Received for publication, July 26, 2002, and in revised form, October 31, 2002
Interleukin (IL)-17 is a pro-inflammatory
cytokine that is produced by activated T cells. Despite increasing
evidence that high levels of IL-17 are associated with several chronic
inflammatory diseases including rheumatoid arthritis, psoriasis, and
multiple sclerosis, the regulation of its expression is not well
characterized. We observe that IL-17 production is increased in
response to the recently described cytokine IL-23. We present evidence
that murine IL-23, which is produced by activated dendritic cells, acts
on memory T cells, resulting in elevated IL-17 secretion. IL-23 also induced expression of the related cytokine IL-17F. IL-23 is a heterodimeric cytokine and shares a subunit, p40, with IL-12. In
contrast to IL-23, IL-12 had only marginal effects on IL-17 production. These data suggest that during a secondary immune response, IL-23 can promote an activation state with features distinct
from the well characterized Th1 and Th2 profiles.
 |
INTRODUCTION |
Interleukin (IL)1-17 is
a T cell-derived pro-inflammatory molecule that stimulates epithelial,
endothelial, and fibroblastic cells to produce other inflammatory
cytokines and chemokines including IL-6, IL-8, G-CSF, and
MCP-1 (1-8). IL-17 also synergizes with other cytokines
including tumor necrosis factor-
and IL-1
to further induce
chemokine expression (7, 9). Interleukin-17 levels are found to be
significantly increased in rheumatoid arthritis synovium (10, 11),
during allograft rejection (12-15), and in other chronic inflammatory
diseases including multiple sclerosis (16) and psoriasis (17-19).
Although clearly produced by activated T cells, previous reports have
not provided clear classification of IL-17 within the paradigm of Th1
and Th2 polarized cytokine profiles.
We have examined the possibility that IL-17 is expressed in
response to signals distinct from those associated with the Th1 or Th2
response. We observe a previously unrecognized activity of the recently
identified cytokine IL-23 (20). IL-23 is a heterodimeric cytokine that
shares one subunit, p40, with IL-12. The initial characterization of
this cytokine has suggested it can promote proliferation within the
memory T cell population. Subsequent work demonstrated that transgenic
over-expression of the second component of IL-23, p19, was sufficient
to induce systemic inflammation and premature death (21). In addition,
the mice had markedly elevated levels of circulating neutrophils.
Interestingly they did not exhibit consistent elevation of IFN-
, a
hallmark effect of IL-12. These data suggest that IL-23 may have a
biological role substantially distinct from that of IL-12. In this
report we present evidence that IL-23 acts to induce a distinct T cell activation state that produces IL-17 as a principle effector cytokine.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture--
Single cell suspensions of spleen were
prepared from C57/BL-6 mice, and mononuclear cells were isolated from
suspended splenocytes by density gradient centrifugation. 2 × 106 cells/ml were cultured with IL-2 (100 units/ml) in the
presence or absence of various stimuli (for times indicated in figure
legends), following which the cells were collected and analyzed for
IL-17 using ELISA (R&D Systems, Minneapolis, MN). Dendritic cells were derived from macrophages (obtained as adherent population from splenocyte suspension) by treating macrophages with rGM-CSF (2 ng/ml) and rIL-4 (1000 units/ml) for 4 days, washing and re-activating using LPS (0.5 µg/ml). Memory and naïve T cells were isolated by staining mononuclear cells isolated from single cell suspension of
murine splenocytes with CyC-CD4 + PE-CD44 or CyC-CD4 + PE-CD62L and
sorting for CD4+ cells that were either
CD44high/CD62Llow for memory phenotype or
CD44low/CD62high for naïve phenotype.
In vitro Induction of T Cell
Differentiation--
CD4+ T cells were purified from
spleen of wild type C57/BL6 mice using anti-CD4 magnetic beads
(Miltenyi Biotech). Purified T cells (2 × 106
cells/ml) were activated for 3 days by plating on plates coated with 5 µg/ml anti-CD3 and 1 µg/ml anti-CD28 antibodies. The cultures were
supplemented with IL-2 and treated with IL-12 (20 nM) + anti-IL-4 (0.5 µg/ml) (for Th1 differentiation), IL-4 (1000 units/ml) + anti-IFN-
(0.5 µg/ml) (for Th2 differentiation), or IL-23 (10 nM)(for IL-17 production). Following initial activation,
the cell cultures were washed extensively and re-stimulated with
anti-CD3 (1 µg/ml) for another 24 h, following which the cell
supernatants were analyzed for various secreted cytokines using
ELISA.
IL-12p40 Antibody Inhibition of IL-17 Induction--
Anti-IL-12
antibody (R&D Systems, cat no. AF-419-NA) or an unrelated control
antibody (anti-FGF-8b (R&D Systems, cat no. AF-423-NA)) were
pre-incubated with IL-23 (100 ng/ml) or conditioned media of
LPS-stimulated dendritic cells (10% v/v) for 1 h at 37 °C and then incubated for another 5-6 days with mononuclear cells isolated from mouse spleen (2 × 106 cells/ml). Supernatants
were collected and levels of IL-17 measured using ELISA.
Purification of IL-23--
Murine IL-23 component was produced
by co-expression of carboxyl-terminal His-tagged p19 and
FLAG-tagged p40 in human embryonic kidney cells (293 cells), and
secreted protein was purified by nickel affinity resin. Endotoxin
levels were undetectable at less than 0.2 endotoxin units per
µg.
 |
RESULTS |
We first examined the ability of various microbial products to
stimulate the production of IL-17. Increased IL-17 has recently been
observed by Infante-Duarte et al. (22) in response to
microbial lipopeptides from a Lyme disease causing spirochete,
Borrelia burgdorferi. We observed that spleen cell
cultures in the presence of various microbial products including LPS
(Gram-negative bacteria), lipoteichoic acid (Gram-positive
bacteria) or lipopeptide (bacterial lipopeptide) resulted in
the production of IL-17 (Fig. 1). Neither purified T cells alone nor purified macrophages themselves produced IL-17. Purified T cells, upon receptor cross-linking using plate-bound anti-CD3 and treatment with supernatants from activated
macrophages/dendritic cells, produced increased IL-17, indicating the
presence of an unidentified factor(s) released by these cells that acts
on T cells to promote IL-17 production.

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 1.
IL-17 production in different cell
types. A, mononuclear splenocytes were cultured in the
presence or absence of microbial lipopeptide (100 ng/ml), LPS (100 ng/ml), or lipoteichoic acid (100 ng/ml) for 3 days, following which
the cells were collected and analyzed for IL-17 using ELISA.
B, purified T cells were obtained from murine splenocytes
following positive selection of fluorescence-activated cell-sorted,
CD4-labeled cells. These cells were cultured (1 × 106
cells/ml) in presence or absence of plate-bound anti-CD3 (5 µg/ml) or
supernatant from activated dendritic cells (LPS-treated) for 3 days and
culture supernatants collected and analyzed for IL-17 levels using an
ELISA kit. Representative results from three independent experiments
are shown.
|
|
In profiling the expression of candidate molecules that might be
responsible for this IL-17 promoting activity, we observed 100-1000-fold increased mRNA expression of the IL-23 (20)
components, p19 and p40, in activated dendritic cells using real-time
RT-PCR (not shown), hence, the effect of IL-23 was examined. Murine
spleen cell cultures, in the presence of IL-23, resulted in high levels of IL-17 production in a dose-dependent manner (Fig.
2A). However, when these cells
were cultured under IL-12-stimulated Th1-inducing conditions,
they resulted in marginal IL-17 production, whereas under Th2-inducing
conditions there was no increased production of IL-17 over controls
(Table I). IL-23 also resulted in higher levels of GM-CSF than observed under Th1-inducing conditions. In
contrast, IFN-
levels were significantly lower than those obtained
under Th1-inducing conditions. Tumor necrosis factor-
levels were
similar to Th1 conditions. IL-12p40 alone did not result in any IL-17
production (data not shown). IL-23 promoted elevated levels of IL-17
mRNA (Fig. 2B). IL-17 mRNA levels were increased
several hundred-fold within 6 h of IL-23 exposure and remained
elevated in the continued presence of IL-23. This effect was not
inhibited by the presence of an antibody against IL-17, suggesting that
the IL-17 itself was not contributing to this process (not shown). In
addition, mRNA for IL-17F, a recently identified IL-17 family
member (23, 24), was also found to be up-regulated in response to IL-23
(Fig. 2C).
IL-23 has been reported to promote the proliferation of memory but not
naïve T cells (20, 25). We therefore examined the effect of
IL-23 on IL-17 production from naïve versus memory T
cell populations. Purified CD4+ T cells were isolated from
splenocytes by fluorescence-activated cell sorting. The memory cell
population was selected as CD4+CD44high (26) or
CD4+CD62Llow (27), and naïve cell
population was selected as CD4+CD44low or
CD4+CD62Lhigh. As seen in (Fig.
3), IL-23 stimulated robust IL-17
production within the memory cell population (CD44high and
CD62Llow) and little or no IL-17 production within the
naïve (CD44low or CD62Lhigh) cell
population.
IL-23-mediated IL-17 production was completely blocked in the presence
of a neutralizing IL-12 antibody (R&D Systems) that interacts with the
p40 subunit shared with IL-23 (used due to unavailability of
neutralizing antibodies specific to IL-23p19 or IL-23) (Fig.
4A and 4B,
left panel). This effect was not due to ligation of Fc
receptors on antigen-presenting cells as there was no change in IL-17
production in the presence of unrelated antibody. This antibody also
inhibited >50% the induction of IL-17 production observed in response
to conditioned media from LPS-stimulated dendritic cells (Fig.
4B, right panel). A marked reduction, but not
abrogation, of IL-17 production was seen in response to ConA stimulation from spleen cell cultures of mice lacking IL-12p40 component (strain B6.129S1-IL12btm1Jm) as compared with
wild type mice or mice lacking IL-12p35 component (strain
B6.129S1-IL12atm1Jm) (Fig. 4B). Thus, IL-23
plays a substantial role in promoting IL-17 production, although it is
clearly not absolutely required.
Finally, to examine the role of IL-12 in IL-17 production, we added
increasing amounts (0.001-1 nM) of murine IL-12 to IL-23 (1 nM) containing cultures. As seen in (Fig.
5A), IL-12 decreased IL-17
levels in a dose-dependent manner. Additionally, we treated splenocytes from mice lacking IL-12 receptor beta chain 2 (IL-12R
2) (28), the specific receptor component of IL-12 (29) with purified IL-23. Splenocytes from IL-12R
2
/
mice responded to
IL-23 stimulus by increasing IL-17 production over the un-stimulated
control (Fig. 5B) without affecting IFN-
levels.
Surprisingly, the background levels of IL-17 in these mice were more
than 10-fold elevated as compared with wild type mice, suggesting a
possible negative regulation by IL-12 of IL-23-induced IL-17
production. However, in contrast to IL-12R
2 knockout mice, we did
not observe increased IL-17 in spleen cultures from IL-12p35 knockout
mice. The reasons for this difference are not known, but could relate
to alteration in IL-12p40 function in the absence of p35, or
differences in genetic background or pathogen exposure.
Taken together these data suggest a role for IL-23 in the
promotion of a distinct T cell activation state that expresses IL-17 as
an effector cytokine. The Th1 and Th2 paradigms have been described as
promoting cell-mediated versus humoral immune responses.
These responses provide important defense for intracellular and
extracellular pathogens, respectively, and defects in either of these
responses are associated with increased susceptibility to specific
pathogens. In contrast, IL-23 may serve to promote an adaptive immune
response to pathogens that is characterized by a heavy reliance on
cells thought to function primarily as mediators of the innate immune response. IL-17, as a principle effector cytokine of this response, is
able to promote the more rapid recruitment of monocytes and neutrophils
through induced chemokine production. In addition, the GM-CSF
production observed in response to IL-23 supports the production of
additional myeloid cells. This is further augmented by high level G-CSF
production from local IL-17-stimulated stromal cells. The character of
this adaptive response is, however, not an exclusive reliance on
phagocytic cells of the myeloid lineage as IL-17 is known to promote
the induction of ICAM thereby providing important co-stimulation
of further T cell responses. The actions of IL-23 appear to be
restricted to memory T cells. However, it remains to be determined
whether there exist co-stimulatory signals that enable action of IL-23
on naïve T cell populations. Further analysis with TCR
transgenic animals will help to clarify these issues.
We thank Andy Chan, Iqbal Grewal, Sherman
Fong, Wenjun Ouyang, and Paul Godowski for their comments and suggestions.
Published, JBC Papers in Press, November 3, 2002, DOI 10.1074/jbc.M207577200
The abbreviations used are:
IL, interleukin;
IFN, interferon;
ELISA, enzyme-linked immunosorbent
assay;
LPS, lipopolysaccharide;
RT, reverse transcription;
ConA, concanavalin A;
KO, knockout;
Ct, cycle threshold.
1.
|
Aggarwal, S.,
and Gurney, A. L.
(2002)
J. Leukoc. Biol.
71,
1-8[Abstract/Free Full Text]
|
2.
|
Yao, Z.,
Fanslow, W. C.,
Seldin, M. F.,
Rousseau, A. M.,
Painter, S. L.,
Comeau, M. R.,
Cohen, J. I.,
and Spriggs, M. K.
(1995)
Immunity
3,
811-821[Medline]
[Order article via Infotrieve]
|
3.
|
Kennedy, J.,
Rossi, D. L.,
Zurawski, S. M.,
Vega, F., Jr.,
Kastelein, R. A.,
Wagner, J. L.,
Hannum, C. H.,
and Zlotnik, A.
(1996)
J. Interferon Cytokine Res.
16,
611-617[Medline]
[Order article via Infotrieve]
|
4.
|
Fossiez, F.,
Djossou, O.,
Chomarat, P.,
Flores-Romo, L.,
Ait-Yahia, S.,
Maat, C.,
Pin, J. J.,
Garrone, P.,
Garcia, E.,
Saeland, S.,
Blanchard, D.,
Gaillard, C.,
Das, M. B.,
Rouvier, E.,
Golstein, P.,
Banchereau, J.,
and Lebecque, S.
(1996)
J. Exp. Med.
183,
2593-2603[Abstract]
|
5.
|
Linden, A.,
Hoshino, H.,
and Laan, M.
(2000)
Eur. Respir. J.
15,
973-977[Abstract/Free Full Text]
|
6.
|
Cai, X. Y.,
Gommoll, C. P., Jr.,
Justice, L.,
Narula, S. K.,
and Fine, J. S.
(1998)
Immunol. Lett.
62,
51-58[CrossRef][Medline]
[Order article via Infotrieve]
|
7.
|
Jovanovic, D. V., Di, B. J. A.,
Martel-Pelletier, J.,
Jolicoeur, F. C., He, Y.,
Zhang, M.,
Mineau, F.,
and Pelletier, J. P.
(1998)
J. Immunol.
160,
3513-3521[Abstract/Free Full Text]
|
8.
|
Laan, M.,
Cui, Z. H.,
Hoshino, H.,
Lotvall, J.,
Sjostrand, M.,
Gruenert, D. C.,
Skoogh, B. E.,
and Linden, A.
(1999)
J. Immunol.
162,
2347-2352[Abstract/Free Full Text]
|
9.
|
Chabaud, M.,
Fossiez, F.,
Taupin, J. L.,
and Miossec, P.
(1998)
J. Immunol.
161,
409-414[Abstract/Free Full Text]
|
10.
|
Kotake, S.,
Udagawa, N.,
Takahashi, N.,
Matsuzaki, K.,
Itoh, K.,
Ishiyama, S.,
Saito, S.,
Inoue, K.,
Kamatani, N.,
Gillespie, M. T.,
Martin, T. J.,
and Suda, T.
(1999)
J. Clin. Invest.
103,
1345-1352[Abstract/Free Full Text]
|
11.
|
Chabaud, M.,
Durand, J. M.,
Buchs, N.,
Fossiez, F.,
Page, G.,
Frappart, L.,
and Miossec, P.
(1999)
Arthritis Rheum.
42,
963-970[CrossRef][Medline]
[Order article via Infotrieve]
|
12.
|
Antonysamy, M. A.,
Fanslow, W. C., Fu, F., Li, W.,
Qian, S.,
Troutt, A. B.,
and Thomson, A. W.
(1999)
Transplant Proc.
31,
1-2
|
13.
|
Antonysamy, M. A.,
Fanslow, W. C., Fu, F., Li, W.,
Qian, S.,
Troutt, A. B.,
and Thomson, A. W.
(1999)
J. Immunol.
162,
577-584[Abstract/Free Full Text]
|
14.
| Loong, C. C., Lin, C. Y., and Lui, W. Y. (2000)
Transplant Proc. 32
|
15.
|
Hsieh, H. G.,
Loong, C. C.,
Lui, W. Y.,
Chen, A.,
and Lin, C. Y.
(2001)
Transpl. Int.
14,
287-298[CrossRef][Medline]
[Order article via Infotrieve]
|
16.
|
Kurasawa, K.,
Hirose, K.,
Sano, H.,
Endo, H.,
Shinkai, H.,
Nawata, Y.,
Takabayashi, K.,
and Iwamoto, I.
(2000)
Arthritis Rheum.
43,
2455-2463[CrossRef][Medline]
[Order article via Infotrieve]
|
17.
|
Albanesi, C.,
Scarponi, C.,
Cavani, A.,
Federici, M.,
Nasorri, F.,
and Girolomoni, G.
(2000)
J. Invest. Dermatol.
115,
81-87[Abstract/Free Full Text]
|
18.
|
Homey, B.,
Dieu-Nosjean, M. C.,
Wiesenborn, A.,
Massacrier, C.,
Pin, J. J.,
and Oldham, E.
(2000)
J. Immunol.
164,
6621-6632[Abstract/Free Full Text]
|
19.
|
Teunissen, M. B.,
Koomen, C. W.,
de Waal Malefyt, R.,
Wierenga, E. A.,
and Bos, J. D.
(1998)
J. Invest. Dermatol.
111,
645-649[Abstract]
|
20.
|
Oppmann, B.,
Lesley, R.,
Blom, B.,
Timans, J. C.,
Xu, Y.,
Hunte, B.,
Vega, F.,
Yu, N.,
Wang, J.,
Singh, K.,
Zonin, F.,
Vaisberg, E.,
Churakova, T.,
Liu, M.,
Gorman, D.,
Wagner, J.,
Zurawski, S.,
Liu, Y.,
Abrams, J. S.,
Moore, K. W.,
Rennick, D.,
de, W.-M. R.,
Hannum, C.,
Bazan, J. F.,
and Kastelein, R. A.
(2000)
Immunity
13,
715-725[Medline]
[Order article via Infotrieve]
|
21.
|
Wiekowski, M. T.,
Leach, M. W.,
Evans, E. W.,
Sullivan, L.,
Chen, S. C.,
Vassileva, G.,
Bazan, J. F.,
Gorman, D. M.,
Kastelein, R. A.,
Narula, S.,
and Lira, S. A.
(2001)
J. Immunol.
166,
7563-7570[Abstract/Free Full Text]
|
22.
|
Infante-Duarte, C.,
Horton, H. F.,
Byrne, M. C.,
and Kamradt, T.
(2000)
J. Immunol.
165,
6107-6115[Abstract/Free Full Text]
|
23.
|
Hymowitz, S. G.,
Filvaroff, E. H.,
Yin, J. P.,
Lee, J.,
Cai, L.,
Risser, P.,
Maruoka, M.,
Mao, W.,
Foster, J.,
Kelley, R. F.,
Pan, G.,
Gurney, A. L.,
de Vos, A. M.,
and Starovasnik, M. A.
(2001)
EMBO J.
20,
5332-5341[Abstract/Free Full Text]
|
24.
|
Starnes, T.,
Robertson, M. J.,
Sledge, G.,
Kelich, S.,
Nakshatri, H.,
Broxmeyer, H. E.,
and Hromas, R.
(2001)
J. Immunol.
167,
4137-4140[Abstract/Free Full Text]
|
25.
| Frucht, D. M. (2002) Sci. STKE 8
|
26.
|
Budd, R. C.,
Cerottini, J. C.,
Horvath, C.,
Bron, C.,
Pedrazzini, T.,
Howe, R. C.,
and MacDonald.
(1987)
J. Immunol.
138,
3120-3129[Abstract/Free Full Text]
|
27.
|
Jung, T. M.,
Gallatin, W. M.,
Weissman, I. L.,
and Dailey, M. O.
(1988)
J. Immunol.
141,
4110-4117[Abstract/Free Full Text]
|
28.
|
Wu, C.-Y.,
Wang, X.,
Gadina, M.,
O'Shea, J. J.,
Presky, D. H.,
and Magram, J.
(2000)
J. Immunol.
165,
6221-6228[Abstract/Free Full Text]
|
29.
|
Chua, A. O.,
Wilkinson, V. L.,
Presky, D. H.,
and Gubler, U.
(1995)
J. Immunol.
155,
4286-4294[Abstract]
|
30.
|
Decken, K.,
Kohler, G.,
Palmer-Lehmann, K.,
Wunderlin, A.,
Mattner, F.,
Magram, J.,
Gately, M. K.,
and Alber, G.
(1998)
Infect. Immun.
66,
4994-5000[Abstract/Free Full Text]
|
31.
|
Cooper, A. M.,
Kipnis, A.,
Turner, J.,
Magram, J.,
Ferrante, J.,
and Orme, I. M.
(2002)
J. Immunol.
168,
1322-1327[Abstract/Free Full Text]
|
32.
|
Elkins, K. L.,
Cooper, A.,
Colombini, S. M.,
Cowley, S. C.,
and Kieffer, T. L.
(2002)
Infection & Immunity
70,
1936-1948[Abstract/Free Full Text]
|
33.
|
Holscher, C.,
Atkinson, R. A.,
Arendse, B.,
Brown, N.,
Myburgh, E.,
Alber, G.,
and Brombacher, F.
(2001)
J. Immunol.
167,
6957-6966[Abstract/Free Full Text]
|