Specific Haptoglobin Expression in Bronchoalveolar Lavage during Differentiation of Circulating Fibroblast Progenitor Cells in Mild Asthma Kristoffer Larsen,† David Macleod,† Kristian Nihlberg,† Eylem Gu1 rcan,† Leif Bjermer,‡ Gyo1 rgy Marko-Varga,§ and Gunilla Westergren-Thorsson*,† Experimental Medical Science, Lund University, BMC C13, S-221 84 Lund, Sweden, Respiratory Medicine and Allergology, University Hospital Lund, S-221 85 Lund, Sweden, and Analytical Chemistry, Lund University, S-221 00 Lund, Sweden Received December 14, 2005
Abstract: Haptoglobin is an acute-phase glycoprotein considered to be involved in tissue repair and is produced by fibroblasts and inflammatory cells. By using a gelbased proteomic approach, we show for the first time a possible role for haptoglobin in the differentiation of fibroblast progenitor cells, termed fibrocytes, in patients with mild asthma. Bronchoalveolar lavage fluid (BALF) was performed to sample circulating fibrocytes from patients with mild asthma and nonasthmatic control subjects. Fibrocytes from the airway lumen were characterized by triple staining of the markers CD34/CD45R0/Rsmooth muscle actin, and subjected to confocal microscopy. The protein expression pattern was analyzed using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight (MALDITOF). Elevated levels of haptoglobin expression in BALF was reported in a sub-group of patients with mild asthma (p < 0.05) when compared to the other subjects. In addition, this increase in haptoglobin was accompanied by differentiation of fibrocytes into fibroblast-like cells. When further analyzing the expression pattern of haptoglobin isoforms, a heterozygous expression was detected in the patients where fibrocyte differentiation could be observed. These data raise the possibility that an acute and specific inflammatory state facilitates the differentiation of fibroblast progenitor cells into activated fibroblasts. Furthermore, this study proposes a novel role for haptoglobin in airway remodeling in patients with asthma. Keywords: asthma • fibrocytes • fibroblasts • haptoglobin • 2-DE • proteomics
Introduction Airway remodeling is a prominent feature of asthma and is characterized by an aberrant tissue repair mechanism leading * To whom correspondence should be addressed. Tel: +46 46 222 85 82. Fax: +46 46 222 31 28. E-mail:
[email protected]. † Experimental Medical Science, Lund University. ‡ Respiratory Medicine and Allergology, University Hospital Lund. § Analytical Chemistry, Lund University. 10.1021/pr050462h CCC: $33.50
2006 American Chemical Society
to structural abnormalities and reduced lung function.1 It is generally accepted that fibroblasts play an important role in airway remodeling due to their key function in modulating tissue repair.2,3 However, the most common asthmatic therapies available today are corticosteroids, which are primarily aimed at regulating the immune response and not the remodeling process per se. Proteomic characterization of lung fibroblasts may therefore be a suitable approach to reveal future biomarkers in airway remodeling, which has been shown in previous studies.4-6 The possible origins of the recruited fibroblasts are still under speculations. Recent findings in several in vivo models and in allergen-induced patients with asthma suggest a selective recruitment of circulating fibroblast progenitor cells termed fibrocytes, which may be recruited from the bone marrow.7-9 These cells have been characterized as antigen presenting cells which in circulation express a specific pattern of markers, including collagen I, CD34, CD45R0, and may extract contractile forces and express R-SMA when recruited into tissue.10,11 By analyzing bronchoalveolar lavage fluid (BALF) from patients with asthma, new insights into the remodeling process can be achieved.12,13 Characterization and optimization of the BALF proteome have been performed in several studies where a previous report demonstrated that activated fibroblasts were obtained from BALF in patients with mild asthma accompanied by elevated levels of eosinophils.14-16 In this pilot study, we hypothesized that the recently discovered emergence of BALF fibroblasts from the airway lumen are responsible for some of the physiological changes associated with asthma, and that we will observe markers of this differentiation process in the BALF proteome from these patients.
Experimental Procedures Patient Selection Criteria. Patients suffering from mild asthma and definite bronchial hyperresponsiveness were included in the pilot study. These patients fulfilled the asthma criteria of the American Thoracic Society. The patients (n ) 6) were further divided in two groups based on whether they showed out-growth of fibroblasts from BALF (n ) 3) or not (n ) 3).15 The nonasthmatic control subjects (n ) 3) were healthy nonsmokers and had no allergic or asthmatic symptoms. The study was fully approved by the Swedish Research Ethical Committee (LU339-00). Journal of Proteome Research 2006, 5, 1479-1483
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Haptoglobin Expression in Mild Asthma
technical notes
Figure 1. Protein expression pattern in BALF obtained from asthmatic patients with/without BALF fibroblasts, where identified proteins are marked with arrows (A). The BALF samples were precipitated in acetone and solubilized in buffer (7 M urea, 2 M thiourea, 2% CHAPS) and were separated by 2-DE in a nonlinear pH-range of 3-10. The zoom-boxes show the differential expression of haptoglobin β-chain. The BALF fibroblast proteome was studied in the pH-range of 4-7 (B). Peaks from the MALDI-TOF spectrum showed peptide fragments covering 33% of the haptoglobin β-domain molecule, including regions near both the N and C termini (C). The expression pattern of haptoglobin β-domain was quantified using image analysis software described in the Methods section and is presented as intensity of optical density (IOD) (D). Values are presented as means ( SEM for n ) 3/group.
Bronchoalveolar Lavage (BAL), Cell Cultures, and Sample Preparation. BAL was performed by instillation of buffered saline solution, divided into 3-4 aliquots with a recovery of more than 70%. Cells and cellular debris were separated by centrifugation. Fibrocytes were isolated from BALF by separating the lavage cells and culturing them until outgrowth of fibroblast-like cells reached confluence. To remove salts from the BALF, proteins were precipitated in acetone and freezedried. Protein quantity was determined using the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL), facilitating equal loading amounts of protein sample for each experiment. Confocal Microscopy. Fibroblast-like cells cultured from BALF were incubated with primary monoclonal mouse antihuman antibodies: CD34, CD45RO (BD Biosciences, Pharmingen, Leiden, The Netherlands) and R-SMA with Cy3 conjugates (Sigma Aldrich, Stockholm, Sweden). Secondary antibodies used for detection of CD34 and CD45R0 were Alexa Fluor 488 and 647 (Molecular Probes, Eugene, OR), respectively. Control experiments were performed with/without primary antibody or with/without secondary antibody to correct for background staining. The cells were analyzed with Leica confocal-scanning equipment TCS SP2 II (Leica, Wetzlar, Germany). Two-Dimensional Gel Electrophoresis (2-DE), Image Analysis, and Identification of Peptides Using MALDI-TOF. Twodimensional gel electrophoresis was performed according to a previously described protocol.17 Gels were stained either with Comassie G250 (Pierce, Rockford, IL) according to instructions 1480
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from the manufacturer, or silver nitrate according to a previously described protocol.18 Gels were scanned using a GS-710 gel scanner (Bio-Rad, Hercules, CA) and image analysis was performed using PDQuest v7.01 computer software (Bio-Rad, Hercules, CA). Excised spots were treated and applied onto a Bruker Anchor-chip target plate according to a previously described protocol15 and was subjected to a Bruker Reflex MALDI-TOF (Bruker-Daltonics, Bremen, Germany). Spectra were analyzed using Bruker Xtof 5.0.1 software. Gels were compared with an online BALF protein database hosted by expasy.org as well as with proteins identified in a previous study.19 Western Blot. Equal amounts of protein samples were loaded for all western blotting which was performed according to a previous described protocol.17 Primary polyclonal sheep anti-human haptoglobin antibody was used, diluted 1:1000 (Abcam Ltd, Cambridge, UK). The secondary antibody used was a polyclonal rabbit anti-sheep antibody, conjugated with horseradish peroxidase (DAKO, Glostrup, Denmark). The intensity of the optical density of the bands on the membrane was measured using Gel-Pro Analyzer 3.0 (Media Cybernetics, Silver Spring, MD). Statistical Analyses. Standard error of the mean (SEM) was calculated. The Mann-Whitney method was used where all values of p < 0.05 were considered to meet the standards for statistical significance.
technical notes
Larsen et al.
Results Patients with BALF Fibroblasts Display a Specific Pattern of Haptoglobin Isoforms. To study the airway lumen of potential key factors involved in the recruitment and differentiation of BALF fibroblasts, we analyzed the protein expression pattern in the BALF from asthmatic patients with/ without BALF fibroblasts and controls. We applied a gel-based proteomic approach of 2-DE in the global nonlinear pH range of 3-10. These experiments were repeated four times. 14 protein spots displayed a statistically significant change in expression pattern in the asthma patients examined where haptoglobin β-domain and tropomyosin displayed the largest regulation in expression (Figure 1A). Of all the gel spots, the four most highly up-regulated represented the haptoglobin β-domain, identification of which has been obtained by MALDI-TOF mass spectrometry. The other proteins identified were transferrin, transthyretin, myosin light chain, IgG heavyand light chain. To ensure that the observed differential haptoglobin expression did not originate from BALF fibroblasts, the proteome for these cells were compared (Figure 1B). The specific haptoglobin expression observed in the BALF could not be seen in these cells. Peaks from the haptoglobin spectrum showed peptide fragments covering 33% of the haptoglobin β-domain, including regions near both the N- and C-termini (Figure 1C). A 2.5-fold increase of haptoglobin β-domain was observed in BALF from asthmatic patients with BALF fibroblasts (p < 0.05) (Figure 1D). To further validate the differential expression pattern of haptoglobin, Western blots of BALF proteins separated by onedimensional gel electrophoresis with polyclonal antibodies for haptoglobin were performed (Figure 2A). The total levels of haptoglobin were elevated in asthmatic patients with BALF fibroblasts when compared to the two other groups (p < 0.05). Furthermore, a differential expression of the haptoglobin β-domain, and the R1, R2 domains in the group of asthmatic patients with BALF fibroblasts were also observed. Interestingly, these patients displayed a heterozygous expression of R1-, R2-, and β-domains of haptoglobin when compared to the other two groups of subjects (Figure 2B). Fibroblasts Derived from BALF Express Specific Fibrocyte Markers. BALF fibroblasts were isolated by separating the cells from the lavage fluid as previously described.15 After 20-30 days in culture, elongated BALF fibroblasts were seen to emerge (Figure 3A). The morphological phenotype of these cells was stable in up to seven passages (data not shown). To study if these cells expressed characteristics of tissue fibrocytes, the cultured BALF fibroblasts were triple stained for the specific fibrocyte markers CD34, CD45R0, and R-SMA and subjected to confocal microscopy. The positive triple stained pattern of CD34, CD45R0, and R-SMA for the BALF fibroblasts suggest that these cells may be derived from circulating fibrocytes (Figure 3B). In addition to the expression of these markers, the cells expressed collagen I and prolyl-4-hydroxylase indicating an active collagen synthesis (data not shown).
Discussion This is the first study that presents differentiated haptoglobin levels in the BALF proteome from asthmatic patients with differentiating fibrocytes derived from the airway lumen. A heterozygous expression of the haptoglobin R-chains was reported in patients with a differentiation of fibrocytes into
Figure 2. Protein expression after Western blotting with polyclonal antibodies for haptoglobin in BALF from controls (lane 1-3), asthmatic patients without BALF fibroblasts (lane 4-6) and asthmatic patients with BALF fibroblasts (lane 7-9) (A). The 39 kDa β-domain and the 17 kDA and 9 kDa R1, R2-domains are marked with arrows, respectively. The expression pattern of total haptoglobin (all domains) was quantified using image analysis software described in the Methods section and is presented as intensity of optical density (IOD) (B). Values are presented as means ( SEM for n ) 3/group. * Significant difference when comparing the haptoglobin expression between patients with/ without BALF fibroblasts (p < 0.05).
BALF fibroblasts. These observations support an inflammatory link to the presence of fibroblast progenitor cells in the airway lumen. Haptoglobin is an acute phase glycoprotein and its production increases greatly during tissue damage, inflammation and infection.20 It has been proposed that haptoglobin has an important biological function in modulating the immune response. Furthermore, haptoglobin is a tetramer with two R-domains and two β-domains. The β-domains are identical, but the R-domains come in allelic forms 1 and 2. It is possible to be homozygous for each of these R-domain alleles, in which case the haptoglobin molecule would have two R-1 domains or two R-2 domains, whereas the heterozygous individuals have one of each of the R-domains. These three forms of haptoglobin are denoted as Hp 1-1, Hp 2-2, or Hp 1-2, respectively. The heterozygous expression of the haptoglobin R-chains in patients with BALF fibroblasts observed in this study, allows speculation of this protein to be involved in the recruitment of these cells. In combination with recent findings where BALF fibroblasts were accompanied by elevated levels of eosinophils, the increased expression of haptoglobin may therefore further emphasize the possibility of inflammation as an important linkage for the presence of fibrocytes in the airway lumen in patients with mild asthma.15 The different isoforms of haptoglobin have distinct physiological properties, including different antioxidant capacities.21 Hp 2-2 is associated with heart disease and atherosclerosis, which interestingly involves fibrosis that is a similar feature in Journal of Proteome Research • Vol. 5, No. 6, 2006 1481
technical notes
Haptoglobin Expression in Mild Asthma
Figure 3. Differentiation of fibroblasts from fibrocytes in BALF. These cells were cultured as described in the method section and outgrowth of BALF fibroblasts was observed after 20-30 days in culture (A). The cultured BALF fibroblasts were stained with antibodies against the fibrocyte markers CD34 (green), CD45R0 (red), and R-SMA (blue) and subjected to confocal microscopy (B). The confocal picture was merged together with a transmission picture.
the airway remodeling process in asthma. Due to its increased expression during inflammation with antioxidant capacity, there remains the potential that haptoglobin could be involved in the pathology of asthma. Previous studies have been performed to characterize a linkage between haptoglobin expression and asthma.22 These studies suggest a correlation between elevated haptoglobin levels and both airway inflammation and hyperresponsiveness. Moreover, it has previously been reported that haptoglobin is increased in specific cells, such as alveolar macrophages and eosinophils in diseased or inflamed tissues, but not in the normal lung.23 However, it is unknown if a particular allelic phenotype of haptoglobin is a predominant feature in patients with asthma, and it would therefore be interesting to screen the genotype and phenotype of haptoglobin in a larger study. The BALF fibroblasts have been characterized as migratory cells with R-SMA expression and a relatively high ECM production.15,24 Absence of haptoglobin expression in haptoglobin knock-out embryonic fibroblasts have been shown to result in decreased cell migration, implicating a potential role for this protein in fibroblast recruitment during tissue repair.25 This may also explain why these fibroblasts may be derived from the airway lumen. The presence of fibroblasts expressing the fibrocyte characteristic markers CD34, CD45R0, and R-SMA in the airway lumen is interesting since it is unknown what consequences the production of ECM components would have for the sub-epithelial fibrosis in asthma. Possible factors involved in the recruitment of these cells to the airway lumen may include angiogenesis and chemotactic recruitment molecules such as chemokines from the epithelium and inflammatory cells in the airway mucosa.26,27 These are processes in where haptoglobin has been suggested to participate.25,28 The results of this pilot study suggest that the differentiation of fibrocytes into BALF fibroblasts in patients with asthma represents a more severe state of inflammation since the 1482
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expression of haptoglobin is increased most dramatically in these patients. This observation, accompanied by the previous findings including elevated levels of eosinophils further support the possibility of an inflammatory link to the finding of these cells. The discovery of BALF fibroblasts emergence raised the question of whether this is a qualitatively distinct manifestation of asthma or if it represents a more progressed condition of disease in terms of inflammation. Future studies including asthma exacerbations and/or allergen exposure may reveal more insights into this field. Abbreviations. (R-SMA) R-smooth muscle actin, 2-DE, Twodimensional gel electrophoresis, BALF Bronchoalveolar lavage fluid, ECM Extracellular matrix, MALDI-TOF Matrix assisted laser desorption/ionization time-of-flight.
Acknowledgment. The authors wish to acknowledge Erik Malmstro¨m for technical assistance and laboratory skills. This work was supported by grants from the Swedish Medical Research Council (11550), Heart-Lung Foundation, CFN Centrala Fo¨rso¨kdjursna¨mden (CFN), Greta and John Kock, Alfred O ¨ sterlund, Anna-Greta Crafoord Foundations, Riksfo¨reningen mot Reumatism, Gustaf V:s 80 Årsfond, and the Medical Faculty, Lund University. References (1) Davies, D. E.; Wicks, J.; Powell, R. M.; Puddicombe, S. M.; Holgate, S. T. J. Allergy Clin. Immunol. 2003, 111, 215-225. (2) Holgate, S. T.; Davies, D. E.; Lackie, P. M.; Wilson, S. J.; Puddicombe, S. M.; Lordan, J. L. J. Allergy Clin. Immunol. 2000, 105, 193-204. (3) Tomasek, J. J.; Gabbiani, G.; Hinz, B.; Chaponnier, C.; Brown, R. A. Nat. Rev. Mol. Cell Biol. 2002, 3, 349-363. (4) Hirsch, J.; Hansen, K. C.; Burlingame, A. L.; Matthay, M. A. Am. J. Physiol. Lung Cell Mol. Physiol. 2004, 287, L1-23.
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