Rapid publication
`
`IL-11 expression is increased in severe
`asthma: Association with epithelial cells
`and eosinophils
`
`Eleanor Minshall, PhD,a Jamila Chakir, PhD,b Michel Laviolette, MD,b Sophie Molet,
`PhD,a Zhou Zhu, MD,c Ron Olivenstein, MD,d Jack A. Elias, MD,c and Qutayba
`Hamid, MD, PhDa Montreal and Sainte Foy, Quebec, Canada, and New Haven, Connecticut
`
`Background: IL-11 is a pleiotropic cytokine produced by a
`variety of stromal cells. Targeted overexpression of this
`cytokine in mice results in a remodeling of the airways and the
`development of airway hyperresponsiveness and airway
`obstruction.
`Objectives: Because these alterations mimic important patho-
`logic and physiologic changes in the airways of some asthmatic
`patients, we investigated the expression of IL-11 messenger
`RNA (mRNA) within the airways of patients with mild to
`severe asthma and nonasthmatic control subjects.
`Methods: Fiberoptic bronchoscopy to obtain bronchial biopsy
`specimens was performed on patients with mild (n = 13), mod-
`erate (n = 10), and severe (n = 9) asthma and on nonasthmatic
`control subjects (n = 9).
`Results: These patients differed in their extent of airway fibro-
`sis with types I and III collagens being noted in greater quanti-
`ties in the biopsy specimens from the severe and moderate
`asthmatics than in those from controls (P < .05). IL-11 mRNA
`expression was observed in the epithelial and subepithelial lay-
`ers of asthmatic and nonasthmatic control subjects. The num-
`ber of cells within the epithelium and subepithelium express-
`ing IL-11 mRNA was greater in those with moderate and
`severe asthma compared with mild asthma and nonasthmatic
`subjects (P < .001). There were also greater numbers of IL-11
`mRNA-positive cells within the subepithelium in severe com-
`pared with moderate asthma (P < .001). Immunostaining for
`IL-11 within the airway tissues confirmed translation of the
`mRNA into IL-11–immunoreactive protein in airway epithelial
`cells. Colocalization of IL-11 mRNA and immunoreactivity
`with resident inflammatory cells demonstrated that this
`cytokine was also expressed by major basic protein–positive
`eosinophils.
`
`From the aMeakins-Christie Laboratories, McGill University, Montreal,
`bLaval Hospital, Sainte Foy, the cMontreal Chest Research Institute, Mon-
`treal, and the dSection of Pulmonary and Critical Care Medicine, Depart-
`ment of Internal Medicine, Yale University School of Medicine, New
`Haven, Conn.
`E.M. and J. C. contributed equally to this work.
`Supported by the Medical Research Council of Canada and Astra Pharm,
`Canada. E. M. is a recipient of a Foulkes Foundation Fellowship. J. M. is
`the recipient of an FRSQ scholarship.
`Received for publication Nov 1, 1999; revised Nov 22, 1999; accepted for
`publication Nov 22, 1999.
`Reprint requests: Qutayba Hamid, MD, PhD, Meakins-Christie Laboratories,
`McGill University, 3626 St Urbain, Montreal, PQ H2X 2P2, Canada.
`Copyright © 2000 by Mosby, Inc.
`0091-6749/2000 $12.00 + 0 1/1/104572
`
`232
`
`Conclusion: These results suggest that IL-11 is involved in the
`chronic remodeling seen in asthmatic airways and is associated
`with increasing severity of the disease. (J Allergy Clin
`Immunol 2000;105:232-8.)
`
`Key words: Severe asthma, IL-11, airway remodeling, airways
`inflammation, collagen deposition
`The pathologic features of asthma are associated with
`airway remodeling as a consequence of hypertrophy/hy-
`perplasia of the airway smooth muscles and of subepithe-
`lial fibrosis.1,2 The latter response is most prominent in
`the lamina reticularis and results from interstitial collagen,
`fibronectin, and proteoglycan deposition.3,4 Although ini-
`tially described in postmortem studies, these structural
`alterations can be observed even in mild and newly diag-
`nosed cases of asthma.3,5 Because any thickening of the
`airway wall will profoundly increase the maximal degree
`of airway narrowing caused by airway smooth muscle con-
`traction, it has been proposed that architectural changes
`similar to those observed in asthmatic patients contribute to
`the development of chronic airway hyperresponsiveness
`and the progressive deterioration in lung function over
`time.6 To date, many studies have focused on the mecha-
`nisms underlying the acute presentations of bronchial asth-
`ma. In contrast, the factors responsible for the more chron-
`ic structural changes within the airway are poorly defined.
`Although myofibroblasts are recognized as the cell type
`responsible for subepithelial collagen deposition in these
`individuals,7 the mechanisms contributing to the onset of
`airway fibrosis in asthma and the role of inflammatory
`cells, in particular eosinophils and epithelial cells, remains
`to be established.
`IL-11 was originally described as a soluble factor
`derived from stromal cells, which was capable of stimu-
`lating plasmacytoma cell proliferation.8 It is a member of
`the IL-6 family of cytokines that share a common recep-
`tor β-subunit gp130 molecule.9 To date, IL-11 has been
`ascribed a variety of functions including the ability to
`regulate hematopoiesis, bone metabolism, and epithelial
`proliferation.10 Consistent with this pleiotropic nature,
`IL-11 is produced by various cell types such as stromal
`cells, fibroblasts, osteoblasts, endothelial cells, and
`epithelial cells.8,11-14 Studies from our own laboratories
`suggest that IL-11 may contribute to the structural airway
`remodeling and alteration in immune functioning evident
`
`Lassen - Exhibit 1069, p. 1
`
`

`

`J ALLERGY CLIN IMMUNOL
`VOLUME 105, NUMBER 2, PART 1
`
`Abbreviations used
`ICC:
`Immunocytochemistry
`ISH:
`In situ hybridization
`MBP: Major basic protein
`mRNA: Messenger RNA
`TGF-β: Transforming growth factor-β
`
`in asthmatic subjects. Thus we have demonstrated that
`IL-11 is produced by human lung fibroblasts and alveo-
`lar and airway epithelial cell lines in response to
`cytokines,11,14,15 histamine,16 eosinophil-derived major
`basic protein (MBP),17 and respiratory viruses.15 We
`have also reported that overexpression of IL-11 within
`the lungs results in subepithelial fibrosis18,19 and pro-
`motes the accumulation of myocytes and myofibro-
`blasts.19 Moreover, the functional sequelae of chronic
`IL-11 expression recapitulate aspects of the abnormal
`pulmonary physiologic features observed in severe asth-
`ma, including the development of airway hyperrespon-
`siveness and baseline airway obstruction.15,19
`Given these actions of IL-11, we hypothesized that the
`expression of IL-11 was increased within the airways of
`asthmatics and that it would be particularly associated
`with the most severely remodeled individuals. Our aim
`was therefore to investigate the expression of IL-11 in a
`range of asthmatic patients with mild to severe disease
`with use of in situ hybridization (ISH) and to colocalize
`IL-11 to inflammatory cell types within the airways.
`These results showed a significantly increased expres-
`sion of IL-11 messenger RNA (mRNA) within the air-
`ways of subjects with severe asthma compared with
`those with mild asthma and with nonasthmatic control
`subjects. The IL-11 mRNA expression within the lungs
`of asthmatic individuals was observed primarily within
`airway epithelial cells and MBP-positive eosinophils.
`
`METHODS
`Subjects studied
`To study the expression of IL-11 mRNA in a range of asthmatic
`subjects, we recruited 32 individuals with mild to severe asthma
`from the asthma clinic at the Laval Hospital (Sainte Foy) and 9 con-
`trol subjects from the asthma clinic at the Montreal Chest Hospital
`(Montreal). Asthmatic severity was defined on the basis of prebron-
`chodilator-measured FEV1 values, with mildly asthmatic subjects
`(n = 13) having an FEV1 value greater than 80% predicted, moder-
`ately asthmatic subjects (n = 10) having FEV1 values between 60%
`and 80% predicted, and severely asthmatic subjects (n = 9) having
`FEV1 values <60% predicted. FEV1 represented the accepted level
`of control with therapy. In moderate asthma the mean FEV1
`improved >30% after β2-agonist administration and further in some
`subjects with oral prednisone, whereas in severe asthma FEV1 was
`less than the historic maximum in some subjects. All patients ful-
`filled the American Thoracic Society criteria for asthma,20 had typ-
`ical clinical symptoms, documented airways reversibility (>15%
`improvement in FEV1), and increased airway responsiveness to
`methacholine (<8 mg/mL) performed only if FEV1 >70%. On enroll-
`ment, the medical history of each patient was taken and a physical
`examination was performed. None of the subjects had a history of
`respiratory tract infection within the previous 6 weeks or
`immunotherapy within the previous 12 months. All subjects were
`
`Minshall et al 233
`
`atopic on the basis of positive skin wheals (>3 mm) to one or more of
`13 common allergens and were currently nonsmokers (2 and 4 ex-
`smokers in the moderate and severe asthma groups, respectively).
`Seven subjects with severe asthma required the regular use of oral
`corticosteroids (mean 42 ± 7.7 mg) to maintain acceptable control of
`symptoms and 2 used inhaled steroids only. In addition, those with
`severe asthma used inhaled β2-agonists and theophyline as neces-
`sary. Those with moderate asthma had their symptoms controlled by
`regular use of β2-agonists and inhaled corticosteroids (mean 1177 ±
`225 µg beclomethasone dipropionate equivalent). Three patients
`also required the regular use of oral corticosteroids. Those with mild
`asthma used inhaled β2-agonists only. Nonsmoker nonatopic control
`subjects volunteered to participate in the study and none had taken
`corticosteroids in the year preceding the study. Informed consent,
`approved by the Montreal Chest Research Institute and Laval Hos-
`pital Ethics Review Committees, was obtained from all patients
`before entry into this study.
`
`Fiberoptic bronchoscopy and tissue
`processing
`The technique of fiberoptic bronchoscopy and the methods for
`processing of bronchial biopsy specimens have been described else-
`where in detail.21
`
`Probe preparation
`A digoxigenin-labeled complementary RNA probe coding for
`IL-11 mRNA was prepared from complementary DNA as described
`previously.22 In brief, complementary DNA was inserted into
`PGEM vectors, linearized, and transcribed in vitro in the presence
`of digoxigenin-11-uridine triphosphate and either SP6 or T7 poly-
`merases. Antisense (complementary to mRNA) and sense probes
`(identical to mRNA) were prepared.
`
`ISH
`Sections of lung tissue were processed for ISH for IL-11 mRNA
`according to Ying et al.22 Briefly, after permeabilization with Triton
`X-100, the tissue sections were then briefly washed in PBS and
`immersed in a proteinase K solution for 20 minutes at 37°C. The
`samples were subsequently fixed in 4% paraformaldehyde, washed,
`and air dried. Hybridization was carried out with use of the
`hybridization mixture containing the appropriate sense or antisense.
`Each section was then covered and incubated overnight at 40°C in
`a humid chamber. Posthybridization, involving a series of high
`stringency washes of the samples in decreasing concentrations of
`saline–sodium citrate buffer at 42°C, was then performed. To
`remove any unbound RNA probes, the samples were washed with
`ribonuclease solution for 20 minutes at 42°C. The hybridization sig-
`nal was visualized by incubating the sections for 4 hours with sheep
`polyclonal antidigoxigenin antibodies (1:1000) conjugated with
`alkaline phosphatase. Color development was achieved by adding
`the freshly prepared substrate (X-phosphate-5-bromo-4-chloro-3-
`indolyl phosphate and nitroblue tetrazolium). Once the reaction was
`completed, the tissue sections were counterstained with hema-
`toxylin, mounted with a coverslip, and examined under a graduated
`microscope for positive signals.
`
`Immunocytochemistry
`Immunostaining for IL-11 immunoreactivity within the tissues to
`confirm translation of the IL-11 mRNA was performed with use of the
`avidin-biotin peroxidase complex method as previously described.23
`Tissue sections (5 µm) from the asthmatic and nonasthmatic subjects
`were incubated overnight at 4°C with the primary goat antihuman IL-
`11 antibody (AB-218-NA, R&D Systems, Minneapolis, Minn) or with
`the primary rabbit antihuman type I or type III collagen antibodies
`
`Lassen - Exhibit 1069, p. 2
`
`

`

`234 Minshall et al
`
`J ALLERGY CLIN IMMUNOL
`FEBRUARY 2000
`
`(Biodesign, Pasadena, Calif). According to the manufacturer’s specifi-
`cations, the IL-11 antibody exhibits no cross-reactivity with other
`cytokines tested, including IL-6 and leukemia inhibitory factor in
`direct ELISA. As a control, sections were processed in the absence of
`the primary antibody with an isotype-matched IgG.
`
`Combined immunocytochemistry and ISH
`To ascertain the expression of IL-11 mRNA by eosinophils, we
`simultaneously applied radiolabeled ISH for IL-11 mRNA with
`MBP immunoreactivity as previously described in detail else-
`where.24
`
`Sequential immunostaining
`Double sequential immunostaining for IL-11 immunoreactivity
`protein and MBP immunoreactivity was performed to localize the
`protein products of the IL-11 mRNA to eosinophils. The method for
`sequential immunostaining has been previously published.25
`
`Quantification
`Slides were coded and positive cells were counted blindly with use
`of ×100 magnification. Cells with positive signal in the subepithelium
`were counted and the results were expressed as the mean number of
`positive cells per square millimeter of submucosa. IL-11 mRNA-pos-
`itive cells within the epithelium were counted by optical analysis and
`expressed as a semiquantitative score on the basis of the percentage of
`the epithelium demonstrating positive signal/total epithelium (0: no
`staining; 1: less than 12.5%; 2: 12.5%-25%; 3: 25%-37.5%; 4: 37.5%-
`50%; 5: 50%-62.5%; 6: 62.5%-75%; 7: 75%-87.5%; 8: 87.5%-100%).
`The within-observer coefficient of variation for repeated measures
`was less than 5%. The extent of collagen staining in the subepithelium
`was measured with an image analysis system. The thickness of the col-
`lagen layer was taken below the basement membrane and these data
`are expressed as the mean of 3 measurements.
`
`Statistical analysis
`Normality and variance assumptions were tested. The numbers
`of cells expressing IL-11 mRNA in normal and asthmatic airways
`were compared with the nonparametric Kruskal-Wallis test. Statis-
`tically significant differences between groups were subsequently
`analyzed with a Mann-Whitney U test (Systat version 7.0, SPSS,
`Chicago, Ill). Correlation coefficients were calculated from Pear-
`son’s moment coefficient and were corrected for multiple compar-
`isons by the use of Bonferroni’s correction factor. Results were con-
`sidered statistically significant for P values <.05.
`
`RESULTS
`IL-11 expression within the airways
`
`IL-11 mRNA expression was visualized as dark purple
`staining of individual cells and was seen in the airway sub-
`mucosa and epithelial cell layer of asthmatic patients and
`nonasthmatic controls (Fig 1, A and B, respectively). No
`positive signals for IL-11 mRNA were observed when the
`sense probe was used or when the tissue sections were
`treated with ribonuclease before hybridization of the anti-
`sense probe. To confirm transcription of the mRNA for IL-
`11, the presence of specific immunoreactivity for this
`cytokine within the tissue sections was confirmed with use
`of polyclonal antihuman IL-11 antibody. The presence of
`specific brown staining after the immunocytochemistry
`(ICC) within the airways of asthmatic patients and nonasth-
`matic control subjects was indicative of IL-11 immunore-
`active protein (Fig 1, C). This staining was not observed
`
`when the isotype-matched control antibody was used (Fig
`1, D).
`
`Expression of IL-11 mRNA in mild, moderate,
`and severe asthma
`To further define the potential role of IL-11 in the
`pathophysiologic mechanisms of asthma, we compared
`the expression of IL-11 mRNA in nonasthmatic and asth-
`matic subjects with varying degrees of airway obstruc-
`tion and fibrosis (Fig 2, A and B). Within the subepithe-
`lial cell layer, the numbers of cells expressing IL-11
`mRNA were significantly greater in moderate (10.8 ± 1.5
`cells per mm2 of submucosal tissue) and severe asthma
`(21.1 ± 2.0 cells per mm2 of submucosal tissue) com-
`pared with mild asthma (2.9 ± 0.7 cells per mm2 of sub-
`mucosal tissue) and nonasthmatic control subjects (1.3 ±
`0.5 cells per mm2 of submucosal tissue, P < .001). With-
`in the subepithelium, there were also significantly greater
`numbers of IL-11 mRNA-positive cells in severe asthma
`compared with moderate asthma (P < .001).
`In the epithelial cell layer there was an increase in the
`epithelial score for IL-11 mRNA expression for those
`with moderate (2.2 ± 0.3) and severe (2.8 ± 0.2) asthma
`compared with those with mild asthma (0.5 ± 0.2) and
`the nonasthmatic control subjects (0.2 ± 0.1, P < .001).
`There were no significant differences between the mod-
`erate and severe asthmatics in their expression of IL-11
`mRNA within the epithelium.
`
`Colocalization of IL-11 mRNA expression
`The studies noted above clearly demonstrate that IL-11
`is expressed at the mRNA level by cells having eosinophil-
`like morphologic features within the airway subepithelium.
`To confirm the identity of these cells, both combined ISH
`and ICC and double-sequential ICC were used (Fig 1, E).
`In the subepithelium of patients with moderate and severe
`asthma IL-11 mRNA was mostly colocalized to MBP-pos-
`itive eosinophils (mean ± SEM, 61% ± 9%, n = 6).
`
`Correlation of IL-11 mRNA expression with
`an index of lung physiology features
`To gain insight into the potential effector functions of
`IL-11, we compared index values of IL-11 production and
`airway physiologic features (FEV1). There were significant
`inverse correlations between the numbers of cells express-
`ing IL-11 mRNA within the epithelium (Fig 3, B, r2 = 0.55)
`and subepithelium (Fig 3, A,, r2 = 0.75) and the FEV1 val-
`ues for the group of asthmatic subjects (P < .05).
`
`Characterization of extent of remodeling
`As noted above, the severity of asthma was initially
`assessed on the basis of pulmonary function parameters. To
`see whether this correlated with the extent of airway remod-
`eling or airway fibrosis, the expression of types I and III col-
`lagens was assessed in the biopsy specimens from the healthy
`and asthmatic subjects. The presence of specific collagen
`immunoreactivity was visualized as brown staining beneath
`the lamina reticularis after the ICC reaction (Fig 1, F). Fig 4
`demonstrates the relationship between asthma severity and
`
`Lassen - Exhibit 1069, p. 3
`
`

`

`J ALLERGY CLIN IMMUNOL
`VOLUME 105, NUMBER 2, PART 1
`
`Minshall et al 235
`
`FIG 1. ISH with digoxigenin for IL-11 mRNA in bronchial biopsy specimens from (A) patient with severe asth-
`ma and (B) nonasthmatic control subject. Note presence of dark brown/purple staining indicative of IL-11
`mRNA within airways epithelium and associated with individual cells in subepithelium. C, Immunostaining
`for IL-11 in individual with severe asthma. D, Primary antibody isotype control for IL-11 immunoreactivity. E,
`Colocalization of IL-11 immunoreactivity and eosinophil specific marker (MBP) in individual with severe asth-
`ma. IL-11 immunoreactivity is shown as brown coloration that localizes to MBP-positive cells (red staining).
`F, Collagen (type III) immunoreactivity, as determined by avidin-biotin peroxidase method, in individual with
`severe asthma is shown as brown staining.
`
`collagen immunoreactivity. It shows that for both type I and
`type III collagen there is increasing expression with the sever-
`ity of the disorder. There was a significantly greater staining
`for collagen type III in severe and moderate asthma compared
`to mild asthma and healthy subjects (P < .05). The extent of
`type I collagen immunoreactivity was also greater in severe
`asthma compared with the mildly asthmatic and healthy sub-
`jects (P < .05) and in moderate asthma compared with healthy
`control subjects (P < .05).
`
`Relationships between IL-11 expression and
`the extent of collagen staining
`Correlational analyses were performed between the
`expression of IL-11 in epithelium, in subepithelium, and
`
`the expression of type I and III collagens for all the sub-
`jects (nonasthmatics, mild, moderate, and severe asth-
`matics) included in the study. No significant correlation
`was found between any of these parameters.
`
`DISCUSSION
`
`Previous studies in mice have demonstrated the capac-
`ity of IL-11 to recapitulate many of the features observed
`in chronic obstructive airway diseases such as severe
`asthma. To investigate the contribution of this cytokine
`to the structural alterations evident in a range of asth-
`matic individuals, we examined the expression of IL-11
`mRNA and immunoreactivity and its association with
`
`Lassen - Exhibit 1069, p. 4
`
`

`

`236 Minshall et al
`
`J ALLERGY CLIN IMMUNOL
`FEBRUARY 2000
`
`A
`
`B
`
`A
`
`B
`
`FIG 2. IL-11 mRNA expression within (A) subepithelium and (B)
`epithelial cell layer of subjects with mild, moderate, and severe
`asthma and of nonasthmatic control subjects. There was signifi-
`cant increase in numbers of cells expressing IL-11 mRNA in mod-
`erate (n = 10) and severe (n = 9) asthma compared with mild asth-
`ma (n = 13) and nonasthmatic control subjects (n = 9, asterisk, P
`< .001 compared with controls and mild asthmatics). Within sub-
`mucosa, patients with severe asthma had significantly increased
`numbers of IL-11 mRNA-positive cells compared with those with
`moderate asthma (pound sign, P < .05).
`
`inflammatory cells with use of a combination of ISH and
`ICC techniques. In addition, we investigated the expres-
`sion of collagen types I and III within the airways of our
`asthmatic and healthy individuals. Our results document-
`ed the increased numbers of cells expressing IL-11 with-
`in the airways of moderate and severe asthmatics and
`localized the majority of this expression to epithelial
`cells and to MBP-positive eosinophils. IL-11 expression
`was directly associated with disease severity but inverse-
`ly correlated with FEV1 measurements, suggesting that
`IL-11 expression is associated with abnormal lung phys-
`iologic features. Immunoreactivity for collagen types I
`and III expression were increased within the airways of
`our asthmatic subjects, and this was related to the sever-
`ity of the disorder, as based on FEV1 values.
`IL-11 is a cytokine with potent immunomodulatory
`properties and the ability to induce substantial remodel-
`ing of the airways; however, there was little evidence
`
`FIG 3. Correlational relationships between percent predicted FEV1
`values and numbers of cells expressing IL-11 mRNA within (A)
`subepithelium and (B) epithelial cell layer. There were significant
`correlations between numbers of IL-11 mRNA-positive cells in
`both airway epithelium and subepithelial regions and this index
`of pulmonary function (P < .05).
`
`concerning the importance of IL-11 in human disorders.
`Therefore the increased numbers of cells expressing IL-
`11 in moderate and severe asthma is an original finding
`that suggests that this cytokine may contribute to the
`sequelae of inflammatory events and structural modifica-
`tions characterizing these asthmatic individuals. Although
`prior reports have shown IL-11 immunoreactivity in nasal
`aspirates from children with viral upper respiratory tract
`infections, particularly those who exhibited wheezing,15
`our study is the first demonstration that inflammatory and
`structural cells express striking amounts of IL-11 in vivo
`in a human disease. It is also the first report showing the
`expression of IL-11 in normal human epithelial cells in
`vivo because previous studies have used alveolar and
`bronchial epithelial cell lines.14,15
`The mechanisms whereby IL-11 exerts its activity in
`the airways are complex. IL-11 has previously been
`shown to have fibrogenic potential in an animal model in
`eliciting subepithelial fibrosis and the local accumulation
`of fibroblasts, myofibroblasts, and smooth muscle
`cells.18,19 Interestingly, IL-11 enhances the accumulation
`of collagen type III (and to a lesser extent type I), which
`
`Lassen - Exhibit 1069, p. 5
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`

`J ALLERGY CLIN IMMUNOL
`VOLUME 105, NUMBER 2, PART 1
`
`Minshall et al 237
`
`is a profile of expression similar to that observed in the
`airways of asthmatic subjects.19 However, in our in vivo
`study we have not been able to show any correlation
`between IL-11 expression in bronchial biopsy specimens
`and types I or III collagens, although tissue fibrosis cor-
`relates with the disease severity. These results might be
`explained by the fact that subepithelial fibrosis and par-
`ticularly collagen deposition is a progressive phenome-
`non, so we cannot exclude the fact that a correlation
`between IL-11 and collagens may be found if bronchial
`biopsies were done at another stage of the disease. To
`date, there is a scarcity of literature documenting the
`mechanisms responsible for the fibrotic response. IL-11
`is known, however, to stimulate the production of tissue
`inhibitor of metalloproteinase-1, which leads to a
`decrease in collagen degradation.26 The resultant colla-
`gen accumulation may be responsible for the tissue fibro-
`sis observed in our moderately and severely asthmatic
`subjects, although the presence of alternative mecha-
`nisms cannot be ruled out.
`In addition to its fibrogenic nature, IL-11 possesses sev-
`eral immunoregulatory functions that are pertinent to the
`pathophysiologic mechanisms of bronchial asthma,
`notably its ability to down-regulate IL-12 production by
`peritoneal and alveolar macrophages27,28 and to polarize T-
`cell responses toward TH2-type cytokine production.29
`Indeed, IL-11 may act to perpetuate the profile of TH2-type
`cytokine expression seen within the lungs of asthmatic sub-
`jects and further studies addressing this action of IL-11 are
`warranted. However, IL-11 also has potent anti-inflamma-
`tory properties and prevents the nuclear translocation of
`nuclear factor-κB, inhibiting the induction of genes depen-
`dent on this transcription factor.30 Whether IL-11 exerts
`predominantly proinflammatory or anti-inflammatory
`actions within the airways remains to be elucidated.
`Regardless of the mechanism of action of IL-11, our
`data showed this cytokine to be particularly associated
`with the airways of moderate and severe asthmatics and
`that both in the epithelium and subepithelium the num-
`bers of cells expressing IL-11 mRNA were inversely cor-
`related to the FEV1 values of the subjects. This suggests
`that the release of IL-11 from epithelium and subepitheli-
`um tissues may contribute both to the development of air-
`way fibrosis and the subsequent decline in lung function.
`Our colocalization data showed the presence of IL-11
`mRNA within MBP-positive eosinophils. Previous data
`from our laboratories have demonstrated the presence of
`transforming growth factor-β1 (TGF-β1) within the asth-
`matic airways, which also localized to eosinophils.31 TGF-
`β1 is a potent stimulator of IL-11 protein production,
`mRNA accumulation, and gene transcription in human
`fibroblasts, alveolar and bronchial epithelial-like cells, and
`smooth muscle cells.11,14,16,32 Therefore it is possible that
`TGF-β1 also exerts an autocrine-paracrine action and
`might be a stimulant for eosinophil IL-11 production with-
`in asthmatic airways. Regardless of whether such a path-
`way exists, eosinophil-derived MBP has been previously
`shown to participate in the production of IL-11 by lung
`fibroblasts.17 The finding of IL-11 expression by eosino-
`
`FIG 4. Immunoreactivity for collagens type I and type III in
`bronchial biopsy specimens from patients with mild, moderate,
`and severe asthma compared with nonasthmatic control subjects.
`There was a significant increase in collagen types I and III
`immunoreactivity in the airways of moderate (n = 10) and severe
`(n = 9) asthmatics compared with mild asthmatics (n = 13) and
`nonasthmatic control subjects (n = 9). Asterisk, P < .05 compared
`with control subjects and those with mild asthma, except for type
`I collagen where increase in collagen immunoreactivity in moder-
`ate asthma was only significant in comparison to healthy control
`subjects; pound sign, P < .05.
`
`phils gives further evidence to support the involvement of
`these cells in the development of structural remodeling of
`the airways in asthma. In addition, the demonstration that,
`like TGF-β1,31 IL-11 expression correlates to asthma
`severity has important implications for biology and for
`disease pathogenesis. This observation raises the possibil-
`ity that some of the biologic effects attributed to TGF-β1,
`especially the fibrogenic response, are in fact mediated by
`IL-11. Neutralization in vitro or in vivo studies with anti-
`IL-11 and anti-TGF-β1 antibodies will need to be per-
`formed to determine whether any effects of TGF-β1 are
`mediated through the induction of IL-11 and vice versa.
`Because the expression of both IL-11 and TGF-β1 seem
`to be closely associated, it would be of interest to compare
`them in the same asthmatic specimens to determine
`whether there is concomitant expression.
`In keeping with the close association of IL-11 with IL-
`6, it is interesting to note that our colocalization of IL-11
`to eosinophils is consistent with the production of IL-6
`by these cells24 and the recent intracellular localization
`of this cytokine to the matrix of the crystalloid granule.33
`IL-6 has also been implicated in the pathogenesis of asth-
`ma and is also produced by epithelial cells.34
`In summary, we have shown increased numbers of cells
`expressing IL-11 mRNA within the bronchial mucosa and
`airway epithelial cells of severely asthmatic subjects.
`Within the subepithelial cell layer this cytokine mRNA
`was colocalized to MBP-positive eosinophils. The patho-
`physiologic features of moderate and severe asthma
`involve the up-regulated expression of IL-11 mRNA, and
`
`Lassen - Exhibit 1069, p. 6
`
`

`

`238 Minshall et al
`
`J ALLERGY CLIN IMMUNOL
`FEBRUARY 2000
`
`therapeutic strategies aimed at regulating the expression of
`IL-11 may be of use in the treatment of asthma.
`
`The thank Ms Lisa Cameron and Dr Rame Taha for their contri-
`butions in the preparation of this manuscript. Special thanks also go
`to Ms Elsa Schotman for technical assistance.
`
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