Thesis
Reference
Dissecting IL-6 responses in inflammation
LISSILAA, Rami
Abstract
L'Interleukin-6 (IL-6) joue un rôle primordial dans le développement de réponses immunitaires locales (exemple: inflammation au niveau des tissus synoviaux) et dans la coordination des réponses immunitaires systémiques (exemple: induction de la sécrétion d'anticorps par les cellules B activées et différenciées, ou induction de la sécrétion de protéines de la phase aïgue par les hépatocytes). Cependant, la contribution relative des deux voies de signalisation activées par l'IL-6 (voie cis et voie trans) dans le développement d'une réponse immunitaire locale et systémique reste à déterminer. Buts de la thèse : - Explorer les rôles respectifs des voies de cis-signalisation et de trans-signalisation dans le développement d'une réponse inflammatoire. Afin de définir les rôles respectifs des voies cis et trans lors d'une réponse inflammatoire locale et systémique, nous avons : - Généré des anticorps monoclonaux ciblant de différentes manières les 2 voies de signalisation activées par l'IL-6. - Utilisé ces anticorps in vivo dans deux modèles murins d'inflammation.
LISSILAA, Rami. Dissecting IL-6 responses in inflammation. Thèse de doctorat : Univ.
Genève, 2010, no. Sc. 4239
URN : urn:nbn:ch:unige-127866
DOI : 10.13097/archive-ouverte/unige:12786
Available at:
http://archive-ouverte.unige.ch/unige:12786
Disclaimer: layout of this document may differ from the published version.
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The main difference between the two domains is that the V-domain contains the antigen- binding properties, whereas the C-domain mediates effector functions (e.g. ADCC, CDC).
This superfamily is composed of proteins that play crucial roles in the immune system.
Among them, the antibody is of particular interest.
An antibody is characterized by a flexible Y-shaped structure, formed by three globular regions. The two antigen-binding sites of the antibody are localized at the tips of the
“Y”- shaped arms of the molecule. Each antigen-binding site is formed by the association of a light chain with the amino-terminal half of a heavy chain. Both light and heavy chains are made up two and four Ig domains, respectively. A flexible stretch, called the hinge region, tethers the three globular regions of the antibody. This hinge region confers flexibility to the antibody, as it allows the arms of the molecule to have independent movement. In addition, the hinge region of the antibody links the two arms of the antibody to the stem of the Y called the constant region (C-region), that confers effector function to the antibody as it interacts with cells or molecules via for example Fc receptors or complement receptors.
Figure 1: Structure of an antibody
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Figure 2: The folded structure of an IgG antibody molecule.
(taken from E.W. Silverton, M.A. Navia, and D.R. Davies, Proc. Natl. Acad. Sci. USA 74:5140, 1977; B, after M. Schiffer, R.L. Girling, K.R. Ely, and A.B. Edmundson, Biochemistry 12:4620, 1973).
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Figure 3: Type I, type II or type III interferon signaling.
(taken from the review “Interferons at age 50: past, current and future impact on biomedecine” Ernest C.
Borden et al., Nature reviews Drug Discovery, 2008)
3) The TNF receptor Superfamily
The tumor necrosis factor (TNF) family is composed of cytokines that are involved in important biological processes such as cell survival, death and differentiation (27), and maintenance of lymphoid homeostasis. The first two members of the TNF family, cloned in 1984, were TNF-! and the lymphotoxin ! (LT-!, also named TNF-") (28). LT-! is usually linked to the cell surface and forms heterotrimers with another member of the TNF family, LT-!. TNFRI and TNFRII, the receptors of both TNF# and LT, form homotrimers when bound to these ligands. More generally, the trimeric structure is a feature of all members of
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Figure 4: Representation of the ligands of the TNF superfamily and their receptors.
(from the review: “signalling pathways of the TNFsuperfamily: a double-edged sword” Bharat B.
Aggarwal.Nature Reviews Immunology, 2003).
The DD initiates the activation of a cascade of cellular proteases, called caspases, responsible for cell death. Among the members of proteins containing a DD in their cytoplasmic tail, CD95 is of particular interest. This protein is also termed fas and is expressed on the surface of various cell types, including activated lymphocytes, and plays a crucial role in regulating lymphocyte homeostasis. The crucial role of CD95 in the immune system is illustrated by the
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Figure 5: Chemokine receptors and their ligands
Chemokines and chemokine receptors are also classified depending on whether their production is constitutive or inducible. The chemokines constitutively secreted play a role in basal leukocyte recruitment and trafficking. Despite their low level of production, these chemokines are involved in the maintenance of homeostasis and immune system. For example, a deficiency for CXCL13 (the ligand of CXCR5) leads to impaired splenic primary follicle development in mice (37). Inducible chemokines, in contrast, are secreted in high levels during inflammatory disorders and their receptors are implicated in disease. Two CXC chemokines have been identified as inducible chemokines: CXCL10 (commonly called IP-10) and CXCL8 (commonly called IL-8). These chemokines and their respective receptors have been shown to be involved in various immune disorders as shown in figure 6.
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Figure 6: Chemokine receptors and diseases.
(from the review:“Chemokine receptors: multifaceted therapeutic targets” Amanda E.I. Proudfoot.
Nature review, 2002).
5) Type I cytokine receptor family
The eythropoietin receptor was the first member of the hematopoietic growth factor receptor superfamily to be identified. A notable feature of this family is their redundancy, that is, many cytokines can bind to a common receptor, and one cytokine can bind to several cytokine receptors. This superfamily of receptors is comprised of class I helical cytokine receptors consisting of homodimers or heterodimers binding to specific ligands. Studies performed on growth hormones have provided new insights into cytokine-receptor interactions (38). The hematopoietic growth factor receptor family, also defined as a type I cytokine receptor family, constitutes the largest group among the cytokine family of receptors. It is composed of three main sub-families based on the sharing of common signal transducing chains containing an
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Figure 7: IL-3, IL-5, and GM-CSF Share a Common Cytokine Receptor ! chain.
As the three cytokines share a common !c subunit, they have significant overlapping functions and play a crucial role in hematopoiesis (43). IL-3, for instance, is considered as a pluripotent stem cell growth factor, IL-5 is involved in differentiation of eosinophils and basophils and GM-CSF is key in differentiation of macrophages and granulocytes. The importance of these cytokines in the immune system is illustrated by the in vivo consequences of disrupting the !c subunit. Targeted mutation of this shared subunit leads to immune disorders including lung lesions with histological resemblance to pulmonary alveolar proteinosis and, more generally, an impaired host defense (44).
5.2) Common " chain receptor
IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 share a common gamma chain ("c) subunit for their receptors. Initially, this "c subunit was identified as a component of the IL-2 receptor complex and was originally termed IL-2R" chain (45)."This name was later changed to "c subunit as
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studies performed on mice deficient for this subunit or the IL-2 cytokine demonstrated different consequences on T and NK cell development. More precisely, mice lacking the “IL- 2R"c” have altered numbers of T and NK cells, whereas a disruption of the IL-2 gene results in normal T and NK cell development (46). These observations suggested that the use of the IL-2"c subunit was not restricted to IL-2 but was probably utilized by receptors of other cytokines. Thus, the IL-2R"c chain was termed the "c subunit, and shown to be expressed by IL-4R, IL-7R, IL-15R and IL-21R, all members of the IL-2 family mediating redundant functions. This redundancy explains the pathogenesis of X-linked Severe combined immunodeficiency (SCID), that is an inherited disorder of the immune system in which the gene encoding the "c subunit is affected (47). Structurally, these cytokines mediate their biological functions through interaction with a specific ligand #-receptor, and the common "c subunit, as indicated in figure 8.
Figure 8: IL-2 family of cytokines.
(from “Receptors for "c family cytokines and TSLP” Rochman et al., Nature Immunology review, 2009)
!
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membrane, there are three conserved motifs termed box1, box2 and box3, whereas IL-12R contains only box1 and box2. A schematic structure of gp130 and related receptor molecules is illustrated in figure 9.
Figure 9: The gp130 subfamily of receptors
Receptor complexes sharing gp130 trigger signaling events through the formation of gp130 homodimers or heterodimers composed of one molecule of gp130 associated to a single LIFR or OSM-R (52) (53).
Gp130 is a remarkably cross-reactive and shared signaling receptor that is activated by more than ten cytokines belonging to the IL-6-type cytokine family (54). This shared receptor explains the overlapping functions of these cytokines, and a concept that will be developed further in the next chapter.
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structural difference between members of IL-6 cytokine family. Indeed, IL-6 and IL-11 show straight #-helices whereas the A helix of LIF, CNTF and OSM is kinked. These structural differences are consistent with receptor recruitment as straight cytokines signal through gp130 homodimers whereas kinked cytokines signal through LIFR-gp130 or OSMR-gp130 heterodimers, respectively.
Figure 10: Structures of IL-6, LIF and CNTF.
(Ribbon representation, from Heinrich et al., Biochem.J., 1998)
Interestingly, although these cytokines are characterized by the recruitment of gp130, certain cytokines bind specifically to an #-receptor before recruiting gp130. For example, IL-6 first binds to IL-6R# before recruiting gp130. These #-receptors are structurally different from gp130 as they contain a short intra-cytoplasmic region devoid of signaling function. In addition, these non-transducing #-subunits show a more restrictive and regulated expression, while gp130 is ubiquitously expressed on most cells. This regulation of the # receptor’s
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expression theoretically reduces the number of cells responding to IL-6-type cytokines. The receptor complexes of the IL-6-type cytokines are illustrated in figure 11.
Figure 11: IL-6 family of cytokines and the domain composition of their receptors
IL-6-type cytokines are involved in many immune functions such as B-cell differentiation and T cell proliferation as well as other biological functions such as haematopoiesis, (56). IL-6 and IL-11 are two cytokines structurally different, yet with overlapping biological functions such as the induction of acute phase proteins (Table 1).
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profiles lead to different isoelectric point (pI) values and charges. Both human and murine IL-6 exhibit post-translational changes that do not interfere with their respective biological functions. The low identity between murine and human IL-6 (42%) results in limited biological cross-reactivity. Interestingly, human IL-6 binds to both human and mouse IL-6R# while murine IL-6 binds only to mouse IL-6R#.
Spectroscopic studies performed on IL-6 have allowed for physicochemical characterization of this cytokine (72). As mentioned previously, IL-6 belongs to a family of cytokines where the main structural feature is a four-#-helix-bundle (73). These four helices are termed A, B, C, and D and are arranged in an up-up-down-down topology as illustrated in the ribbon representation below:
Figure 12: Ribbon structure of the interleukin-6 cytokine.
(adapted from PDB database PDB accession number: 1CNT)
Helices A and B are parallel and connected by a long loop, whereas helices B and C are anti- parallel and connected by a shorter loop. Helices C and D are connected by a long loop.
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Figure 13: Schematic representation of the IL-6R#.
(adapted from the review “gp130 and the interleukin-6 family of cytokines, Taga T., Annu. Rev. Immunol. 1997)
2.3) Soluble IL-6R# (sIL-6R#)
In addition to membrane-bound IL-6R# (mIL-6R#), a naturally soluble form of this protein (sIL-6R#) has been described (90). IL-6 binds to (sIL-6R#) with an affinity similar to that of the membrane IL-6R# (0.5 to 2nM). Interestingly, this soluble protein can be found in various body fluids including serum and urine and has been suggested to prolong IL-6’s plasma half- life (91). Whereas in many cases soluble receptors compete for ligand binding with their membrane bound counterparts, the complex formed by IL-6 and sIL-6R# is agonistic, and thus, activates cells that express gp130 even in the absence of mIL-6R#. This process is termed IL-6 trans-signaling and allows sIL-6R# to widen the repertoire of cell types responding to IL-6. Generation of the sIL-6R# is regulated by two distinct mechanisms:
! mRNA splicing: In vitro and in vivo studies have demonstrated the existence of the sIL-6R# form (90,92). This"process leads to the generation of a transcript that lacks 94
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terminal domain. These amino acids delineate the ligand-binding pocket of the receptor, keeping its structure intact (86).
! A membrane-proximal region composed of three FNIII domains (D4, D5 and D6 domains).
In its intracellular membrane proximal region, gp130 contains conserved Box1 and Box2 that are critical for association of Janus activated kinases (JAKs) with gp130.
Figure 14: Schematic representation of gp130.
(adapted from the review “gp130, a shared cytokine signal transducer” Taga.T,. Journal of Neurochemistry, 1996).
An important feature of gp130 is its ubiquitous pattern of expression. Indeed, gp130 mRNA has been shown to be expressed in almost all organs, including spleen, lung, liver, heart, kidney and brain (98). However, expression of gp130 has been shown to be regulated by many factors including IL-6 (99,100).
Interestingly, gp130 occurs also in a soluble form (sgp130) generated by alternative splicing.
At least three distinct isoforms have been identified and detected in human serum with
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step consists of the association of IL-6 to IL-6R#. In the first model, this binary complex recruits a gp130 molecule leading to the formation of a ternary complex. A dimerization of this ternary complex allows the formation of a hexameric structure. In the second model, the IL-6/IL-6R# binary complex recruits two gp130 molecules, leading to the formation of a tetrameric structure that can then bind to another IL-6/IL-6R# complex, to form the hexamer.
These models are illustrated in figure 15.
Figure 15: Schematic representation of IL-6R assembly.
(adapted from “Dynamics of the gp130 cytokine complex: A model for assembly on the cellular membrane ” Schroers, A et al., Protein Science, 2005)
Based on combined structural and biochemical studies performed with human IL-6, sIL-6R# and sgp130 molecules, Boulanger and colleagues have estimated the affinity of IL-6 hexameric complex association (110). The following affinity predictions were made from these studies:
- 9nM for the formation of the binary IL-6/ sIL-6R# complex association.
- 38nM between the binary complex and the sgp130.
- 0.8nM for the formation of the two IL-6/ sIL-6R#/sgp130 trimers.
A B
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Figure 16: Kinetic and affinities of hexameric complex assembly.
(adapted from “Hexameric structure and assembly of the interleukin-6/IL-6 alpha-receptor/gp130 complex”
Boulanger, MJ et al., Science, 2003)
The binary complex (IL-6/IL-6R#) is mediated by interactions between the site I of the IL-6 cytokine and the CBD of IL-6R#. These interactions involve aromatic residues such as Phe74 and Phe229 expressed by IL-6 and IL-6R#, respectively. Electrostatic interactions are also important in this binary complex.
Site II of IL-6 interacts with the CBD of gp130, D2 and D3 (site IIa). Tyr31 and Ph169, expressed by IL-6 and gp130, respectively, are important residues involved in this interaction (111). IL-6R# and gp130 interact (site IIb) through their domain D3. This interaction involves
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Figure 17: Schematic representation of the IL-6 hexameric complex.
(adapted from “Hexameric structure and assembly of the interleukin-6/IL-6 alpha-receptor/gp130 complex”
Boulanger, MJ et al., Sciences 2003).
3.2) IL-6 signaling through gp130
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Figure 18: IL-6 biological activities
1) IL-6 in immune responses
Cytokines play an important role in the regulation of immune responses and in B-cell maturation. Among these cytokines, IL-6 is of particular interest. As mentioned in chapter 2, IL-6 was originally assigned the name of B-cell stimulatory factor 2 or BSF-2 in 1986, having been found to induce terminal differentiation of B cells (118). This role was further extended to Ig production without a preferential effect on certain isotypes or on cell proliferation (67).
In vivo, antibody responses are significantly mediated through secondary structure formed during responses to pathogens in the primary B cell follicle, referred to as germinal centers (GCs). GCs are, thus, dynamic structures located in secondary lymphoid organs, and provide
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Figure 19: IL-6 is important in the development and function of TFH.
(from the review: “With a Little Help from Their Friends: Interleukin-21, T Cells, and B Cells” Jonathan S.
Silver and Christopher A.Hunter. Immunity Previews, 2008)
1.2) Roles of the IL-6 signaling complex in B cell-related diseases
Regarding the role of IL-6 in terminal B cell differentiation, this cytokine has been suggested to play a role in the pathogenesis of polyclonal and monoclonal plasma cell abnormalities.
Two decades ago, Suematsu and colleagues generated transgenic mice that carried the human IL-6 genomic gene fused with a human Ig heavy chain enhancer (120). These mice showed a massive plasmacytosis in the thymus and in secondary lymphoid organs, as well as an infiltration of plasma cells in the lung. Nevertheless, these IL-6 transgenic mice did not develop plasmacytomas. One year later, the laboratory of Jacques Van Snick studied the link between IL-6 and the development of plasmacytomas in mice (131). This study provided for the first time a correlation between IL-6 and the development in vivo of plasmacytomas. Mice immunized with a plasmacytoma cell line transfected with an IL-6 expression vector
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Figure 20: sIL-6Ra orchestrates transition between innate and acquired immunity.
(from “transgenic blockade of Interleukin-6-trans-signalingabrogates inflammation”. Rabe B et al., Blood 2007)
In their model, the resolution of acute inflammation process occurs in three main phases:
! In the first phase (A): tissue-resident mononuclear cells respond to challenge by secreting pro-inflammatory cytokines, including IL-6, TNF# and IL-1! that stimulate the secretion by endothelial cells of CXC-chemokines, including KC which is the
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Figure 21: Representation of the different protein constructs generated.
To generate biotinylated sIL-6R# and IL-6/sIL-6R# linked complex, the HindIII/NheI fragments were ligated with an XbaI / MfeI fragment containing IRES, LS BirA and an IRES- EGFP, as illustrated in figure 22.
Figure 22: Representation of the biotinylated sIL-6R# and IL-6/sIL-6R# linked complex constructs.
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IL-6-induced macrophage differentiation of M1 cells
It was shown previously that the functional cytokines Leukemia induced factor (LIF) and IL-6 acted as hematopoietic differentiation inducers that induce terminal differentiation and growth arrest of the mouse myeloid leukemia M1 cells (182). Upon IL-6 stimulation, M1 cells form colonies of macrophage-like cells which results in increased expression of F4/80 antigen. For the experiment, M1 cells were cultured in RPMI 1640 (Sigma) supplemented with 10% fetal calf serum (FCS), 2mM of glutamine and 50#g/ml of gentamicin. Cells were washed with serum-free RPMI 1640 medium and seeded into 96-well tissue culture plate at a density of 2x104 cells per well, in the presence of 1ng/ml of murine recombinant IL-6 (406-ML, R&D Systems). After incubation for 72hr at 37°C, cells were washed and stained for F4/80 antigen, by Flow cytometry (FACSCalibur, BD Biosciences), using a fluorescin isothiocyanate isomer 1 (FITC)-coupled rat anti-mouse F4/80 antigen (MCA497FA, AbD serotec). A FACS profile of M1 staining in the presence of IL-6 is shown in figure 23.
Figure 23: M1 Leukemia cell differentiation in response to IL-6
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Figure 24: Overview of the fusion and screening processes used to generate IL-6R# mAbs
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Figure 25: Generation of IL-6/sIL-6R# linked complex.
PEAK cells were transfected with PEAK8 expression vector containing the cDNA encoding IL-6/sIL-6R#. Supernatant of transfected and non transfected cells were harvested, analyzed by ELISA (A) and after
purification by SDS PAGE (B).
The IL-6/sIL-6R # linked complex is active in IL-6 trans-signaling assay
To test the functional activity of the IL-6/sIL-6R# linked complex, a STAT-3-dependent luciferase assay was used. In the assay, HEK 293-derived PEAK cells that express gp130 but do not express mIL-6R# were used. These cells were transfected with a STAT-3-dependent firefly luciferase reporter plasmid and the constitutive renilla reporter plasmids pRL-TK.
Cells were stimulated with medium, 200ng/ml of IL-6, 200ng/ml of IL-6 in the presence of 600ng/ml of sIL-6R#, or with 500ng/ml of IL-6/sIL-6R# linked complex (figure 26). Results show that IL-6/sIL-6R# is fully active on gp130-expressing PEAK cells, as it induces a strong luciferase activity, as compared to IL-6 alone. To determine whether this activation was
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specific to complex, 10µg/ml of the recombinant soluble gp130 protein (R&D Systems), that inhibits IL-6/sIL-6R# activity, was used in the assay.
Figure 26: IL-6 trans-signaling assay: STAT-3 luciferase assay.
HEK-293 derived cells were transfected with STAT-3-responsive firefly luciferase reporter gene and constitutively expressing renilla luciferase plasmid. Transfected cells were stimulated with either medium, IL-6
±sIL-6R#, or IL-6/sIL-6R#±sgp130. 18hr later, luciferase activity was calculated as a ratio of firefly to renilla luciferase luminescence.
Generation of CHO cell lines expressing IL-6, mIL-6R # , and IL-6/mIL-6R # linked complex
CHO cells were transfected with linearized plasmids encoding IL-6, mIL-6R# or the IL-6/mIL-6R# linked complex, as described in the material and methods section. Transfected cells were then stained with anti-IL-6 (MAB406, R&D Systems) and anti-IL-6R (MAB1830, R&D Systems) as primary antibodies, and allophycocyanin-conjugated goat anti-rat IgG, Fc"
specific (112-136-071, Jackson Immunoresearch) as a secondary antibody. Then, an enrichment of CHO expressing high levels of IL-6, mIL-6R# or IL-6/mIL-6R# was
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performed by magnetic cell separation (MACS) using anti-APC Microbeads. Results are shown in figure 27.
Figure 27: Flow cytometry analysis of CHO expressing IL-6, mIL-6R# and IL-6/mIL- 6R# linked complex.
CHO cells were stably transfected with either IL-6, mIL-6R# or IL-6/mIL-6R# and stained with anti-IL-6 and anti-IL-6R# comercial mAbs. Cell staining was analyzed by flow cytometry.
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Figure 28: Generation of mAbs targeting IL-6R# (A) and IL-6/sIL-6R# (B).
Wistar rats were immunized with a combination of CHO-mIL-6R# cells and sIL-6R# protein (A) or CHO- IL-6/mIL-6R# and IL-6/sIL-6R#(B).
Two fusions were assessed following the immunization schedule A, and three following the schedule B. Results are shown in table 3.
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Table 3: Hybridoma fusion summary.
Positive clones bind specifically mIL-6R#, while neutralizing clones inhibit IL-6-responses mediated either by mIL-6R# or sIL-6R# in IL-6 cis-and trans- signaling assays.
The supernatants produced from hybridomas were screened for binding to CHO-transfectants via flow cytometric analysis. Clones that are positive for CHO-mIL-6R# and CHO-IL6/ mIL- 6R# were expanded and tested for their capacity to neutralize in vitro IL-6 functional activity.
Two clones, 2B10 and 25F10, were chosen for further characterization. 2B10 was generated following immunization schedule A, and 25F10 from the B. Binding profiles of 2B10 and 25F10 hybridoma are shown in figure 29.
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Figure 29: Binding profiles of 2B10 (left panels) and 25F10 (right panels) producing hybridomas on CHO wt and CHO cells expressing IL-6, mIL-6R# and IL-6/mIL-6R#.
The supernatants produced from hybridomas, generated from the fusion of splenocytes and Sp2/0 myeloma cells, were screened for binding to CHO-wt (A) or expressing IL-6 (B), mIL-6R# (C) or IL-6/mIL-6R# (D) via the
8200 cellular detection system. Two clones were selected: 2B10 and 25F10.
After two rounds of sub-cloning, 2B10 and 25F10 mAbs were purified from hybridoma supernatants expanded in serum-free medium (Hybridoma SFM; Invitrogen Life Technologies) using a Protein G affinity column (GE Healthcare). Both mAbs were identified as rat IgG1 isotype.
Characterization of 2B10 and 25F10
Binding properties of 2B10 and 25F10 mAbs
The specificity of 2B10 and 25F10 was first evaluated by ELISA. In this experiment, mAbs were tested for their ability to bind either biotinylated sIL-6R# protein or IL-6/sIL-6R# linked protein. Results are shown in figure 30 and demonstrated that both 2B10 and 25F10 are able to capture biotinylated sIL-6R# with equivalent potencies. However, only 25F10 demonstrated a binding activity for IL-6/sIL-6R# linked complex.
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Figure 32: Binding profiles of 2B10 and 25F10 to mouse splenocytes as analyzed by flow cytometry.
Splenocytes from naïve C57/Bl6 mice were stained with FITC-conjugated anti-CD4+ antibody and 2B10, or 25F10 or Rat IgG1 conjugated to FMAT Blue dye (FL-4 channel). Plots were gated on lymphocytes. CD4+ T cells that are positive for 2B10, 25F10 or rat IgG1 are gated and percentage of different populations on gated
viable lymphocytes are indicated.
Data in figure 32 show that 2B10 and 25F10 bind to the native mIL-6R# expressed on CD4+ T cells, and probably on other subsets of lymphocytes as 17% and 18% of CD4- cells are positive for 2B10 and 25F10, respectively.
To further characterize the functional properties of 2B10 and 25F10, both mAbs were tested in IL-6 cis- and trans-signaling assays.
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Functional properties of 2B10 and 25F10 mAbs
IL-6 cis-signaling assay
To evaluate the capacity of 2B10 and 25F10 to inhibit in vitro IL-6 responses mediated by the mIL-6R#, two different in vitro assays mimicking IL-6 cis-signaling were assessed, namely the T1165 proliferation and M1 differentiation assays. Results are shown in figure 33.
Figure 33: 2B10 and 25F10 in IL-6 cis-signaling assays.
(A) T1165 cells were incubated with 1ng/ml of recombinant IL-6 for 48hr, in the presence of various concentrations of either 2B10, 25F10 or rIgG1 control mAb. T1165 proliferation was evaluated using the
colometric WST-1 reagent. (B) M1 cells were incubated with 1ng/ml of recombinant IL-6 (to induce differentiation into macrophages) in the presence of 10µg/ml of mAbs. 72hs later, cells were stained with
antibody against F4/80 antigen and analysed by flow cytometry.
Results, shown in figure 33, indicate that only 2B10 is able to neutralize the IL-6-dependant proliferation of the mIL-6R#-bearing T1165 mouse plasmacytoma cell line as well as M1 differentiation.
118 IL-6 trans-signaling assay
To evaluate the ability of 2B10 and 25F10 to neutralize IL-6 trans-signaling in vitro, both mAbs were tested in a IL-6/sIL-6R# complex induced STAT-3 activity assay using HEK 293-derived PEAK cells. In the assay, 2B10 and 25F10 were tested in the presence of either the IL-6+sIL-6R# complex or the IL-6/sIL-6R# linked protein. Results are shown in figure 34.
Figure 34: 2B10 and 25F10 in IL-6 trans-signaling assay.
HEK-293-derived cells were transfected with the STAT-3 responsive firefly reporter gene and constitutively expressing the renilla luciferase construct. Transfected cells were stimulated by exogenous IL-6+sIL-6R# (A) or
IL-6/sIL-6R# linked complex (B) for 18hr, following addition of mAbs at the indicated concentrations.
Data, shown in figure 34, demonstrate that 2B10 and 25F10 inhibit the STAT-3 luciferase activity induced by the native IL-6+sIL-6R# complex as compared to rat IgG1 antibody.
However, 2B10 is not able to inhibit the activity of IL-6/sIL-6R# linked complex.
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Taken together, the in vitro data suggest that 2B10 and 25F10 recognize mIL-6R# but show distinct functional properties, as summarized and illustrated in figure 35.
Figure 35: 2B10 and 25F10 show distinct function properties in vitro
or the IL-6/sIL-6RaFP (25F10). Taken together, the data suggest distinct modes of action for the two mAbs targeting IL-6Ra, and a schematic representation is shown to illustrate the dichotomy (Fig. 2).
IL-6trans-signaling is sufficient to drive local immune responses in AIA
Prior experiments using AIA have shown that IL-6 influences both the induction and effector phase of inflammatory arthritis (12, 18–
20). These investigations have pointed toward a role for IL-6 trans-signaling in the local regulation of IL-6 activity within the inflamed synovium (12, 21, 22). Based on these observations, initial in vivo studies aimed at testing the potential of 2B10 and 25F10 to block the induction of inflammatory arthritis, the AIA model was used first. The i.a. administration of either 2B10 or 25F10 to mBSA-challenged C57BL/6 mice resulted in a signifi- cant decrease in joint swelling (Fig. 3A). Histological evaluation of joint sections showed that parameters of disease activity were significantly less severe in mice receiving 2B10 and, to a lesser extent, 25F10 as compared with the C57BL/6 mice treated with the rIgG1 isotype control mAb (Fig. 3B). Indeed, administration of 2B10 or 25F10 reduced synovial hyperplasia, synovial infiltrate, cellular exudate, and joint damage (Fig. 3C). Thus,
blocking IL-6trans-signaling locally was sufficient to avoid ex- cessive inflammation of the synovium in AIA.
Classical IL-6 signaling is obligate and sufficient for disease induction in CIA
To differentiate IL-6–driven mechanisms of inflammation in a T cell-mediated disease model, 2B10 and 25F10 were tested for their capacity to protect mice from CIA. Male DBA/1J mice were immunized with bovine CII in CFA (day 0) and treated with 1 mg mAb (i.p.) on days 0, 2, and 5. Following a boost of CII in IFA at day 21, mice developed clinical signs of disease that presented inflammation of individual paws. Administration of 2B10 pro- tected mice by significantly delaying the onset of disease and decreasing incidence and disease severity (Fig. 4A, 4B) as com- pared with rIgG1 and 25F10. To establish the severity of local inflammation and cartilage destruction, histological sections were prepared from knee joints obtained at day 39. To assess cellular infiltration and joint destruction, serial sections were stained with H&E or safranin O/fast green, respectively. Joints from mice treated with rIgG1 and 25F10 showed significant destruction of cartilage characterized by a loss of safranin O staining (Fig. 4C, upper panels), associated hyperplasia of the synovial tissue, and
FIGURE 3. IL-6 signaling regulates disease severity in AIA. AIA was established in C57BL/6 mice. To block IL-6 signaling locally, mBSA was administered with 100mg of either 25F10, 2B10, or the control rIgG1.A, Joint inflammation was assessed by measurement of knee joint swelling.
Results presented are mean6SEM (pp,0.05 for control versus 2B10 and rIgG1 versus 25F10). B, Differences in arthritis indices were de- termined by histological assessment of synovial infiltrate, exudate, hy- perplasia, and erosion from tissue taken 3 d post-Ab treatment.C, Rep- resentative H&E-stained midsagittal joint sections taken on day 3 after Ab treatment are shown for each group as well as sham joint receiving no mBSA, included as control of healthy tissue. The white asterisk indicates a region with substantial pannus formation and associated femoral bone erosion. Arrows indicate synovial areas with infiltrates (yellow) and exu- date (blue) (original magnification340). AI, arthritis index.
FIGURE 4. Neutralization of IL-6 classical versustrans-signaling in CIA. Mice (n= 10 per group) were treated with 1 mg/mouse 2B10, 25F10, or rIgG1. Each group received three injections administrated on day 0, 2, and 5 postprimary immunization with CII in CFA.A, Incidence. Results of three independent experiments.B, Clinical score. Results are the mean6 SEM of three independent experiments.C, Representative safranin O/fast green-stained (upper panels) and H&E-stained joint sections (lower pan- els) from experimental mice sacrificed on day 39 (experimental end point) or from nonimmunized mice (sham). Three mice were analyzed per group for each experiment. Results are representative of two independent experiments (original magnification35).D, Cartilage damage was eval- uated as the percentage of loss of safranin O/fast green staining of the joints in the 2B10- or 25F10-treated CIA mice.E, Histological score was assessed, as described inMaterials and Methods, for the joints in the 2B10- or 25F10-treated CIA mice.DandE, Results are the mean6SEM of two independent experiments.pp,0.05;ppp,0.01;pppp,0.001.
The Journal of Immunology 5
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An investigation of IL-6R # domains recognized by mAbs 2B10 &
25F10
As 2B10 and 25F10 show distinct binding and functional activities, it seemed feasible to think that both mAbs recognized different epitopes on IL-6R#. To address this question an epitope mapping study was performed. To demonstrate the absence of cross reactivity of 2B10 and 25F10 with human IL-6R#, PEAK cells were transfected transiently with cDNA encoding human IL-6R#. FACS analysis demonstrated that neither 2B10 nor 25F10 bound to human IL-6R# as compared to the anti-human IL-6R# control mAb. Based on this observation, expression constructs encoding human-mouse hybrid versions of IL-6R# were generated to determine the region of mouse IL-6R# containing the epitope recognized by 2B10 and 25F10.
The extracellular region of IL-6R# was nominally divided into three regions. Human-mouse hybrids were constructed, HMM, HHM, HMH, MHH, MMH and MHM where H and M correspond to a human and murine fragments of IL-6R#, respectively. A schematic representation of these constructs is shown in figure 36.
Figure 36: Schematic representation of human/mouse IL-6R# hybrids.
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Robust T cell-dependent B cell responses are controlled by IL-6 cis- signaling.
As mentioned in introduction, the first function assigned to IL-6 was to induce differentiation of activated B cells into Ig producing cells without cell proliferation (186). IL-6 plays a crucial role in humoral immunity, as IL-6 deficient mice exhibit impaired immune and acute- phase response (83).
To dissect the relative contribution of the IL-6 signaling pathways in antigen-specific humoral responses, C57BL/6 mice were immunized subcutaneously with 20µg of NP-OVA emulsified in Alum, and co-administered with either 2B10, 25F10 or ratIgG1 control antibody at the time of NP-OVA/Alum immunization and again at day 2 and day5. At day7, serum was collected from mice and NP-specific titers for each group of mice were analyzed by ELISA. These titers were significantly decreased in 2B10 treated mice as compared to both 25F10 and rat IgG1 administered mice (figure 37), indicating a prominent role of IL-6 cis-signaling in primary humoral response.
Figure 37: Differential contribution of IL-6-signaling pathways in primary humoral responses.
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A question of whether the altered NP-specific humoral response observed in 2B10 treated mice (figure 37) was correlated with an alteration of GCB and TFH numbers was then studied.
In this regard, draining lymph nodes from C57/BL6 treated mice were harvested at day 7 following NP-OVA immunization, and analyzed for GCB and TFH cells. Results are shown in figure 38.
Figure 38: Blockade of IL-6 signaling pathways fails to alter TFH and GCB numbers.
Draining LNs from C57/BL6 mice treated with 2B10, 25F10 or rat IgG1 mAb were harvested at day 7 following immunization with NP-OVA antigen emulsified in Alum. Cells were analyzed by flow cytometry for their contents
in CD19hiPNAhi cells (=GCB cells) or CD4+CD44+CXCR5+BTLA+cells (=TFH).
The blockade of IL-6 signaling fails to modify established disease in arthritic mice.
As demonstrated previously, prophylactic neutralization of IL-6 cis-signaling decreased arthritis severity in CIA model. The impact of blocking IL-6 signaling pathways during the effector phase of the disease was also tested. In this regard, DBA/1J mice were treated with 1mg of 2B10, 25F10 or rat IgG1 isotype control, at day 21 coinciding with the secondary CII
126
immunization. Results shown in figure 39 demonstrate that blocking IL-6 pathways after disease onset failed to alter the disease pathogenesis.
Figure 39: Blockade of IL-6 pathways in prophylactic (A) and therapeutic (B) settings.
Male DBA/J mice were immunized with bovine type II collagen (CII) in CFA and co-treated with 1mg of mAbs i.p. on d0, 3 and 5 (A) or on d21, 23 and 25 (B). Results are average of clinical scores of treated mice.
130
higher. When the cells are pre-incubated with 25F10, binding of IL-6 is not inhibited, and therefore when 25F10 dissociates from IL-6R#, the IL-6/IL-6Ra complex can interact with gp130 in the membrane plan with high affinity (pM range).
This hypothesis explaining the mode of action of 25F10 is illustrated in figure 40.
Figure 40: Hypothetical mode of action of 25F10 in IL-6 trans-signaling (A) and IL-6 cis- signaling (B).
Hypothesis 2:
Most of the data concerning the kinetic of assembly for the IL-6 hexameric complex were generated from studies with soluble proteins (i.e. IL-6, sIL-6R# and sgp130)(110) (106,193).
To date, the stoichiometry and kinetics of assembly of the IL-6/mIL-6R# receptor complex in mouse remain unclear. More precisely, no study has compared the respective affinities of the IL-6/mIL-6R# and the IL-6/sIL-6R# complexed to gp130. As IL-6R# and gp130 are in close proximity and in the same “plane” (i.e. plasma membrane) one could hypothesize that the IL- 6/mIL-6R# complex interacts with gp130 with a higher affinity than does IL-6/sIL-6R#
complex for 25F10. If this postulate is true, the prediction would be that the affinity of 25F10
131
for IL-6R# (nM range) would be too low to interfere with the formation of the high affinity IL-6/mIL-6R#/gp130 complex (pM range) as illustrated in figure 41.
Figure 41: Hypothetical mode of action of 25F10 in IL-6 trans-signaling (A) and IL-6 cis- signaling (B).
These two hypothesis are not mutually exclusive and could be further explored by increasing the affinity of 25F10 for IL-6R# (i.e. to the pM range) by antibody affinity maturation in vitro for example. The resulting increase in 25F10 affinity would strengthen the interaction between the antibody and the membrane-bound protein. 25F10 could then interfere with the binding of IL-6/IL-6R#-membrane-bound complex to gp130 and, thereby, inhibit IL-6 cis- signaling.
Role of IL-6 signaling pathways in systemic immune responses
T follicular helper cells (Tfh) play a central role in the development of the germinal center reaction (GC)(130,194). Indeed, GC generates long-lived plasma cells and memory B cells. It has been shown in several studies that IL-6 plays a central role in these processes as illustrated in figure 42.
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Figure 42: Role of IL-6 in GC reaction. Generation of TFH occurs during the first encounter.
Following this contact between naive CD4+T cell and DC, CD4+T cell receives the instruction to differentiate into TFH that migrates into B cell follicle of secondary lymphoid organs, where it provides instructions to GC B cell to induce their differentiation into plasma cells or long-lived memory B cells. GCB undergo class switch recombination (CSR) and somatic hypermutation (SHM) during their development. IL-21 and IL-6 play key roles
in the orchestration of these two molecular processes.
Interestingly, in vivo data herein demonstrated that despite an alteration of NP-specific IgG titers in mice treated with 2B10, no alteration of GC B cell and TFH numbers in draining LNs was observed. Additionally, histological analysis confirmed the presence of GC in all treated groups. In this respect, one can hypothesize that mice treated with 2B10 are probably prone to alterations in the quality of humoral response generated following immunization with a T- dependent antigen. In this respect, two molecular processes involved directly in the generation of class switched antibodies could be altered, i.e. class switches recombination (CSR) and somatic hypermutation (SHM).
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Figure 43: Mapping the in vivo dichotomy between IL-6 pathways in systemic and local immune reponses.
of October 29, 2010
This information is current as
doi:10.4049/jimmunol.0901464 online Nov 23, 2009;
2009;183;7692-7702; originally published J. Immunol.
Jean-François Gauchat
Cognet, Walter Ferlin, Greg Elson, Pascale Jeannin and Ulrick Mavoungou-Bigouagou, Fouad Lefouili, Isabelle
Guilhot, Tormo, Dorothée Duluc, Rami Lissilaa, Florence
Sandrine Crabé, Angélique Guay-Giroux, Aurélie Jeanne
Signaling
Activities Requiring IL-6R for Cytokine Regulating NK and T Cell Cytokine-Like Factor 1 to Form a The IL-27 p28 Subunit Binds
http://www.jimmunol.org/cgi/content/full/183/12/7692
Data
Supplementary
C1
http://www.jimmunol.org/cgi/content/full/jimmunol.0901464/D
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