetano{at}nih.gov
Received for publication 5 September 1997 and in revised form 6 October 1997.
C. Reis e Sousa is supported by a Visiting Fellowship from the Fogarty International Center, National Institutes of Health.We are grateful to Drs. G. Trinchieri and M. Wysocka for providing antibodies against IL-12, and to R. Dreyfuss and S. Everett for photography.
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