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*
Institute for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, New York, NY 10029;
Department of Molecular Genetics and Microbiology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ 08854; and
Department of Molecular Biology, DNAX Research Institute, Palo Alto, CA 94304
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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hIL-10R1 is species specific and does not bind the mouse IL-10 (mIL-10), whereas mIL-10R1 binds both mIL-10 and hIL-10 (1, 2). Parenthetically, the homologue of the EBV BCRF I gene product, known as viral IL-10 (vIL-10), is active on both murine and human cells (9, 10, 11, 12, 13). Despite the evidence that vIL-10 shares many immunosuppressive activities with cellular IL-10 (cIL-10) (9, 10, 11, 12, 13), vIL-10 lacks several cIL-10 activities on certain cell types. vIL-10 neither up-regulated MHC class II expression on mouse B cells, nor stimulated mouse thymocyte or mast cell proliferation (11, 14, 15). The findings in vivo that vIL-10, but not cIL-10, prolongs allograft transplant survival while cIL-10, but not vIL-10, accelerates tumor rejection confirmed their differential activities (16, 17, 18). Further analysis of IL-10R1 function showed that vIL-10 binds to IL-10R with at least 1000-fold lower affinity than cIL-10 (18, 19). Despite its poorer receptor-binding ability, vIL-10 has similar specific activities on certain cells (9, 10, 11, 12, 13, 18, 19). The existence of additional IL-10R component(s) besides IL-10R1 that may complement the deficiency in binding and/or mediate enhanced IL-10R activation by vIL-10 has been proposed (1, 2, 19). The discovery of IL-10R2 has made it possible to investigate whether IL-10R2 differentially regulates vIL-10 binding and/or signal transduction, and whether differentially regulated receptor binding and/or signal transduction lead to changes in the spectrum of biological activities.
The binding of IL-10 to its cell surface receptors activates the Janus kinase (JAK)-STAT signal transduction pathway. Following the ligand-receptor interaction, Jak1 (associated with IL-10R1) and Tyk2 (associated with IL-10R2), members of the receptor-associated Janus tyrosine kinases (JAK) family, are phosphorylated. A family of latent cytoplasmic transcription factors, STATs, are then activated (20, 21, 22, 23, 24, 25). Among the seven mammalian family members, Stat1, Stat3, and, in some cells, Stat5 are activated by IL-10 (3, 23). Further studies proved that Stat3 activation is essential for all known IL-10-mediated immune responses, although the activation of Stat3 alone is not sufficient for IL-10-mediated anti-inflammatory response (26, 27). However, signal transduction initiated by vIL-10 has not been fully investigated.
To address these issues, we compared the roles of IL-10R2 in receptor binding and activation for hIL-10 and vIL-10. Consistent with previous findings (3, 18, 19), we demonstrate here that although IL-10R2 does not bind either hIL-10 or vIL-10, its presence is essential for both hIL-10- and vIL-10-mediated signal transduction. Furthermore, we found that hIL-10 and vIL-10, despite having different binding affinities, mediate identical signal transduction in cells in which they induce similar biological responses. However, the inability to stimulate immune responses in MC/9 cells by vIL-10, compared with hIL-10, is largely due to the complete inability to initiate any signal activation in these cells. The inability to initiate signaling by vIL-10 is mainly but not wholly due to low level expression of cell surface IL-10R1. Higher level IL-10R1 expression on stably transfected MC/9 cells allows vIL-10 to have hIL-10-like activity in terms of signal transduction and biological responses. This suggests that cell surface IL-10R1 expression is a determining factor in cellular responses to vIL-10 and implies that limited activity on certain cell types may account for the differential immune regulation profile between cIL-10 and vIL-10. These results also indicate that through modulation of cell surface IL-10R1, IL-10 could have immunostimulatory or immunosuppressive profiles in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The 16–9 CHO cells transfected with an HLA-B7 reporter
construct and expressing hIL-10R1/hIFN-
R1 chimera, hIL-10R2, or
hIL-10R1/hIFN-R1 plus hIL-10R2 (3) were maintained in
complete F12 medium containing 450 µg/ml G418. The mouse pro-B cell
line Ba/F3 expressing recombinant mIL-10R1 was maintained in RPMI 1640
medium containing 10% FCS, 5 ng/ml murine IL-3 (PeproTech, Rocky Hill,
NJ), and 1 mg/ml G418 (Life Technologies, Grand Island, NY)
(1). The mouse mast cell line MC/9 was purchased from
American Type Culture Collection (Manassas, VA) and maintained in DMEM
medium containing 10% FCS and 5% Con A supernatant prepared from CBA
mouse spleen cells. Thymocytes and splenocytes were prepared from adult
CBA mouse thymus and spleen as previously described
(18).
Proteins and Abs
hIL-10, hIL-10I87A, vIL-10, and vIL-10A87I were prepared as previously described (18). The 1B1.2 rat anti-mouse IL-10R1 hybridoma (26) was grown and culture supernatants were purified by MabTrap G II columns (Amersham, Uppsala, Sweden). Biotin-labeled anti-mIL-10R1 was purchased from BD PharMingen (San Diego, CA). Anti-phosphorylated Stat1, anti-phosphorylated Stat3, anti-phosphorylated Stat5, anti-Stat1 and anti-Stat3 (New England Biolabs, Beverly, MA), and anti-Stat5 (BD PharMingen) were purchased.
125I-labeled hIL-10 binding/competitive displacement
125I-labeled hIL-10 (75–140 µCi/µg) was purchased from NEN Life Science Products (Boston, MA). 125I-labeled hIL-10 binding and competitive displacement was assessed as described elsewhere (18, 28, 29). CHO cells (1 x 106) expressing various IL-10R component(s) were incubated in 200 µl as duplicate samples in various concentrations of 125I-labeled hIL-10 (binding) or 100 pM 125I-labeled hIL-10 in the presence of between 0.6 pM and 200 nM hIL-10 or vIL-10 (competitive) for 4 h at 4°C in RPMI 1640, 2% BSA, and 0.02% sodium azide (binding buffer), with shaking. Reaction mixtures were overlaid onto 150 µl of dibutyl phtalate:dioctyl phtalate (3:2) in conical bottom tubes and centrifuged for 1 min (Eppendorf 5413). The pellets were cut off with a razor blade and analyzed in a COBRA II gamma counter (Packard Instrument, Downers Grove, IL).
EMSA of STATs
CHO cells (2 x 106) expressing
different IL-10R component(s) were treated with either hIL-10 or vIL-10
for 15 min. The cells were lysed and nuclear extracts obtained for
binding to 32P-labeled 22-bp IFN--activation
sequence element in the promoter region of the human IRF-1 gene
(3, 18). For supershift assays, 1 µl of anti-Stat1
or anti-Stat3 Abs was added to the incubation mixture, incubated
for 20 min at 22°C, and then 4 µl of the reaction mixture was
electrophoresed at 200 V for 4 h at 4°C on a 16 x 16-cm
5% polyacrylamide (19:1 acrylamide:bisacrylamide) gel. The dried gel
was exposed to Kodak XAR-5 film (Kodak, Rochester, NY) with an
intensifying screen for 1 day at -80°C. When Ba/F3-mIL-10R1 or MC/9
cell lines were used, 5 x 106 cells were
plated in complete RPMI 1640 medium, cultured for 4 h, and then
stimulated with hIL-10 or vIL-10 at various times. Nuclear extracts and
EMSAs were performed as previously described by using the
32P-labeled 22-bp probe based on the IFN-
response region (GRR) within the promoter of the FcRI gene
(25).
Northern blot hybridization analysis
Total RNA was isolated from Ba/F3, Ba/F3 transfected with mIL-10R1, and MC/9 cell lines by an acid guanidium thiocyanate-phenol-chloroform extraction method, and poly(A)+ RNA was purified using a RiboSep mRNA isolation kit (Collaborative Research, Bedford, MA). Northern blot hybridization was performed as recommend by protocol with the PerfectHyb Plus hybridization buffer system (Sigma-Aldrich, St. Louis, MO). Full-length mIL-10R1 and mIL-10R2 probes were generated by RT-PCR.
Flow cytometric analysis
Cells were washed three times in PBS supplemented with 1% BSA (PBS/BSA) and then stained with 5 µg/ml purified or biotin-labeled rat anti-mIL-10R1 mAb in PBS/BSA at 4°C for 45 min. After washing three times with PBS/BSA, cells were then stained with goat anti-rat IgG conjugated with FITC or streptavidin-PE for 45 min at 4°C (1:30 dilution; BD Biosciences, Mountain View, CA). The cell surface expression of the mIL-10R1 was analyzed in a BD Biosciences FACScan using CellQuest software. Controls included unstained cells or secondary reagent alone.
Generation of stable MC/9-mIL-10R1 cell lines
mIL-10R1 cDNA was cloned into the pcDNA3.1 vector (Invitrogen, Carlsbad, CA) by RT-PCR from Ba/F3-mIL-10R1 cells and sequenced to confirm fidelity. pcDNA3.1-mIL-10R1 plasmid was linearized by SspI enzyme digestion. DNA constructs were then transfected into 1 x 107 MC/9 cells at 600 V in a cuvette with a 0.2-cm electrode gap in 200 µl of DMEM medium by Gene Pulser apparatus (Bio-Rad, Hercules, CA). Two days later, cells were cultured with selective medium containing 1.2 mg/ml G418 and 2 wk later flow cytometry was performed to verify mIL-10R1 expression. Cell sorting was conducted on single-cell suspensions stained with mIL-10R1 held on ice during sorting. The selected populations expressing different levels of mIL-10R1 were sorted directly into tubes with DMEM medium with 5% Con A supernatants. All cells were routinely monitored by flow cytometry to ensure stable expression of mIL-10R1.
Proliferative responses
MC/9 cells were rested in RPMI 1640 with 10% FCS medium
overnight, then 5 x 104 cells were plated
in 96-well round-bottom plates (Corning, Corning, NY) at a density of
5 x 104/well in 200 µl. hIL-10 or vIL-10,
with or without 5 µg/ml anti-mIL-10R1 mAbs, was added as
indicated at a range of concentrations. Cells were incubated with
stimuli for 24 h, and proliferation was assessed 6 h after
adding 1 µCi/well [3H]thymidine (Amersham
Life Sciences, Arlington Heights, IL) by measuring radioactivity using
a MicroBeta TriLux 
counter (Wallac, Turku, Finland). Thymocyte
proliferation assay was performed as previously described
(18).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether IL-10R2 binds or enhances the binding of
IL-10 to IL-10R1, 16-9 CHO cells expressing various IL-10R components
were used. IL-10-induced signaling and biological responses are cell
restricted and CHO cells will not transduce IL-10R and Stat3 events,
but will transduce IFN--like signaling and Stat1-dependent MHC class
I up-regulation (3). Therefore, cells were transfected to
express hIL-10R2 alone; hIL-10R1 chimeric receptor, in which the
transmembrane and intracellular domains of the hIFN-R1 chain are
substituted for the transmembrane and intracellular domains of the
hIL-10R1 chain; or both receptors. Nontransfected 16-9 CHO cells were
included as a control. As shown in Fig. 1
A, CHO cells and cells
expressing IL-10R2 do not bind 125I-labeled
hIL-10. In contrast, cells expressing IL-10R1 alone or IL-10R1 along
with IL-10R2 bind 125I-labeled hIL-10. These data
demonstrate that IL-10R1 but not IL-10R2 binds IL-10. Scatchard
analysis showed that the calculated Kd
for hIL-10 is 
56 pM on cells expressing IL-10R1/R1 alone or both
receptors (Fig. 1B). These data indicate that IL-10R2 does
not enhance the binding affinity of hIL-10 for IL-10R1.
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C). Cells were incubated with a constant
concentration of 125I-labeled hIL-10 along with
various amounts of unlabeled hIL-10 or vIL-10. As expected, hIL-10 is
an effective competitor while vIL-10 is a very weak competitor. The
receptor binding affinity for hIL-10 is at least 1000-fold higher than
vIL-10. The data showed that there are no binding differences for
either hIL-10 or vIL-10 on cells expressing IL-10R1 alone or both
receptors, indicating that IL-10R2 does not bind to or enhance binding
affinity of vIL-10 to the IL-10R complex.
Previous studies revealed that only cells expressing both IL-10Rs could
mediate cIL-10-induced signal transduction and immune responses
(3). To determine whether the functional difference
between cIL-10 and vIL-10 is due to a differential utilization of the
IL-10R complex, we examine the signal transduction of vIL-10 in CHO
cells expressing IL-10R2 alone, IL-10R1/IFN-R1 chimeric receptor
alone, or both receptors. We previously showed that hIL-10 stimulates
IFN--like responses only in those cells that express both receptors,
inducing both Stat1 activation and MHC class I Ag reporter gene
up-regulation (3, 18). Fig. 1D shows that both
vIL-10 and hIL-10 induced STAT activation only in cells expressing both
receptors, and the DNA-binding complex can be completely supershifted
by anti-Stat1 mAb. This indicates that despite affinity
differences, vIL-10 induces the same STAT protein activation as hIL-10,
and this activation requires expression of both receptors. Flow
cytometric analysis of MHC class I Ag expression on the same cells
showed that both hIL-10 and vIL-10 induced similar class I MHC
up-regulation (data not shown). This further supports the finding that
vIL-10 induces signal transduction and immune responses through the
same receptor complex as does cIL-10 in these cells.
Both hIL-10 and vIL-10 induce STAT activation in Ba/F3 cells
hIL-10 and vIL-10 have similar specific activities despite
1000-fold differences in receptor binding affinity in Ba/F3 cells
(16, 17). To investigate whether vIL-10 induces different
signals compared with hIL-10, Ba/F3 cells stably transfected with
mIL-10R1 were used to examine IL-10-mediated JAK-STAT activation. It
was previously reported that cIL-10 treatment of these IL-10R1-bearing
Ba/F3 cells led to activation of Stat1, Stat3, and Stat5 (25, 26). Fig. 2A shows that
vIL-10 and hIL-10 induce similar DNA-binding complexes, and the
DNA-binding complexes appeared within 5 min, were maximal by 15 min,
and disappeared 90 min after stimulation (Fig. 2A). Fig. 2B shows that the dose response of STAT activation to hIL-10
and vIL-10 are also similar. These data indicate that hIL-10 and vIL-10
are concordant in their abilities to induce JAK-STAT in this B cell
line. Fig. 3C further shows that both
ligands similarly activate Stat1, Stat3, and Stat5 in this cell
line.
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Since cIL-10, but not vIL-10, is a potent stimulator of murine
MC/9 mast cell line proliferation (4, 6), we next examined
STAT activation by hIL-10 and vIL-10 using EMSAs in this discordant
cell line. In hIL-10-stimulated cells, STAT activation occurred within
10 min (Fig. 3A). However, in vIL-10-treated cells, STAT
activation was not detected, even after prolonged stimulation (Fig. 3A). The supershift experiment shows that the DNA-binding
complexes in MC/9 cells consist of at least Stat1 and Stat3 (Fig. 3B). Western blotting is consistent with these findings and
shows that IL-10 activates only Stat1 and Stat3 in MC/9 cells (Fig. 3C). Fig. 3C also demonstrates that hIL-10 but
not vIL-10 induced STAT activation in MC/9 cells; vIL-10-induced STAT
phosphorylation only occurred in Ba/F3 cells (Fig. 3C).
These results demonstrate that proliferative responses correlate with
STAT activation, and that hIL-10 induces normal STAT activation,
whereas vIL-10-induced signal transduction appears to be completely
impaired in MC/9 cells.
Impaired signal transduction by vIL-10 in MC/9 cells is due to differential IL-10R1 but not IL-10R2 expression
IL-10 and IL-10Rs share many structural and functional features
with IFN- and IFNRs (1, 2, 30, 31, 32, 33, 34, 35, 36). In addition,
IFN- activates signal transduction in Th2 but not Th1 cells, due to
an absence of IFN-R2 in the Th1 cells (37, 38).
Therefore, we determined whether the unresponsiveness of MC/9 cells to
vIL-10 is due to lack of expression of IL-10R2. Northern blot analysis
shows that there are similar levels of IL-10R2 mRNA expression in Ba/F3
and MC/9 cells (Fig. 4A). The
result is consistent with the fact that hIL-10 can mediate normal
signal transduction in these cells and suggests that differential
IL-10R2 expression likely does not play a role in the differential
activities between hIL-10 and vIL-10 in MC/9 cells.
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A),
consistent with the fact that Ba/F3-mIL-10R1 was established by stably
transfecting mIL-10R1 into Ba/F3 cells (1, 19). Flow
cytometric analysis to measure cell surface mIL-10R1 protein expression
(Fig. 4B) confirms that mIL-10R1 is expressed at much higher
levels on Ba/F3-mIL-10R1 than MC/9 cells. Scatchard analysis further
confirms there are 3000 receptors per Ba/F3-mIL-10R1 cell with an
affinity of 50 pM (23), compared with 200 receptors per
MC/9 cell with the same affinity (Fig. 4C). High-level IL-10R1 expression can render MC/9 cells responsive to vIL-10
To determine whether the differential expression of IL-10R1
between Ba/F3-mIL-10R1 and MC/9 cells could account for the
differential signal transduction and proliferative responses by hIL-10
and vIL-10, we generated a series of MC/9 cells expressing different
levels of mIL-10R1. Full-length mIL-10R1 cDNA was generated by RT-PCR
from Ba/F3-mIL-10R1 cells and cloned into the pcDNA3.1 expression
vector. This plasmid encoding mIL-10R1 was transfected into MC/9 cells
by electroporation, and stable transfectants were then selected in the
presence of G418. We were able to establish MC/9 cells expressing
different levels of mIL-10R1 by FACS sorting (Fig. 5A). The higher level of
expression in MC/9 cells was comparable to that in Ba/F3-mIL-10R1
cells, while medium and low-level mIL-10R1 expression were
considerably less.
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B, the intensity of STAT activation by
hIL-10 in MC/9 is dependent upon cell surface mIL-10R1 expression. In
medium-level mIL-10R1-expressing cells, STAT activation by hIL-10 is
stronger than in MC/9 or MC/9-neo, which express low levels of
mIL-10R1, while the activation was strongest in cells expressing high
levels of mIL-10R1. No vIL-10-induced STAT activation was observed in
MC/9 cells or MC/9 cells expressing the empty pcDNA3.1 vector. However,
in vIL-10-stimulated MC/9 cells expressing medium or high levels of
mIL-10R1, activation of STATs was observed. The intensity of STAT
activation is also dependent on the cell surface mIL-10R1 expression;
it is much stronger in MC/9 expressing high levels of mIL-10R1 compared
with cells with medium levels of mIL-10R1 expression. Supershift
experiments demonstrated that the DNA-binding complexes activated by
hIL-10 in MC/9 cells expressing higher level mIL-10R1 were mostly Stat3
along with some Stat1 (Fig. 5C). In vIL-10-stimulated cells,
the major DNA-binding complex was also Stat3 (Fig. 5C), and
Stat1 activation could be observed after longer film exposure of
72 h instead of 36 h (data not shown). The combination of
anti-Stat1 and anti-Stat3 supershifted the majority of
DNA-binding complexes, suggesting that Stat1 and Stat3 activation were
the most dominant events in these cells, consistent with the Western
blotting results of Fig. 3C. We next determined whether
higher level mIL-10R1 expression could lead to proliferative responses
in MC/9 cells by vIL-10. Fig. 5D demonstrates that vIL-10
could induce MC/9 proliferation only in those cells expressing higher
densities of cell surface mIL-10R1. Anti-mIL-10R1 mAb completely
abrogated proliferation, proving that the proliferative response
requires a specific receptor-ligand interaction, rather than the
overexpression of the mIL-10R1 acting as a coreceptor in a novel
fashion. These data show that cell surface receptor density determines
both the threshold and the magnitude of response to high- and
low-affinity ligands. However, despite the three orders of magnitude
differences in ligand affinity, receptor densities among the cell lines
differ only 10-fold (Figs. 4C and 5A) and
signaling responses also differ by far less than 1000 (Fig. 5B), once threshold is reached. Murine IL-10R1 expression is associated with the biologic responses of primary cells to cIL-10 and vIL-10
cIL-10 but not vIL-10 enhances IL-2 plus IL-4-driven proliferation
of thymocytes, while other cells (e.g., splenocytes) respond similarly
to cIL-10 or vIL-10 (1, 14, 18, 19). To investigate
whether the lack of a biological response to vIL-10 in thymocytes is
also due to insufficient mIL-10R1 expression, mouse thymocytes and
splenocytes were isolated and flow cytometric analysis was performed.
As predicted, thymocytes express much lower levels of mIL-10R1 compared
with splenocytes (Fig. 6A).
Northern blotting on thymocytes and splenocytes also showed that
IL-10R1 mRNA is expressed at much higher levels in splenocytes compared
with thymocytes, while IL-10R2 mRNA is expressed at similar levels
(Fig. 6B). We next investigated Stat3 activation in both
splenocytes and thymocytes. As shown in Fig. 6C, hIL-10
activates Stat3 phosphorylation in both splenocytes and thymocytes, and
the activation is weaker in thymocytes. In contrast, vIL-10 induces
Stat3 activation only in splenocytes but not thymocytes, and induces a
weaker phosphorylation compared with hIL-10, probably due to its lower
receptor affinity. These data support the notion that levels of IL-10R1
expression are also critical in determining IL-10-mediated immune
responses of primary cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Expression of IL-10R2 did not change the IL-10R1-binding affinity
for hIL-10 or vIL-10, showing that IL-10R2 is not involved in binding
to the ligand (Fig. 1). Next, comparing signaling events, only cells
expressing both IL-10R1 plus IL-10R2 can respond to vIL-10, consistent
with previous findings with hIL-10 (Fig. 1D; Ref.
3). These data demonstrate that IL-10R2 is an essential
receptor subunit for both cIL-10- and vIL-10-mediated signal
transduction. vIL-10 and hIL-10 induce Stat1, Stat3, and Stat5
activation in the pro-B cell line Ba/F3 cells transfected with mIL-10R1
(Figs. 2 and 3). Collectively, these studies demonstrate that vIL-10 is
capable of inducing the same signals and biological responses as
cIL-10.
To investigate the mechanism for vIL-10 unresponsiveness in certain
cells, the STAT activation mediated by hIL-10 and vIL-10 in the MC/9
mast cell line was studied. It has been previously shown that hIL-10
but not vIL-10 induces proliferation in these cells (11, 14). Here, we found that there is no detectable STAT activation
after vIL-10 stimulation, indicating severely impaired signal
transduction (Fig. 3). This signal transduction defect was not due to
defects in JAKs or STATs, since hIL-10 can mediate normal JAK-STAT
activation in these cells (Fig. 3).
Differential receptor component expression on different cell types is
an important mechanism for cytokines to selectively regulate their
biological responses. For example, like IL-10, the functional IFN-R
complex is also composed of two chains, IFN-R1 and IFN-R2
(34, 35, 36). Although IFN-R1 binds IFN- with high
affinity, only cells expressing both receptor components can mediate
IFN--exerted signal transduction and biological activity. IFN-
inhibits the proliferation on Th2 cells but not on Th1 or Tc1 cells.
Unresponsiveness to IFN- on Th1 cells is due to lack of IFN-R2
expression on Th1 or Tc1 (37, 38). Down-regulation of the
IFN-R2 expression in Th1 cells may allow the immune system to
selectively inhibit Th2 cells and promote Th1 cell phenotypes.
Furthermore, Novelli et al. (39) showed that proliferation
or apoptosis of T cells was dependent on the ratio of the IFN-R1 and
IFN-R2 chains on the cell surface. Because IL-10 bears close
topological resemblance to IFN- and their receptor complexes also
belong to the same cytokine receptor family, we investigated whether
IL-10R2 plays the same role in determining whether cells respond to
vIL-10. The results show that IL-10R2 is expressed at similar
transcriptional levels in both Ba/F3 and MC/9 cells, and indicate that
it is unlikely that IL-10R2 determines differential responses to vIL-10
(Fig. 4).
We next investigated whether the lack of signal transduction in MC/9
cells is due to insufficient mIL-10R1 expression. Consistent with this
hypothesis, vIL-10 induced STAT activation and proliferation in MC/9
cells expressing higher levels of mIL-10R1 (Fig. 5), and an increase in
intensity of DNA-binding complexes results from an increase in the
density of cell surface mIL-10R1 (Fig. 5B). Thus, upon
increasing cell surface IL-10R1 expression, vIL-10 mediates similar
signal transduction and immune responses as does hIL-10; cell surface
receptor expression is a critical factor in determining whether cells
are responsive to the low-affinity ligand.
The finding that the density of IL-10R1 expression on the cell surface
plays a critical role in determining whether certain cells are
activated by an IL-10 molecule may explain the differential in vivo
immune regulatory profiles among various IL-10 molecules (26, 27). vIL-10, which has 1000-fold lower affinity, may only
activate cells with a high level of receptors. On the other hand,
cIL-10 may activate cells expressing both low and high levels of
IL-10R1. Depending on the method of administration (local or systemic),
dose, immune mediators present, and the precise mixture of cells
present, this could explain why vIL-10 is generally immunosuppressive,
while cIL-10 is more pleiotropic in its effects. Our results also
indicate that modulating IL-10R1 expression on certain cells might be
an important mechanism for regulating IL-10-mediated immune responses.
In a recent study on different types of dendritic cells, it was found
that rheumatoid dendritic cells are resistant to the immunosuppressive
effect of IL-10 in vitro due to insufficient expression of cell surface
IL-10R1 (40). As noted above, similarly the ratio of the
IFN-R1 and IFN-R2 chains on the cell surface can modulate the
response of T cells from proliferation to apoptosis
(39).
Although the data explain why vIL-10 mediates cell proliferation in
Ba/F3 cells but not in MC/9 cells, there is not a strict correlation
among affinities, receptor densities, and signaling responses, either
within or between cell lines. Thus, hIL-10 and vIL-10 induce STAT
activation and cell proliferation with the same specific activities in
Ba/F3 cells, despite a 1000-fold difference in receptor-binding
affinities. Similarly, once threshold is reached in MC/9 cells,
receptor affinity, density, and signaling responses do not strictly
correlate (Fig. 5). Thus, the signaling and biological responses do not
represent linear correlates to ligand-receptor occupancy, indicating
that additional mechanisms might regulate between Ba/F3 and MC/9 cell
line responses.
First, it is possible that additional molecules are activated in one cell line but not the other. For example, beside Stat1 and Stat3 activation, Stat5 is activated in Ba/F3 cells expressing mIL-10R1 (25). This additional Stat5 activation appears to be cell restricted and recent findings suggested that Stat5 has pleiotropic functions regulating cell proliferation, differentiation, and apoptosis in the Ba/F3 cell line (41). Therefore, Stat5 might lower the threshold of proliferative signaling in Ba/F3 cells, resulting in similar responses, despite the 1000-fold difference in affinity. It is also possible there are differences in signal transduction molecules downstream of STAT activation between Ba/F3 and MC/9 cells.
A second possibility is that there is differential activity by negative regulation in Ba/F3 cells vs MC/9 cells. The suppressors of cytokine signaling (SOCS) family of molecules is characterized by an Src homology 2 domain and a carboxyl-terminal, unique conserved motif referred to as the SOCS box (42). There are at least eight SOCS proteins (SOCS1–7 and cytokine-inducible SH2-containing protein) and SOCS1 and SOCS3 appear to be the most potent inhibitors of cytokine signaling. It has been recently shown that SOCS3 plays a role in negatively regulating IL-10 signaling (43). Thus, it is possible that SOCS3 is regulated differently in Ba/F3 than in MC/9 cells. Our ongoing studies show that induction of SOCS3 expression by cIL-10 and vIL-10 is concordant in Ba/F3 cells but discordant in MC/9 cells (Y. Ding and J. S. Bromberg, unpublished observation) just as the other second messengers examined here. Thus, SOCS3 expression probably does not determine the differences between these two cell lines.
Third, there may be an additional component or components of the IL-10R
complex. This component may complement the deficiency in binding and/or
mediate enhanced IL-10R activation by vIL-10. Additionally, it must be
noted that although we proved there are similar levels of IL-10R2 mRNA
expressed in both Ba/F3 and MC/9 cells (Fig. 4C), we did not
determine the levels of IL-10R2 protein due to lack of Abs against
IL-10R2. Very recently, it was shown that IL-10R2 is involved in IL-22
binding and signal transduction (44, 45). Although these
reports did not provide evidence for receptor subunit competition,
IL-22 and the IL-22R complex may affect IL-10-mediated signal
transduction and biological responses. It must be noted that our Ba/F3
and MC/9 lines do not express IL-22 transcripts (our unpublished
data).
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2 Address correspondence and reprint requests to Dr. Jonathan S. Bromberg or Dr. Yaozhong Ding, Institute for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1496, New York, NY 10029-6574. E-mail addresses: jon.bromberg@mountsinai.org or yaozhong.ding{at}mssm.edu
3 Abbreviations used in this paper: hIL-10, human IL-10; CHO, Chinese ovary cell; mIL-10, mouse IL-10; vIL-10, viral IL-10; cIL-10, cellular IL-10; JAK, Janus kinase; SOCS, suppressors of cytokine signaling.
Received for publication May 7, 2001. Accepted for publication October 11, 2001.
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