ARTICLE
In early June this year, the traditional meeting on Cytokines organised
by the French Society of Immunology (namely M. Dy, A. Vasquez, H. Gascan,
Y. Jacques) did not take place in Paris as usual, but close to the sea
side in a very charming town on the Brittany coast, Le Croisic. Several
sessions were organised to give an update on cytokines, their receptors
and their signalling pathways, in various contexts such as immunology,
haematology, apoptosis and cellular differentiation. Beside plenary sessions,
selected posters were presented as short communications.
The opening session focused on the IL-12 cytokine family. G. Trinchieri
(Schering-Plough, Dardilly, France) gave an overview of the biological
effects of IL-12. This cytokine is a heterodimeric, proinflammatory cytokine
consisting of a light chain, defined as p35, disulfide-linked to a heavier
chain, p40. The p35 subunit is similar to other alpha-helix hematopoeitin
cytokines, whereas the p40 subunit is similar to the extracellular portion
of a hematopoeitin receptor, and is most closely related to the IL-6 or
CNTF alpha receptor. IL-12 is inducible by the activation of toll-like
receptors 2, 4 and 9 and other receptors such as CD44, RANKL, CD40 ligands.
G. Trinchieri emphasized the immunoregulatory function of IL-12 and the
ability of this cytokine to promote the secretion of IL-2 and IFN-gamma,
and to inhibit the production of IL-4, IL-5, IL-6 and IL-10. The highlight
of this presentation was the control of IL-12 secretion. It has been reported
that the p35 alpha-chain is the limiting factor in the control of IL-12
heterodimer production. New results concerning the post-translational
control of p35 by glycosylation were presented. Deglycosylation experiments
using neuraminidase and Endo-F to examine intracellular versus
extracellular IL-12 heterodimer revealed that the p35 chain was modified
with sialic acid adducts to N-linked oligosasccharides before secretion.
Inhibition of N-glycosylation by tunicamycin altered the formation and
the secretion of the heterodimer p70. There was no association of the
alpha and beta chain in the absence of glycosylation of p35, however secretion
of p40 was not altered. This modification of p35 during maturation affected
the cleavage of leader peptide. There are two cleavage events of p35,
one independent of glycosylation and one where glycosylation is absolutly
necessary [1]. Lastly, data about inactivation of p35/p40 and beta1/beta2
receptor chains were presented. These experiments showed a major deficit
in mice deficient for p40 or beta1, but no defects were observed in the
case of inactivation of p35 or beta2.
O. Devergne (INSERM U. 131, Paris, France) presented an overview on
EBI3 protein (Epstein-Barr virus-induced gene 3). EBI3 is a soluble hematopoietin
component related to the p40 subunit of IL-12. This protein shares 30%
homology with CNTFR and p40. EBI3 is localized in the endoplasmic reticulum
in association with the molecular chaperone calnexin. In cells transfected
with an expression vector coding for EBI3 and p35, EBI3 co-immunoprecipitates
with the p35 subunit of IL-12. This association is not covalent. In
vivo, EBI3 extract from the trophoblast component of a human full-term
normal placenta, co-immunoprecipitates with p35, indicating that EBI3
forms a heterodimer with p35. The biological effects of this heterodimer,
and in particular the antagonist effect on IL-12, was suggested but no
data were presented to support this idea [2, 3]. Finally, O. Devergne
presented new data on the localisation of EBI3. It is expressed in placenta
cells, trophoblast and some populations of the extra-villous trophoblast.
During pregnancy, a soluble form of EBI3 is detected in serum, but is
not associated with any known function. At the end of her talk, some questions
about the inactivation of EBI3 and its potential effects on pregnancy
were raised, but this work is in progress.
The third speaker, R. Kastelein (DNAX, Palo Alto, USA) started his talk
with the presentation of a new cytokine, IL-50 or human thymic stromal
lymphopoietin (hTSLP). This new cytokine acts via the IL-7 receptor
alpha chain and an orphan receptor, hRdelta2. This orphan receptor is
related to the gamma2 chain and murine TSLP receptor. The activation of
this complex, IL-7Ralpha and hTSLPR, by IL-50 leads to phosphorylation
of STAT3 and STAT5. IL-50 or human TSLP acts on myeloid and dendritic
cells. It induces the release of T cell-attracting chemokines (TARC) by
monocytes and CD11c+ dendritic cells. hTSLP enhances the maturation
of CD11c+ dendritic cells (induction of the co-stimulatory
molecules CD40 and CD80) and displays enhanced capacity to elicit proliferation
of naive T cells [4]. In the second part, he presented the new cytokine
IL-23. It is composed of a novel subunit, p19 and the p40 subunit of IL-12
and shows biological activities similar to IL-12 but is also distinct
from IL-12. IL-23 is secreted by activated dendritic cells and bound to
the beta1 chain of the IL-12 receptor but fails to engage the IL-12 Rbeta2.
After binding to the beta1 chain and the novel receptor subunit, IL-23
induces the phosphorylation of STAT4. The biological activities of this
new heterodimeric cytokine are restricted to T cells. IL-23 induces Th1
differentiation of T cell CD4+ from cord blood and induces
a strong proliferation of mouse and human T memory cell. This last activity
is unique, as IL-12 has no effect on this cell type. Like IL-12, IL-23
induces the secretion of IFN-gamma by T cell [5]. Transgenic mice overexpressing
IL-23 die rapidly, with a chronic inflammation. Short oral presentations
closed this session on the IL-12 cytokine family. The work of Leite-de-Moraes
et al., described the ability of IL-12 to induce in vivo
the production of INF-gamma and TNF-alpha in NK T cells. Data presented
by L. Derré et al., demonstrated that IL-12 induces the
receptor CD94/NKG2A on a T cell clone CD8+. A short presentation
focused on CLC (cardiotrophin-like cytokine), the new member of IL-6 cytokine
family. H. Plun-Favreau et al., demonstrated that co-expression
of CLC and soluble CNTF receptor in mammalian cells generated a secreted
composite cytokine, which displayed activities on cells expressing LIF
receptor and gp130. The last short presentation by J. Bitard et al.,
focused on the binding site of LIF on the LIF receptor. The data presented
showed that LIF bound to the Ig domain of the LIF receptor.
The second session of this meeting focused on two cytokine families,
the IL-10 and the IL-2/IL-15 family. J.C. Renauld (Ludwig Institute for
Cancer Research, Brussel, Belgium) started this session by presenting
a new cytokine related to IL-10, called IL-TIF or IL-22. Isolation of
this new molecule was performed by a cDNA substraction after stimulation
of mouse T cells by IL-9. The cDNA regulated by IL-9 encoded a gene of
180 amino acids and revealed 22% identity with IL-10. This novel cytokine
was designated IL-TIF for IL-10-related T cell-derived inducible factor
or IL-22 and is mainly expressed in the thymus and brain. In vivo,
transgenic mice for IL-9 did not modify the expression of IL-22. The biological
activities of IL-22 are mediated by a heterodimeric receptor. This receptor
is composed of the beta chain of the IL-10 receptor and CRF2-9. CRF2-9
was originally described as an orphan receptor called ZCYTOR11. The activation
of this receptor leads to phosphorylation of Jak1, Tyk2 and STAT 1,3.
Other signalling pathways are recruited by this receptor, as MAP kinase,
p90 RSK and p38. IL-22 acts on mesengial, melanoma, intestine, lung, HUVEC,
and hepathoma cells but does not activate monocyte cells. In vivo,
IL-22 up-regulates acute phase reactant production by liver cells, indicating
that this cytokine has a potential role in the inflammatory process [6].
The end of this talk focused on cloning of a new receptor, CRF2-10. This
soluble receptor shares 33% amino acid identity with the extracellular
domain of the IL-22 receptor. In fact, this new soluble receptor is able
to bind to IL-22 and inhibits the activity of this cytokine. The IL-22
binding protein, IL-22BP, is expressed in various tissues with maximal
expression in breast cells [7]. Concerning interleukin-15 and its receptors,
S. Bulfone-Pons (University of Lübeck, Germany) presented new data
concerning IL-15 and its role in the immune system. Beside the known role
of this cytokine in Th1 development, she described the implication of
IL-15 in Th2 response by using a murine model of allergic sensitisation
to ovalbumin (OVA). Intraperitoneal injection of IL-15 enhances production
of IgE in response to OVA injections and induces IL-4 and IL-5 production
whereas injection of a soluble alpha chain of the IL-15 receptor, acting
as an IL-15 antagonist, suppresses the production of IgE and prevents
the OVA-induced allergic pulmonary inflammation. These data highlighted
a new role of IL-15 in enhancing a Th2 response. New results concerning
the role of the different chains of the IL-15 receptor were presented.
IL-15 receptor is made up of three chains alpha (specific for IL-15),
beta and gamma (shared by IL-2). On memory CD8 T cells, IL-15-induced
proliferation requires the beta and gamma chain, whereas its anti-apoptotic
effect can be mediated by the alpha chain, in the absence of beta and
gamma chain (M. Berard et al.). This latter observation was also
confirmed in a model of ceramide-induced apoptosis in a human B cell line,
where there is no implication of the beta chain (S. Bulfone-Paus). The
role of IL-15 in T cells has also been described in an IL-15 overexpressing
transgenic model (S. Dubois et al.), emphasing the role of IL-15
in blocking IL-2-induced activation induced cell death (AICD). IL-15 seems
to set activate T cells in a quiescent state similar to anergy, blocking
AICD. These results clearly demonstrate that IL-15 and IL-2 act differentially
and favour opposing actions, which tend to emphasise two competing major
goals of the immune response: maintenance of the peripheral self-tolerance
for IL-2 and maintenance and survival of CD4 and CD8 T cells for IL-15.
In the context of hematopoeisis, B. Azzarone et al. showed new
data identifying a hybrid receptor GM-CSFRbeta/IL-15Rgamma involved in
the GM-CSF dependant activation of Stat5.
The last plenary session focused on cytokine implication in apoptosis
and cell differentiation. Apoptosis plays a central role in development
and homeostasis of metazoans. In the immune system, apoptosis is essential
as it leads to destruction of infected, autoreactive or cancer cells,
and to deletion of activated T lymphocytes at the end of the immune response.
The signalling pathways of apoptosis can be divided into two components,
involving either the mitochondria or death receptors (Fas, TNF-R, DR3,
4 et 5). Activation of one of those pathways ultimately results in activation
of caspases, proteases leading to morphological characteristics of apoptotic
cells. In plenary sessions, an update and new data on apoptosis in the
immune and hematopoietic system were presented, including several short
communications, mainly on apoptotic pathways in immune pathologies. O.
Hermine (URA CNRS 1461, Paris, France) emphasised the role of caspases
in the erythroid maturation, demonstrating that these proteins, transiently
activated via the mitochondrial pathway during erythroblast differentiation,
activate proteins involved in DNA and nucleus condensation. Activity of
caspases may then account for the nuclear structural changes associated
with the maturation of erythroblasts [8]. Such activation of caspases
has been demonstrated in other enucleation systems (keratinocytes, crystalline
lens cells). P. Auberger (INSERM U. 526, Nice, France) presented new data
on the role of Src kinases in apoptosis in T cells. He identified the
protein Fyn as a substrate of caspases in death receptor-mediated apoptosis
in the Jurkat cell line. Cleavage of this protein increases its kinase
activity and its translocation from the plasma membrane to the cytoplasm
[9]. His data also support a role for Fyn in TCR-induced activation of
the caspases in a murine T cell model. Another set of data presented by
J. Bertoglio (INSERM U. 461, Chatenay-Malabry, France) illustrated the
implication of small GTPases Rho in apoptosis. One of the Rho effectors,
the serine/threonine kinase ROCK possesses a DETD motif, a motif recognised
for cleavage by caspases. Like PAK, a Rac effector, ROCK is also cleavable
by these proteases, and this cleavage induces its activation [10]. One
may then postulate that caspases reorganise cell structures indirectly
by activating proteins involved in actin cytoskeleton regulation. A. Atfi
(INSERM U. 482, Paris, France) presented new data on TGF-beta signalling
pathway and highlighted the role of the Jun kinase cascade both in apoptosis
induced by the protein Smad7 and in the suppression of the Smad2 transcriptional
activity [11, 12]. Several short oral communications focused on apoptosis
in pathologies, as dysregulation of apoptotic pathways is often associated
with defects in maintaining homeostasis in the immune and hematopoetic
compartments. The work of E. Schneider et al. demonstrated that
the Fas/Fas-L pathway can confer an immune privilege to thyrocytes in
the context of experimental autoimmune thyroiditis (EAT) and that this
Fas-dependent protection required IL-10. In another immune pathology,
the autoimmune lymphoproliferative syndrome type II, C. Vonarbourg et
al. suggested that the functional defect in apoptosis associated with
this disease could be due to a defect in the Fas signalling pathway and
illustrated a Fas-induced cell death dependent of FADD but independent
of caspases. Data on signalling pathways involved in TGF-beta-induced
apoptosis were presented by M.F. Bourgeade. She described the MAPK/p38
kinase pathway as necessary in TGF-beta-induced activation of caspases
in a model of Burkitt's lymphoma, highlighting differences in caspases
activation induced by TGF-beta or by death receptors, which require adaptor
proteins like FADD, TRADD. E. Schneider et al., assessed the implication
of Fas/Fas-L in murine hematopoiesis. She presented data supporting the
implication of a subset of NK cells in Fas-mediated apoptosis of hematopoietic
progenitors, not directly but via TGF-beta secretion.
In conclusion, characterisation of new cytokines and dissection of signalling
pathways have been the main focus of this meeting. Data presented were
of a high quality and a number have been published in international journals
since this meeting. The common interests of the participants lead to exciting
and enriching debates and everybody agreed to meet again next year at
the same place to share new results.
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