Targeted Expression of IL-11 in the Murine Airway Causes Lymphocytic
`Inflammation, Bronchial Remodeling, and Airways Obstruction
`
`Weiliang Tang,* Gregory P. Geba,* Tao Zheng,* Prabir Ray,* Robert J. Homer,*§ Charles KuhnIll," Richard A. Flavell,|
`and JackA. Elias*
`*Departmentof Internal Medicine, Section of Pulmonary and Critical Care Medicine, ‘Department of Pathology, and Department of
`Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520;8VA-CT Health Care System, West Haven,
`Connecticut 06516; and \Department of Pathology, Brown University School of Medicine, Memorial Hospital of Rhode Island,
`Pawtucket, Rhode Island 02860
`
`D3
`
`Abstract
`
`Interleukin-11 is a pleotropic cytokine produced by lung
`stromalcells in response to respiratory viruses, cytokines,
`and histamine. To further define its potential effector func-
`tions, the Clara cell 10-kD protein promoter wasused to ex-
`press IL-11 and the airwaysof the resulting transgene mice
`were characterized. In contrast to transgene (— ) littermates,
`the airways of [L-I| transgene (+) animals manifest nodu-
`lar peribronchiolar mononuclearcell infiltrates and impres-
`sive airways remodeling with subepithelial fibrosis. The in-
`flammatory foci contained large numbers of B220(+) and
`MHC Class II(+) cells and lesser numbers of CD3(+),
`CD4(+), and CD8(+) cells. The fibrotic response contained
`increased amounts of types III and I collagen, increased
`numbers of « smooth muscle actin and desmin-containing
`cells and a spectrum of stromal elements including fibro-
`blasts, myofibroblasts, and smooth muscle cells. Physiologic
`evaluation also demonstrated that 2-mo-old transgene (+)
`mice had increased airways resistance and non-specific air-
`ways hyperresponsiveness to methacholine when compared
`with their transgene (—) littermates. These studies demon-
`strate that the targeted expression of IL-11 in the mouseair-
`way causes a B and T cell—predominant inflammatory re-
`sponse, airway remodeling with increased types III and I
`collagen, the local accumulation of fibroblasts, myofibro-
`blasts, and myocytes, and obstructive physiologic dysregu-
`lation. IL-11 may play an importantrole in the inflamma-
`tory and fibrotic responses in viral and/or nonviral human
`airway disorders. (J. Clin. Invest. 1996. 98:2845-2853.) Key
`words: epithelial cell + fibrosis « cytokine « myofibroblast +
`collagen
`
`Introduction
`
`Obstructive airways disorders are a major cause of morbidity
`and mortality, with asthma affecting ~ 9-12 million people
`(1, 2), chronic obstructive pulmonary disease (COPD)! affect-
`ing 12-14 million people (3), and bronchiolitis and bron-
`chiectasis affecting large numbers of people (4, 5)
`in the
`
`Address correspondence to Jack A. Elias, Yale University School of
`Medicine, Section of Pulmonary and Critical Care Medicine, Depart-
`ment of Internal Medicine, 333 Cedar Street, 105 LCI, New Haven,
`CT 06520. Phone: 203-785-4163; FAX: 203-785-3826.
`Received for publication 30 May 1996 and accepted in revised form
`10 October 1996.
`
`The Journal of Clinical Investigation
`Volume 98, Number 12, December 1996, 2845-2853
`
`United States alone. Chronic airway inflammation and airway
`remodeling (defined as fibrosis, matrix alterations, and/or
`changes in structural or resident cells of the airway wall) are
`important features of these disorders (3-11). However, the
`pathogenetic mechanisms that generate these responses and
`the relationship between these responses and the physiologic
`dysregulation characteristic of these disorders are poorly un-
`derstood.
`
`Respiratory viruses play an important role in obstructive
`disorders of the human airway. Viruses are important precipi-
`tants of asthmatic exacerbations (6, 12-15) and may similarly
`exacerbate COPD (16). In addition, epidemiologic investiga-
`tions have demonstrated important associations between infan-
`tile viral infections and the existence of asthma (12, 13, 17, 18),
`and pediatric infections and COPD (16, 19) in laterlife. These
`viral effects are felt to be mediated via a number of mecha-
`
`nisms, including the induction and modulation of local inflam-
`mation (10, 12, 20). Virus-stimulated cytokine productionis
`increasingly understoodto play a prominentrole in the gener-
`ation of these inflammatory abnormalities (6, 21-23). The con-
`tribution(s) that each virus-stimulated cytokine makes to the
`pathologic and physiologic abnormalities characteristic of viral
`infections in hosts with normal and obstructed airways has,
`however, been inadequately investigated. In addition, we
`knowlittle about the mechanism(s) by which pediatricviral in-
`fections and virus-induced cytokines predispose to airways dis-
`ordersin laterlife.
`
`IL-11 was initially discovered as a plasmacytomaprolifera-
`tion stimulating activity in supernatants from transformed
`matrow fibroblasts (24, 25). In accordance with this finding,
`most studies of IL-11 have focused onits roles in hematopoie-
`sis (25). IL-11 has, however, been shown to have a variety of
`other bioactivities, including the ability to stimulate the acute
`phase response (25), augment the production of metallopro-
`teinase inhibitors (26, 27), increase immunoglobulin produc-
`tion (25, 28), and alter neural phenotype (29). Previous studies
`from our laboratory demonstrated that human lungfibroblasts
`and epithelial cells produce IL-11 in response to cytokines (IL-1
`and TGF-B,), histamine, and viruses that have been epidemio-
`logically associated with asthmatic exacerbations (rhinovirus,
`respiratory syncytial virus [RSV], and parainfluenza virus type
`3, [PIV3]) (21, 30, 31). Our studies have also demonstrated
`that IL-11 can be foundin the nasal secretions of children with
`
`viral upper respiratory tract infections and that IL-11 induces
`
`1. Abbreviations used in this paper: AHR,airways hyperresponsive-
`ness; BAL, bronchoalveolar lavage; CC10, clara cell 10-kD protein;
`COPD, chronic obstructive pulmonary disease; MCh, methacholine;
`PIV3, parainfluenza virus type 3; RSV, respiratory synctial virus.
`
`Transgenic Expression of Interleukin-11 in the Mouse Airway
`
`2845
`
`Lassen - Exhibit 1009, p. 1
`
`Lassen - Exhibit 1009, p. 1
`
`

`

`airways hyperresponsiveness (AHR) wheninhaled in a tran-
`sient fashion into murine lungs (23). As a result of these obser-
`vations, we postulated that IL-11 plays an importantrole in the
`pathogenesis of human airway disorders, particularly those
`that are associated with viral infections.
`
`In keeping with the chronic nature of disorders such as
`asthma, COPD, bronchiectasis, and bronchiolitis, we addressed
`this hypothesis by defining the respiratory tract manifestations
`of IL-11 when chronically present in the respiratory tract. This
`was done by generating and evaluating the airways of trans-
`genic mice in which the Clara cell 10-kD protein (CC10) pro-
`moter was used to target IL-11 to the respiratory tree. These
`studies demonstrate that IL-11 causes impressive airway alter-
`ations with transgene (+) animals manifesting a nodular B and
`T cell-predominant peribronchiolar inflammatory response,
`bronchial remodeling with subepithelial fibrosis, and physiologic
`dysfunction characterized by airways obstruction and nonspe-
`cific AHR.
`
`Methods
`
`Production and identification of transgenic mice. To study the effec-
`tor functions of IL-11 in the airway, we took advantage of the fact
`that the murine respiratory tract epithelium contains 50-60%clara
`cells (32, 33). As previously described (32), we used the promoter of
`the CC10 geneto target the expression of human IL-11 to airwaytis-
`sues. The rat CC10 promoter wasa gift of Drs. Barry Stripp and Jef-
`frey Whitsett (34). It was isolated as a 2.3-kb HindIII fragment and
`subcloned into the HindIII site of construct pKS-SV40, yielding con-
`struct pKS-CC10-SV40. pKS-SV40 had been previously prepared by
`inserting a 0.85-kb BglII/BamHI fragment containing S$V40 intronic
`and polyadenylation sites into the BamHIsite in construct pBlue-
`script ITKS (StratageneInc., La Jolla, CA). The cDNA encoding hu-
`man IL-11 was a generousgift of Dr. Paul Schendel (Genetics Insti-
`tute, Cambridge, MA). It was isolated as a 1.2-kb EcoRI fragment,
`endfilled with Klenow enzyme and subcloned into the EcoRVsite in
`pKS-CC10-SV40 using standard techniques. All constructs were
`checked for correct orientation of the inserts by restriction enzyme
`digestion, and junction sequences were confirmed by sequencing. The
`resulting CC10-IL-11-SV40 construct waspurified, digested with Asp
`718 and BamHIto generate the CC10-IL-11-SV40 fragment(Fig. 1),
`separated by electrophoresis through 1% agarose, and isolated by
`electroelution into dialysis tubing. The DNA fragment was then puri-
`fied through Elutip-D columns following the manufacturer’s instruc-
`tions (Schleicher and Schuell, Inc., Keene, NH) and dialyzed against
`injection buffer (0.5 mM Tris-HClI/25 mM EDTA,pH 7.5). Trans-
`genic mice were prepared in (CBA X C57 BL/6) F, eggs using stan-
`dard pronuclear injection as previously described (32, 35). The pres-
`ence or absence of the transgene was evaluated in offspring animals
`using tail-dertved DNA. This was initially done by Southern blot
`analysis using **P-labeled IL-11 cDNA as a probe. Similar results
`were obtained by PCR using 5’-CGACTGGACCGGCTGCTGC-3’
`and 5'-CTAACTAGGGGGAGATAATGGCGGGGGGA-3’ as up-
`per and lowerprimers, respectively. 35 cycles were performed. Each
`cycle was heated at 95°C for 1 min, annealed at 63°C for 1 min, and
`elongated at 72°C for 2 min.
`
`Asp 718
`
`
`
`
`Tithee | oot 0 euOMeUris
`rat CC10 promoter
`
`Is
`>|
`2.3 kb
`*
`1.2 kb
`0.85 kb
`
`Bam HI
`
`Figure 1. Schematicillustration of CC10-IL-11 construct used in the
`preparation of the transgenic mice described in this manuscript.
`
`Bronchoalveolar lavage and quantification of IL-11 levels. Mice
`were killed via cervical dislocation, a median sternotomy was per-
`formed, blood was obtained via right heart puncture and aspiration,
`and serum was prepared. The trachea wasthen isolated via blunt dis-
`section and small caliber tubing was inserted and secured in the air-
`way. Three successive washes of 0.75 ml PBS with 0.1% BSA were
`then instilled and gently aspirated. Each bronchoalveolar lavage
`(BAL)aliquot was centrifuged and the supernatants were harvested
`andstored individually at —70°C until ready to be used. Thelevels of
`IL-11 in the BAL fluid and serum were quantitated immunologically
`via ELISAandbiologically using the B9.11 plasmacytomaprolifera-
`tion bioassay. The ELISA was performed as previously described by
`our laboratory (21, 30) using antibodies 11h3/15.6.1 and 11h3/19.6.1
`provided by Dr. Edward Alderman (Genetics Institute). The bioas-
`say was also performedas previously described by our laboratory
`(36, 37) using B9.11 cells also provided by Genetics Institute. Since
`both IL-6 and IL-11 can stimulate B9.11 cell proliferation, this assay
`was performed in the presence and absence of neutralizing antibodies
`against IL-11 (a gift of Dr. Alderman) and IL-6 (a gift of Dr. Pravin
`Sehgal, New York Medical College, Valhalla, NY) to assess the rela-
`tive contribution of each of these moieties.
`
`Northern analysis. Total cellular RNA from a variety of mouse
`tissues was obtained using guanidine isothiocyanate extraction and
`formaldehyde-agarose gel electrophoresis as previously described
`(21, 30). IL-11 gene expression was assessed by probing with ”P-
`labeled IL-11 cDNA. Equality of sample loading and efficiency of
`transfer were assessed via ethidium bromidestaining.
`Histologic evaluation. Animals were killed via cervical disloca-
`tion, median stenotomies were performed, and right heart perfusion
`was accomplished with calctum and magnesium-free PBSto clear the
`intravascular space. The heart and lungs were then removed en bloc,
`inflated with 1 cc neutral buffered 10% formalin, fixed overnight in
`10%formalin, embedded in paraffin, and sectioned and stained. He-
`matoxylin and eosin, and Mallory’s trichrome stains were performed.
`Morphometric analysis. Morphometric study was carried out on
`mice aged 15 d, 1 mo, and 2 mo. The thickness of the walls of small
`airways from the base of the columnarepithelium to the outerlimit of
`the adventitia was measured using an eye-piece reticle. Bronchioles
`< 250 xm in diameter that presented a closed circular or oval profile
`were selected and all measurements were madeat 400 magnification
`to the nearest whole micrometer. The wall thickness was routinely
`evaluated at two points on opposite sides of the short axis of theellip-
`tical profiles and measurements were madeat locations where cell
`borders appeared sharp to minimize tangential sectioning. 5-12 air-
`ways were measured per mouse, mean 8.4. The presence or absence
`of lymphoid nodules was recorded for each bronchiolar profile,
`whether or not it was considered appropriate for measuring wall
`thickness. Statistical evaluations of the morphometric results were
`performed by the Bonferonni multiple comparisons test using Instat
`software for the Macintosh.
`
`the vascular tree
`Immunohistochemistry. Animals were killed,
`was perfused, and the heart and lungs were removeden bloc as de-
`scribed above. The tissues were then processed using a numberof ap-
`proaches. For evaluations of cell surface markers and subepithelial
`airway cellularity, lungs were inflated with 1 x PBS/33% (vol/vol)
`OCTtissue-tek compound (Miles Laboratories, Inc., Elkhart, IN)
`and snap frozen in OCT by submersion into 2-methylbutane cooled
`with dry ice. Tissue sections werecut, transferred onto silane-treated
`glass slides, fixed with acetone for 15 min, and stained with various
`antibody reagents
`as previously described (32). Sections were
`blocked with avidin-blocking kit (Vector Laboratories, Inc., Burlin-
`game, CA) and BSA before reaction with the desired biotinylated
`primary antibody. The slides were then washedthree times (in 0.1 M
`Tris buffer, pH 7.5) and the tissue sections were incubated with a pre-
`diluted streptavidin-alkaline phosphatase solution (Vector Laborato-
`ries, Inc.) for 1 h. The sections were washed and developed using
`Vector red staining (Vector Laboratories, Inc.) in accordance with
`the manufacturer’s instructions. The slides were counter stained with
`
`2846=Tang et al.
`
`Lassen - Exhibit 1009, p. 2
`
`Lassen - Exhibit 1009, p. 2
`
`

`

`Meyer’s hematoxylin, and then mounted with aqueous histologic
`mounting medium (Zymed Laboratories, Inc., So. San Francisco, CA).
`For types I and III collagen immunostaining, mouse lungs were
`chilled in acetone containing protease inhibitors (20 mM iodoaceta-
`mide and 2 mM phenylmethyl sulfonyl fluoride) at —20°C for 16-20 h.
`The lungs were then chopped into ~ 2 x 5 X 6 mm?pieces and im-
`mersed three times in glycol methacrylate (GMA) monomerat 4C
`for 6 h. The samples were then embedded in GMAaccordingto the
`manufacturer’s instructions (JB4 Embedding Kit; Polysciences Inc.,
`Warrington, PA) and 2-ym sections were cut and transferred ontosi-
`lane-treated glass slides. Immunohistochemical staining was under-
`taken as described above except that sections were treated with 1 M
`citric acid (pH 3.0) for 2 h before staining, and primary antibody incu-
`bation took place at 4°C overnight.
`The antibodies that were employed and their sources are listed
`below. They included antibodies to CD3, CD4, CD8 (Gibco Labora-
`tories, Grand Island, NY), Mac-1 (Pharmingen, San Diego, CA),
`B220 (Pharmingen), MHC Class II (a gift from Dr. Kim Bottomly,
`Yale University), type I collagen (Chemicon International, Inc., Te-
`mecula, CA), type III collagen (agift from Dr. J. Madri, Yale Univer-
`sity), a-smooth muscle actin (Sigma Chemical Co., St. Louis, MO),
`and desmin (Sigma Chemical Co.).
`Electron microscopy. Fragments of lung from three age- and sex-
`matched littermate pairs were fixed in 3%gluteraldehyde, postfixed
`in osmium tetroxide, and embedded in Epox 812 (Ernest F. Fulham,
`Inc., Latham, NY). Tissues were then cut onto grids, stained with ura-
`nyl acetate and lead nitrate, and examined in a Philips 300 micro-
`scope (Philips Electronic Instruments, Inc., Mahwah, NJ).
`Physiologic evaluation of transgenic mice. Age, sex, and weight
`matched littermate mice were anesthetized with pentobarbital (90
`mg/kg) and tracheostomized with an 18-gauge angiocatheter. Air-
`ways resistance was then measured using a modification of the tech-
`niques described by Martin et al. (38) as previously described (32).
`With these techniques, the changes in the lung volumes of anesthe-
`tized and tracheostomized mice were measured plethysmographically
`by determining the pressure in a Plexiglass chamber using an inline
`microswitch pressure transducer. Flow was measured by differentia-
`tion of the volume signal and transpulmonary pressure was deter-
`mined via a second Microswitch pressure transducer placed in line
`with the plethysmograph and animal ventilator. Resistance was then
`calculated using the method of Amdur and Mead(39). Theresistance
`of the tracheostomy catheter was routinely eliminated. Baseline mea-
`surements of pulmonary resistance were obtained by ventilating the
`mouse in the plethysmograph at volumes of 0.4 ml at a rate of 150
`breaths per minute (settings previously shown to produce normalar-
`terial blood gases in this species) (38). Bronchial reactivity was also
`assessed using noncumulative methacholine challenge procedures as
`previously described by our laboratory (32). In this procedure, in-
`creasing concentrations of methacholine (MCh) in PBS were admin-
`istered by nebulization (20 one-ml breaths) using a Devilbiss Aerosonic
`nebulizer (Model 5000; DeVilbiss Health Care, Somerset, PA) that
`producesparticles 1-3 .M in diameter. Pulmonaryresistance wascal-
`culated precisely 1 min later. Stepwise increases in MCh dose were
`then given until the pulmonary resistance, in comparison with the
`baseline level, had at least doubled. All animals received serial three-
`fold increases in MCh from 1 to 100 mg/ml. The data are expressed as
`the PCyo) (provocative challenge 100), the dose at which pulmonary
`resistance was 100%abovethe baseline level as calculated by linear
`regression analysis.
`Statistical analysis. Values are expressed as means+SEM.Unless
`otherwise noted, group means are compared with the Student’s two
`tailed unpaired ¢ test using the StatView software for the Macintosh.
`
`Results
`
`Generation of transgenic mice. To generate transgenic mice in
`which IL-11 is overproduced in a lung-specific/selective fash-
`
`Founders
`
`copy # controls
`
`(-}littermate
`~@—1.2 kb
`
`1234567 8 9101112
`
`Figure 2. Southern blot analysis of CC10-IL-11 mice. Tail DNA was
`obtained and the presence or absence of the CC10-IL-11 construct
`was determined using Southern blot analysis as described in Methods.
`Theresults obtained using tail DNA from transgene (+) founderani-
`mals (lanes 2-8) are compared with a transgene (—) littermate (lane
`1) and copy numbercontrol (lanes 9-22).
`
`ion, pronuclear microinjections of the CC10-IL-11-SV40 con-
`struct were performed on two separate occasions. From these
`microinjections, seven animals with transgene copy numbers
`varying between 1 and 70 were obtained (Fig. 2). These
`founder animals were bred with C57 BL/6 mice andthe trans-
`gene status of these offspring were similarly analyzed. This
`analysis demonstrated that the transgenes passed onto the off-
`
`Mouse Tissue RNA
`
`
`| HuIL-11contrel
`et
`
`ine
`*
`Ez
`Sz
`
`Kidney Liver
`
`Lung Muscle
`
`Pancrea Skin
`
`Spleen Uterus
`
`Figure 3. Northern blot analysis of IL-11 mRNAexpression in mouse
`organs. Total cellular RNA wasisolated from the noted organs of
`transgene (+) CC10-IL-11 mice andthe levels of IL-11 mRNAchar-
`acterized using Northern blot analysis as described in Methods. The
`IL-11 mRNAintotal cellular RNA from the different tissues is com-
`
`pared with the IL-11 mRNAin TGF-f, (10 ng/ml)-stimulated human
`lung fibroblasts (Hu IL-11 control). Ethidium bromide controls are in
`bottom panel.
`
`Transgenic Expression of Interleukin-11 in the Mouse Airway
`
`2847
`
`Lassen - Exhibit 1009, p. 3
`
`Lassen - Exhibit 1009, p. 3
`
`

`

`(ng/ml)
`
`IL-11 Positive
`
`Negative
`Transgene Status
`
`Figure 4. Levels of immunoreactive IL-11 in BAL fluid and serum of
`transgene (+) and (—) littermates. The noted values represent the
`mean+SEMofthe evaluations of four separate pairs of transgene
`(+) and (—) littermates (*P < 0.01 vs. serum of transgene (+) ani-
`mals and BALand serum oftransgene (—) animals; paired ¢ test).
`
`spring of these founder animals in a Mendelian fashion. Of
`these founders, lines 2-12 and 3-2 were chosen for more exten-
`sive analysis. Since they manifest similar pathologic, immuno-
`logic, and physiologic abnormalities, they will be discussed in a
`unified fashion.
`
`Organ specificity and intensity ofIL-1] gene expression and
`protein production. To determineif the CC10-IL-11 transgene
`was appropriately expressed, Northern analysis was used to
`compare the levels of IL-11 mRNAin the lungs and extrapul-
`monary organs of transgene (+) and (—) littermates. IL-11
`mRNAwasreadily detected in the lungs of transgene (+) ani-
`mals, but could not be appreciated in the lungs of transgene
`(—) animals (Fig. 3 and data not shown). In accordance with in
`vitro studies using fibroblasts and epithelial cells (21, 30), this
`IL-11 mRNAappeared to have one major and one minortran-
`script. IL-11 gene expression also appeared to be appropri-
`ately targeted to the lung since human IL-11 mRNA wasnot
`detectable in the RNA from a variety of extrapulmonary organs
`(Fig. 3). In all cases, IL-11 mRNA appeared to be appropri-
`ately translated since IL-11 was easily detected immunologically
`and biologically in the BAL fluid of the transgene (+) animals,
`but not in the serum of transgene (+) animals or the serum or
`BALfluid of transgene (—) littermates (Fig. 4 and TableI).
`
`Table I. IL-11 Bioactivity in BAL Fluids From Transgene (+)
`and (—) Animals
`
`(SH]-Tdrincorporation?
`
`Incubation conditions*
`
`No antibody
`
`+ Anti-IL-11
`
`BAL (-)
`BAL(+)
`
`BAL(+)
`Negative control
`
`3,178 £3,708
`57,383+5,351
`52,960+44,717
`1,896+101
`
`1,705 + 166
`5,568+378
`6,189+5,507
`_—
`
`*BAL were performed on transgene (+) and (—) littermate F, progeny
`of IL-11 transgenic mice. *B9.11 plasmacytoma[7H]-Tdr incorporation
`assessed in the presence and absence of anti-IL-11. BAL plasmacy-
`toma-stimulating activities are compared with the proliferation (FH]-Tdr
`incorporation) of B9.11 cells incubated in medium alone (negative con-
`trol). Values represent the mean+SEMoftriplicate determinations.
`
`
`
`Figure 5. Histologic abnormalities in airways of CC10-IL-11 trans-
`genic mice. The lungs of transgene (+) and (—) animals were re-
`moved,fixed, and evaluated using hematoxylin and eosin and
`trichromestains. The histologic appearance of the transgene (— )
`mouse lung (A) is compared with the peribronchiolar lymphocytic in-
`filtrates and bronchiolar thickening in the transgene (+) animals (B
`and C). The collagen content of the lungs of transgene (— ) animals
`appears in green in D. This contrasts with the impressive subepithe-
`lial fibrosis seen in the airways of transgene (+) animals (£). (Origi-
`nal magnification 67.5.)
`
`Effect ofIL-11 on murine airways. Progeny mice were killed
`at various intervals between 0.5 and 2 mo ofage, and the air-
`ways of transgene (+) and (—) littermates were compared. A
`total of 78 age and sex matched littermate pairs were evalu-
`ated. In contrast with their transgene (—)
`littermates, the
`transgene (+) animals manifest an impressive airway pheno-
`type composed of: (a) nodular collections of lymphocyte-like
`cells next to bronchi and bronchioles, and (6) airway wall
`thickening and remodeling (Fig. 5, A-C). The collections of
`lymphocytes were appreciated less often in the 0.5-mo-old ani-
`mals, but were prominently noted in the 1- and 2-mo-old ani-
`mals (data not shown). The impressive and progressive effects
`of IL-11 on the thickness of the airway wall were easily seen in
`the morphometric evaluations (Fig. 6). Insight into the cause
`of this thickening and remodeling was obtained [rom the
`trichrome evaluations. These stains demonstrated only small
`amounts of collagen in the lungs of transgene (—) animals.
`This contrasted with the extensive subepithelial fibrosis seen in
`the airways of the [L-1| transgene (+) animals (Fig. 5, D and 2).
`Composition ofperibronchiolar nodules. The results noted
`above demonstrate that IL-11 overexpression in the murine
`airway generates nodular collections of lymphocyte-like mono-
`nuclearcells. The phenotype of these cells was, therefore, ana-
`lyzed by immunohistochemistry using frozen lung sections.
`These studies demonstrated that the majority of the cells in
`these nodules were MHCClass II (+) and B220 (+) (Fig. 7).
`Collections of CD3(+), CD4(+), and CD8(+) cells were also
`noted (Fig. 6). Significant Mac-1 immunoreactivity was not
`
`2848=Tanget al.
`
`Lassen - Exhibit 1009, p. 4
`
`Lassen - Exhibit 1009, p. 4
`
`

`

`
`
`10
`
`8
`
`*
`
`*
`
`22
`
`2 —a
`
`n»
`
`g2 £
`
`3 B
`
`Transgene(+)
`———O—__
`6
`_ —+#—_Transgene(-)
`
`
`
`4
`
`2
`
`;
`
`:
`
`s
`
`2=
`
`eo
`
`Figure 8. Immunohistochemical analysis of fibrotic response in
`CC10-IL-11 transgenic mice. Immunohistochemistry was used to
`evaluate the type I collagen in the airways of transgene (+) (A) and
`(—) (C) animals and the type ITI collagen in the airways of transgene
`(+) (B) and (—) (D) animals. (Original magnification 50x.)
`
`Composition of the subepithelial fibrotic response. Studies
`were undertaken to determine if types I or II collagen were
`increased in the airways of the transgene (+) animals. These
`immunohistochemical evaluations demonstrated modest in-
`
`creases in type I collagen and impressive increases in type III
`collagen in transgene (+) vs. (—) animals (Fig. 8). Thus, the
`subepithelial fibrosis seen in CC10-IL-11 transgenic animals
`results, at least in part, from the increased accumulation of
`type III and, to a lesser extent, type I collagens.
`Immu-
`Structural characterization of the transgenic airway.
`nohistochemistry and electron microscopy were used to fur-
`ther characterize the cellular and structural alterations in the
`airways of the CC10-IL-11 transgene (+) animals. The immu-
`nohistochemical evaluations demonstrated an increase in the
`
` ‘eA
`
`Figure 9. Immunohistochemical evaluation of subepithelial cellular-
`ity in CC10-IL-11 mice. Immunohistochemistry was used to evaluate
`the cellular componentsof the subepithelial fibrotic response in
`CC10-IL-11 transgene (+) and (—) animals. A and C represent the
`a-smooth muscle actin immunoreactivity in transgene (—) and (+)
`animals, respectively. B and D represent the desmin immunoreac-
`tivity in transgene (—) and (+) animals, respectively. (Original
`magnification 50x.)
`
`Transgenic Expression of Interleukin-11 in the Mouse Airway
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`Figure 6. Morphometric analysis of airway wall thickening of
`CC10-IL-11 animals. The thickness of the bronchioles of 0.5-, 1-, and
`2-mo-old transgene (+) and (—) littermates were measured as de-
`scribed in Methods. Values represent the mean+SEMofatleast
`three pairs of animals at each time point (*P < 0.001 Bonferonni
`Multiple comparisonstest).
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`appreciated with only a rare cell staining with this antibody
`(Fig. 7). None of the antibodies that were used reacted with
`the airways in sections of lung from transgene (—) mice, in
`great extent because of the lack of airway inflammation in
`these animals (data not shown). These observations demonstrate
`that these nodules are composed of large numbers of B lym-
`phocytes and lesser numbers of CD4(+) and CD8(+) T cells.
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`Figure 7. Immunohistochemistry of peribronchiolar nodularinfil-
`trates. Immunohistochemical techniques were used to evaluate the
`cellular composition of the peribronchiolar infiltrates seen in the
`transgene (+) CC10-IL-11 animals. Antibodies against B220 (A),
`MHCclassIT (B), CD3 (C), CD4 (D), CD8 (£), and Mac-1 (F) were
`employed. (Original magnification 50x.)
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`Figure 10. Electron microscopic ap-
`pearance of peripheral airway of
`CC10-IL-11 mice. (A) Low power
`view of airway wall with epithelial
`cells (EP), myocytes (M), and fibro-
`blastic cells (F). Note intact base-
`ment membrane (open arrows) and
`collagen deposition (C) (4,000).
`(B) Smooth muscle cell with well
`formed Golgi (G). Note myofilaments
`(MF) (18,000). (C) Fibroblast with
`rough endoplasmic reticulum (RER)
`(18,000X). (D) Myofibroblast with
`subplasmalemmal dense body (open
`arrow) and basal lamina (closed
`arrow) (36,000). (E) Myofibro-
`blast with myofilaments (MF) and
`subplasmalemmalvesicles (open
`arrow) (36,000x).
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`number of a smooth muscle actin and desmin staining cells in
`the walls of the transgene (+) vs. (—) animals (Fig. 9). This
`staining was not uniform, however, with some stromalcells
`failing to manifest either of these markers. The electron micro-
`graphs demonstrated that the CC10-IL-11 transgene (+) ani-
`mals had normal basement membranesand enhanced striated
`collagen deposition (Fig. 10). They also demonstrated the ac-
`cumulation of a variety of stromalcells in these fibrotic loca-
`tions. Ultrastructurally, many cells appeared to be fibroblasts.
`Others appeared to be myofibroblasts based on their fibro-
`blast-like appearance and the presence of myofilaments, dense
`bodies, subplasmalemmal vesicles, and/or identifiable basal
`lamina (Fig. 10 and data not shown). Smooth musclecells with
`abundant myofilaments were also appreciated. Some of these
`cells showed morphologic evidence of increased synthetic ac-
`tivity with increased rough endoplasmic reticulum and promi-
`nent Golgi (Fig. 10). Thus, the subepithelial response in the
`airways of the CC10-IL-11 transgene (+) animals occursin the
`absence of basement membranethickening and is character-
`ized by increased interstitial collagen deposition and height-
`Airway inflammation and fibrosis are prominentfeatures of a
`ened stromal cellularity with the local accumulation of fibro-
`blasts, myofibroblasts, and smooth muscle cells.
`variety of disorders including asthma, COPD, bronchiectasis,
`and bronchiolitis (3-11). In these disorders, the relationship(s)
`Effect of IL-1] on airway physiology. Airways obstruction
`and hyperresponsiveness to nonspecific stimuli such as MCh
`between inflammation, fibrosis, and physiologic dysregulation,
`and the contribution that individual mediators make to the
`are prominentfeatures of asthma, COPD, andavarietyofdis-
`eases characterized by chronic airways inflammation and/or
`pathogenesis of these abnormalities are poorly understood.
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`fibrosis. Thus, studies were undertaken to characterize the
`physiologic profile of IL-11 transgene (+) and (—) animals.
`Overall, 18 age, sex, and weight matched littermate pairs were
`evaluated. The baseline airways resistance of IL-11 transgene
`(+) animals greatly exceeded that of IL-11 transgene (—) ani-
`mals. At 2 mo of age, the airways resistance of the transgene
`(+) animals was approximately threefold greater than the
`resistance of the transgene (—) animals (745.34227.5 vs.
`227.5+6.4 cm H,O/liters per s; P < 0.05). In addition, 1.5-2-
`mo-old transgene (+) animals manifest exaggerated sensitivity
`to methacholine since they achieved a 100% increase in air-
`waysresistance at 1/10 to 1/100 the dose of methacholine re-
`quired by their transgene (—) littermate controls (Fig. 11).
`When viewed in combination, these studies demonstrate that
`IL-11 transgene (+) animals manifest increased airways resis-
`tance and airways hyperresponsiveness to MCh when com-
`pared with transgene (—) littermate controls.
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`Discussion
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`2850=Tang et al.
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`together as IL-6-type cytokines based on their overlapping
`functional profiles and shared use of the gp130 molecule in
`their multimeric receptor complexes (42). In keeping with these
`findings, a comparison of the phenotype of the CC10-IL-11 an-
`imals described in this report and CC10-IL-6 mice described
`previously by our laboratories (32), shows interesting similari-
`ties and differences. The inflammatory response seen in both
`the IL-11 and IL-6 transgenic lines was almost exclusively lym-
`phocytic and contained significant numbersof B cells. This
`is in keeping with the knownBcell stimulatory activities of
`both of these cytokine moieties (28, 42, 43). Interestingly, how-
`ever, inflammation were more prominentin the CC10-IL-6 an-
`imals and airway remodeling and subepithelial fibrosis was
`more prominent in CC10-IL-11 animals. In addition, marked
`physiologic differences were noted with CC10-IL-6 animals
`manifesting normal baseline airways resistance and airways
`hyporesponsiveness to methacholine, while CC10-IL-11 ani-
`mals demonstrated increased baseline airways resistance and
`ARRto methacholine. These findings clearly support