American Journal of Respiratory and Critical Care Medicine

Rationale: Transforming growth factor (TGF)-β has a central role in driving many of the pathological processes that characterize pulmonary fibrosis. Inhibition of the integrin αvβ6, a key activator of TGF-β in lung, is an attractive therapeutic strategy, as it may be possible to inhibit TGF-β at sites of αvβ6 up-regulation without affecting other homeostatic roles of TGF-β.

Objectives: To analyze the expression of αvβ6 in human pulmonary fibrosis, and to functionally test the efficacy of therapeutic inhibition of αvβ6-mediated TGF-β activation in murine bleomycin–induced pulmonary fibrosis.

Methods: Lung biopsies from patients with a diagnosis of systemic sclerosis or idiopathic pulmonary fibrosis were stained for αvβ6 expression. A range of concentrations of a monoclonal antibody that blocks αvβ6-mediated TGF-β activation was evaluated in murine bleomycin–induced lung fibrosis.

Measurements and Main Results: αvβ6 is overexpressed in human lung fibrosis within pneumocytes lining the alveolar ducts and alveoli. In the bleomycin model, αvβ6 antibody was effective in blocking pulmonary fibrosis. At high doses, there was increased expression of markers of inflammation and macrophage activation, consistent with the effects of TGF-β inhibition in the lung. Low doses of antibody attenuated collagen expression without increasing alveolar inflammatory cell populations or macrophage activation markers.

Conclusions: Partial inhibition of TGF-β using αvβ6 integrin antibodies is effective in blocking murine pulmonary fibrosis without exacerbating inflammation. In addition, the elevated expression of αvβ6, an activator of the fibrogenic cytokine, TGF-β, in human pulmonary fibrosis suggests that αvβ6 monoclonal antibodies could represent a promising new therapeutic strategy for treating pulmonary fibrosis.

Scientific Knowledge on the Subject

Transforming growth factor (TGF)-β is an important driver of pulmonary fibrosis and therapeutic strategies to inhibit its actions are sought. However, TGF-β has other homeostatic roles that could make therapeutic inhibition problematic.

What This Study Adds to the Field

Monoclonal antibodies against a tissue-restricted activator of TGF-β, αvβ6, can inhibit fibrosis at doses that do not exacerbate inflammation, thus avoiding potential risks associated with global TGF-β inhibition.

Transforming growth factor (TGF)-β is a key factor in the initiation and maintenance of fibrosis, the pathological deposition of excess extracellular matrix (ECM) in injured or diseased tissue. TGF-β promotes fibroblast activation and proliferation, and the expression and secretion of ECM components (16). Adenoviral overexpression studies show that TGF-β is unusual in its ability to promote lung fibrosis in vivo in the absence of prominent inflammation (7). In addition, knockout mice deficient for Smad3, a mediator of TGF-β signaling, are resistant to the development of lung fibrosis (8). Several studies with anti–TGF-β agents have shown protection from fibrosis in disease models (917). Consequently, the TGF-β pathway has been identified as a potential therapeutic target for treatment of diseases associated with the pathology of fibrosis. In human disease, TGF-β is highly expressed in lungs of patients with idiopathic pulmonary fibrosis (IPF) (18), and global transcript profiling in IPF has shown the overexpression of a number of known TGF-β target genes, including many ECM components (19).

In addition to its profibrotic activities, TGF-β plays a number of other homeostatic roles in inflammation, immune tolerance, and cancer biology (20). Thus, therapeutic inhibition of TGF-β would ideally block fibrosis without the adverse effects associated with total loss of TGF-β function. One strategy to selectively modulate TGF-β activity would be to inhibit tissue-restricted activators of TGF-β, such as the αvβ6 integrin. TGF-β is synthesized as a latent protein that is unable to bind to its cognate receptor until converted to the active form by one of several mechanisms, including cleavage by proteases, exposure to low pH or ionizing radiation, or conformational changes in the latent complex allowing it to bind to its cognate receptors (2124). The αvβ6 integrin binds to an arginine–glycine–aspartic acid (RGD) motif in latent TGF-β (specifically, the TGF-β1 and TGF-β3 isoforms) and converts it to an active form (21, 25). Although other activation mechanisms have been identified, studies in β6 integrin–deficient mice (β6 null mice) suggest that αvβ6-mediated activation of TGF-β is necessary for development of fibrosis in lung and kidney disease models (21, 26). αvβ6 is expressed at low or undetectable levels in normal adult tissues, but is strongly up-regulated in inflammatory/fibrotic disease, and is generally restricted to epithelial cells (21, 2730). Thus, the up-regulated expression of αvβ6 in epithelial cells during tissue injury provides a mechanism for increased local activation of TGF-β and subsequent TGF-β–mediated effects on surrounding cells. Selectively blocking αvβ6 binding to ligand with monoclonal antibodies (mAbs) (31) provides a method for localized inhibition of TGF-β activation, specifically in tissues where there is up-regulated expression of αvβ6. This approach offers the potential to decrease risks associated with global inhibition of TGF-β.

Comparison of the genetic null phenotypes for αvβ6 and TGF-β1 further supports the hypothesis that blocking αvβ6-mediated TGF-β activation may be preferable to global targeting of TGF-β in lung disease. Mice completely deficient for αvβ6 function live a normal lifespan and are resistant to bleomycin-induced lung fibrosis. They have mild inflammation that is limited to the lung and late-onset emphysema. In contrast, TGF-β1–deficient mice show severe inflammation in multiple organ systems, resulting in death at 3 to 4 weeks of age (32, 33). The more severe phenotype of the TGF-β1 null mice implies that activation of latent TGF-β1 in other tissues does not require αvβ6 during normal development. The lung inflammation in β6 null mice and the low expression of αvβ6 in normal lung led to the speculation that a low level of homeostatic TGF-β signaling might be required to prevent pulmonary inflammation. A partial inhibition of the elevated TGF-β signaling that is characteristic of pulmonary fibrosis might, therefore, be desirable to prevent fibrosis without promoting inflammation. This could be evaluated with function blocking αvβ6 mAbs in an in vivo pulmonary fibrosis model.

In the studies described here, we show that αvβ6 is significantly upregulated in human lung diseases associated with inflammatory and fibrotic pathology. Evaluating a range of concentrations of a function-blocking αvβ6 mAb in mice, we demonstrate that high doses produce mRNA changes in the lung that are consistent with the inflammation and macrophage activation seen in the αvβ6-deficient mice, but are reversible after treatment is stopped. In addition, we show that low doses of αvβ6 mAbs attenuate bleomycin-induced fibrosis in vivo without significantly altering alveolar inflammatory cell populations or expression of genes associated with macrophage activation.


αvβ6 mAbs were generated as previously described (31). Recombinant soluble murine TGF-β receptor type II–Fc fusion protein (rsTGF-βRII–Fc) was generated as previously described (34) and purchased from R&D Systems (532-R2; Minneapolis, MN). Purified, endotoxin-free preparations of αvβ6 mAbs 3G9, 8G6, 4B4 (previously described as 6.3G9, 6.8G6, and 6.4B4) (31), isotype control mAb 1E6, and rsTGF-βRII–Fc (purified protein in phosphate-buffered saline [PBS]) were used in in vivo experiments.

3G9 treatment in normal mice.

C57Bl6 mice (7–10 weeks old; Jackson Laboratories, Bar Harbor, ME) were dosed by intraperitoneal injection once per week for 4 weeks with 5 mg/kg of rsTGF-βRII–Fc, PBS, or the following doses of 3G9: 0.3, 1, 3, 10, and 30 mg/kg.

Bleomycin-induced pulmonary fibrosis models.

SV129 mice were used for experiments with lung hydroxyproline as an endpoint. C57Bl6 mice (7–10 wk old; Jackson Laboratories) were used for experiments with mRNA or bronchoalveolar lavage (BAL) collection and analysis as endpoints. For quantitative analysis of collagen gene expression as an endpoint, transgenic mice carrying a luciferase reporter gene under the control of a 17-kb region of the colIα2 gene promoter were used (35). All animal studies were performed using the protocols and guidelines of the institutional animal care and use committee.

Bleomycin Instillation and mAb Administration

Mice were instilled with bleomycin or saline in the trachea as previously described (21). In studies where hydroxyproline was measured, mAbs were administered at 4 mg/kg three times per week. In the reporter gene, Smad2/3 phosphorylation, and BAL studies, mAbs were administered once per week. Treatments were initiated relative to bleomycin delivery, as described in the Results. In transcript profiling studies, a single dose of mAb was given 1 day before bleomycin instillation. All injections were administered by intraperitoneal injection. There were 5 to 11 mice per group for all experiments, except the 3G9 group from the day 1 to 30 treatment study, for which the sample size was 3. Total lung hydroxyproline was measured as previously described (21). The specific mouse strains used in each study are described in the Results section.

RNA Preparation, Expression Profiling, and Analysis

RNA isolation, sample labeling, hybridization, and staining were performed as previously described (36). For samples from PBS- and 3G9-treated normal mice, significance analysis of microarrays was used to identify probe sets with signal intensities altered by experimental treatment compared with the PBS-treated group, with the false discovery rate threshold set not to exceed 0.01 at the 0.95 confidence limit. Further filtering was done by selecting the significantly affected (false discovery rate < 0.01; confidence limit, 0.95) probe sets showing at least a twofold change in signal intensity relative to PBS-treated control mice. Virtual pathway analysis was performed using the Ingenuity Pathway Analysis database (Ingenuity Systems, Redwood City, CA). For samples from bleomycin-challenged mice, statistical and clustering analyses were done using GeneSpring (Agilent, Santa Clara, CA) software. Transcripts affected by bleomycin injury, identified using analysis of variance (ANOVA), were probe sets showing a twofold or greater change in signal intensity (P < 0.01), and subsets of transcripts affected by 1 mg/kg 3G9 treatment were further selected as probesets showing significant (P < 0.01) change in signal intensity relative to 1 mg/kg 1E6 treatment.

BAL and Differential Staining of BAL Cells

Two lavages of 0.8 ml PBS each were collected per mouse, total cells counted by hemocytometer, and cytospun aliquots were stained with DiffQuik (Fisher Scientific, Waltham, MA). Cell types were categorized by morphology (21). There were 12–19 mice per treatment group for Day 8, and 7–10 mice per treatment group for each of Days 2, 5, and 11, with the exception of 3 mg/kg, Day 11 (n = 4).


Paraffin tissue sections of human lung disease were obtained from G. Davis (University of Vermont), C. Feghali-Bostwick (University of Pittsburgh), and R. Lafyatis (Boston University Medical School). All pathologies were reviewed and pathological diagnoses confirmed by the same board-certified pathologist (Dr. Carl O'Hara) experienced in evaluating pulmonary pathology. Immunohistochemistry for αvβ6 was performed as previously described (36). αvβ6 staining intensity was scored using an analog visual scale (0–100), as previously described (37). The entire biopsy was examined by a blinded scorer. The degree of brown cellular staining for αvβ6 was used to assign a staining score using a 10-cm visual analog scale, left-anchored (0 cm = 0) for no stain and right-anchored (10 cm = 100) for diffuse stain. All human tissue samples were obtained with local institutional review and patient approval.

Western Analysis and Luciferase Assay

Western blots with 30 μg of protein per lane from lung nuclear extracts (38) were incubated with pSmad2/3 primary antibody (no. 3101L; Cell Signaling Technologies) and horseradish peroxidase–labeled donkey anti-rabbit secondary antibody, and then visualized by West Femto substrate (Pierce Biotechnology, Rockford, IL). For luciferase assays, lungs were collected, homogenized, and luciferase activity measured per manufacturer's protocol (no. 6016911; Perkin Elmer, Waltham, MA). The number of mice per luciferase treatment group was as follows: 11–17 for PBS, 0.1, 0.3, and 1.0 mg/kg 3G9; 5–6 for other treatment groups.

Statistical Analysis

Statistical comparisons were made between vehicle control and/or isotype control and test article using ANOVA. When statistically significant differences were established at a probability of P less than 0.05 using ANOVA, significant differences between groups were evaluated by Dunnet's test.

Expression of αvβ6 in Human Lung Disease and in the Bleomycin Lung Fibrosis Model

Up-regulated TGF-β expression and signaling has been described in a variety of human lung diseases involving fibrotic or inflammatory pathology (18, 39). However, analysis of αvβ6 expression has only been reported for a small sample of fibroinflammatory lung disease and was limited to frozen tissue samples (28). Using the 2A1 mAb, which selectively recognizes an αvβ6 epitope on paraffin tissue sections (31), we have evaluated the expression of αvβ6 in 32 lung tissue samples from patients with fibrotic changes. These included 11 with a diagnosis of systemic sclerosis lung disease and 21 with IPF. In addition, we immunostained a lung tissue array from normal regions of lung biopsies from cancer patients. Expression of αvβ6 in normal lung tissue was nearly undetectable by immunohistochemistry (Figure 1A). However, in all disease samples, fibrotic regions of lung showed strong αvβ6 expression (Figures 1B–1F). The αvβ6 was localized to epithelial cells overlying regions of overt fibrosis, or in regions adjacent to inflammatory infiltrates. Intense staining was seen within both type II and type I pneumocytes lining the alveolar ducts and alveoli, whereas large airways were largely negative, and intraalveolar macrophages were negative. When comparing the αvβ6 staining in systemic sclerosis samples, a trend was noted toward higher intensity staining in samples showing usual interstitial pneumonitis (UIP; Figures 1C and 1D), a pathology associated with prominent fibrosis and progressive disease, as compared with nonspecific interstitial pneumonitis (NSIP; Figure 1B), a pathology associated with less fibrosis and a better prognosis. To quantify this observation, the relative intensity of αvβ6 staining in each of the NSIP and UIP samples was scored (Figure 1G). The intensity of staining scored higher in the UIP samples than in all but one of the four NSIP samples. The only NSIP sample showing high-intensity staining for αvβ6 was an NSIP fibrosing variant, which is a pathology associated with more fibrosis than typical NSIP, and may be more closely related to UIP pathology (40). The difference did not reach statistical significance, possibly due to the small sample size. In 11 of the 21 IPF samples, a clear pathological diagnosis was available, and all 11 of these pathologies were UIP. The αvβ6 staining in these IPF samples (Figures 1E and 1F) was also scored for staining intensity and was found to be comparable to the intense staining seen in the patients with systemic sclerosis with UIP (Figure 1G). In summary, expression of αvβ6 was up-regulated in all IPF and systemic sclerosis lung disease samples examined and trended higher in samples with NSIP fibrosing variant or UIP pathology as compared with NSIP.

Effect of Anti-αvβ6 mAbs in Normal Mice

β6 null mice, which genetically lack the αvβ6 integrin and have compromised TGF-β signaling in the lung, develop mild pulmonary inflammation, characterized by enlarged, foamy macrophages and occasional foci of perivascular or peribronchiolar inflammation (41, 42). The effect of αvβ6 mAb treatment was evaluated in normal C57Bl6 adult mice administered increasing doses of the 3G9 mAb ranging from 0.3 to 30 mg/kg as weekly intraperitoneal injections for 4 weeks. 3G9 is a murine mAb that binds to both human and mouse αvβ6 with picomolar affinity and has potent function-blocking activity (31). Mice were killed 1 week after the last dose (treatment phase), and RNA from lung tissue was prepared for microarray analysis. In addition, a second cohort of mice was treated with the same doses of 3G9 for the same 4-week period, and lungs were collected 8 weeks after the last dose of antibody (recovery phase) to assess the reversibility of 3G9 treatment–induced changes. Mice dosed for 4 weeks with no recovery phase showed no significant changes in gene expression in the lung with treatment up to 3 mg/kg. However, in the 10 and 30 mg/kg treatment groups, a number of probe sets were significantly changed relative to PBS-treated control mice (Figure 2A), and those with greater than twofold changes are identified in Table E1 in the online supplement. High-dose 3G9–induced changes in gene expression largely resolved during the recovery period, which was reflected in the virtual absence of differences between transcript profiles of 3G9- and PBS-treated mice (Figure 2B). The absence of significant changes in gene expression in the recovery groups suggests that changes in gene expression induced by high-dose 3G9 were reversible when treatment was ceased. Transcript profiling in a second strain of mice, CD-1, yielded highly similar results (data not shown), indicating that these effects were not strain specific.

Functional annotation of the genes affected by 3G9 using the Ingenuity Pathway Analysis database showed strong association of these genes with immune response and immunoregulatory cytokine signaling (see Figure E1A in the online supplement). The two highest scoring networks obtained by virtual regulatory pathway analysis suggest that high-dose 3G9–induced changes in gene expression are characterized by alterations in chemokine, TGF-β, and IFN signaling (Figures E1B and E1C). Importantly, there was substantial concordance between transcripts up-regulated by high-dose 3G9 treatment and those that are up-regulated in lungs of β6 null mice (43). Three of the five most up-regulated transcripts in lungs of β6 null mice (matrix metalloproteinase [MMP]-12, serum amyloid A [SAA]3 and lipocalin-2) were also among the four transcripts most highly up-regulated by 3G9 treatment at 10 mg/kg.

The genes up-regulated by high-dose 3G9 treatment included several markers of macrophage activation (e.g., MMP-12, MMP-13, cathepsin K, CCL9, and CCL12). These genes are up-regulated in β6 null mice and other disease models involving macrophage activation (43, 44). MMP-12 shows the greatest fold up-regulation of any transcript in lungs from both β6 null and high-dose 3G9-treated mice, and is functionally required for the development of emphysema in β6 null mice (42). To determine the dose at which 3G9 treatment increases expression of this macrophage activation marker, relative levels of MMP-12 transcript were assayed by quantitative polymerase chain reaction analysis of lung RNA isolated from mice treated with different doses of 3G9. A dose-dependent increase in MMP-12 transcript was detected that was significant at 10 and 30 mg/kg, but not at doses of 3 mg/kg or less (Figure 2C). The fold change in MMP-12 expression at 10 and 30 mg/kg was comparable to that seen in the β6 null mice (data not shown). In summary, high-dose 3G9 treatment of normal mice induced gene expression changes in lung that are highly similar to those seen in β6 null mice, consistent with 3G9 acting as an efficient inhibitor of αvβ6 function in vivo.

αvβ6 mAb Treatment Attenuates Bleomycin-induced Fibrosis

αvβ6 up-regulation in the lungs of SV129 mice at 10 days after bleomycin instillation has been previously reported (21). In the time points evaluated here, αvβ6 was not up-regulated in the lungs at Day 1, was diffusely up-regulated at Day 5, and demonstrated strong focal up-regulation at Days 10, 14, and 28 (data not shown). The antifibrotic efficacy of blocking αvβ6 function with 3G9 was evaluated using prophylactic and therapeutic treatment regimens in the SV129 model of bleomycin-induced pulmonary fibrosis. SV129 mice were chosen because this was the background strain on which β6 null mice had demonstrated protection from bleomycin-induced fibrosis (21). SV129 mice are known to have a slower fibrotic reaction than C57Bl6 mice (45), and increased hydroxyproline content is seen at 30 and 60 days, whereas C57Bl6 mice are typically evaluated at 14 days. Mice were treated at 12 mg/kg/week to assess efficacy, a dose that produces gene expression changes similar to those of the β6 null mice. In the first set of studies, mice were challenged with intratracheal administration of bleomycin (or intratracheal saline as a control) and dosed intraperitoneally with 3G9 or 1E6, an isotype control mAb (46), beginning at the time of bleomycin instillation. Mice were then killed at Day 30 and lungs were evaluated for hydroxyproline content. 3G9 treatment resulted in a 65% inhibition of the bleomycin-induced increase in lung hydroxyproline relative to 1E6 treatment (Figure 3A). Having mAbs that block αvβ6 function in vivo enabled us to evaluate the efficacy of αvβ6 blockade in established lung fibrosis, which could not otherwise be tested using β6 null mice. In these studies, treatment was delayed until 15 days after bleomycin installation, a time point by which collagen accumulation in the lung has already begun. At Day 30, the increase in hydroxyproline was again attenuated in 3G9-treated mice (Figure 3B). A second delayed treatment study was performed in which treatment was initiated at Day 15, and lungs were analyzed at Day 60. Three different αvβ6 mAbs, 3G9, 8G6, and 4B4, were tested for efficacy compared with PBS control–treated mice. The 4B4 mAb was included because it binds with high affinity to the αvβ6 integrin but has weak ligand-blocking activity, whereas 8G6 is a function-blocking αvβ6 mAb of similar potency to 3G9 and belongs to a different biochemical class (31). At Day 60 after bleomycin instillation, treatment with the weak-blocking mAb, 4B4, resulted in a nonsignificant 46% inhibition, whereas treatment with the more potent αvβ6 blocking antibodies, 3G9 or 8G6, yielded significant inhibition of 57 and 78%, respectively (Figure 3C). Thus, function-blocking αvβ6 mAbs were effective, not only in prophylactic prevention of bleomycin-induced lung fibrosis, but also in therapeutically attenuating fibrotic progression when treatment was delayed until after the initiation of fibrosis.

Effects of αvβ6 mAb Treatment on Gene Expression in the Bleomycin Fibrosis Model: Collagen-Reporter Mice and Transcript Profiling

To better characterize the effects of αvβ6 mAb treatment on bleomycin-induced lung collagen expression, we sought a more sensitive endpoint that would be better suited to generating a dose–response effect of 3G9 mAb treatment. Transgenic mice carrying a transgene in which a luciferase reporter gene is expressed under the control of the collagen Iα2 promoter have previously been used to provide a quantitative readout of collagen expression in fibrosis models, and was found to correlate with other endpoints of fibrosis (35, 47). However, use of these mice in the bleomycin-induced lung fibrosis model has not been previously published. Lung luciferase levels in transgenic reporter mice receiving bleomycin were 10-fold greater than saline-instilled controls analyzed 14 days after instillation, providing a more sensitive in vivo system for dose–response studies. A dose titration of the 3G9 antibody was evaluated for efficacy and compared with a positive control, the rsTGF-βRII–Fc. rsTGF-βRII–Fc contains the extracellular domain of the TGF-βRII, which directly binds active TGF-β1 and TGF-β3 and inhibits their interaction with endogenous TGF-βRII. This protein has demonstrated efficacy as an inhibitor of TGF-β in a variety of disease models (9, 12, 34, 48). 3G9 treatment of bleomycin-challenged reporter mice produced a dose-dependent decrease in lung luciferase levels (Figure 4). Significant efficacy was achieved at a weekly dose of 0.3 mg/kg, with near maximal efficacy at 1 mg/kg and higher. The efficacy of 3G9 at 3 mg/kg was comparable to the soluble TGF-β receptor at 5 mg/kg. It is noteworthy that, although αvβ6 is not the only potential activator of TGF-β, blockade of αvβ6 function in this model achieved efficacy equivalent to that of a direct inhibitor of TGF-β. The collagen Iα2/luciferase mice were generated and maintained on a C57Bl6/DBA2 hybrid background strain. The fibrotic response to bleomycin in DBA2 mice is similar to that of C57Bl6 mice (49), and thus the C57Bl6/DBA2 hybrids were expected to have a robust fibrotic response at 14 days, which was evidenced by the significant induction of the collagen reporter gene in this strain. In addition, we have performed bleomycin-induced lung fibrosis studies in C57Bl6 mice using trichrome staining at Day 14 as a measure of collagen production, and found the 3G9 mAb to have effective antifibrotic activity in this strain of mice (data not shown). A similar dose–response study with the 3G9 mAb was also performed in a radiation-induced lung fibrosis model in C57Bl6 mice, and near maximal antifibrotic efficacy was seen at a weekly dose of 1 mg/kg (see Puthawala and colleagues, this issue, pages 82–90 [50]). Taken together, these studies confirm that the efficacy of 3G9 is neither strain specific nor disease model specific, and that near-maximal protection is achieved at 1 mg/kg dosed once weekly.

Transcript profiling of bleomycin-challenged lungs collected from control and αvβ6 mAb treatment groups was performed on C57Bl6 mice to characterize the effects of αvβ6 blockade on molecular endpoints of the disease. A group of genes with altered expression in bleomycin-challenged lungs was identified as probe sets showing at least a twofold mean increase in normalized signal intensity (P < 0.01). In addition, a group of transcripts affected by 1 mg/kg 3G9 treatment with bleomycin challenge was identified as probe sets showing a significant (P < 0.01) change in signal intensity compared with mice treated with 1E6 and bleomycin challenge. We identified 211 probe sets representing transcripts significantly affected by bleomycin injury and attenuated by 3G9 treatment, but not by the isotype control mAb, 1E6 (Figure 5). Functional annotation of these probe sets using the Ingenuity Pathways Analysis database revealed a gene regulatory network showing association of αvβ6 blockade with decreased TGF-β signaling (Figure E2). Accordingly, the transcripts that were increased by bleomycin and significantly changed by 1 mg/kg 3G9 treatment included genes previously identified as targets of TGF-β signaling and/or molecules previously shown to play a role in bleomycin-induced fibrosis (Table E2).

αvβ6 mAb Treatment Blocks Bleomycin-induced Increases in Phospho-Smad2/3 In Vivo

Smad proteins are important intracellular mediators of TGF-β signaling. Both the level of phosphorylation and the degree of nuclear localization of Smad proteins are increased during bleomycin-induced pulmonary fibrosis (38). Lung tissue was collected from bleomycin-challenged, 3G9- and rsTGF-βRII–Fc–treated C57Bl6 mice 7 days after challenge and evaluated for nuclear phospho-Smad2/3 levels. Bleomycin induced a substantial increase in phospho-Smad2/3 levels. The bleomycin-induced increase in phospho-Smad2/3 was completely blocked by 3G9 treatment at 3 mg/kg (Figure 6). Complete inhibition of phospho-Smad2/3 increases was not required for antifibrotic efficacy, because efficacious doses of 3G9 (1 mg/kg) and rsTGF-βRII–Fc (5 mg/kg) showed partial inhibition that did not reach significance using semiquantitative densitometry (Figure 6B). These findings are the first to demonstrate in vivo decreased phosphorylation of Smad2/3, a key downstream mediator of TGF-β signaling, through the blockade of αvβ6 function in lung. These results, together with the transcript profiling results described previously here, indicate that the mechanism of action of αvβ6 mAbs in vivo is related to inhibition of the TGF-β signaling pathway.

Analysis of BAL Cell Composition with αvβ6 mAb Treatment

BAL cell populations were analyzed to determine if αvβ6 inhibition was preventing fibrosis via alterations in the major subpopulations of inflammatory cells in the lung. A course of four different time points were analyzed (Days 2, 5, 8, and 11). As expected, there were elevations in BAL cell counts at Days 5, 8, and 11 due to the intratracheal administration of bleomycin when compared with saline-instilled C57Bl6 mice. However, throughout the time course, there was no significant difference in the total number of BAL cells, nor in the numbers of macrophages, neutrophils, or lymphocytes in the BAL, in mice treated with efficacious doses of 3G9 (0.3, 1.0, and 3 mg/kg) when compared with mice treated with an isotype control antibody, 1E6 (Figure 7). At one time point, Day 8, there was a nonsignificant increase (P = 0.074 [t test] compared with 1E6-treated control animals) in neutrophils in the 3 mg/kg–treated group. Thus, there are no significant alterations in BAL cell populations in this disease model at doses of 3G9 that are effective in blocking bleomycin-induced increases in phospho-Smad3 and collagen expression, suggesting that the efficacy of αvβ6 mAbs is not likely mediated by gross antiinflammatory mechanisms.

Inhibition of αvβ6-mediated TGF-β activation shows promise as a novel therapeutic modality for treating pulmonary fibrosis. Here, we have demonstrated up-regulation of αvβ6 expression in IPF lung and in a spectrum of fibrotic pulmonary pathology associated with systemic sclerosis. The consistent up-regulation of αvβ6 expression in all samples tested and in lung fibrosis with different etiology suggests that up-regulated expression of αvβ6 may be a common phenomenon in a variety of clinical indications involving pulmonary fibrosis. In systemic sclerosis lung disease, there was a trend toward more intense staining in patients with UIP, generally associated with a poorer prognosis, than in those with NSIP pathology. In the context of previous data and data shown here, the highly up-regulated expression of αvβ6 strongly supports a pivotal role of this integrin in TGF-β activation and subsequent fibrosis in patients with pulmonary fibrosis.

The results presented here, together with those of Puthawala and colleagues (this issue, pages 82–90), are the first to demonstrate the efficacy of blocking antibodies to the αvβ6 integrin in a model of pulmonary fibrosis. Antibodies that block αvβ6-mediated TGF-β activation were shown to be effective in blocking fibrosis using a variety of different endpoints: total lung hydroxyproline content, collagen reporter transgene expression, and transcript profiling. The attenuation of phospho-Smad2/3 increases and the down-regulation of TGF-β–inducible target genes that are overexpressed in lungs of bleomycin-challenged mice are evidence that αvβ6 mAbs are effective in inhibiting the TGF-β pathway in this disease model in vivo. Although protection from fibrosis in genetically αvβ6-deficient mice has been previously reported, the demonstration of efficacy using blocking mAbs in vivo is an important step in establishing the therapeutic utility of inhibiting this pathway in the adult. The efficacy of αvβ6 mAbs does not appear to be inferior to soluble TGF-βRII–Fc, a more global inhibitor of TGF-β, suggesting that alternative mechanisms of TGF-β activation are not able to promote fibrosis in the presence of αvβ6 blocking antibodies.

TGF-β has pleiotropic effects in the initiation and maintenance of fibrosis. This cytokine has been shown to induce alveolar epithelial cell apoptosis, which may be a key event in the initiation of fibrosis. TGF-β is also well established as a potent activator of fibroblasts, and strongly induces the expression of ECM components. In addition, recent data points toward a critical role of αvβ6-mediated TGF-β activation in promoting epithelial-to-mesenchymal cell transition, which may be an important source of fibroblasts in lung injury (51). These and other findings contribute to an overall picture of pulmonary fibrosis in which epithelial injury is a key factor in driving fibrotic progression. αvβ6 is commonly up-regulated on injured epithelial cells, leading to increases in local activation of TGF-β and fibrosis. The efficacy of αvβ6 blockade in preventing pulmonary fibrosis is thus likely to be mediated by attenuating the profibrotic effects of TGF-β on multiple biologies that contribute to fibrotic progression.

Our use of transgenic collagen reporter mice in the bleomycin model represents a novel endpoint for evaluating efficacy. Collagen reporter gene expression was a more sensitive endpoint than hydroxyproline and better suited to an analysis of dose-dependent effects of antibody treatment. In these reporter gene mice, the αvβ6 antibody, 3G9, demonstrated significant, near-maximal efficacy in attenuating bleomycin-induced changes in collagen expression and in normalizing the gene expression profile at a dose of 1 mg/kg weekly. In radiation-induced pulmonary fibrosis (Puthawala and colleagues, this issue, pages 82–90), near maximal efficacy was seen in the same 1–3 mg/kg dose range as in the collagen reporter model. The similar efficacy and dose response in these two models, which are very different in both the nature of the injury that initiates fibrosis and the time course over which fibrosis develops, suggests that αvβ6-mediated TGF-β activation may be an important driver of pulmonary fibrosis initiated by different mechanisms. These results, together with the finding that αvβ6 is up-regulated in human disease associated with fibrotic pathology, suggest that αvβ6 could be an effective therapeutic target in pulmonary fibrosis.

Although low-dose treatment shows efficacy in preventing pulmonary fibrosis, only high-dose treatment (⩾10 mg/kg) of normal mice produced significant changes in gene expression profiles. The profile of transcript changes induced by 3G9 treatment at these doses was consistent with the inflammatory changes seen in lungs of β6 null (αvβ6-deficient) mice, including up-regulation of MMP-12, the most highly up-regulated transcript in β6 null mice. Because β6 null mice develop emphysema late in life, treatment for longer than the 4-week period described here would be required to evaluate the possibility of inducing emphysematous changes. β6 null mice crossed onto an MMP-12 null background do not develop emphysema, demonstrating that MMP-12 is functionally required for emphysema development in these mice (42). Because MMP-12 is not induced at low doses of 3G9 mAb treatment, it is likely that emphysematous changes would not be induced by low-dose treatment. It is therefore possible to achieve near-maximal efficacy in normalizing bleomycin-induced changes at mAb doses that are lower than those required to produce αvβ6-mediated inflammatory changes in the lung. Furthermore, pharmacokinetic data (unpublished) shows that, similar to other integrin antibodies, 3G9 has a significantly shorter half-life at 1 mg/kg than it does at 10 mg/kg, and the difference in exposure (area under the curve) at these two doses is approximately 80-fold, considerably higher than the 10-fold increase in dose. Thus, the window between the near-maximal antifibrotic efficacy seen at 1 mg/kg and the inflammation seen at 10 mg/kg is larger when measured on the basis of drug exposure. In this dose–response window, Smad activation was strongly inhibited at 3 mg/kg, whereas, at 1 mg/kg, inhibition of Smad activation was not significant. It is possible that only small decreases in Smad phosphorylation are needed for attenuating fibrosis, or, alternatively, that Smad-independent pathways downstream of TGF-β activation are mediating efficacy at low doses of mAb. The importance of Smad-independent pathways in mediating TGF-β–dependent fibrosis has been demonstrated (52, 53), but additional studies will be required to evaluate their role in mediating the therapeutic effects of αvβ6 mAbs. Finally, these results suggest that low-dose partial inhibition of αvβ6-mediated TGF-β activation is sufficient to block fibrosis, whereas significantly higher levels of inhibition are required to induce inflammatory changes that are similar to those seen in αvβ6-deficient mice.

In IPF, the role of inflammation in disease progression is controversial. Although some level of inflammation is a consistent feature of the disease, the lack of efficacy of immune-suppressive treatment and the apparent development and progression of fibrosis in the absence of significant inflammation in some patients have led some to propose that IPF is not the result of chronic inflammation, but rather is due to aberrant wound healing resulting from ongoing epithelial damage and/or endothelial cell activation (54, 55). Regardless of the relative contribution of inflammation to disease progression, the development of therapeutics that specifically target the fibrotic process could be an important new avenue for treatment. Antibodies to αvβ6 block fibrosis independently of any antiinflammatory action. Although a large number of agents have shown efficacy in inhibiting bleomycin-induced fibrosis, anti-αvβ6 mAbs are among a small subset of these agents that have demonstrated efficacy without concomitant antiinflammatory effects.

In summary, the up-regulation of αvβ6, a key activator of TGF-β, in fibrotic human lung diseases suggests that it may play an important role in driving pathological fibrosis. There is a key therapeutic advantage in inhibiting αvβ6-mediated TGF-β activation rather than inhibiting TGF-β globally. The requirement for αvβ6 function is clearly more tissue-specific than TGF-β, as αvβ6 knockout mice do not show the inflammation in multiple organ systems associated with TGF-β deficiency. This is likely due to redundant mechanisms for TGF-β activation that are unaffected by loss of αvβ6 function. This redundancy, however, does not appear to diminish the efficacy of αvβ6 blockade in models of fibrotic disease, as we have consistently seen robust efficacy in blocking fibrosis in multiple lung and kidney disease models. These findings warrant further development of αvβ6 blocking mAbs as important therapeutics for treating fibrotic disease.

1. Roberts AB, Sporn MB, Assoian RK, Smith JM, Roche NS, Wakefiled LM, Heine UI, Liotta LA, Falanga V, Kehrl JH, et al. Transforming growth factor type β: rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc Natl Acad Sci USA 1986;83:4167–4171.
2. Roberts AB, Sporn MB. Regulation of endothelial cell growth, architecture, and matrix synthesis by TGF-β. Am Rev Respir Dis 1989;140:1126–1128.
3. Varga J, Rosenbloom J, Jimenez SA. Transforming growth factor β (TGF β) causes a persistent increase in steady-state amounts of type I and type III collagen and fibronectin mRNAs in normal human dermal fibroblasts. Biochem J 1987;247:597–604.
4. Grande JP, Melder DC, Zinsmeister AR. Modulation of collagen gene expression by cytokines: stimulatory effect of transforming growth factor-β1, with divergent effects of epidermal growth factor and tumor necrosis factor-α on collagen type I and collagen type IV. J Lab Clin Med 1997;130:476–486.
5. Walker GA, Masters KS, Shah DN, Anseth KS, Leinwand LA. Valvular myofibroblasts activation by transforming growth factor-β: implications for pathological extracellular matrix remodeling in heart valve disease. Circ Res 2004;95:253–260.
6. Eickelberg O, Kohler E, Reichenberger F, Bertschin S, Woodtli T, Erne P, Perruchoud AP, Roth M. Extracellular matrix deposition by primary human lung fibroblasts in response to TGF-β1 and TGF-β3. Am J Physiol 1999;276:L814–L824.
7. Sime PJ, Xing Z, Graham FL, Csaky KG, Gauldie J. Adenovector-mediated gene transfer of active transforming growth factor-β1 induces prolonged severe fibrosis in rat lung. J Clin Invest 1997;100:768–776.
8. Bonniaud P, Kolb M, Galt T, Robertson J, Robbins C, Stampfli M, Lavery C, Margetts PJ, Roberts AB, Gauldie J. Smad3 null mice develop airspace enlargement and are resistant to TGF-β-mediated pulmonary fibrosis. J Immunol 2004;173:2099–2108.
9. George J, Roulot D, Koteliansky VE, Bissell DM. In vivo inhibition of rat stellate cell activation by soluble transforming growth factor type II receptor: a potential new therapy for hepatic fibrosis. Proc Natl Acad Sci USA 1999;96:12719–12724.
10. Sharma K, Jin Y, Guo J, Ziyadeh FN. Neutralization of TGF-β by an anti-TGF-β antibody attenuates kidney hypertrophy and the enhanced extracellular matrix gene expresssion in STZ-induced diabetic mice. Diabetes 1996;45:522–530.
11. Bonniaud P, Margetts PJ, Schroeder JA, Kapoun AM, Damm D, Murphy A, Chakravarty S, Dugar S, Higgins L, Protter AA, et al. Progressive TGF-(β)1–induced lung fibrosis is blocked by an orally active ALK5 kinase inhibitor. Am J Respir Crit Care Med 2004;171:889–898.
12. Zheng H, Wang J, Koteliansky V, J. GP, Hauer-Jensen M. Recombinant soluble transforming growth factor β type II receptor ameliorates radiation enteropathy in mice. Gastroenterology 2000;119:1286–1296.
13. Kasuga H, Ito Y, Sakamoto S, Kawachi H, Shimizu F, Yuzawa Y, Matsuo S. Effects of anti-TGF-β type II receptor antibody on experimental glomerulonephritis. Kidney Int 2001;60:1745–1755.
14. Ziyadeh FN, Hoffman BB, Han DC, Iglesias-de la Cruz MC, Hong SW, Isono M, Chen S, McGowan TA, Sharma K. Long-term prevention of renal insufficiency, excess matrix gene expression, and glomerular mesangial matrix expansion by treatment with monoclonal antitransforming growth factor-β antibody in db/db diabetic mice. Proc Natl Acad Sci USA 2000;97:8015–8020.
15. Laping NJ. ALK5 inhibition in renal disease. Curr Opin Pharmacol 2003;3:204–208.
16. Miyajima A, Chen J, Lawrence C, Ledbetter S, Soslow RA, Stern J, Jha S, Pigato J, Lemer ML, Poppas DP, et al. Antibody to transforming growth factor-β ameliorates tubular apoptosis in unilateral ureteral obstruction. Kidney Int 2000;58:2310–2313.
17. Wang Q, Wang Y, Hyde DM, Gotwals PJ, Koteliansky VE, Ryan ST, Giri SN. Reduction of bleomycin induced lung fibrosis by transforming growth factor β soluble receptor in hamsters. Thorax 1999;54:805–812.
18. Broekelmann TJ, Limper AH, Colby TV, McDonald JA. Transforming growth factor β1 is present at sites of extracellular matrix gene expression in human pulmonary fibrosis. Proc Natl Acad Sci USA 1991;88:6642–6646.
19. Selman M, Pardo A, Barrera L, Estrada A, Watson SR, Wilson K, Aziz N, Kaminski N, Zlotnik A. Gene expression profiles distinguish idiopathic pulmonary fibrosis from hypersensitivity pneumonitis. Am J Respir Crit Care Med 2006;173:188–198.
20. Li MO, Wan YY, Sanjabi S, Robertson AK, Flavell RA. Transforming growth factor-β regulation of immune responses. Annu Rev Immunol 2006;24:99–146.
21. Munger JS, Huang X, Kawakatsu H, Griffiths MJD, Dalton SL, Wu J, Pittet JF, Kaminski N, Garat C, Matthay MA, et al. The integrin αvß6 binds and activates latent TGFß1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell 1999;96:319–328.
22. Gleizes PE, Munger JS, Nunes I, Harpel JG, Mazzieri R, Noguera I, Rifkin DB. TGF-β latency: biological significance and mechanisms of activation. Stem Cells 1997;15:190–197.
23. Khalil N. TGF-β: from latent to active. Microbes Infect 1999;1:1255–1263.
24. Barcellos-Hoff MH. Latency and activation in the control of TGF-β. J Mammary Gland Biol Neoplasia 1996;1:353–363.
25. Annes JP, Rifkin DB, Munger JS. The integrin αvß6 binds and activates latent TGFß3. FEBS Lett 2002;511:65–68.
26. Ma LJ, Yang H, Gaspert A, Carlesso G, Barty MM, Davidson JM, Sheppard D, Fogo AB. Transforming growth factor-β–dependent and independent pathways of induction of tubulointerstitial fibrosis in β6−/− mice. Am J Pathol 2003;163:1261–1273.
27. Breuss JM, Gillett N, Lu L, Sheppard D, Pytella R. Restricted distribution of integrin β6 mRNA in primate epithelial tissues. J Histochem Cytochem 1993;41:1521–1527.
28. Breuss JM, Gallo J, DeLisser HM, Klimanskaya IV, Folkesson HG, Pittet JF, Nishimura S, Aldape K, Landers DV, Carpenter W, et al. Expression of the β6 subunit in development, neoplasia and tissue repair suggests a role in epithelial remodeling. J Cell Sci 1995;108:2241–2251.
29. Zambruno G, Marchisio PC, Marconi A, Vaschieri C, Melchiori A, Giannetti A, De Luca M. Transforming growth factor-β1 modulates β1 and β5 integrin receptors and induces the de novo expression of the αvß6 heterodimer in normal human keratinocytes: implications for wound healing. J Cell Biol 1995;129:853–865.
30. Hakkinen L, Koivisto L, Gardner H, Saarialho-Kere U, Carroll JM, Lakso M, Rauvala H, Laato M, Heino J, Larjava H. Increased expression of β6-integrin in skin leads to spontaneous development of chronic wounds. Am J Pathol 2004;164:229–242.
31. Weinreb PH, Simon KJ, Rayhorn P, Yang WJ, Leone DR, Dolinski BM, Pearse BR, Yokota Y, Kawakatsu H, Atakilit A, et al. Function-blocking integrin αvβ6 monoclonal antibodies. J Biol Chem 2004;279:17875–17887.
32. Christ M, McCartney-Francis NL, Kulkarni AB, Ward JM, Mizel DE, Mackall CL, Gress RE, Hines KL, Tian H, Karlsson S. Immune dysregulation in TGF-β 1–deficient mice. J Immunol 1994;153:1936–1946.
33. Martin JS, Dickson MC, Cousins FM, Kulkarni AB, Karlsson S, Akhurst RJ. Analysis of homozygous TGF β 1 null mouse embryos demonstrates defects in yolk sac vasculogenesis and hematopoiesis. Ann N Y Acad Sci 1995;752:300–308.
34. Cosgrove D, Rodgers K, Meehan D, Miller C, Bovard K, Gilroy A, Gardner H, Kotelianski V, Gotwals P, Amattucci A, et al. Integrin α1β1 and transforming growth factor-β1 play distinct roles in alport glomerular pathogenesis and serve as dual targets for metabolic therapy. Am J Pathol 2000;157:1649–1659.
35. Inagaki Y, Truter S, Bou-Gharios G, Garrett LA, de Crombrugghe B, Nemoto T, Greenwel P. Activation of proα2(I) collagen promoter during hepatic fibrogenesis in transgenic mice. Biochem Biophys Res Commun 1998;250:606–611.
36. Hahm K, Lukashev ME, Luo Y, Yang WJ, Dolinski BM, Weinreb PH, Simon KJ, Chun Wang L, Leone DR, Lobb RR, et al. Alphav β6 integrin regulates renal fibrosis and inflammation in alport mouse. Am J Pathol 2007;170:110–125.
37. Kissin EY, Merkel PA, Lafyatis R. Myofibroblasts and hyalinized collagen as markers of skin disease in systemic sclerosis. Arthritis Rheum 2006;54:3655–3660.
38. Venkatesan N, Pini L, Ludwig MS. Changes in Smad expression and subcellular localization in bleomycin-induced pulmonary fibrosis. Am J Physiol 2004;287:L1342–L1347.
39. Khalil N, O'Connor RN, Flanders KC, Unruh H. TGF-β1, but not TGF-β2 or TGF-β3, is differentially present in epithelial cells of advanced pulmonary fibrosis: an immunohistochemical study. Am J Respir Cell Mol Biol 1996;14:131–138.
40. Travis WD, Matsui K, Moss J, Ferrans VJ. Idiopathic nonspecific interstitial pneumonia: prognostic significance of cellular and fibrosing patterns: survival comparison with usual interstitial pneumonia and desquamative interstitial pneumonia. Am J Surg Pathol 2000;24:19–33.
41. Huang XZ, Wu JF, Cass D, Erle DJ, Corry D, Young SG, Farese RV Jr, Sheppard D. Inactivation of the integrin β6 subunit gene reveals a role of epithelial integrins in regulating inflammation in the lungs and skin. J Cell Biol 1996;133:921–928.
42. Morris DG, Huang X, Kaminski N, Wang Y, Shapiro SD, Dolganov G, Glick A, Sheppard D. Loss of integrin α(v)β6-mediated TGF-β activation causes MMP12-dependent emphysema. Nature 2003;422:130–131.
43. Kaminski N, Allard JD, Pittet JF, Zuo F, Griffiths MJ, Morris D, Huang X, Sheppard D, Heller RA. Global analysis of gene expression in pulmonary fibrosis reveals distinct programs regulating lung inflammation and fibrosis. Proc Natl Acad Sci USA 2000;97:1778–1783.
44. Woodruff PG, Koth LL, Yang YH, Rodriguez MW, Favoreto S, Dolganov GM, Paquet AC, Erle DJ. A distinctive alveolar macrophage activation state induced by cigarette smoking. Am J Respir Crit Care Med 2005;172:1383–1392.
45. Keogh KA, Standing J, Kane GC, Terzic A, Limper AH. Angiotensin II antagonism fails to ameliorate bleomycin-induced pulmonary fibrosis in mice. Eur Respir J 2005;25:708–714.
46. Abraham WM, Sielczak MW, Ahmed A, Cortes A, Lauredo IT, Kim J, Pepinsky B, Benjamin CD, Leone DR, Lobb RR, et al. α4-integrins mediate antigen-induced late bronchial responses and prolonged airway hyperresponsiveness in sheep. J Clin Invest 1994;93:776–787.
47. Denton CP, Zheng B, Shiwen X, Zhang Z, Bou-Gharios G, Eberspaecher H, Black CM, de Crombrugghe B. Activation of a fibroblast-specific enhancer of the proα2(I) collagen gene in tight-skin mice. Arthritis Rheum 2001;44:712–722.
48. Smith JD, Bryant SR, Couper LL, Vary CPH, Gotwals PJ, Koteliansky VE, Lindner V. Soluble transforming growth factor-β type II receptor inhibits negative remodeling, fibroblast transdifferentiation, and intimal lesion formation but not endothelial growth. Circ Res 1999;84:1212–1222.
49. Schrier DJ, Kunkel RG, Phan SH. The role of strain variation in murine bleomycin-induced pulmonary fibrosis. Am Rev Respir Dis 1983;127:63–66.
50. Puthawala K, Hadjiangelis N, Jacoby SC, Bayongan E, Zhao Z, Yang Z, Devitt ML, Horan GS, Weinreb PH, Lukashev ME, et al. Inhibition of integrin αvβ6, an activator of latent transforming growth factor-β, prevents radiation-induced lung fibrosis. Am J Respir Crit Care Med 2008;177:82–90.
51. Kim KK, Kugler MC, Wolters PJ, Robillard L, Galvez MG, Brumwell AN, Sheppard D, Chapman HA. Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix. Proc Natl Acad Sci USA 2006;103:13180–13185.
52. Dai C, Yang J, Liu Y. Transforming growth factor-β1 potentiates renal tubular epithelial cell death by a mechanism independent of Smad signaling. J Biol Chem 2003;278:12537–12545.
53. Wang S, Wilkes MC, Leof EB, Hirschberg R. Imatinib mesylate blocks a non-Smad TGF-β pathway and reduces renal fibrogenesis in vivo. FASEB J 2005;19:1–11.
54. Selman M, Pardo A. Role of epithelial cells in idiopathic pulmonary fibrosis: from innocent targets to serial killers. Proc Am Thorac Soc 2006;3:364–372.
55. Thannickal VJ. Idiopathic interstitial pneumonia: a clinicopathological perspective. Semin Respir Crit Care Med 2006;27:569–573.
Correspondence and requests for reprints should be addressed to Gerald S. Horan, Ph.D., Biogen Idec, 12 Cambridge Center, Cambridge, MA 02142. E-mail:


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