IMMUNOLOGY
`
`Glatiramer acetate increases IL-1 receptor antagonist
`but decreases T cell-induced IL-1␤ in human
`monocytes and multiple sclerosis
`
`Danielle Burgera,1, Nicolas Molnarfia,b, Martin S. Weberb,c, Karim J. Brandta, Mahdia Benkhouchad, Lyssia Gruaza,
`Michel Chofflone, Scott S. Zamvilb, and Patrice H. Lalived,e,f
`
`aDivision of Immunology and Allergy, Clinical Immunology Unit (Hans Wilsdorf Laboratory), Department of Internal Medicine, University Hospital and
`Faculty of Medicine, University of Geneva, 1211 Geneva, Switzerland; bDepartment of Neurology and Program in Immunology, University of California, San
`Francisco, CA 94143; cDepartment of Neurology, Technische Universita¨ t Mu¨ nchen, D-80538 Munich, Germany; dDepartment of Pathology and Immunology,
`Faculty of Medicine, University of Geneva, 1211 Geneva, Switzerland; and eDepartment of Neurosciences, Division of Neurology, Neuroimmunology
`Laboratory, and fDepartment of Genetics and Laboratory Medicine, Laboratory Medicine Service, University Hospital of Geneva, 1211 Geneva, Switzerland
`
`Edited by Michael Sela, Weizmann Institute of Science, Rehovot, Israel, and approved January 26, 2009 (received for review December 1, 2008)
`
`Mechanisms of action as well as cellular targets of glatiramer
`acetate (GA) in multiple sclerosis (MS) are still not entirely under-
`stood. IL-1␤ is present in CNS-infiltrating macrophages and micro-
`glial cells and is an important mediator of inflammation in exper-
`imental autoimmune encephalitis (EAE), the MS animal model. A
`inhibitor of IL-1␤, the secreted form of IL-1 receptor
`natural
`antagonist (sIL-1Ra) improves EAE disease course. In this study we
`examined the effects of GA on the IL-1 system.
`In vivo, GA
`treatment enhanced sIL-1Ra blood levels in both EAE mice and
`patients with MS, whereas IL-1␤ levels remained undetectable. In
`vitro, GA per se induced the transcription and production of sIL-1Ra
`in isolated human monocytes. Furthermore,
`in T cell contact-
`activated monocytes, a mechanism relevant to chronic inflamma-
`tion, GA strongly diminished the expression of IL-1␤and enhanced
`that of sIL-1Ra. This contrasts with the effect of GA in monocytes
`activated upon acute inflammatory conditions. Indeed, in LPS-
`activated monocytes, IL-1␤and sIL-1Ra production were increased
`in the presence of GA. These results demonstrate that, in chronic
`inflammatory conditions, GA enhances circulating sIL-1Ra levels
`and directly affects monocytes by triggering a bias toward a less
`inflammatory profile, increasing sIL-1Ra while diminishing IL-1␤
`production. This study sheds light on a mechanism that is likely to
`participate in the therapeutic effects of GA in MS.
`
`experimental autoimmune encephalitis 兩 cellular contact 兩
`inflammation 兩 autoimmune disease
`
`Glatiramer acetate (GA; copolymer-1; Copaxone) is composed
`
`of a mixture of synthetic peptides of 50 to 90 aa randomly
`composed of L-glutamic acid (E), L-lysine (K), L-alanine (A), and
`L-tyrosine (Y). Initially developed to mimic the myelin basic
`protein, a major component of the myelin sheath, and to induce
`experimental autoimmune encephalitis (EAE), GA unexpectedly
`inhibited EAE in both rodents and monkeys (1). In subsequent
`clinical trials, GA reduced relapse rate and progression of disability
`in patients with relapsing–remitting multiple sclerosis (MS; RRMS)
`leading to its approval in 1995 (2).
`A number of investigations in MS and EAE addressed the
`immunological basis of GA clinical effects. However, the mech-
`anisms of GA action are still elusive. Initial
`investigations
`attributed most GA activity to a preferential Th2-polarization of
`myelin-specific T cells, thus focusing on its effects on the
`adaptive immune response (3). However, recent reports indi-
`cated that GA treatment also exerts immunomodulatory activity
`on cells of the monocytic lineage, i.e., monocytes/macrophages
`and dendritic cells (4–9). For instance, monocytes from GA-
`treated patients with MS secrete less IL-12 and TNF in response
`to LPS stimulation compared with monocytes from healthy
`controls and untreated patients with MS (4). Accordingly,
`dendritic cells and monocytes isolated from GA-treated patients
`
`produce more anti-inflammatory IL-10 and less pro-inflamma-
`tory IL-12 (5, 9). Furthermore, GA promotes the development
`of anti-inflammatory type II monocytes in EAE, accompanied
`by induction of regulatory T cells and increased secretion of both
`IL-10 and TGF-␤ (10).
`IL-1␤ is a pleiotropic pro-inflammatory cytokine whose pro-
`duction is tightly controlled at several levels (11). Indeed, as
`recently reviewed, there are several roadblocks to the release of
`IL-1␤ beginning with the transcription of the IL1B gene and
`ending with the exit of the active cytokine from the cell. In the
`extracellular space, IL-1␤activity is mainly ruled by the secreted
`IL-1 receptor antagonist (sIL-1Ra), which binds type I IL-1
`receptor without triggering signals (12). As it potently inhibits
`the various effects of IL-1, sIL-1Ra is considered an important
`regulator of the inflammatory and overall immune response
`mediated by IL-1 (13). Because of its extreme efficacy as a
`pro-inflammatory mediator, if these intracellular and extracel-
`lular roadblocks are not enough to limit IL-1␤ activity, it may
`also be reduced by the preferential binding to the cell surface or
`soluble form of type II IL-1 receptor, preventing it from trig-
`gering the signal-transducing type I receptor (11). Finally, the
`facilitation of IL-1␤ processing by the caspase 1 inflammasome
`through ATP activation of the P2X7 receptor can also be viewed
`as a potential roadblock to the activity of IL-1␤ (11).
`IL-1␤ is mainly produced upon activation of cells of the mono-
`cytic lineage. In chronic/sterile immuno-inflammatory diseases, the
`factors triggering pro-inflammatory cytokine production are still
`elusive. T cells may exert a pathological effect through direct
`cellular contact with monocytes/macrophages, inducing massive
`up-regulation of IL-1␤ and TNF (14). Besides triggering pro-
`inflammatory cytokine production, contact-mediated activation of
`monocytes induces the production and/or shedding of cytokine
`inhibitors such as sIL-1Ra and soluble receptors of IL-1 and TNF
`(15). The importance of T cell contact-mediated activation of
`monocytic cells in MS was further demonstrated in vitro in co-
`cultures of T cells and microglial cells (16, 17).
`In MS, IL-1␤ is mainly expressed by microglial cells and
`infiltrating monocyte/macrophages throughout the white matter
`and in acute lesions (18). This assertion was further confirmed
`in EAE studies. Indeed, dark agouti rats treated with sIL-1Ra
`during the induction of EAE, or after adoptive transfer with
`myelin antigen-primed lymph node cells, develop milder signs of
`
`Author contributions: D.B., N.M., M.S.W., M.C., S.S.Z., and P.H.L. designed research; N.M.,
`M.S.W., K.J.B., M.B., and L.G. performed research; D.B. and P.H.L. analyzed data; and D.B.
`and P.H.L. wrote the paper.
`
`The authors declare no conflict of interest.
`
`This article is a PNAS Direct Submission.
`
`Freely available online through the PNAS open access option.
`1To whom correspondence should be addressed. E-mail: danielle.burger@hcuge.ch.
`
`www.pnas.org兾cgi兾doi兾10.1073兾pnas.0812183106
`
`PNAS 兩 March 17, 2009 兩 vol. 106 兩 no. 11 兩 4355– 4359
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`Table 1. Clinical characteristics of patients with MS and healthy
`controls
`
`Sex
`
`Age (y)
`
`Disease
`duration
`(y)
`
`EDSS
`
`GA treatment
`duration (mo)
`
`31
`F
`34
`F
`36
`F
`29
`M
`24
`M
`24
`F
`30
`F
`35
`F
`44
`F
`40
`M
`— 32.7 ⫾ 6.4
`
`—
`—
`—
`—
`—
`—
`—
`—
`—
`—
`—
`
`—
`—
`—
`—
`—
`—
`—
`—
`—
`—
`—
`
`45
`F
`31
`F
`15
`M
`23
`F
`41
`F
`36
`F
`34
`F
`27
`M
`31
`F
`46
`F
`41
`M
`— 33.6 ⫾ 9.6
`
`17
`10
`1
`1
`6
`3
`5
`3
`10
`6
`6
`6.2 ⫾ 4.7
`
`3.0
`1.5
`1.5
`2.0
`7.0
`2.0
`2.0
`1.5
`1.0
`2.5
`4.0
`2.5 ⫾ 1.7
`
`—
`—
`—
`—
`—
`—
`—
`—
`—
`—
`—
`
`—
`—
`—
`—
`—
`—
`—
`—
`—
`—
`—
`—
`
`39
`M
`33
`F
`26
`M
`45
`F
`32
`F
`27
`F
`23
`M
`39
`M
`25
`F
`— 32.1 ⫾ 7.6
`
`6
`7
`8
`15
`11
`4
`2
`9
`5
`7.4 ⫾ 3.9
`
`2.5
`2.0
`1.5
`1.5
`2.0
`0
`1.0
`2.0
`1.0
`1.5 ⫾ 0.8
`
`56
`18
`60
`27
`13
`44
`29
`36
`19
`33.6 ⫾ 16.8
`
`Clinical
`category
`
`Healthy
`controls
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`Mean ⫾
`SD
`Untreated
`RRMS
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`11
`Mean ⫾
`SD
`GA-
`treated
`RRMS
`1
`2
`3
`4
`5
`6
`7
`8
`9
`Mean ⫾
`SD
`
`EDSS: Expanded Disability Status Score; RRMS: relapsing-remitting multiple
`sclerosis; GA: glatiramer acetate. Data expressed at time of sampling.
`
`were activated by increasing doses of GA. The production of
`sIL-1Ra was enhanced by GA in a dose-dependent manner,
`reaching a plateau at 25 ␮g/mL (Fig. 3A). The latter dose was used
`for the in vitro experiments described later. Noticeably, GA did not
`induce IL-1␤ production, demonstrating that GA triggers an anti-
`inflammatory bias in human monocyte cytokine production.
`To confirm that GA affected the IL-1 system, we assessed its
`effect on human monocytes activated upon chronic/sterile and
`acute/infectious inflammatory conditions as mimicked by direct
`cellular contact with stimulated T cells and LPS, respectively.
`Studies of cell-cell interactions such as those occurring in T cell
`contact activation of human monocytes are usually complicated
`by the simultaneous presence of at least 2 viable cell types. To
`obviate this problem, and possible interferences caused by the
`
`sIL-1Ra levels are elevated in sera of EAE mice treated with GA. (A) GA
`Fig. 1.
`ameliorates EAE. C57BL/6 mice were injected s.c. daily with GA (150 ␮g) or
`vehicle (PBS solution) 7 d before immunization with 10 ␮g myelin oligoden-
`drocyte glycoprotein 35–55 peptide (dpi, day post-immunization). (B) EAE
`mice treated (GA) or not (vehicle) were killed at disease peak and their serum
`analyzed for IL-1␤ and sIL-1Ra content. IL-1␤ was not detected (not shown).
`
`the disease (19). sIL-1Ra delivered by non-replicative HSV-1
`vectors in EAE C57BL/6 mice delays disease onset and decreases
`disease severity (20). In addition, IL-1␣/␤ double deficient
`(IL-1⫺/⫺) mice exhibit significant resistance to EAE induction
`with reduction in disease severity, whereas IL-1Ra⫺/⫺ mice are
`highly susceptible to EAE induction in the absence of pertussis
`toxin administration (21). These observations demonstrate that
`the IL-1/IL-1Ra system is crucial for autoantigen-specific T cell
`induction in mice and that sIL-1Ra efficiently blocks IL-1␤
`effects and ameliorates EAE disease course (19–22).
`In this study we addressed the question of the effects of GA
`on IL-1 system in vivo and in vitro. The results show that
`GA-treatment increases the circulating levels of sIL-1Ra in both
`EAE mice and patients with MS. This is reflected in vitro by the
`direct effect of GA on human blood monocytes. Indeed, GA
`induces the production of the cytokine inhibitor sIL-1Ra and
`diminishes the production of IL-1␤ in conditions related to
`chronic inflammation, i.e., in monocytes activated by direct
`contact with stimulated T cells.
`
`Results
`sIL-1Ra Serum Levels Are Elevated in GA-Treated EAE Mice. To assess
`whether GA-treatment affected sIL-1Ra levels in the MS animal
`model, EAE was induced in mice treated either with GA or PBS
`solution (i.e., vehicle). As shown in Fig. 1A, EAE severity was
`reduced in GA-treated mice, as previously demonstrated (10).
`At peak disease, mouse sera were analyzed for levels of sIL-1Ra
`and IL-1␤. IL-1␤ was not detectable in any of the sera (not
`shown). However, sIL-1Ra was significantly elevated in mice
`treated with GA (3,336 ⫾ 1,190 pg/mL sIL-1Ra, mean ⫾ SD)
`compared with animals that received vehicle as a control (1,296 ⫾
`657 pg/mL sIL-1Ra; Fig. 1B). This demonstrates that GA-treatment
`enhanced sIL-1Ra concentration in EAE mouse serum.
`
`sIL-1Ra Levels Are Elevated in Sera of Patients with MS Treated with
`GA. sIL-1Ra circulating levels in MS have been shown to vary as
`a function of clinical status and treatment, so we examined
`whether GA-treatment would affect sIL-1Ra levels in patients
`with MS. IL-1␤ and sIL-1Ra levels were measured in sera of
`patients with RRMS treated with GA or untreated, and in
`healthy controls (Table 1). IL-1␤was not detectable in any of the
`sera. As shown in Fig. 2, sIL-1Ra was significantly increased in
`serum of patients treated with GA (434 ⫾ 265 pg/mL sIL-1Ra)
`whereas there was no significant difference between untreated
`patients (218 ⫾ 60 pg/mL sIL-1Ra) and healthy controls (188 ⫾
`65 pg/ml sIL-1Ra). This demonstrates that GA treatment en-
`hances sIL-1Ra in the serum of patients with MS.
`
`GA Differentially Regulates IL-1␤ and sIL-1Ra Production in Human
`Monocytes. To assess whether GA per se would affect the IL-1
`system in human monocytes, freshly isolated human monocytes
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`IMMUNOLOGY
`
`Fig. 4. GA affects sIL-1Ra and IL-1␤ mRNA in both CEsHUT-activated and
`resting monocytes. (A) Monocytes (2 ⫻ 106 cells/well/3 mL) were preincubated
`for 1 h with the indicated dose of GA and then cultured for 3 h in thepresence
`of CEsHUT (6 ␮g/mL). Total mRNA was isolated and analyzed by duplex real-time
`quantitative PCR (see Materials and Methods) for the presence of IL-1␤(open
`circles) and sIL-1Ra (filled circles) transcripts. Results are presented as percent-
`age of mRNA expression level, 100% being transcript expression measured
`after 3 h ofmonocyte activation by CE sHUT in the absence of GA; mean ⫾ SD
`of 3 different experiments. (B) Monocytes (2 ⫻ 106 cells/well/3 mL) were
`activated for 18 h with the indicated dose of GA. Total mRNA was isolated and
`analyzed by duplex real-time quantitative PCR (see Materials and Methods)
`for the presence of IL-1␤ (open circles) and sIL-1Ra (filled circles) transcripts.
`Results are presented as percentage of mRNA expression level, 100% being
`the transcript level at 50 ␮g/mL GA; mean ⫾ SD of 3 different experiments, i.e.,
`monocytes prepared from 3 different blood donors.
`
`level, monocytes were incubated for 1 h withincreasing doses of
`GA and then activated by CEsHUT or not activated. As shown in
`Fig. 4A, GA,
`in the absence of other stimuli,
`induced the
`expression of sIL-1Ra transcript in a dose-dependent manner,
`whereas that of IL-1␤ was not induced. When monocytes were
`activated by CEsHUT, GA diminished IL-1␤mRNA expression by
`30% whereas it enhanced sIL-1Ra mRNA expression by 25% in
`monocytes activated by CEsHUT (Fig. 4B), corroborating the
`effects of GA on protein production (Fig. 3). Together, these
`data suggest that GA displays opposite activity toward IL-1
`system members in monocytes/macrophages by directly inducing
`sIL-1Ra expression and production and by modulating both
`IL-1␤ and sIL-1Ra expression/production induced by CEsHUT.
`
`Discussion
`The present study sheds light on mechanisms by which GA might
`exert beneficial effects in MS. GA treatment enhances sIL-1Ra
`blood levels in patients with MS and in EAE mice. This is likely
`to be the consequence of the direct triggering effect of GA on
`monocytic production of sIL-1Ra. In addition, GA diminishes
`monocytic IL-1␤ production induced by direct contact with
`stimulated T cells. Thus, through different mechanisms, GA
`dampens IL-1␤ activity, which correlates with disease severity.
`Recent insights derived from studies on the mechanism of
`action of GA show a pivotal role of monocytes in the modulation
`of the immune system and highlight the importance of these cells
`as a target for pharmacologic intervention in autoimmune
`diseases (4–10, 24). These results suggest that GA might be
`useful in autoimmune diseases other than MS, as suggested by its
`beneficial effect in animal models of autoimmune diseases such
`as uveoretinitis (25) and inflammatory bowel disease (26), and
`graft rejection (27), whereas its efficacy has not been demon-
`strated in animal models of systemic lupus erythematosus (28)
`and collagen-induced arthritis (29).
`The premise that GA enhances sIL-1Ra levels in treated
`patients with MS is reminiscent of observations made with
`another immunomodulator used in MS. Indeed, IFN␤ also
`increases circulating serum levels of sIL-1Ra in patients with MS
`(30). Interestingly, with both immunomodulators, the circulating
`levels of sIL-1Ra are doubled in treated patients compared with
`untreated individuals. Together these observations suggest that
`the enhancement of sIL-1Ra might be relevant to therapeutic
`effects of both GA and IFN␤. Indeed, sIL-1Ra is transported and
`expressed into the CNS, where it could inhibit
`the pro-
`
`sIL-1Ra levels are elevated in sera of patients with MS treated with GA.
`Fig. 2.
`The levels of sIL-1Ra and IL-1␤ were measured in sera of patients with RRMS
`treated with GA or not treated, and age-matched healthy controls, as de-
`scribed in Table 1. IL-1␤ was under the detection limit (15 pg/mL) in all
`individuals. There was no significant difference between healthy controls and
`untreated patients with RRMS. Results are presented as a box plot (GraphPad
`Prism 4).
`
`fact that target cells are potentially phagocytic, isolated mem-
`branes from stimulated HUT-78 cells were solubilized with
`CHAPS and used as a stimulus, referred to as CEsHUT (23). As
`shown in Fig. 3B, GA enhanced the production of sIL-1Ra in
`monocytes activated by CEsHUT and LPS in a similar manner,
`and GA-induced sIL-1Ra production was additive to that trig-
`gered by CEsHUT or LPS. In contrast, the production of IL-1␤
`induced by CEsHUT was inhibited by GA, whereas LPS-induced
`production of IL-1␤ was enhanced in the presence of GA (Fig.
`3C). These observations suggest that GA displays opposite
`effects on signaling events downstream of LPS and CEsHUT.
`
`GA Affects the Expression of Cytokine Transcripts. To assess whether
`GA affected the production of cytokines at the transcriptional
`
`Fig. 3. GA differentially regulates IL-1␤ and sIL-1Ra production in human
`monocytes. (A) Monocytes (5 ⫻ 104 cells/200 ␮L/well; 96-well plates) were
`activated with the indicated dose of GA. After 48 h, supernatants were
`harvested and the production of IL-1␤(open circles) and sIL-1Ra (filled circles)
`were measured in triplicate wells and represented as mean ⫾ SD. Results are
`representative of 3 different experiments. (B) Monocytes (5 ⫻ 104 cells/200
`␮L/well; 96-well plates) were preincubated for 1 h with or without 25 ␮g/mL
`GA and then cultured for 48 h in the presence or absence of CEsHUT (1 ␮g/mL)
`or LPS (100 ng/mL). sIL-1Ra was measured in culture supernatants (mean ⫾ SD,
`n ⫽ 3 different experiments). (C) Monocytes (5 ⫻ 104 cells/200 ␮L/well; 96-well
`plates) were preincubated for 1 h with or without 25 ␮g/mL GA and then cultured
`for 48 h in the presence or absence of CEsHUT (6 ␮g/mL) or LPS (100 ng/mL). IL-1␤
`was measured in culture supernatants (mean ⫾ SD, n ⫽ 3 different experiments,
`i.e., monocytes prepared from 3 different blood donors).
`
`Burger et al.
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`PNAS 兩 March 17, 2009 兩 vol. 106 兩 no. 11 兩 4357
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`inflammatory activities of IL-1␤, whose expression is increased
`in MS lesions (18, 31). The efficiency of sIL-1Ra treatment was
`demonstrated in EAE animals, in which it results in delayed and
`milder disease (19, 22). Besides, polymorphisms encoded within
`the IL-1 gene cluster were associated with MS (32). In particular,
`mild/moderate disease has been correlated to allele 2 of the
`IL-1Ra gene (IL1RN) variable number of tandem repeats ge-
`notype, which favors the production of sIL-1Ra (12, 33). In
`addition, families displaying high innate IL-1␤/sIL-1Ra ratio are
`at increased risk to have a relative who develops MS (34).
`Together, these studies reinforce the potential clinical benefit of
`GA to selectively induce sIL-1Ra secretion by monocytes in MS,
`as demonstrated in the present study. Of note, direct treatment
`with the commercially available form of sIL-1Ra (Anakinra)
`may represent an alternative treatment for MS, although its short
`lifespan once injected in humans may limit its efficacy (13).
`Nevertheless, as demonstrated here, the enhancement of intrin-
`sic production of sIL-1Ra might be a mediator of the beneficial
`effects of GA in MS.
`Most studies have focused on the effects of GA treatment on
`the adaptive immune system, in particular on T cells. Recent data
`favor the view that primary immune modulation of APC directs
`T cell immune modulation as a secondary step. Indeed, in mice
`lacking mature B and T cells, the GA treatment effect on
`monocytes is unbowed as indicated by an anti-inflammatory
`‘‘type II’’ cytokine shift (10). This indicates that GA does not
`require T cells or T cell products to alter monocytic cytokine
`production. Furthermore, adoptive transfer of highly purified,
`GA-induced type II monocytes into mice with EAE triggers T
`cell immune modulation and ameliorates the disease course of
`recipient mice. However, MHC II-deficient type II monocytes
`were unable to mediate this effect on T cells or disease severity
`(10). Taken together, these observations indicate that in vivo GA
`treatment exerts a direct effect on APC, which rules T cell
`immune modulation as the effector arm of GA clinical benefit
`in CNS autoimmune disease. The present study confirm the
`direct effect of GA on cells of the monocytic lineage by
`demonstrating that it down-regulates T cell contact-induced
`IL-1␤ production and directly triggers the production of sIL-
`1Ra. Thus, GA directly affects both the antigen presentation and
`cytokine production of monocytic cells.
`Direct cellular contact with stimulated T cells is a major
`pathway for the production of IL-1␤ and TNF in monocytes/
`macrophages under sterile conditions (17, 35, 36). Indeed,
`contact-mediated activation of monocytes/macrophages by stim-
`ulated T lymphocytes is as potent as optimal doses of LPS to
`inducing IL-1␤and TNF production in monocytes (37, 38). This
`model was recently used to assess the potency of kinase inhib-
`itors in acute and chronic inflammatory conditions (39). It is thus
`likely that this mechanism is highly relevant to the pathogenesis
`and persistence of chronic/sterile inflammation in diseases such
`as MS. The effect of GA on cytokine production induced by
`contact with stimulated T cells in human microglial cells was
`previously demonstrated (16). Stimulated T cells that were
`pretreated with GA induced lower levels of TNF, IL-1␤, and IL-6
`in human microglial cells and phorbol 12-myristate 13-acetate
`(PMA)/IFN␥-treated U937 monocytic cells. However, in the
`latter study, GA was absent during microglial cell activation,
`implying that GA rather inhibited the ability of T cells to activate
`cytokine production by cells of the monocytic lineage. The
`present study demonstrates that GA influences cytokine pro-
`duction by acting directly on human monocytes. Together, these
`studies suggest that GA affects the activation stage of both T cells
`and monocytes/macrophages to diminish contact-induced pro-
`inflammatory cytokine production.
`GA displays opposite effects on monocytes activated by LPS
`and T cell contact. Indeed, in contrast with CEsHUT-activated
`
`monocytes, the production of the pro-inflammatory cytokine
`IL-1␤ was up-regulated when cells were activated by LPS. This
`result is in agreement with a previous study showing that GA
`enhances the production of IL-1␤ in the human monocytic cell
`line THP-1 when activated by LPS (40). Thus, GA displays
`opposite effects on cytokine production by monocytes activated
`upon acute/infectious (i.e., LPS) and chronic/sterile (i.e., CE-
`sHUT) inflammatory conditions. Therefore, the use of LPS as an
`in vitro stimulus should be used with caution to mimic inflam-
`matory conditions when chronic/sterile inflammatory diseases
`are investigated (39).
`In conclusion, this study demonstrates that GA directly affects
`monocytes/macrophages by triggering the production of the
`anti-inflammatory cytokine sIL-1Ra. As sIL-1Ra can be both
`transported through the blood–brain barrier and induced within
`the CNS, it might exert immunomodulatory effects in both
`systemic and CNS compartments. In the latter, GA may also
`dampen the production and activity of IL-1␤. Finally, the present
`data strengthen recent demonstrations that, in addition to the
`modulation of the adaptive immune system, GA significantly
`affects the innate immune system.
`
`Materials and Methods
`Patients. Patients and healthy volunteers were recruited at the University
`Hospital of Geneva in accordance with institutional guidelines, and ap-
`proval of the local ethical committee was obtained. Blood was drawn from
`10 healthy controls, 11 untreated patients with RRMS, and 9 GA-treated
`patients with RRMS (Table 1). Sex, age, clinical score, and disease duration
`were matched between groups. All enrolled patients had definite RRMS
`according to revised McDonald criteria (41). At the time of blood sampling,
`GA-treated patients received 20 mg of GA s.c. daily for at least 1 year, with
`mean treatment duration of 33.6 ⫾ 16.8 months (Table 1). None of the
`patients were receiving an immunomodulatory or immunosuppressive
`drug in addition to GA. Patients from the untreated group did not receive
`any immunosuppressive or immunomodulatory drug for at least 6 months
`preceding the study.
`
`EAE Induction and GA Treatment. EAE was induced in 6 C57BL/6 mice using 10
`␮g myelin oligodendrocyte glycoprotein 35–55 peptide in complete Freund
`adjuvant. After immunization and 48 h later, mice received an i.v. injection of
`200 ng pertussis toxin. Mice were scored as follows: 0, no symptoms; 1,
`decreased tail tone; 2, mild monoparesis or paraparesis; 3, severe paraparesis;
`4, paraplegia and/or quadriparesis; and 5, moribund condition or death. Mice
`received daily s.c. injections of 150 ␮g GA suspended in PBS solution (n ⫽ 3) or
`PBS solution alone (n ⫽ 3) starting 7 days before EAE induction as described
`elsewhere (42). All experiments were carried out in accordance with guide-
`lines prescribed by the Institutional Animal Care and Use Committee at the
`University of California, San Francisco.
`
`Materials. FCS, streptomycin, penicillin, L-glutamine, RPMI-1640, and PBS
`solution free of Ca2⫹ and Mg2⫹ were purchased from Gibco; purified phyto-
`hemagglutinin from EY Laboratories; Lymphoprep from Axis-Shield; PMA,
`polymyxin B sulfate, and mouse anti-␤-tubulin antibody from Sigma; and GA
`from Sanofi-Aventis. Other reagents were of analytical grade or better.
`
`Monocytes. Peripheral blood monocytes were isolated from buffy coats of
`blood of healthy volunteers as previously described (43). To avoid activation
`by endotoxin, polymyxin B sulfate (2 ␮g/mL) was added in all solutions during
`isolation procedure.
`
`T Cell Stimulation and Membrane Isolation. HUT-78, a human T cell line, was
`obtained from the American Type Culture Collection. Cells were maintained
`in RPMI-1640 medium supplemented with 10% heat-inactivated FCS, 50
`␮g/mL streptomycin, 50 IU/mL penicillin, and 2 mM L-glutamine in 5% CO2-air
`humidified atmosphere at 37 °C. HUT-78 cells (2 ⫻ 106 cells/mL) were stimu-
`lated for 6 h byphytohemagglutinin (1 ␮g/mL) and PMA (5 ng/mL). Plasma
`membranes of stimulated HUT-78 cells were prepared as previously described
`and solubilized in 8 mM CHAPS (23, 44). CHAPS extract of membranes of
`stimulated HUT-78 cells was referred as to CEsHUT. Previous studies demon-
`strated that fixed, stimulated HUT-78 cells, plasma membranes of the latter
`
`4358 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0812183106
`
`Burger et al.
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`4 of 5
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`YEDA EXHIBIT NO. 2061
`MYLAN PHARM. v YEDA
`IPR2015-00644
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`

`
`IMMUNOLOGY
`
`cells, and CEsHUT display similar ability to induce cytokine production in human
`monocytes (15). To activate monocytes, CEsHUT was used at either 1 ␮g/mL or
`6 ␮g/mL proteins as previously determined (38, 44).
`
`mRNA Quantification. Monocytes (2 ⫻ 106 cells/well/3 mL) were cultured in
`6-well plates with the indicated stimulus for 3 h or 18 h.Total RNA was isolated
`and analyzed by quantitative real-time PCR as previously described (23).
`
`Cytokine Production. Monocytes (5 ⫻ 104 cells/well/200 ␮L) were activated with
`the indicated stimulus in RPMI medium 1640 supplemented with 10% heat-
`inactivated FCS, 50 ␮g/mL streptomycin, 50 U/mL penicillin, 2 mM L-glutamine,
`and 5 ␮g/mL polymyxin B sulfate in 96-well plates and cultured for 48 h. The
`production of sIL-1Ra and IL-1␤ was measured in culture supernatants and
`patients’ serum by commercially available enzyme immunoassay: IL-1␤ (Im-
`munotech) and sIL-1Ra (Quantikine; R&D Systems). IL-1␤and sIL-1Ra concen-
`trations in serum of patients with RRMS and healthy controls were determined
`by triplicate measurements of the same sample.
`
`Statistics. When required, significance of differences between groups was
`evaluated using the Student t test.
`
`ACKNOWLEDGMENTS. This work was supported by Swiss National Science
`Foundation Grants 320000–116259 (to D.B.) and 310000–113653 (to P.H.L.); Swiss
`Society for Multiple Sclerosis grants (to D.B. and P.H.L.); a Hans Wilsdorf Foun-
`dation grant (to D.B.); Swiss National Science Foundation Advanced Researcher
`Fellowship PA00A-119532 (N.M.); National Multiple Sclerosis Society Fellowship
`(M.S.W.); National Institutes of Health Grants RO1 AI509709 and RO1 AI073737
`(to S.S.Z.); National Multiple Sclerosis Society Grants RG 4124 and RG 3913 (to
`S.S.Z.); Maisin Foundation (S.S.Z.); and the Dana Foundation (S.S.Z.).
`
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