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Secretion and not Restricted to T and NK Cells
Preclinical Research and Development, Genetics Institute, Andover, MA 01810
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, indicating that receptor expression and hence
IL-12 responsiveness may be regulated, at least in part, by the local
cytokine milieu. In this study, we report that cellular expression of
both IL-12Rß1 and ß2 mRNA is increased in the lymph nodes of naive
mice following systemic administration of murine rIL-12 (rmIL-12).
Changes in IL-12R mRNA were associated with increased IFN- secretion
following ex vivo activation of lymph node cells with rmIL-12,
indicating the presence of a functional receptor complex. Expression of
IL-12R mRNA was not restricted to lymph node T cells, and its autocrine
regulation was independent of secondary IFN- secretion. Data from
fractionated lymph node cells as well as rmIL-12-treated B
cell-deficient mice suggest that IL-12-responsive B cells may represent
an alternative cellular source for IFN- production. However, the
strength of the biological response to rmIL-12 is not governed solely
by receptor expression, as rmIL-12-induced IFN- secretion from
cultured lymph node cells is accessory cell dependent and can be
partially blocked by inhibition of B7
costimulation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The biological response to IL-12 is mediated through specific binding
to a high affinity receptor complex that is present on NK cells and
activated lymphocytes (12, 13, 14). Scatchard analysis of
iodinated rhIL-123
binding to human PHA blasts indicates the presence of at least two
binding sites with high (5–20 pM) and low (2–6 nM) affinities,
respectively (15). Consistent with this observation, cDNAs
encoding two distinct IL-12R proteins (designated IL-12Rß1 and
IL-12Rß2) have been identified by expression cloning. IL-12Rß1 is a
member of the gp130 receptor family and binds IL-12 with relatively low
affinity. However, IL-12Rß1 is required for signaling, as mAbs
against this subunit inhibit rhIL-12-induced activation
(12), and IL-12Rß1-deficient mice are unresponsive to
rmIL-12 (16). A second component of the IL-12R was
subsequently identified (IL-12Rß2) that by itself binds IL-12 with
low affinity, but confers high affinity binding (55 pM) and IL-12
responsiveness when coexpressed in cells with IL-12Rß1
(15). Regulation of IL-12R expression in the context of
Th1 and Th2 differentiation has been studied in vitro using both murine
and human T cells (17, 18). In both instances, Th2 cell
development that results in abrogation of IL-12 responsiveness is
associated with the specific loss of IL-12Rß2 mRNA expression. In
these in vitro systems, coincubation of murine and human T cells with
either IFN- or IFN-
/ß respectively during Th2 differentiation
results in maintenance of IL-12Rß2 expression and hence the ability
to respond to IL-12 stimulation.
Although Abs against IL-12Rß1 bind to resting PBMCs, activation with
anti-CD3 Ab or PHA is required to promote high affinity binding of
labeled rhIL-12 (12) and responsiveness to IL-12
stimulation (19, 20). Despite the presence of the IL-12R
complex on activated T cells, depletion of monocytes from PHA blasts
partially abrogates the ability of IL-12 to induce IFN-. These data
suggest that expression of the IL-12R complex alone is not sufficient
to confer maximum biological responsiveness to IL-12. Further analysis
demonstrated that this monocyte-dependent T cell response to IL-12
stimulation was mediated predominantly via the CD58/CD2 interaction
(21, 22). The observation that T cell responsiveness to
IL-12 is dependent on signals provided by accessory cells is supported
further by reports demonstrating synergy between IL-12 and B7 for
IFN- secretion (23, 24). Taken together, these
observations indicate that expression of both chains of the IL-12R is a
prerequisite for IL-12-mediated activation; however, the strength of
the biological response can be influenced by additional factors.
Administration of rmIL-12 to mice has been shown to result in the rapid
induction of high levels of circulating IFN- that can be detected
within 24 h of a single injection of rmIL-12 (25). As
IL-12Rß2 expression is absent on resting T cells (18, 26), it is likely that this early production of IFN-
following rmIL-12 administration comes from activated NK and NK/T cells
that constitutively express the IL-12R (27, 28). In
tumor-bearing mice, however, systemic treatment with rmIL-12 renders
lymph node cells (LNC) sensitive to rmIL-12-mediated IFN- secretion
(29), suggesting that treatment with rmIL-12 is capable of
regulating its own receptor. This hypothesis is supported by the recent
observations that rmIL-12 administration to
Leishmania-infected BALB/c mice enhanced both lymph node
IL-12Rß2 expression as well as the sensitivity to ex vivo stimulation
with rmIL-12 (26, 30). The current studies were designed
to further explore IL-12R expression/regulation as well as the pathways
that confer rmIL-12 responsiveness and subsequent IFN- secretion
following systemic rmIL-12 treatment. In this study, we report that
mRNA for both IL-12Rß1 and ß2 is increased in the lymph nodes of
rmIL-12-treated C57BL/6 mice and that changes in receptor expression
correlate with the responsiveness of cultured LNC to ex vivo activation
with rmIL-12. Receptor expression was not restricted to lymph node T
cells, as mRNA for both IL-12Rß1/ß2 as well as IFN- were readily
detected in CD4- and CD8-depleted LNC that consisted primarily of B
cells. The possibility that IL-12-responsive B cells may represent an
alternative source for IFN- secretion was substantiated by studies
in B cell-deficient mice, in which diminished IFN- production in
response to systemic rmIL-12 administration was observed relative to
wild-type treated controls. Autocrine regulation of the IL-12R complex
was, however, shown to be independent of secondary IFN- secretion at
both the level of IL-12R mRNA expression and functional responsiveness
to rmIL-12 stimulation. The strength of the biological response to
IL-12 is not, however, governed solely by IL-12R expression, as
rmIL-12-induced IFN- secretion from LNC is accessory cell dependent
and can be partially blocked by inhibition of B7 costimulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, which is apparent as early as day 2 and increases
further by day 5 (29, 31). In both mouse strains, the
magnitude and kinetics of IFN- secretion from cultured LNC
stimulated with rmIL-12 correlate with changes in circulating levels of
IFN-. Thus, the studies performed to further explore regulation of
IL-12R expression and pathways that govern IL-12 responsiveness
utilized LNC from rmIL-12-treated mice. IL-12 administration and tissue preparation
Female C57BL/6 mice were purchased from Taconic (Germantown,
NY). IFN- knockout mice, B cell-deficient mice (both on the C57BL/6
background), and wild-type littermates were purchased from The Jackson
Laboratory (Bar Harbor, ME). rmIL-12 (1 µg/mouse) or vehicle control
was administered by s.c. injection in a volume of 100 µl. Control and
rmIL-12-treated mice (n = 5) were sacrificed after
either two or five injections of rmIL-12, as indicated in
Results.
In some experiments, blood was collected by cardiac puncture under
methoxyflurane anesthesia for serum cytokine analysis. Lymph nodes
(popliteal, axial, and inguinal) were collected into complete RPMI 1640
media (10% FBS (HyClone, Logan, UT), 5 x
10-5 M 2-ME (Life Technologies, Grand Island,
NY), 100 µg/ml streptomycin, and 100 U/ml penicillin (Sigma, St.
Louis, MO)). Single cell suspensions were prepared by standard
techniques, and pooled LNC were resuspended at a final concentration of
5 x 106 cells/ml in complete RPMI. Cells
were stimulated with rmIL-12 (1 ng/ml) for 48 h at 37°C and 5%
CO2, after which time culture supernatant was
collected for subsequent analysis of cytokine secretion by ELISA
assays. IFN- was measured using R46A2 and biotinylated XMG1.2
(PharMingen, San Diego, CA) Abs for capture and detection,
respectively. Murine IL-10 and IL-13 were assayed using commercially
available ELISA kits (R&D Systems, Minneapolis, MN), according to the
manufacturer’s instructions.
Fractionation of LNC from IL-12-treated mice
Lymph node T cells from rmIL-12-treated mice were enriched by
either accessory cell depletion (for functional studies) or positive
selection (RNA analysis). Accessory cell depletion was achieved by
passing single cell suspensions of LNC over columns containing sterile
prewashed nylon wool (Cellular Products, Buffalo, NY). Columns were
incubated at 37°C for 15 min, after which time the T cells were
eluted from the column with 15 ml of RPMI with 5% FBS. The T
cell-enriched fraction was washed twice in complete RPMI, and an
aliquot was removed for FACS analysis before stimulation with rmIL-12.
Either unfractionated LNC (5 x 106/ml) or
the enriched T cell fraction (2.5 x 106
cells/ml to adjust for accessory cell depletion) was stimulated with
rmIL-12 (1 ng/ml), and IFN- secretion was determined after
48 h.
For RNA analysis, CD4 and CD8 T cells were positively selected by incubating single cell suspensions of LNC with Ab-coated magnetic particles (Miltenyi Biotec, Auburn, CA) for 15 min on ice. Procedures for cell staining, column preparation/loading, as well as T cell recovery were performed according to the manufacturer’s instructions. Separated cells were washed twice with ice-cold PBS and then pelleted and stored at -70°C for subsequent RNA extraction and mRNA analysis. In all instances in which LNC were fractionated, FACS analysis was performed to check the relative purity of the isolated cells. The following conjugated Abs were purchased from PharMingen: CD4 (L3T4), CD8 (Ly-2), B220 (RA3-6B2), Mac-1 (M1/70), CD3e (145-2C11), and NK1.1 (PK136) together with the appropriate isotype controls.
Inhibition of B7 signaling in IL-12-stimulated LNC
Purified Abs against murine B7.1 (16-10A1) and B7.2 (GL1) were
purchased from PharMingen. LNC from rmIL-12-treated mice were isolated
after either 2 or 5 days of treatment. Single cell suspensions of
unfractionated LNC were cultured at a density of 5 x
106 cells/ml in the presence of anti-B7.1 (5
µg/ml), anti-B7.2 (5 µg/ml), or a combination of the two Abs.
Cells stimulated with rmIL-12 and purified isotype-matched control Abs
(10 µg/ml, equivalent to the combination of B7.1 and 2) served as a
control. IFN- secretion was determined after 48 h of
culture.
IL-12R expression
RNA was isolated from either whole lymph nodes (snap frozen in
liquid nitrogen), or fractionated cells using a total RNA isolation
kit, according to the manufacturer’s instructions (Promega, Madison,
WI). Purified RNA was treated with DNase and adjusted to a
concentration of 50 ng/µl before mRNA analysis by TaqMan PCR.
Gene-specific primer pairs and probes for murine IL-12Rß1,
IL-12Rß2, IFN-, CD40 ligand, CD40, IL-10, IL-13, and cyclophilin
were designed using PrimerExpress software, and the labeled probes were
prepared by Perkin-Elmer Applied Biosystems (Foster City, CA). Standard
curves for each gene were generated with RNA from rmIL-12-treated mice
using the relevant probe and primer sets. mRNA expression in control
and IL-12-treated mice (or fractionated cells) was normalized based on
cyclophilin expression in each sample and is presented as relative
units of mRNA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production and IL-12R expression following
systemic treatment with rmIL-12
Treatment of naive C57BL/6 mice with rmIL-12 for 5 consecutive
days resulted in marked increase in lymph node cellularity that was
predominantly due to the expansion of B220+ cells
(data not shown). Similar phenotypic changes have been described
following systemic administration of rmIL-12 to tumor-bearing mice
(29). In addition to the marked increase in cellularity,
LNC from rmIL-12-treated mice displayed an activated phenotype with
increased expression of CD69 and to a greater extent Ly6 A/E, on both T
and B cells (data not shown). Consistent with previously published
findings, serum levels of IFN- increased following systemic
administration of rmIL-12 at a dose of 1 µg/mouse (Fig. 1
a). Quantitative analysis of
lymph node RNA from rmIL-12-treated mice demonstrated an increase in
mRNA for both IL-12Rß1 and ß2 compared with vehicle-treated
controls (Fig. 1b). Consistent with changes in receptor
expression, restimulation of LNC isolated from treated mice, but not
controls, with rmIL-12 for 48 h resulted in a marked increase in
IFN- secretion (Fig. 1c). Similar changes in lymph node
IL-12R expression and IFN- secretion following rmIL-12 treatment
have been observed in more than four separate studies. In addition to
the data generated using TaqMan PCR, increased expression of IL-12Rß1
and IL-12Rß2 in rmIL-12-treated mice has been demonstrated using
RNase protection, although IL-12R mRNA expression was not detected in
LNC from saline-treated mice using this methodology (data not
shown).
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b). In
contrast, of the nonselected cells, 92% stained positively for
B220+ (Fig. 2c) and 6% were positive
for Mac-1 (not shown). Relative expression of IL-12ß1 and IL-12Rß2
mRNA was determined after normalizing cyclophilin in the fractionated
cells to the value obtained for unfractionated LNC isolated from
saline-treated controls. Consistent with the data presented in Fig. 1
b, increased expression of IL-12Rß2 was observed in
single cell suspensions of unfractionated LNC isolated from
rmIL-12-treated mice (Fig. 3b). Separation of the LNC
into highly purified CD4 and CD8 T cells further amplified the relative
expression of both IL-12Rß1 and to a greater extent IL-12Rß2 mRNA
(Fig. 3, a and b). However, in the non-T cell
fraction, both IL-12Rß1 and IL-12Rß2 mRNA were readily detected at
levels close to those observed in the unfractionated LNC from
rmIL-12-treated mice (Fig. 3, a and b). As an
additional control for the purity of sorted cells, CD40 ligand mRNA,
which is expressed almost exclusively on murine T cells, was assessed
in the fractionated cells by quantitative PCR. Although CD40 ligand
mRNA expression decreased following systemic rmIL-12 treatment, its
expression was enriched in positively selected CD4 and CD8 T cells and
almost undetectable in the non-T cell fraction (Fig. 4a). Conversely, CD40 mRNA
expression was found to be preferentially enriched in RNA isolated from
the non-T cell fraction and substantially reduced in positively
selected T cells (Fig. 4b). Despite the significant
enrichment for accessory cells based on flow-cytometric analysis and
distribution of CD40 ligand/CD40 mRNA, there was abundant mRNA for
IFN- detected in the non-T cell compartment of fractionated LNC
isolated from rmIL-12-treated mice (Fig. 4c).
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production in B cell-deficient mice treated with rmIL-12
Data from fractionated LNC isolated from rmIL-12-treated mice
raised the possibility that B cells, which made up the majority of the
non-T cell fraction, may represent an alternative source of IFN-
production. To explore this further, B cell-deficient mice (and
wild-type littermates) were treated with rmIL-12 and serum and lymph
node cytokine production determined after 2 or 5 days of treatment.
Based on FACS analysis of LNC, staining for
NK1.1+ NK cells was comparable in wild-type and B
cell-deficient mice (1.05% vs 0.89%, respectively). Although the
early (day 2) serum IFN- response in rmIL-12-treated wild-type and
knockout mice was similar, serum IFN- levels were substantially
reduced in B cell-deficient mice on day 5 of treatment relative to
controls (Fig. 5a). Assessment
of IFN- production from cultured LNC from rmIL-12-treated knockout
mice demonstrated a similar attenuation of cytokine secretion following
ex vivo stimulation with rmIL-12 (Fig. 5, b and
c). This lack of responsiveness to stimulation with rmIL-12
was apparent despite comparable expression of IL-12Rß1 and IL-12Rß2
mRNA in the lymph nodes of rmIL-12-treated wild-type and knockout mice
(Fig. 6). This finding is consistent with
data from fractionated LNC, in which IL-12R mRNA expression was similar
in the T and non-T cell (primarily B cell) fractions. If IL-12R
expression was restricted to lymph node T/NK cells, one would predict
overrepresentation of IL-12R mRNA transcripts in lymph nodes isolated
from B cell-deficient mice.
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knockout mice
As in vitro studies suggest that IFN- is an important positive
regulator of IL-12Rß2 expression during Th2 differentiation of naive
T cells, studies were performed in IFN- knockout mice to determine
whether early production of this cytokine in response to IL-12 is
required for up-regulation of IL-12Rß2 mRNA expression and subsequent
rmIL-12 responsiveness. As shown in Fig. 7, compared with saline-treated controls,
increased expression of IL-12Rß1 and ß2 mRNA was observed as early
as day 2 in wild-type rmIL-12-treated mice. Despite continued rmIL-12
dosing, comparable expression of lymph node IL-12R mRNA was observed on
day 5. In two separate experiments, a modest delay in the kinetics of
IL-12R up-regulation was observed in rmIL-12-treated IFN- knockout
mice (day 2; Fig. 7); however, by day 5, expression of IL-12Rß2 mRNA
was comparable in both IFN- knockout and wild-type mice treated with
rmIL-12 (Fig. 7). Lymph node cytokine mRNA expression (Fig. 8) as well as protein secretion from
cultured LNC stimulated ex vivo with rmIL-12 (Fig. 9) confirmed the presence of a functional
IL-12R complex in rmIL-12-treated IFN- knockout mice. Furthermore,
in the absence of IFN-, the secretion of both IL-10 and IL-13 from
cultured cells activated with rmIL-12 was enhanced compared with
wild-type controls, suggesting that IFN- may negatively regulate
cytokine secretion in this in vitro assay system.
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from IL-12-treated mice is accessory cell
dependent
To determine whether IL-12R expression on purified T cells was
sufficient to confer IL-12 responsiveness, lymph node T cells from
rmIL-12-treated mice were enriched by nylon wool depletion of accessory
cells before restimulation with rmIL-12 in vitro. The resulting T cell
population was found to be greater than 95% positive for cell surface
CD3 expression (not shown). These highly enriched T cells were cultured
at a density of 2.5 x 106 cells/ml and
IFN- production in response to ex vivo stimulation with rmIL-12
compared with unfractionated LNC (5 x 106
cells/ml) from either control or rmIL-12-treated mice. Consistent with
previous findings, rmIL-12-mediated IFN- secretion from
unfractionated cells was enhanced by in vivo treatment with rmIL-12
(Fig. 10a). In contrast, in
the absence of accessory cells, IFN- secretion from the enriched T
cells stimulated with rmIL-12 was negligible, indicating that cytokine
production was dependent on the presence of accessory cells. In an
attempt to elucidate the nature of the accessory cell-dependent IFN-
response, unfractionated LNC from mice treated with rmIL-12 for either
2 or 5 consecutive days were stimulated ex vivo with rmIL-12 in the
presence of anti-murine B7.1 and/or B7.2 Abs. Stimulation of both
day 2 and day 5 LNC cultures with rmIL-12 alone resulted in marked
IFN- secretion; however, the magnitude of this in vitro response
increased with continued rmIL-12 dosing (Fig. 10, b and
c). This is despite the fact that comparable expression of
lymph node IL-12R mRNA was seen on both days 2 and 5 of dosing (Figs. 6 and 7, wild-type rmIL-12). IFN- production from day 2 LNC stimulated
with rmIL-12 was partially inhibited by the addition of an
anti-murine B7.2 Ab (Fig. 10b). Although the
anti-B7.1 Ab had no effect on rmIL-12-mediated IFN- secretion
from day 2 LNC, essentially complete inhibition of rmIL-12-mediated
IFN- secretion was observed when the B7.1 and B7.2 Abs were added in
combination (Fig. 10b). A similar pattern of cytokine
inhibition was observed following the addition of anti-B7 Abs to
day 5 LNC; however, at this later time point, blocking B7 costimulation
was considerably less effective at inhibiting the rmIL-12-induced
secretion of IFN- (Fig. 10c).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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),
and driving the generation of Ag-specific Th1-type CD4 T cells
(reviewed in Ref. 10). The biological response to IL-12 is
mediated through specific binding to a high affinity receptor complex
that is expressed on NK cells and activated T cells (11).
More recently, the pathways that lead to IL-12R expression on T cells
and subsequent IL-12 responsiveness have been explored in a variety of
in vitro systems. In the context of T cell differentiation, IL-12Rß2
expression is selectively down-regulated on Th2 cells, rendering them
resistant to IL-12-induced IFN- secretion (17, 18).
Similarly, activation of human T cells with PHA in the presence of IL-4
attenuates the response to subsequent IL-12 stimulation and is
associated with the loss of high affinity binding of radiolabeled IL-12
(32). Consistent with these in vitro observations,
IL-12Rß2 mRNA expression in isolated CD4 T cells is rapidly
down-regulated in vivo following infection with Leishmania
major (26). This selective loss of receptor
expression and IL-12 responsiveness can be prevented by appropriate in
vivo treatment with either IL-12 or IFN- (26, 30),
which may explain the therapeutic effects of IL-12 in this model of
parasite infection (33, 34). Although IL-12R expression is a prerequisite for IL-12 responsiveness, the strength of the biological response to IL-12 is influenced by additional signals, including costimulatory molecules and/or cytokines, many of which are supplied by accessory cells. In this context, CD58, CD28, IL-2, IL-15, and IL-18 have all been shown to enhance the response of T or NK cells to IL-12-mediated cellular activation. Whether or not the potentiation of IL-12 responsiveness is mediated solely at the level of IL-12R expression remains to be determined. However, activation of naive murine T cells with anti-CD3 Ab in the presence of B7.2-expressing CHO cells was found to enhance expression of both IL-12Rß1 and IL-12Rß2 mRNA (35). This observation could account for the observed synergistic effects of IL-12 and CD28 Abs for T cell activation (23).
We previously demonstrated that systemic administration of rmIL-12 to
tumor-bearing mice enhanced the responsiveness of LNC to
rmIL-12-mediated IFN- secretion. We demonstrate in this study that
mRNA expression of both IL-12Rß1 and IL-12Rß2 is increased in lymph
nodes following administration of rmIL-12 to naive C57BL/6 mice.
Consistent with the changes in receptor expression, stimulation of LNC
from rmIL-12-treated mice, but not naive animals, with rmIL-12 ex vivo
promoted IFN- secretion. Importantly, these changes were observed in
the absence of exogenously administered Ag, and thus demonstrate the
existence of an autocrine pathway for amplification of IL-12
responsiveness in vivo. Although NK cells may represent a constitutive
source of IL-12R mRNA, the percentage of NK1.1-positive cells in lymph
nodes of naive mice is low (<1.5%), and changes in IL-12R mRNA in
rmIL-12-treated mice occurred in the absence of any increase in
NK1.1-positive cells. In fact, due to the large expansion of B cells
after rmIL-12 treatment, the percentage of NK1.1 cells in the lymph
nodes decreased following treatment with rmIL-12 (1.4% and 0.3% in
control and rmIL-12-treated mice, respectively). Thus, it would seem
unlikely that changes in NK cell number could account for either the
increased expression of IL-12R mRNA or the enhanced secretion of
IFN- following activation with rmIL-12 in these studies. Additional
support for this statement comes from the observation that there was
abundant mRNA for both IL-12R and IFN- detected in highly purified T
cells isolated from the lymph nodes of rmIL-12-treated mice.
Studies in IFN- knockout mice established that the secondary
production of IFN-, which mediates many of the in vivo activities of
IL-12, is not required for up-regulation of lymph node IL-12R mRNA
expression following systemic rmIL-12 treatment. However, in the
absence of IFN-, there was a modest delay in up-regulation of
IL-12Rß2 mRNA compared with wild-type mice treated with rmIL-12.
Given the observation that receptor expression is not restricted to
lymph node T cells (see below), additional studies to determine the
cellular source of IL-12Rß1 and ß2 mRNA expression in
rmIL-12-treated IFN--deficient mice are warranted. Nonetheless,
functional activity of the IL-12R was confirmed in IFN- knockout
mice, as IL-10 and IL-13 secretion in response to ex vivo stimulation
with rmIL-12 were increased relative to saline-treated IFN- knockout
controls. The ability of IL-12 to induce IL-10 secretion in the absence
of IFN- in our current study is consistent with early findings using
neutralizing Abs to IFN- (36). Thus, although we have
demonstrated that IL-12 treatment can increase the expression of a
functional IL-12R complex in the absence of IFN-, the underlying
mediator of this response remains to be determined. As these results
were obtained from naive IFN- knockout mice, it indicates that
regulation of the IL-12R can occur in the absence of either TCR
engagement or secondary IFN- secretion.
Analysis of RNA from fractionated LNC demonstrated abundant mRNA for
both IL-12R chains in the non-T cell fraction following IL-12
treatment. Based on both flow-cytometric analysis as well as the
distribution of CD40/CD40 ligand mRNA, T cell contamination of the
accessory cell fraction was minimal at best. This observation that
IL-12R mRNA expression is not restricted to T cells is consistent with
the findings of Jones et al. (30), in which IL-12 binding
to B220+ cells isolated from IL-12-treated
Leishmania-infected BALB/c mice was demonstrated by
flow-cytometric analysis. Similar results were also obtained using
freshly isolated murine B cells (37); however, in both of
these studies, the expression of IL-12Rß2 in the isolated
B220+ cells was not confirmed by RNA analysis. As
mRNA for IFN- was readily detected in the non-T cell fraction that
consisted primarily of B cells, additional studies were performed in B
cell-deficient mice to determine whether they contribute to the
production of IFN- following systemic treatment with rmIL-12. In
these studies, the early IFN- response to rmIL-12, which has been
attributed primarily to NK cell activation, was comparable in wild-type
and knockout mice. However, with continued administration of rmIL-12,
no further increase in serum IFN- was observed in the B
cell-deficient mice. The change in serum cytokines correlated with
IFN- secretion from cultured LNC stimulated ex vivo with rmIL-12, as
this response was also attenuated in B cell-deficient mice. This
diminished response to ex vivo activation was apparent despite similar
expression of receptor mRNA in the lymph nodes of rmIL-12-treated
wild-type and B cell knockout mice. This finding is consistent with the
observation that IL-12R mRNA expression was similar in the T and non-T
cell (predominantly B cells) fractions of separated LNC, as one would
predict increased expression of IL-12R mRNA in B cell-deficient mice if
IL-12R expression was restricted to T and NK cells. Although the
ability of B cells to secrete IFN- has been somewhat controversial,
highly purified B220+ B cells stimulated with
IL-12 and IL-18 express IFN- mRNA and secrete immunoreactive IFN-
protein (38). Preliminary analysis of lymph node IL-18
mRNA indicates a modest but reproducible increase following
administration of IL-12 (J. P. Leonard, unpublished observation).
As in vitro studies have shown that IL-12 is capable of up-regulating
IL-18R expression on B cells (39), it is conceivable that
a similar in vivo response to IL-12 might lead to appropriate B cell
activation, enabling IFN- secretion. Although we cannot rigorously
exclude the possibility that the attenuated production of IFN-
observed in rmIL-12-treated B cell-deficient mice may be due in part to
a lack of accessory cell-dependent T cell costimulation, quantitative
PCR analysis demonstrated comparable expression of B7.1 and B7.2 mRNA
in the lymph nodes of rmIL-12-treated wild-type and B cell knockout
mice (data not shown). In addition, the proliferative response to
soluble anti-CD3 Ab that is accessory cell dependent was not
diminished in B cell-deficient mice (data not shown), suggesting that
there is sufficient help for T cell activation from resident
professional APCs in the lymph node. Collectively, the findings that
IL-12R and IFN- mRNA were readily detected in the B cell-enriched
fraction of LNC isolated from rmIL-12-treated mice, together with the
diminished production of IFN- protein observed in similarly treated
B cell-deficient mice, support the hypothesis that rmIL-12-responsive B
cells represent an alternative source of IFN- production.
Lymph node cultures from treated mice were used to determine whether
IL-12-induced IFN- secretion was dependent solely on appropriate
IL-12R expression. Neutralizing Abs against murine B7.2, but not B7.1,
partially inhibited the ability of IL-12 to induce IFN- secretion
from unfractionated LNC, indicating that costimulation through this
pathway is required for maximal IL-12 responsiveness. As Abs against
B7.1 alone had little effect on cytokine secretion, it would appear
that B7.2 plays the dominant role in mediating effects of IL-12 in this
system. However, synergistic inhibition of IFN- secretion from
rmIL-12-stimulated LNC was observed when the Abs were combined in
vitro. Although B7.2 has been shown to enhance IL-12R expression on
anti-CD3-activated T cells (35), B7.2 engagement is
not required for in vivo receptor regulation, as expression of
IL-12Rß1 and ß2 mRNA in rmIL-12-treated B7.2 knockout mice was
comparable with that seen in wild-type controls (J. P. Leonard and
J. P. Sypek, unpublished observation).
Although the magnitude of IFN- secretion from cultured LNC increased
with continued rmIL-12 treatment, this heightened responsiveness to
rmIL-12 did not correlate with changes in IL-12R mRNA, as the
expression of IL-12Rß1 and ß2 was similar on both days 2 and 5 of
the study. In contrast, blockade of B7 signaling had a greater
inhibitory effect on rmIL-12-induced IFN- secretion when added to
day 2 cultures compared with day 5, suggesting a B7-independent pathway
for rmIL-12-induced IFN- secretion at this later time point.
However, as purified T cells isolated after 5 days of treatment
remained unresponsive to ex vivo stimulation with rmIL-12, it would
argue that the B7-independent production of IFN- at this later time
point was still dependent on resident accessory cells. The precise
nature of the signals required is yet to be resolved, but could
potentially involve alternate pathways of costimulation or accessory
cell-derived cytokines. If B cells do indeed represent an alternative
source of IFN- secretion, as is suggested by our data, one potential
explanation is that the kinetics of ex vivo cytokine secretion reflects
the time course for appropriate B cell activation in vivo.
The data in this report define a complex pathway of cellular activation
and regulation of IL-12R expression and subsequent IFN- secretion
following systemic administration of rmIL-12. The increased expression
of IL-12Rß1 and ß2 mRNA in rmIL-12-treated mice correlated with the
sensitivity to ex vivo activation with rmIL-12 and was independent of
secondary IFN- secretion. These results are in contrast to in vitro
studies that utilized murine T cells in which IFN- was required for
maintenance of IL-12Rß2 expression during Th2 T cell differentiation.
In addition, results from fractionated LNC as well as B cell-deficient
mice suggest that IL-12-responsive B cells may represent an alternative
source of IFN- production. Although the underlying mechanism that
mediates the observed increase in IL-12R expression remains undefined,
the ability of IL-12 to up-regulate its own receptor represents a
potential autocrine pathway that could lead to amplification of the
biological response. Although this finding may account for the marked
and sustained elevation of serum IFN- observed after IL-12
administration, widespread activation in the absence of exogenously
administered Ag could potentially contribute to the toxicity associated
with systemic IL-12 treatment. The in vitro studies also indicate that
additional signals including costimulation through the B7 pathway can
regulate the magnitude of the biological response to IL-12. This
finding, which is consistent with the CD58-dependent induction of
IFN- from human PHA blasts activated with IL-12, represents an
additional level for regulating IL-12 responsiveness
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. J. P. Leonard, Genetics Institute, Preclinical R&D, One Burtt Road, Andover, MA 01810. E-mail address:
3 Abbreviations used in this paper: rh, recombinant human; LNC, lymph node cell; rm, recombinant murine.
Received for publication April 12, 1999. Accepted for publication August 31, 1999.
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