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`Immunology
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`.
`IL-11 Receptor 0c in the Pathogenesis of
`IL-13-Induced Inflammatlon and Remodellng
`Qingsheng Chen, Lesley Rabach, Paul Noble, Tao Zheng,
`Chun Geun Lee, Robert J. Homer and Jack A. Elias
`
`Jlmmunol 2005; 174230523 13; ;
`doi: 10.4049/jimmunol.174.4.2305
`http ://www.j immunol.org/content/ 1 74/4/23 05
`
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`
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`References
`
`This article cites 59 articles, 18 of which you can access for free at:
`http ://www.j immunolorg/content/ 1 74/4/23 05 .full#ref—list- 1
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`The Journal ofImmunology is published twice each month by
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`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. l
`
`
`
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`
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`
`Lassen - Exhibit 1007, p. 1
`
`

`

`The Journal of Immunology
`
`IL-11 Receptor a in the Pathogenesis of IL-13-Induced
`Inflammation and Remodeling1
`
`Qingsheng Chen,2* Lesley Rabach,2* Paul Noble,* Tao Zheng, Chun Geun Lee,*
`Robert J. Homer,H and Jack A. Elias3*
`
`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 IL-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-llRa, 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-llRa on the tissue effects of transgenic IL-13. These studies demonstrate that IL-13 is a potent stimulator of IL-11 and
`IL-llRa. 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-
`llRa loci and that these alterations are ameliorated in the absence of IL-llRa. 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-Bl via IL-llRa-dependent pathways. When viewed in combination, these studies demonstrate that
`IL-llRa plays a key role in the pathogenesis of IL-13-induced inflammation and remodeling. The Journal of Immunology, 2005,
`174: 2305—2313.
`
`I nterleukin—l3 is a pleiotropic 12—kDa product of a gene on
`
`chromosome 5 at q3l that is produced in large quantities by
`stimulated Th2 cells. It was originally described as an IL—4—
`like molecule based on shared eifector properties, including the
`ability to stimulate IgE production. Subsequent studies demon—
`strated that IL—13 and E—4 often play distinct roles in biology. A
`prominent aspect of this distinction is the appreciation that E—4
`plays a key role in Th2 cell diiferentiation and response generation,
`whereas IL—l3 contributes as the major eifector of Th2 inflamma—
`tion and tissue remodeling (1—4). In accord with these observa—
`tions, IL—l3 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 lDepartment of Pathology,
`Yale University School of Medicine, New Haven, CT 06520; and 1: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.
`1This work was supported by National Institutes of Health Grants HL64242,
`HL78744, HL66571, and HL56389 (to J.A.E.).
`
`2 Q.C. and LR. made equal contributions to this work.
`3 Address correspondence and 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)4 (1—11). Studies
`from our laboratory and others have demonstrated that IL—13 me—
`diates its tissue eifects by activating a broad array of downstream
`target genes,
`including chemokines, matrix metalloproteinases
`(MMPs), TGF—Bl, and chitinases (12—16). The importance of Ur
`6—type cytokines in the generation of the eifects of IL—l3, however,
`have not been investigated.
`IL—11 is a multifunctional E—6—type cytokine with diverse bio—
`logic properties, including the ability to stimulate hemopoiesis,
`thrombopoiesis, megakaryocytopoiesis, and bone resorption; reg—
`ulate macrophage diiferentiation; and confer mucosal protection
`after chemotherapy and radiation therapy (17—22). These eifects
`are mediated by a multimeric receptor that contains a ligand—bind—
`ing a subunit, IL—llRa, and the ubiquitous B subunit, gpl30, that
`triggers intracellular signaling (18, 23, 24). Previous studies from
`our laboratory and others demonstrated that, like IL—l3, IL—11 is
`expressed in an exaggerated fashion in the dysregulated Th2 re—
`sponse in the asthmatic airway (25). Although IL—ll can inhibit
`Thl responses,
`inhibit the production of Thl—related cytokines
`such as E—l2, and shift inflammation in a Th2 direction (22, 26—
`29), little else is known about the 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 HIE—induced pathologies has not been
`established.
`
`We hypothesized that HIM signaling plays a key role in Hf
`l3—induced Th2 inflammation. To test this hypothesis, we charac—
`terized the expression of IL—1 1, IL—1 lRa, and gpl30 in lungs from
`wild—type (WT) mice and mice in which IL—13 was overexpressed
`in a lung—specific fashion. We also characterized the eifects of a
`null mutation of E—llRa on the tissue eifects of transgenic IL—l3.
`These studies demonstrate that HIE is a potent stimulator of
`IL—11 and IL—llRa. They also demonstrate that E—llRa plays a
`
`key role in E—l3—induced inflammation, :fi 0,9,;7hyaluronic acid
`
`Copyright © 2005 by The American Association of Immunologists, Inc.
`
`0022-1767/05/$02.00
`
`Lassen — Exhibit 1007, p. 2
`
`
`
`“oz‘z.19querNno189113[{q,1310'10unu1uuf‘mmm,l:duq11101;pepeommoq
`
`
`
`
`
`Lassen - Exhibit 1007, p. 2
`
`

`

`2306
`
`IL—11 AND PATHOGENESIS OF E—13 PHENOTYPE
`
`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 was evaluated. Samples were
`compared with a simultaneously performed standard curve.
`
`TGF— B bioassay
`
`The levels of total and bioactive TGF—Bl were evaluated by ELISA (R&D
`Systems) using untreated and acid—treated BAL fluids according to the
`manufacturer’s instructions.
`
`Murine 100% 02 exposure
`
`Adult 6— to 8—wk—old Tg’ and TgJr mice with WT or null mutant IL—11Ra
`loci were exposed to room air (controls) or continuously to 100% O2 in a
`Plexiglas chamber as previously described (19, 36). All protocols were
`reviewed and approved by the institutional animal care and use committee
`at Yale University School of Medicine.
`
`Statistics
`
`Normally distributed data are expressed as the mean : SEM and assessed
`for significance by Student’s t test or ANOVA as appropriate. Data that
`were not normally distributed were assessed for significance using the Wil—
`coxon rank—sum test.
`
`Results
`Efiect of IL—13 on IL—11 and IL—1 1R expression
`Studies were undertaken to define the eifects 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 HIM and IL—11Ra. These eifects 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 E—6—type cytokines,
`E—6 and LIF (Fig. 1). A modest increase 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—1 1Ra were also at least partially specific, because
`
`
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`(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—Bl via
`HI 1 1Ra—dependent pathways.
`
`Materials and Methods
`Overexpression transgenic mice
`
`CC10—IL—13 transgenic mice were generated in our laboratory, bred onto a
`C57BL/6 background, and used in these studies. These mice use the Clara
`cell 10—kDa protein (CC10) promoter to 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—11Ra—null mice (IL—11111147) 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+I+ and null’/’ IL—11Ra
`loci were generated by breeding the IL—13 transgenic (TgT) mice with the
`IL—11Ra”’ animals. Genotyping was accomplished as previously de—
`scribed (30, 32). Littermate control WT mice with (+/+) or without (—/—)
`IL—11Ra loci were used as controls.
`
`Bronchoalveolar lavage (BAL)
`
`Lung inflammation was assessed by BAL as previously described (13, 33).
`The BAL samples 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 removed en bloc, inflated at 25 cm pressure with
`neutral—bulfered 10% formalin, fixed in 10% formalin, embedded in par—
`aflin, sectioned, and stained. H&E, Mallory’s Trichrome, and periodic ac—
`id—Schilf 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 Rionuant; BD Biosciences) as
`described by the manufacturer. Other mRNA levels were evaluated by
`RT—PCR analysis 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
`
`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 comparing the staining of serial sections that were incubated in
`the presence and the absence of the primary Ab.
`
`IL—13 regulation of IL—11Ra and IL—11. Lungs were ob—
`FIGURE 1.
`tained from 2—mo—old CC10—IL—13 Tg’ and Tg+ mice, and the levels of
`mRNA encoding the noted cytokines, proteins, and receptors were evalu—
`ated using RNase protection. Each lane represents an individual animal.
`
`Lassen — Exhibit 1007, p. 3
`
`
`
`
`
`
`
`“oz‘zJQQUIQAONno189113[{q,1310'[ounmturf'mmn/,I:duq11101;pepBOIHMOG
`
`Lassen - Exhibit 1007, p. 3
`
`

`

`The Journal of Immunology
`
`2307
`
`comparable alterations in the expression of E—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—1 1R signaling in IL—I3—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 HJ—llRoF/T mice. The inflammatory responses in HIE
`Tg+ mice with WT and null E—llRa loci were then compared. As
`previously reported (12, 30), IL—13 was a potent stimulator of tis—
`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 E—llRa loci.
`In the absence of E—llRa, 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 and tissues, compensatory increases in
`neutrophils were not noted (Fig. 2).
`
`Role of IL—IIRa in IL—I3—induced chemokine elaboration
`
`Previous studies from our laboratory demonstrated that IL—13 in—
`duces its tissue alterations in part via the induction of a wide array
`
`of CC chemokines (12). To investigate the mechanism by which
`E—llRa deficiency diminished HIE—induced inflammation, we
`compared the expression of selected chemokines in IL—13 Tg+
`mice with WT and null E—llRa loci. In Tg’ mice with WT or
`null IL—11Ra loci, the levels of mRNA encoding MCP—l/CCL2,
`MCP—2/CCL8, MCP—3/CCL7, MIP—la/CCL—3, MIP—lB/CCL4,
`MIP—2/CXCL—2/3, MIP—3ot/CCL20, C10/CCL—6, eotaxin/CCL—11,
`eotaxin—2/CCL21, and thymus and 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 E—llRa loci
`(Fig. 3A). In contrast, in the absence of E—llRa, the ability of
`IL—13 to induce MCP—l/CCL2, MCP—2/CCL8, MCP—3/CCL7,
`MIP—la/CCL3, MIP—lB/CCL4, MIP—2/CXCL2—3, MIP—3a/
`CCL20, C10/CCL6,
`eotaxin/CCL11,
`eotaxin—2/CCL21,
`and
`TARC/CCL17 was markedly diminished (Fig. 3A). In accord with
`these mRNA alterations, comparable alterations in BAL MCP—l/
`CCL2, MIP—la/CCL—3, and eotaxin/CCL—11 protein were ob—
`served (Fig. 3, B—D). Thus, E—llRa plays an essential role in the
`stimulation of selected chemokines by IL—13.
`
`
`myofibroblast accumulation
`
`Quantitative morphometric, biochemical, and immunohistochem—
`ical approaches were used to define the role of IL—11Ra in HIE—
`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 and null E—llRa
`loci. Similar amounts of collagen and BAL HA and similar num—
`bers of anti—smooth muscle actin—staining parenchymal cells were
`noted in lungs from WT littermate control mice and IL—11RoF/T
`animals (Fig. 4). In WT mice, HIE caused an impressive increase
`in lung collagen content (Fig. 4, A and B) and BAL HA levels (Fig.
`4C) that could be easily determined by histochemical and bio—
`chemical measurement techniques. In addition, HIE 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 HIE—induced collagen and HA were significantly
`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—11R0F/’ mice compared with Tg+/
`HJ—llRotH+ animals (Fig. 4D).
`Interestingly,
`the anti—smooth
`muscle actin staining of vascular smooth muscle cells was not
`altered in the absence of IL—1 1Ra (Fig. 4D). Thus, HIM signaling
`plays a critical role in HIE—induced tissue 11
`3
`
`myofibroblast accumulation.
`
`Role of IL—IIRa in IL—I3—induced production and activation of
`TGF431
`
`Previous studies from our laboratory demonstrated that the fibrotic
`eflects of IL—13 are mediated by its ability to induce and activate
`TGF—Bl and that 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—Bl 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—Bl, TGF—B2, and TGF—B3 (Fig. 5A). IL—13 also augmented
`MMP—9 mRNA accumulation (Fig. 5A). In accord with these ob—
`servations, IL—13 increased the levels of spontaneously activated
`and total TGF—Bl protein in BAL fluids from these animals (Fig.
`5, B and C). In all cases, these inductive eflects appeared to be
`
`Lassen — Exhibit 1007, p. 4
`
`
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`
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`The BAL cell recoveries of Tg/IL— 11Ra+l+ mice (
`), Tgi/IL— llRa’I’
`
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`mice (E33513), Tg+/IL— llRa Jr” mice (I), and Tg+/IL— llRa 7” mice
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`
`

`

`2308
`
`IL—ll AND PATHOGENESIS OF E—13 PHENOTYPE
`
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`IL—llRa—dependent, because the levels of mRNA encoding TGF—
`61, +32, and 433 and MMP—9 and the production and activation of
`TGF— [31 were significantly decreased in E—llRa—null mutant mice
`(Fig. 5). Thus, HIE stimulates and activates TGF—Bl and induces
`production of the TGF—Bl activator, MMP—9, via an IL—l 1Ra—de—
`pendent mechanism.
`
`Role of IL—IlRa in IL—I3—induced alveolar remodeling
`
`To define the role(s) of E—llRa in the pathogenesis of IL—13—
`induced alveolar remodeling, we compared the alterations in lung
`volume and alveolar size in IL—13 Tg+ mice with WT and null
`E—llRa loci. In accord with previous observations (13), HIE
`caused an impressive increase in these parameters in lungs from
`mice with WT E—llRa loci (Fig. 6, A and B). In contrast, these
`eifects of HIE were significantly diminished in mice with null
`E—llRa loci (Fig. 6, A and B). Thus, E—llRa plays a key role in
`this remodeling response.
`
`Efiects of IL—IlRa deficiency on lung proteases
`
`To determine whether a deficiency of IL—1 1Ra could modulate the
`HIE—induced alveolar phenotype by decreasing the production of
`respiratory proteases, we compared the levels of mRNA encoding
`lung—relevant MMPs and cathepsins in WT and HJ—llRoF/T mice.
`As noted above (Fig. 5A), HIE is a potent stimulator of MMP—9,
`and this inductive event was mediated via an IL—l 1Ra—dependent
`pathway. As shown in Fig. 6C, HIE was also a potent stimulator
`of MMP—2, MMP—12, tissue inhibitor of MMP (Timp)—1, Timp—Z,
`Timp—3, Timp—4, cathepsin K, cathepsin S, cathepsin B, and ca—
`thepsin L.
`Interestingly,
`the induction of MMP—2, MMP—12,
`
`Timp—l to —4, cathepsin K, and cathepsin B was decreased in the
`absence of IL—11Ra (Fig. 6C). Thus, in the setting of a deficiency
`of IL—1 1Ra, IL—13 is unable to optimally stimulate lung proteases.
`
`Role of IL—IlRa in IL—I3—induced mucus metaplasia
`Studies were next undertaken to determine whether E—llRa
`
`played an important role in the pathogenesis of HIE—induced mu—
`cus metaplasia. In these studies we compared mucin gene expres—
`sion in Tg+ mice with WT and null IL—l 1Ra loci. The expression
`of gob—5, a calcium—activated chloride channel involved in the mu—
`cus response (38), was also evaluated. In lungs from Tg’ mice
`with WT or null E—llRa loci, the levels of expression of Muc—5 ac
`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—SAC and
`gob—5 in murine lung (Fig. 7). Interestingly, the stimulation of
`muc5AC and gob—5 gene expression were diminished in Tg+ mice
`with null mutant E—llRa loci (Fig. 7). These studies demonstrate
`that HIM plays an important role in the pathogenesis of HIE
`stimulation of mucin and gob—5 gene expression.
`
`Role of IL—IlRa in IL—I3—induced respiratory death
`
`In CClO—E—l3 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 E—llRa in this fatal
`response, we compared the survival of HIE Tg+ mice with WT
`and null E—llRa loci. Tg+ mice with IL—llRozH+ 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 shown in Fig. 8, a deficiency of E—llRa
`
`Lassen — Exhibit 1007, p. 5
`
`Lassen - Exhibit 1007, p. 5
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`FIGURE 4. Role of IL—11Ra in IL—13—induced fibro—
`sis and HA and myofibroblast accumulation. The colla—
`gen content of 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
`Tgir mice with WT and null IL—11Ra 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 of five mice. D, Representa—
`tive of four similar evaluations. *, p < 0.05; **, p <
`0.01. N.D., none detected.
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`significantly improved the survival of these animals, with Tg+/IL—
`llRoF/T animals beginning to die at ~55 mo of age and many
`animals living to 7.8 mo of age (Fig. 8). Thus, E—llRa plays a
`critical role in the pathogenesis of the HIE—induced pathologies
`that lead to the death of these animals.
`
`Role of IL—IIRa in IL—I3—indnced protection in 100% 02
`
`Previous studies from our laboratory demonstrated that HIE con—
`fers an impressive level of cytoprotection in the context of hyper—
`oxia—induced acute lung injury (19). To define the role of E—llRa
`in this protective response, we compared the survival of Tg+ and
`Tg’ mice with WT and null E—llRa loci in 100% 02. WT mice
`died after 4—6 days of exposure to 100% 02 (Fig. 9). Interestingly,
`HJ—llRa’/’ mice were more susceptible to 100% 02 than their
`WT littermate controls, dying after 2—3 days of exposure to 100%
`02. As described previously (36), IL—13 Tg+ mice with WT Hf
`llRa 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 E—llRa 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.
`
`Efiect of IL—IIRa deficiency on IL—13 production and receptor
`expression
`
`A deficiency of E—llRa could modify the HIE—induced pheno—
`type by altering IL—13 production or the expression of the subunits
`that make up the E—13R. To address the former, we compared the
`levels of IL—13 in BAL from Tg+ mice with WT and null E—llRa
`loci. As shown in Fig. 10A, a deficiency of E—llRa did not alter
`the levels of transgenic HIE protein. To address the receptor is—
`sue, we compared the levels of expression of IL—4Ra and HI
`13Ra1, which make up the signaling E—13R complex, and the
`decoy receptor E—13Ra2 in mice with WT and null IL—l 1Ra loci.
`The levels of mRNA encoding IL—4Ra, IL—l3Ra1, and IL—13 R012
`in Tg’ mice with WT and null IL—l 1Ra loci were comparable and
`
`were at or below the limits of detection of our assays (Fig. 103).
`As previously reported (39), HIE was a potent stimulator of each
`of these moieties (Fig. 103). In these experiments a deficiency of
`E—llRa caused only modest alterations in the levels of expression
`of IL—4Ra and IL—13Ra1 (Fig. 103). Importantly, in the absence of
`E—llRa,
`the levels of expression of IL—13Ra2 were not aug—
`mented (Fig. 103). 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 HIE phenotype that is seen in
`E—llRa—null mice is not due to a decrease in HIE production, a
`decrease in IL—13Ra1—IL—4Ra receptor expression, or an increase
`in expression of the E—13Ra2 decoy receptor.
`
`Discussion
`Because HIE and HIM 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—1 1Ra
`L
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`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—Bl in the absence of Ur
`llRa. These studies define a previously unappreciated mechanism
`of regulation of IL—11 and E—llRa. Because IL—11 is the only
`known ligand for the E—llRa—gpl30 receptor complex,
`these
`studies also define
`
`
` 1n the pathogenes1s of HIE—induced
`tissue responses.
`IL—11 was discovered as an E—6—like molecule that stimulated
`
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`the proliferation of E—6—dependent plasmacytoma cells (40). Sub—
`sequent investigation has focused to a great extent on the eifects of
`exogenously administered rIL—ll and its role as a potential ther—
`apeutic agent. These studies highlighted impressive eifects 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 treatment that fosters
`
`Lassen — Exhibit 1007, p. 6
`
`Lassen - Exhibit 1007, p. 6
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`FIGURE 5. Role of IL—llRa in lL—13 stimulation of TGF—B moieties and
`MMP—9. Lungs were obtained from Tg’ and TgJr mice with +/+ and —/—
`IL—llRa 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—Bl were evaluated by ELISA. A, Representative of four similar eval—
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`minimum of five mice that were either 2 (E33513) or 4 (I) mo of age.
`
`platelet reconstitution after bone marrow ablative therapy (17, 18).
`They also defined the ability of recombinant and transgenic HIM
`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 HIM and HIM
`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 eflects of high concentrations of exogenous
`IL—11, endogenous HIM has important proinflammatory eflects at
`sites of HIE—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 E—llRa, IL—13 was
`also unable to optimally stimulate the production of the proinflam—
`matory chemokines
`(MCP—l/CCL—Z, MCP—Z/CCLS, MCP—3/
`CCL7, MIP—la/CCL3, MIP—lB/CCIA, MIP—