`
`
`
`
`
`
`
`IL-11 Receptor o in the Pathogenesis of
`IL-13-Induced Inflammation and Remodeling
`
`D2
`
`BioLegend’
`
`This information is current as
`of November2, 2017.
`
`Qingsheng Chen, Lesley Rabach, Paul Noble, Tao Zheng,
`Chun Geun Lee, Robert J. Homer and Jack A. Elias
`J Immunol 2005; 174:2305-2313; ;
`doi: 10.4049/jimmunol.174.4.2305
`http://www.jimmunol.org/content/174/4/2305
`
`Why The JI?
`
`¢ Speedy Publication! 4 weeks from acceptance to publication *average
`
`¢ Rapid Reviews! 30 days* from submissionto initial decision
`
`¢ No Triage! Every submission reviewed by practicing scientists
`
`References
`
`Thisarticle cites 59 articles, 18 of which you can accessforfree at:
`http://www.jimmunol.org/content/174/4/2305 full#ref-list-1
`
`Subscription
`
`Information about subscribing to The Journal ofImmunology is onlineat:
`http:/jimmunol.org/subscription
`
`Permissions
`
`Submit copyright permission requestsat:
`http://www.aai.org/About/Publications/JI/copyright.html
`
`Email Alerts Receive free email-alerts when new articles cite this article. Sign up at:
`http://jimmunol.org/alerts
`
`The Journal ofImmunology is published twice each month by
`The American Association of Immunologists,Inc.,
`1451 Rockville Pike, Suite 650, Rockville, MD 20852
`Copyright © 2005 by The American Association of
`Immunologists All rights reserved.
`Print ISSN: 0022-1767 Online ISSN: 1550-6606.
`
`
`
`Lassen - Exhibit 1007, p. 1
`
`
`
`LIOT‘JJAQWAAONUojsan3Aq/Sio'jou!mam//:dyyyWoy,papeopumog
`
`
`
`
`
`Lassen - Exhibit 1007, p. 1
`
`

`

`The Journal of Immunology
`
`IL-11 Receptor a in the Pathogenesis of [L-13-Induced
`Inflammation and Remodeling’
`
`Qingsheng Chen,”* Lesley Rabach,”* Paul Noble,* Tao Zheng, Chun Geun Lee,*
`Robert J. Homer,** and Jack A. Elias**
`
`IL-13 is a major stimulator of inflammation and tissue remodeling at sites of Th2 inflammation. In Th2-dominant inflammatory
`disorders such as asthma, IL-11 is simultaneously induced. However, the relationship(s) between IL-11 and IL-13 in these re-
`sponses has not been defined, and the role(s) of IL-11 in the genesis of the tissue effects of IL-13 has not been evaluated. We
`hypothesized that IL-11, signaling via the [L-11Ra-gp130 receptor complex, plays a key role in IL-13-induced tissue responses.
`To test this hypothesis we compared the expression of IL-11, IL-11Ra, and gp130 in lungs from wild-type mice and transgenic mice
`in which IL-13 was overexpressed in a lung-specific fashion. We simultaneously characterized the effects of a null mutation of
`IL-11Re on the tissue effects of transgenic IL-13. These studies demonstrate that IL-13 is a potent stimulator of IL-11 and
`IL-11Ra. They also demonstrate that IL-13 is a potent stimulator of inflammation, fibrosis, hyaluronic acid accumulation, myo-
`fibroblast accumulation, alveolar remodeling, mucus metaplasia, and respiratory failure and death in mice with wild-type IL-
`11Raloci and that these alterations are ameliorated in the absence of IL-11Ra. Lastly, they provide insight into the mechanisms
`of these processes by demonstrating that IL-13 stimulates CC chemokines, matrix metalloproteinases, mucin genes, and gob-5 and
`stimulates and activates TGF-f1 via IL-11Ra-dependent pathways. When viewed in combination, these studies demonstrate that
`IL-11Raplays a key role in the pathogenesis of IL-13-induced inflammation and remodeling. The Journal of Immunology, 2005,
`174: 2305-2313.
`
`I nterleukin-13 is a pleiotropic 12-kDa product of a gene on
`
`chromosome5 at q31 that is produced in large quantities by
`stimulated Th? cells. It was originally described as an IL-4-
`like molecule based on shared effector properties, including the
`ability to stimulate IgE production. Subsequent studies demon-
`strated that IL-13 and IL-4 often play distinct roles in biology. A
`prominent aspect of this distinction is the appreciation that IL-4
`plays a key role in Th2 cell differentiation and response generation,
`whereas IL-13 contributes as the major effector of Th2 inflamma-
`tion and tissue remodeling (1-4). In accord with these observa-
`tions, IL-13 dysregulation has been documented, and IL-13 has
`been implicated in the pathogenesis of a variety of diseases char-
`acterized by inflammation and tissue remodeling,
`including
`asthma, idiopathic pulmonary fibrosis, scleroderma, viral pneumo-
`nia, hepatic fibrosis, nodular sclerosing Hodgkin’s disease, and
`
`*Section of Pulmonary and Critical Care Medicine and ‘Department of Pathology,
`Yale University School of Medicine, New Haven, CT 06520; and *Pathology and
`Laboratory Medicine Service, Veterans Affairs-Connecticut Health Care System,
`West Haven, CT 06516
`Received for publication September 13, 2004. Accepted for publication November
`22, 2004.
`The costs of publication of this article were defrayed in part by the payment of page
`charges. This article must therefore be hereby marked advertisement in accordance
`with 18 U.S.C. Section 1734 solely to indicate this fact.
`!' This work was supported by National Institutes of Health Grants HL64242,
`HL78744, HL66571, and HL56389 (to J.A.E.).
`
`?Q.C. and LR. made equal contributions to this work.
`3 Address correspondenceand reprint requests to Dr. Jack A. Elias, Section of Pul-
`monary and Critical Care Medicine, Yale University School of Medicine, 300 Cedar
`Street (S441 TAC), P.O. Box 208057, New Haven, CT 06520-8057. E-mail address:
`jack.elias @yale.edu
`4 Abbreviations used in this paper: COPD, chronic obstructive pulmonary disease;
`BAL, bronchoalveolar lavage; HA, hyaluronic acid; MMP, matrix metalloproteinase;
`Tg, transgenic; Timp, tissue inhibitor of MMP; WT, wild type; TARC, thymus and
`activation-regulated chemokine.
`
`chronic obstructive pulmonary disease (COPD)* (1-11). Studies
`from our laboratory and others have demonstrated that IL-13 me-
`diates its tissue effects by activating a broad array of downstream
`target genes,
`including chemokines, matrix metalloproteinases
`(MMPs), TGF-B1, and chitinases (12-16). The importance of IL-
`6-type cytokines in the generation of the effects of IL-13, however,
`have not been investigated.
`IL-11 is a multifunctional IL-6-type cytokine with diverse bio-
`logic properties, including the ability to stimulate hemopoiesis,
`thrombopoiesis, megakaryocytopoiesis, and bone resorption; reg-
`ulate macrophage differentiation; and confer mucosal protection
`after chemotherapy and radiation therapy (17-22). These effects
`are mediated by a multimeric receptor that contains a ligand-bind-
`ing a subunit, IL-11Ra, and the ubiquitous B subunit, gp130, that
`triggers intracellular signaling (18, 23, 24). Previous studies from
`our laboratory and others demonstrated that, like IL-13, IL-11 is
`expressed in an exaggerated fashion in the dysregulated Th? re-
`sponse in the asthmatic airway (25). Although IL-11 can inhibit
`Thi responses,
`inhibit the production of Thl-related cytokines
`such as IL-12, and shift inflammation in a Th? direction (22, 26—
`29), little else is known aboutthe role(s) of IL-11 in the generation
`and/or expression of Th2 tissue responses. In particular, interac-
`tions between IL-11 and IL-13 have not been defined, and a role
`for IL-11 in the genesis of IL-13-induced pathologies has not been
`established.
`
`Wehypothesized that IL-11 signaling plays a key role in IL-
`13-induced Th2 inflammation. To test this hypothesis, we charac-
`terized the expression of IL-11, IL-11Ra, and gp130 in lungs from
`wild-type (WT) mice and mice in which IL-13 was overexpressed
`in a lung-specific fashion. We also characterized the effects of a
`null mutation of IL-11Ra onthetissue effects of transgenic IL-13.
`These studies demonstrate that IL-13 is a potent stimulator of
`IL-11 and IL-11Ra. They also demonstrate that IL-11Ra plays a
`
`key role in IL-13-induced inflammation, fi 5,hyaluronic acid
`
`Copyright © 2005 by The American Association of Immunologists, Inc.
`
`0022-1767/05/$02.00
`
`Lassen - Exhibit 1007, p. 2
`
`
`
`
`
`LIOT‘JJAqWAAONUojsan3Aq/21o0'Tountu!ammn//:dyyyWoypapeopumog
`
`Lassen - Exhibit 1007, p. 2
`
`

`

`2306
`
`IL-11 AND PATHOGENESIS OF IL-13 PHENOTYPE
`
`(HA) accumulation, myofibroblast accumulation, alveolar remod-
`eling, mucus metaplasia, and respiratory failure and death. Lastly,
`they provide insights into the mechanisms of these processes by
`demonstrating that IL-13 stimulates CC chemokines, MMPs, mu-
`cin genes, and gob-5 and stimulates and activates TGF-B1 via
`IL-11Ra-dependent pathways.
`
`Materials and Methods
`Overexpression transgenic mice
`
`CC10-IL-13 transgenic mice were generated in our laboratory, bred onto a
`CS7BL/6 background, and used in these studies. These mice use the Clara
`cell 10-kDa protein (CC10) promoterto target IL-13 to the lung. The meth-
`ods used to generate and characterize these mice were described previously
`(30). In this modeling system, IL-13 caused a mononuclear cell- and eosi-
`nophil-rich tissue inflammatory response, alveolar enlargement, subepithe-
`lial and parenchymal fibrosis, mucus metaplasia, and respiratory failure
`and death, as previously described (12, 13, 30).
`IL-11Re-null mice (IL-11Ra‘~) were provided by Drs. L. Robb and C.
`Glenn Begley (Walter and Eliza Hall Institute, Victoria, Australia) (31,32).
`These mice were bred for more than eight generations onto a C57BL/6
`genetic background. CC10-IL-13 mice with WT*’* and null~’~ IL-11Ra
`loci were generated. by breedingthe IL-13 transgenic (Tg*) mice with the
`IL-11Ra~’~ animals. Genotyping was accomplished as previously de-
`scribed (30, 32). Littermate control WT mice with (+/+) or without (—/—)
`IL-11Rq@ loci were used as controls.
`
`Bronchoalveolar lavage (BAL)
`
`Lunginflammation was assessed by BALas previously described (13, 33).
`The BALsamples from each animal were then pooled and centrifuged. The
`number and types of cells in the cell pellet were determined as previously
`described (12, 13). The supernatants were stored at —20°C until used.
`
`Lung volume and morphometric assessments
`
`Animals were anesthetized, the trachea was cannulated, and the lungs were
`removed and inflated with PBS at 25 cm. The size of each lung was eval-
`uated via volume displacement, and alveolar size was estimated from the
`mean chord length of the airspace, as previously described by our labora-
`tory (13). Chord length increases with alveolar enlargement.
`
`Histologic evaluation
`
`Animals were killed, a median sternotomy was performed, and right heart
`perfusion was accomplished with calcium- and magnesium-free PBS. The
`heart and lungs were then removedenbloc, inflated at 25 cm pressure with
`neutral-buffered 10% formalin, fixed in 10% formalin, embedded in par-
`affin, sectioned, and stained. H&E, Mallory’s Trichrome, and periodic ac-
`id-Schiff with diastase stains were performed at Yale University School of
`Medicine.
`
`mRNA analysis
`
`The levels of mRNA encoding IL-11 and IL-11Ra were evaluated with a
`commercial RNase protection assay (BD RiboQuant; BD Biosciences) as
`described by the manufacturer. Other mRNA levels were evaluated by
`RT-PCRanalysis as previously described (13). The primers used have been
`described previously (12, 13, 15, 16). For each cytokine, the optimal num-
`bers of cycles that will produce a quantity of cytokine product that is
`directly proportional to the quantity of input mRNA was determined ex-
`perimentally. B-Actin was used as an internal standard. Amplified PCR
`products were detected using ethidium bromide gel electrophoresis, quan-
`titated electronically, and confirmed by nucleotide sequencing.
`
`Quantification of IL-13 and chemokines
`
`BAL IL-13 and chemokine levels were quantitated using commercial
`ELISA kits (R&D Systems) according to the manufacturer’s instructions.
`
`Immunohistochemistry
`
`Quantification of lung collagen
`
`Collagen content was determined biochemically by quantifying total sol-
`uble collagen using the Sircol collagen assay kit (Biocolor) according to
`the manufacturer’s instructions (15). The data are expressed as the collagen
`content of the entire right lung. Collagen was also assessed morphometri-
`cally using picosirius red staining, performed as described previously by
`our laboratory (15). These data are expressed as the percentage of the
`histologic section with picosirius red staining.
`
`Quantification of HA
`
`The levels of BAL HA were measured using a competitive ELISA using
`biotinylated HA-binding protein as described previously (34, 35). Micro-
`titer plates were coated with HA by combining rooster comb HA, carbo-
`diimide HCl, and HCl. Samples were incubated with biotinylated HA-
`binding protein for 1 h and then added to the wells. The plate was then
`agitated, washed, developed with HRP-streptavidin, and exposed to per-
`oxidase substrate for 30 min. OD at 405 nm wasevaluated. Samples were
`compared with a simultaneously performed standard curve.
`
`TGF-B bioassay
`
`Thelevels of total and bioactive TGF-81 were evaluated by ELISA (R&D
`Systems) using untreated and acid-treated BAL fluids according to the
`manufacturer’s instructions.
`
`Murine 100% O, exposure
`
`Adult 6- to 8-wk-old Tg” and Tg* mice with WTornull mutant IL-11Ra
`loci were exposed to room air (controls) or continuously to 100% O, ina
`Plexiglas chamber as previously described (19, 36). All protocols were
`reviewed and approved bythe institutional animal care and use committee
`at Yale University School of Medicine.
`
`Statistics
`
`Normally distributed data are expressed as the mean + SEM andassessed
`for significance by Student’s ¢ test or ANOVA as appropriate. Data that
`were not normally distributed were assessed for significance using the Wil-
`coxon rank-sum test.
`
`Results
`Effect of IL-13 on IL-1] and IL-I1R expression
`Studies were undertakento define the effects of IL-13 on IL-11 and
`
`its receptor components in murine lung. These studies demon-
`strated that transgenic IL-13 is a potent stimulator of the expres-
`sion of IL-11 and IL-11Ra. These effects were readily apparent at
`all time points evaluated (1—4 mo; Fig. 1 and data not shown). The
`induction of IL-11 was associated with similar increases in the
`
`including
`levels of mRNA encoding other IL-6-type cytokines,
`IL-6 and LIF (Fig. 1). A modestincrease in gp130 expression was
`also observed (Fig. 1). Similar alterations in M-CSF, GM-CSF,
`stem cell factor, L32, and GAPDH, however, were not found. The
`alterations in IL-11Rawerealso atleast partially specific, because
`
`
`
`
`
`LIOT‘JJAqWAAONUojsan3Aq/21o0'Tountu!ammn//:dyyyWoypapeopumog
`
`wom [ETE R
`
`www HtRee
`somo: trSE
`
`oe TENRice
`ae LENGRG
`
`celOIh-13 ~ vee TE DR,
`
`
`
`
`
`soos LAE
`ooAPE
`
`a-Smooth muscle actin and myosin H chain staining cells were evaluated
`by immunohistochemistry as previously described by our laboratory (15).
`The primary Abs were obtained from DakoCytomation. Specificity was
`assessed by comparingthe staining of serial sections that were incubated in
`the presence and the absence of the primary Ab.
`
`IL-13 regulation of IL-11Rq@ and IL-11. Lungs were ob-
`FIGURE 1.
`tained from 2-mo-old CC10-IL-13 Tg~ and Tg* mice, and the levels of
`mRNAencoding the noted cytokines, proteins, and receptors were evalu-
`ated using RNase protection. Each lane represents an individual animal.
`
`Lassen - Exhibit 1007, p. 3
`
`Lassen - Exhibit 1007, p. 3
`
`

`

`The Journal of Immunology
`
`2307
`
`comparable alterations in the expression of IL-6Ra and IFN-yRa
`were not observed (Fig. 1). These studies demonstrate that IL-13 is
`a potent stimulator of IL-11 and the a subunit of its receptor in
`murine lung.
`
`Role of IL-11R signaling in IL-13-induced inflammation
`
`To address the importance of IL-11 in the pathogenesis of IL-13-
`induced tissue inflammation, CC10-IL-13 transgenic mice were
`bred with IL-11Ra~’~ mice. The inflammatory responses in IL-13
`Tg* mice with WT andnull IL-11Rq@ loci were then compared. As
`previously reported (12, 30), IL-13 was a potent stimulatoroftis-
`sue inflammation that caused a progressive increase in the accu-
`mulation of macrophages, lymphocytes, and eosinophils in the tis-
`sues and BAL fluids of IL-13 Tg* mice with normal IL-11Reloci.
`In the absence of IL-11Ra, an impressive decrease in this inflam-
`matory response was noted. In 2- and 4-mo-old mice, impressive
`decreases in BAL total cell, macrophage, and eosinophil recovery
`were noted (Fig. 2, A and B). A similarly, impressive decrease in
`tissue inflammatory cell accumulation was apparent (Fig. 2C and
`data not shown). In BAL andtissues, compensatory increases in
`neutrophils were not noted (Fig. 2).
`
`Role of IL-11Ra in IL-13-induced chemokine elaboration
`
`Previous studies from our laboratory demonstrated that IL-13 in-
`ducesits tissue alterations in part via the induction of a wide array
`
`of CC chemokines (12). To investigate the mechanism by which
`IL-11Rea deficiency diminished IL-13-induced inflammation, we
`compared the expression of selected chemokines in IL-13 Tg*
`mice with WT and null IL-11Raq@ loci. In Tg™ mice with WT or
`null IL-11Rq loci, the levels of mRNA encoding MCP-1/CCL2,
`MCP-2/CCL8, MCP-3/CCL7, MIP-la/CCL-3, MIP-1B/CCL4,
`MIP-2/CXCL-2/3, MIP-3a/CCL20, C10/CCL-6, eotaxin/CCL-11,
`eotaxin-2/CCL21, and thymusand activation-regulated chemokine
`(TARC)/CCL17 were comparable and in many cases were near or
`below the limits of detection of our assays (Fig. 3A). As previously
`reported (12, 37), IL-13 increased the levels of mRNA encoding
`these chemokine moieties in Tg* mice with WT IL-11Ra loci
`(Fig. 3A). In contrast, in the absence of IL-11Ra, the ability of
`IL-13 to induce MCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7,
`MIP-la/CCL3, MIP-1B/CCL4, MIP-2/CXCL2-3, MIP-3a/
`CCL20, C10/CCL6,
`eotaxin/CCLI11,
`eotaxin-2/CCL21,
`and
`TARC/CCL17 was markedly diminished (Fig. 3A). In accord with
`these mRNAalterations, comparable alterations in BAL MCP-1/
`CCL2, MIP-la/CCL-3, and eotaxin/CCL-11 protein were ob-
`served (Fig. 3, B—D). Thus, IL-11Ra plays an essential role in the
`stimulation of selected chemokines by IL-13.
`
`
`myofibroblast accumulation
`
`
`
`‘te
`rev
`
`*
`evy
`
`|
`
`LoL _ ee
`Totalecis
` Macraphager
`Eaxvinnghily
`Lasephocytas
` Kewbrephith,
`
`
`
`
`
`LIOT‘JJAqWAAONUojsan3Aq/21o0'Tountu!ammn//:dyyyWoypapeopumog
`
`Quantitative morphometric, biochemical, and immunohistochem-
`ical approaches were used to define the role of IL-11Ra in IL-13-
`induced pulmonary fibrosis and HA and myofibroblast accumula-
`tion.
`In these studies, we compared these collagen, HA, and
`cellular parameters in IL-13 Tg* mice with WT andnull IL-11Ra
`loci. Similar amounts of collagen and BAL HAand similar num-
`bers of anti-smooth muscle actin-staining parenchymal cells were
`noted in lungs from WT littermate control mice and IL-11Ra7/~
`animals (Fig. 4). In WT mice, IL-13 caused an impressive increase
`in lung collagen content (Fig. 4, A and B) and BAL HAlevels (Fig.
`AC) that could be easily determined by histochemical and bio-
`chemical measurement techniques. In addition, IL-13 increased the
`accumulation of parenchymal myofibroblast-like cells that con-
`tained anti-smooth muscle actin, but did not stain with Abs against
`smooth muscle myosin (Fig. 4D and data not shown). In contrast,
`the levels of IL-13-induced collagen and HA weresignificantly
`reduced in lungs from Tg* mice with null vs WT IL-11Ra loci
`(Fig. 4, A-C). Myofibroblast accumulation was similarly decreased
`in lungs from IL-13 Tg*/IL-11Ra~’~ mice compared with Tg*/
`IL-11Ra*’* animals (Fig. 4D).
`Interestingly,
`the anti-smooth
`muscle actin staining of vascular smooth muscle cells was not
`altered in the absence of IL-11Ra (Fig. 4D). Thus, IL-11 signaling
`
`
`playsacritical role in IL-13-induced tissue {i »and HA and
`.
`oped a
`
`Lymphueyter Neutropiih
`Total cells Macrophage: Encinophis
`myofibroblast accumulation.
`
`A *
`ah a
`ae
`Sat.yo5
`eoso
`2
`:
`=Bes
`2M ]
`Tad
`:
`i
`al
`}
`a i
`
`i ,
`
`a
`
`#
`m
`
`«
`rey
`
`B ay
`a0
`fo
`= 70.
`
`&
`roi
`
`:
`=
`ooo
`a
`:
`Sao.
`2°)Be}a
`
`a a
`
`HoT} op
`
`TLS
`
`
`FIGURE 2. Role of IL-11Ra in IL-13-induced inflammation. A and B,
`
`
`
`
`
`The BALcell recoveries of Tg”/IL-11Ra*’* mice (1), Tg/AIL-11Ra7/~
`
`
`
`
`
`
`
`
`
`mice (), Tg‘/IL-11Ra *’* mice @@), and Tg*/IL-11Ra ~~ mice
`(I) at
`2 (A) and 4 (B) moofage are compared. The histologic effects in 4-mo-old
`mice are illustrated in C (original magnification, X10). *, p < 0.01.
`
`Role of IL-11Ra in IL-13-induced production and activation of
`TGF-B1
`
`Previous studies from our laboratory demonstratedthatthe fibrotic
`effects of IL-13 are mediated by its ability to induce andactivate
`TGF-B1 andthat this activation is mediated to a great extent by
`MMP-9 (15). To define the importance of IL-11Ra in these re-
`sponses, we evaluated the TGF-81 production of Tg* mice with
`WT and null IL-11Ra loci. In mice with a WT IL-11Ra locus,
`IL-13 was a potent stimulator of the levels of mRNA encoding
`TGF-B1, TGF-62, and TGF-63 (Fig. 5A). IL-13 also augmented
`MMP-9 mRNAaccumulation (Fig. 5A). In accord with these ob-
`servations, IL-13 increased the levels of spontaneously activated
`and total TGF-61 protein in BAL fluids from these animals (Fig.
`5, B and C). In all cases, these inductive effects appeared to be
`
`Lassen - Exhibit 1007, p. 4
`
`Lassen - Exhibit 1007, p. 4
`
`

`

`2308
`
`IL-11 AND PATHOGENESIS OF IL-13 PHENOTYPE
`
`A
`~
`-
`CClW-IL-1i:
`To-llRe: 4/40-/-
`
`
`
`2000
`
`
`MCP-1
`isag
`MCP.2
`inde
`MCP-3
`Mip- Ter
`14g
`Mip- 1B
`Ling
`Mip-2
`Teed
`Mip-Sce
`cue
`
`B
`
`|
`
`|
`
`MCP-1(pe/ml)
`
`
`
`
`
`FIGURE 3. Role of IL-11Ra in
`IL-13-induced chemokine stimula-
`Eotaxin-2
`tion. A, Comparison of the levels of
`TARC
`mRNA encoding the noted chemo-
`
`kines in lungs from Tg” and Tg*
`RANTES [oeselae
`mice with +/+ and —/— IL-11Ra
`COMM13:
`-
`-
`+
`+
`B- Actio
`
`iL-LiRa:
`alte
`id
`hpi
`salen
`
`loci. B-D, Levels
`of MCP-1,
`MIP-1a, and eotaxin protein were as-
`
`sessed by ELISA in BALfluids from
`
`
`
`2-mo-old (4)
`and 4-mo-old (i)
`mice. The evaluations in A are repre-
`sentative of four similar evaluations.
`|
`ie |
`2500
`B-D,Each value is the mean + SEM
`afT
`of evaluations in a minimum offive
`350)
`I
`|
`2 Me
`mice. *, p < 0.05; *#, p < 0.01.
`ae
`
`
`
`
`
`
`
`LIOT‘JJAQWAAONUojsan3Aq/Sio'Tout!Mam//:dyyyWoy.papeopumog
`
`100
`
`He
`
`&=
`
`acd
`
`= a
`
`O Rig
`
`
`
`MIP-1a(pg/ml)
`
`200
`
`130
`
`10
`
`3)
`
`a
`COE Eas
`WetiRee
`
`>
`+/+
`
`-
`af
`
`ate
`
`
`
`ni
`
`CCWHL-13:
`TLL Re:
`
`=
`4/4
`
`-
`aio
`
`+
`He
`
`+
`~}-
`
`IL-11Ra-dependent, because the levels of mRNA encoding TGF-
`B1, -62, and -63 and MMP-9 andthe production and activation of
`TGF-B1 were significantly decreased in IL-11Ra-null mutant mice
`(Fig. 5). Thus, IL-13 stimulates and activates TGF-B1 and induces
`production of the TGF-61 activator, MMP-9, via an IL-11Ra-de-
`pendent mechanism.
`
`Role of IL-11Ra in IL-13-induced alveolar remodeling
`
`To define the role(s) of IL-11Ra in the pathogenesis of IL-13-
`induced alveolar remodeling, we comparedthe alterations in lung
`volume and alveolar size in IL-13 Tg* mice with WT andnull
`IL-11Ra loci. In accord with previous observations (13), IL-13
`caused an impressive increase in these parameters in lungs from
`mice with WT IL-11Ra loci (Fig. 6, A and B). In contrast, these
`effects of IL-13 were significantly diminished in mice with null
`IL-11Ra loci (Fig. 6, A and B). Thus, IL-11Ra plays a key role in
`this remodeling response.
`
`Effects of IL-1IRa deficiency on lung proteases
`
`To determine whethera deficiency of IL-11Ra could modulate the
`IL-13-induced alveolar phenotype by decreasing the production of
`respiratory proteases, we compared the levels of mRNA encoding
`lung-relevant MMPsandcathepsins in WT and IL-11Ra~‘~ mice.
`As noted above (Fig. 5A), IL-13 is a potent stimulator of MMP-9,
`and this inductive event was mediated via an IL-11Ra-dependent
`pathway. As shown in Fig. 6C, IL-13 wasalso a potent stimulator
`of MMP-2, MMP-12,tissue inhibitor of MMP (Timp)-1, Timp-2,
`Timp-3, Timp-4, cathepsin K, cathepsin S, cathepsin B, and ca-
`thepsin L.
`Interestingly,
`the induction of MMP-2, MMP-12,
`
`Timp-1 to -4, cathepsin K, and cathepsin B was decreased in the
`absence of IL-11Ra (Fig. 6C). Thus, inthe setting of a deficiency
`of IL-11Ra, IL-13 is unable to optimally stimulate lung proteases.
`
`Role of IL-11Ra in IL-13-induced mucus metaplasia
`Studies were next undertaken to determine whether IL-11Ra
`
`played an importantrole in the pathogenesis of IL-13-induced mu-
`cus metaplasia. In these studies we compared mucin gene expres-
`sion in Tg* mice with WTand null IL-11Raloci. The expression
`of gob-5, a calcitum-activated chloride channel involved in the mu-
`cus response (38), was also evaluated. In lungs from Tg” mice
`with WTor null IL-11Raloci, the levels of expression of Muc-Sac
`and gob-5 were at or near the limits of detection in our assay (Fig.
`7). In contrast, IL-13 was a potent stimulator of muc-5AC and
`gob-5 in murine lung (Fig. 7). Interestingly, the stimulation of
`mucSAC and gob-5 gene expression were diminished in Tg* mice
`with null mutant IL-11Ra loci (Fig. 7). These studies demonstrate
`that IL-11 plays an important role in the pathogenesis of IL-13
`stimulation of mucin and gob-5 gene expression.
`
`Role of IL-11Ra in IL-13-induced respiratory death
`
`In CC10-IL-13 Tg* mice, progressive lung pathology is noted. As
`a result, these mice die prematurely from a fibrodestructive, in-
`flammatory alveolar filling process that abrogates normal respira-
`tory function (12). To define the role of IL-11Raq in this fatal
`response, we compared the survival of IL-13 Tg* mice with WT
`and null IL-11Re loci. Tg* mice with IL-11Ra*’* loci started to
`die at ~ 100 days of age, and 100% of these animals were dead by
`4.1 mo of age (Fig. 8). As shownin Fig. 8, a deficiency of IL-11Ra
`
`Lassen - Exhibit 1007, p. 5
`
`Lassen - Exhibit 1007, p. 5
`
`

`

`=
`ev
`
`B is
`e
`Ee Vb
`
`2309
`
`a
`ay
`
`
`
`
`
`
`
`LIOT‘JJAQWAAONUojsan3AqBio"jountuul!ama/:dyyWoypapeopumog
`
` -
`aie
`
`=
`ai
`
`+
`ae
`
`4
`=f
`
`sbieM
`sm
`Git
`
`aii
`
`aa
`
`=
`=2
`=
`
`o S
`
`2a
`
`ati
`SCWHLE
`TE1 Rin
`
`D
`
`EL-tiea GM)
`
`lira G9
`
`— HelNe)
`
`1-130)
`
`a&aai
`
`COTE Le
`Wake
`
`>
`4s
`
`-
`mf
`
`+
`sate
`
`*
`ae
`
`‘
`ey
`
`Cxseo
`
`:
`ms me
`= 2600 |
`2 20001
`<i
`i
`= ison:
`+000
`
`
`_=sell :oon
`
`+a
`
`nf
`
`Leal oo
`
`H-libe:
` 4fe
`
`-
`“fn
`
`+
`fe
`
`
`
`
`
`FibroticareavsTotalarea(a) ie
`
`The Journal of Immunology
`
`FIGURE 4. Role of IL-11Raqin IL-13-inducedfibro-
`sis and HA and myofibroblast accumulation. The colla-
`gen contentof lungs from 4-mo-old IL-13 Tg” and Tg*
`mice with +/+ and —/— IL-11Ra loci were compared
`using Picosirius Red (A) and Sirchol (B) collagen eval-
`uations. C, HA content of BAL fluids from Tg” and
`Tg* mice with WT andnull IL-11Rq@ loci. D, Compar-
`ison of a-smooth muscle actin staining of lungs from
`4-mo-old IL-13 Tg* mice with +/+ and —/— IL-11Ra@
`loci. A-C, Each value represents the mean + SEM of
`evaluations in a minimum offive mice. D, Representa-
`tive of four similar evaluations. *, p < 0.05; **, p <
`0.01. N.D., none detected.
`
`significantly improved the survival of these animals, with Tg */IL-
`11Ra~’~ animals beginning to die at ~5.5 mo of age and many
`animals living to 7.8 mo of age (Fig. 8). Thus, IL-11Ra plays a
`critical role in the pathogenesis of the IL-13-induced pathologies
`that lead to the death of these animals.
`
`Role of IL-1IRa in IL-13-induced protection in 100% O>
`
`Previous studies from our laboratory demonstrated that IL-13 con-
`fers an impressive level of cytoprotection in the context of hyper-
`oxia-induced acute lung injury (19). To define the role of IL-11Ra
`in this protective response, we compared the survival of Tg~ and
`Tg mice with WTand null IL-11Re loci in 100% O3. WT mice
`died after 4—6 days of exposure to 100% O,(Fig. 9). Interestingly,
`IL-11Ra~‘~ mice were more susceptible to 100% O, than their
`WTlittermate controls, dying after 2-3 days of exposure to 100%
`O,. As described previously (36), IL-13 Tg* mice with WT IL-
`11Ra loci lived for an extended interval, with many of these an-
`imals living for 8-12 days in hyperoxia (Fig. 9). Interestingly, a
`deficiency of IL-11Ra did not significantly alter this protective
`response (Fig. 9). Together,
`these studies demonstrate that IL-
`11Ra plays an important role in regulating the response of other-
`wise normal mice to hyperoxia. They also demonstrate that IL-
`11Ra does not play a significant role in the cytoprotection that is
`conferred by IL-13.
`
`Effect of IL-1]Ra deficiency on IL-13 production and receptor
`expression
`
`A deficiency of IL-11Ra@ could modify the IL-13-induced pheno-
`type by altering IL-13 production or the expression of the subunits
`that make up the IL-13R. To address the former, we compared the
`levels of IL-13 in BAL from Tg~ mice with WTandnull IL-11Ra@
`loci. As shown in Fig. 10A, a deficiency of IL-11Ra did notalter
`the levels of transgenic IL-13 protein. To address the receptor is-
`sue, we compared the levels of expression of IL-4Ra and IL-
`13Ra1, which make up the signaling IL-13R complex, and the
`decoy receptor IL-13Ra2 in mice with WTandnull IL-11Raloci.
`The levels of mRNA encoding IL-4Ra, IL-13Ra1, and IL-13 Ra2
`in Tg” mice with WTand null IL-11Raq loci were comparable and
`
`were at or below the limits of detection of our assays (Fig. 108).
`Aspreviously reported (39), IL-13 was a potentstimulator of each
`of these moieties (Fig. 10B). In these experiments a deficiency of
`IL-11Ra caused only modestalterations in the levels of expression
`of IL-4Ra and IL-13Ra1 (Fig. 10B). Importantly, in the absence of
`IL-11Ra,
`the levels of expression of IL-13Ra2 were not aug-
`mented (Fig. 108). In fact, modest decreases in the levels of ex-
`pression of this decoy receptor were noted. These studies demon-
`strate that the amelioration of the IL-13 phenotype that is seen in
`IL-11Ra-null mice is not due to a decrease in IL-13 production, a
`decrease in IL-13Ra1-IL-4Ra receptor expression, or an increase
`in expression of the IL-13Ra2 decoy receptor.
`
`Discussion
`Because IL-13 and IL-11 are juxtaposed in inflammatory tissues,
`studies were undertaken to define the relationship(s) between these
`
`important regulatory moieties. These studies de
`that
`IL-13 is potent stimulator of IL-11 and IL-11Ra.’
`
`
`
`
`
`Lastly they demonstrate that IL-13 is unable to optimally stimulate
`inflammatory chemokines, proteases, and mucin genes and is un-
`able to fully stimulate and activate TGF-B1 in the absence of IL-
`11Ra. These studies define a previously unappreciated mechanism
`of regulation of IL-11 and IL-11Ra. Because IL-11 is the only
`known ligand for the IL-11Ra-gp130 receptor complex,
`these
`studies also define
`
`
` in the pathogenesis of IL-13-induced
`tissue responses.
`IL-11 was discovered as an IL-6-like molecule that stimulated
`
`the proliferation of IL-6-dependent plasmacytomacells (40). Sub-
`sequent investigation has focused to a great extent on the effects of
`exogenously administered rIL-11 and its role as a potential ther-
`apeutic agent. These studies highlighted impressive effects of
`IL-11 on platelets, which is the basis for the approval of IL-11 by
`the U.S. Food and Drug Administration as a treatmentthat fosters
`
`Lassen - Exhibit 1007, p. 6
`
`Lassen - Exhibit 1007, p. 6
`
`

`

`2310
`
`IL-11 AND PATHOGENESIS OF IL-13 PHENOTYPE
`
`A =o
`
`e
`—e
`
`a=-ess
`
`l
`
`CCPL:
`Wether
`
`=
`+e
`
`=
`af
`
`+
`+
`
`+
`ae
`
`B
`
`~
`=
`=Bh£Fa
`
`é
`
`-
`
`sel
`
`P
`
`|
`
`S=
`we
`
`
`
`‘ow | |
`
`
`iene a
`+
`+
`CCRML-Las
`-
`
`Th-liRe:94/4 ape afe spin
`
`
`co10-11. ia:
`=
`o
`+
`*
`Hike: as
`of
`bit
`afm
`my
`
`A CCI TLL:
`-
`_
`+
`+
`Tdike:
`af
`canon
`se
`sof
`TGF. BL a.fs.os
`
`TOP: 62
`
`
`TGR 63
`MMPS
`
`f- Actin
`
`= 14a
`12a
`1s
`SO}
`Gi}
`400
`
`
`
`
`
`ActiveTGP-B1(petal)
`
`
`ate ®
`
`oO
`
`ei
`S000
`
`Si
`
`
`
`TotalTGF-Bl(pg/mb)
`
`COM-IL-d:
`IL-l Res
`
`~
`a
`
`-
`jf
`
`#
`
`|
`
`
`
`
`*
`aye
`
`
`
`+
`+f
`
`FIGURE 5. Role of IL-11Ra in IL-13 stimulation of TGF-B moieties and
`MMP-9, Lungs were obtained from Tg” and Tg* mice with +/+ and —/—
`IL-11Rq@loci. The levels of mRNA encoding TGF-B moieties and MMP-9
`were assessed by RT-PCR(A), and the levels of bioactive (B) and total (C)
`TGF-£1 were evaluated by ELISA. A, Representative of four similar eval-
`
`uations. B and C, Values represent the mean + SEM ofevaluation in a
`
`
`
`minimum of five mice that were either 2 (4) or 4 (HD) moofage.
`
`platelet reconstitution after bone marrow ablative therapy (17, 18).
`They also defined the ability of recombinant and transgenic IL-11
`to confer cytoprotection and inhibit inflammation during states of
`mucosal/tissue injury (27, 28, 33, 41-43). These studies did not,
`however, address in detail the roles of endogenous IL-11 and IL-11
`signaling in the generation of tissue inflammatory and extraosse-
`ous remodeling responses. The present studies provide a new level
`of insight into the biology of IL-11 by demonstrating that in ad-
`dition to the protective effects of high concentrations of exogenous
`IL-11, endogenous IL-11 has important proinflammatory effects at
`sites of IL-13-mediated tissue inflammation. In the absence of
`
`IL-11 signaling, the ability of IL-13 to induce lymphocytic and
`eosinophilic tissue inflammation was markedly diminished. In ac-
`cord with these findings, in the absence of IL-11Ra, IL-13 was
`also unable to optimally stimulate the production of the proinflam-
`matory chemokines
`(MCP-1/CCL-2, MCP-2/CCL8, MCP-3/
`CCL7, MIP-la/CCL3, MIP-1B/CCL4, MIP-2/CXCL2-3, MIP-
`3a/CCL20, C10/CCL6, eotaxin/CCL11, eotaxin 2, an