BBA Clinical 1 (2014) 2–11
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BBA Clinical j o u r n a l h o m e p a g e : h t t p : / / w w w . j o u r n a l s . e l s e v i e r . c o m / b b a - c l i n i c a l /
Cells from the skin of patients with systemic sclerosis secrete chitinase 3-like protein 1☆ Yuen Yee Ho a, Murray Baron b, Anneliese D. Recklies a, Peter J. Roughley a, John S. Mort a,⁎ a b
Shriners Hospital for Children, Department of Surgery, McGill University, 1529 Cedar Avenue, Montréal, Quebec H3G 1A6, Canada Department of Rheumatology, Jewish General Hospital, 3755 Cote Ste Catherine Road, Montréal, Quebec H3T 1E2, Canada
a r t i c l e
i n f o
Article history: history: Received 4 November 2013 Received in revised form 18 Decembe Decemberr 2013 Accepted 19 December 2013 Available online 8 January 2014 Keywords: Scleroderma Systemic sclerosis Stem cell Chitinase Chitinas e 3-like protein 1 Cytokine Oncostatin M
a b s t r a c t
Background: The chit chitinas inase-li e-like ke prot protein, ein, Chi3 Chi3L1, L1, is asso associat ciated ed wit with h incr increas eased ed �bro broticacti ticactivit vity y as wel welll as in�ammatory processes. The capacity of skin cells from systemic sclerosis (SSc) patients to produce Chi3L1, and the stimulation stimula tion of its synthes synthesis is by cytokine cytokiness or growth factors known to be associat associated ed with SSc, was investig investigated. ated. Methods: Cell Cellss wer were e isol isolated ated fro from m for forearmand/or earmand/or abdo abdomen men skinbiopsie skinbiopsiess take taken n fro from m SSc pati patient entss and norm normal al individual divi dualss and sti stimula mulated ted wit with h cyto cytokin kines es and grow growth th fact factors ors to asse assess ss Chi Chi3L1 3L1 expr expressi ession. on. Chi3 Chi3L1L1-expr expressi essing ng cell cellss were characterized by immunohistochemical staining. Results: Chi Chi3L1was 3L1was no nott sec secret reted ed by sk skin in cel cellsfromnorm lsfromnormalindi alindivid vidual ualss no norr wa wass it itss syn synthe thesi siss in induc duced ed by anyof th the e cytokines cytokin es or growth factors investigated. investigated. In contrast, Chi3L1 secretion was induced by OSM or IL-1 in cells from allforearm allforear m bio biopsie psiess of SSc pati patient ents, s, andendogen andendogenoussecreti oussecretion on in theabsenceof cyto cytokin kines es wasdetecte wasdetected d in sever several al specimen spec imens. s. Pat Patien ients ts wit with h Chi3 Chi3L1-p L1-produ roducingcells cingcells at bot both h thearm andabdomenhad a dise diseaseduratio aseduration n of les lesss than 3 years. Endogenous Chi3L1 production was not a property of the major �broblast population nor of myo�broblasts, but rather was related to the presence of stem-like cells not present in normal skin. Other cells, however, contributed contributed to the upregulat upregulation ion of Chi3L1 by OSM. Conclusions: The Conclusions: The emergence of cells primed to respond to OSM with increased Chi3L1 production production appears to be associated associa ted with patholo pathological gical processes active in SSc. �cance: Th General signi � The e pre presen sence ce of pro progen genit itor or cel cellsexpre lsexpress ssin ing g th the e chi chilec lecti tin n Chi Chi3L 3L1 1 in SScskin app appea ears rs to pla play ya role in the initiat initiation ion of the disease process. © 2013 The Authors Authors.. Published by Elsevier B.V. All rights reserved.
1. Introduction
Systemic System ic scler sclerosis osis (scleroderma, (scleroderma, SSc) is a comple complex x autoimm autoimmune une disease with a highly highly variable variable array of clinical clinical features, the the most characcharacteristic being an overproduction and excessive deposition of collagen in the skin and intern internal al organs, with a progres progressive sive course and often fatal outcome. It is relatively rare, affecting between 50,000 and 100,000 North Nor th Am Ameri erica cans,and ns,and up to 250 250,00 ,000 0 Eur Europe opeans ans[1,2] [1,2].. Al Altho though ugh le less ss co commmon than other rheumatic diseases, it has one of the highest mortality rates [3] rates [3]..
Abbreviat Abbre viations ions:: Chi3L1, chitinase 3-like protein 1; DAPI, 4′,6-diamidino-2-phenylindole; ECM, extracellular matrix; IL, interleukin; mRSS, modi �ed Rodnan skin score; OSM, oncostatin M; PDGF, platelet-derived growth factor; αSMA, α-smooth muscle actin; SBTI, soybean trypsin inhibitor; SSc, systemic sclerosis (scleroderma); TIE2, tyrosine kinase with Ig and EGF homology domains-2; TGF β, transforming growth factor-β ☆ This is an o pen-acc pen-access ess article distributed distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ⁎ Corresponding author at: Genetics Unit, Shriners Hospital for Children, 1529 Cedar Avenue Ave nue,, Mon Montr tréal éal,, Que QuebecH3G becH3G 1A6 1A6,, Ca Canad nada. a. Tel Tel.: .: +1 51 514 4 28 282 2 716 7166; 6; fa fax: x: +1 51 514 4 84 842 2 55 5581 81.. E-mail address:
[email protected] address:
[email protected] (J.S. (J.S. Mort).
Disturbances of both the immune and vascular systems are thought to contribute to the development of SSc. Endothelial alterations often occur early in the diseas disease, e, followed by vascul vascular ar damage that leads to a cascade of stimulatory changes culminating in tissue � brosis brosis [4] [4].. This processs involv proces involves es T lymph lymphocytes ocytes [5] [5],, monocy monocytes, tes, macrop macrophages hages [6] and mast cells [7] cells [7] as as well as � broblasts broblasts [8] [8].. The activated cells secrete a variety of products, including growth factors, cytokines and their antagonists [9] antagonists [9].. These substances cause in �ammation and increased deposition deposi tion of extrac extracellul ellular ar matrix (ECM) components, components, leadin leading g to progressiv progr essive e and widesp widespread read tissue �brosi brosis. s. The hetero heterogenei geneity ty of vario various us forms of SSc and the dif �culty in discriminating between disease activity activit y (aspects of the disease that vary over time and are potential potentially ly reversible spontaneously or with drug treatment) and disease damage (irrev (ir revers ersibl ible e tis tissue sue inj injury ury tha thatt res result ultss fro from m the dis diseas ease) e) [10] complicate studies of SSc. At present there are no validated biomarkers which can be use used d to mon monito itorr dis diseas ease e pro progre gressi ssion. on. The There re is an ext extens ensive ive lit litera eratur ture, e, however, howev er, investigatin investigating g the relationshi relationship p between many of the cytok cytokines ines and effector molecules implicated implicated in the various pathological processes associated with the development and progression of SSc [11 SSc [11–15] 15].. The chitinase-like protein, Chi3L1 (YKL40, HCgp39), has been shown to be associated with increased �brotic activity as well as in �ammatory process proc esses. es.Chi Chi3L1is 3L1is upr upregu egulate lated d in man many y path patholog ologica icall con conditi ditions ons[16 [16–23] 23]..
2214-6474/$ – see front matter © 2013 The Authors Authors.. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbacli.2013.12.001
Y.Y. Ho et al. / BBA Clinical 1 (2014) 2 –11
Elevated serum levels of Chi3L1 are associated with poor prognosis, shorter recurrence-free interval and low overall survival [16,17] survival [16,17] in in patients with a broad range of cancers cancers,, including breast [16] breast [16] and and colorectal cancers [17] cancers [17].. Patients with diseases characterized by in �ammation and tissue � brosis, including rheumatoid arthritis [18] arthritis [18],, osteoarth osteoarthritis ritis [19] [19],, pneumonia [20] pneumonia [20],, liver cirrhosis [21] cirrhosis [21] and and systemic sclerosis (SSc) [22 (SSc) [22–24] are also reported to have elevated serum Chi3L1 levels. Chi3L1 belongs to the family of mammalian mammalian chitin chitinase-li ase-like ke proteins, proteins, which share share primary sequence homology and three-dimensional structure with the family 18 glycohydrolases [25] glycohydrolases [25] but but lack chitinolytic chitinolytic activity. The lack of catalyti catalyticc activity activi ty is due to two amino acid substi substitutions tutions in the activ active e site region of the protein, the most critical one being substitution of the catalytic glutamate residue with leucine [26] leucine [26].. Chi3L1 Chi3L 1 is produ produced ced by macro macrophage phages, s, synov synovial ial cellsand chond chondrocyte rocytess from arthritic joints [27] joints [27],, and neutro neutrophils phils [28] [28].. It ha hass been been sh show own n to ac actt � synergistically with insulin-like growth factor (IGF-1) in broblasts to stimulate cell growth [29] growth [29].. The protein also has mitogenic effects on chondrocytes chondroc ytes and synovial cells and promotes proteoglycan proteoglycan synthesis in these cells [30]. [30]. Furthermore, Chi3L1 has been shown to be a migration and adhesion factor for vascular smooth muscle cells, which suggests a role in angiogenesis [31] angiogenesis [31].. Previous work has demonstrated that Chi3L1 can modulate the response of connective tissue cells to in�amm ammato atory ry cyt cytoki okines nes,, suc such h as ILIL-1 1 or TNF TNF--α [32] [32].. Chi Chi3L1 3L1−/ − mice micedisdisplay pla y an exa exagge ggerat rated ed in�amm ammator atory y res respon ponse se in a lun lung g inj injury ury mod model el [33] [33],, supporting this observation. The detailed molecular mechanisms by which Chi3L1 exerts its biological effects are not known. In SSc, dermal �broblasts are one of the main effector cells involved in the development of � brotic lesions, and their biological activity is regulated by a variety of in �ammatory cytokines and growth factors. Chi3L1 had been implicated in other pathologies leading to excessive �brosis brosis [34] [34],, and thus its production might be upregulate upregulated d in affected tissue tis suess in pat patien ients ts wit with h SSc SSc.. Thegoals of the pre presen sentt wor work k wer were e to inv invesestigatethe tig atethe cap capaci acity ty of ski skin n cel cells ls fro from m SScpatie SScpatientsand ntsand hea health lthy y ind indivi ividua duals ls to synthesize Chi3L1 and to assess the regulation of this process by growth factors and cytokines shown to be associated with this disease. 2. Materials and methods
2.1. Patients, Patients, control controls, s, and skin biopsies biopsies Full-thick Fullthickness ness biopsi biopsies es were obtain obtained ed by an exper experience ienced d rheuma rheumatoltologist (MB) from the skin of the distal forearm and abdomen of 41 SSc patients recruited from the Canadian Scleroderma Research Group (CSRG) Registry. Similar skin biopsies were also obtained from 10 healthy control individuals. To be eligible for the Registry, patients must have a diagnosis of SSc made by the referring rheumatologist, be age ≥ 18 years years,, an and d be �uen uentt in Eng Englis lish h or Fre French nch.. Reg Regist istry ry patients patients undergo an extensive clinical history, physical evaluation, and laboratory investigations, and complete a series of self-report questionnaires. Clinical sclerodermatous involvement of skin from biopsy sites was determine deter mined d by an expe experience rienced d rheumatologist rheumatologist (MB). Patients Patients and individual vid ualss for con contro troll bio biopsi psies es pro provid vided ed wri writte tten n inf inform ormed ed con conse sent, nt, and the sample collection collection and analysis protocols protocols were approved by the McGil McGilll University Unive rsity Insti Institutiona tutionall Revi Review ew Board. Board.Patien Patients ts were class classii�ed as ha havi ving ng limited cutaneous SSc (lcSSc) or diffuse cutaneous SSc (dcSSc) according in g to th the e cl clas assi si�cat cation ion by LeR LeRoy oy et al. [35] [35].. Dis Diseas ease e dur duratio ation n was calcucalculated from the date of appear appearance ance of the � rst non-Raynaud's symptom of SSc. This is based on both cutaneous and systemic symptoms, including includ ing respir respiratory atory symptom symptoms, s, �nge ngerr ulc ulcers ers,, in�ammator ammatory y arthri arthritis, tis, telangiectasia, angiectasi a, skin tightening anywhere, fatigue, puffy extremities (hands or feet), weight loss, heartburn or dysphagia and erectile dysfunction. 2.2. SSc clinical clinical outcomes outcomes Usingstandar Using standardized dizedde de�nitions nitions,, the recrui recruiting tingrheuma rheumatologis tologistt (MB) (MB)rereported whether or not the patients had active or healed digital ulcers,
3
interstitial lung disease and pulmonar interstitial pulmonary y hyperten hypertension. sion. Disease activity was measured using the Valentini Scleroderma Scleroderma Disease Activity Index (SDAI) [36,37] (SDAI) [36,37],, consisting of 10 variables with weights ranging from 0.5 to 2.0 and resulting in a total score ranging from 0 to 10. Variables being measured with the SDAI include modi�ed Rodnan skin score ( N 14), scleredema, sclere dema, change in skin symptoms in the last month, digital necrosis, change in vascular symptoms in the last month, arthritis, lung diffusion capacity b 80% pre predic dicted ted,, cha change nge in car cardiop diopulm ulmona onary ry sym symptom ptoms, s, ery erythr throocyte sedimentation rate N 30 mm/h and hypocomplementemia [36,37] hypocomplementemia [36,37].. Disease severity was measured using physicia physician n global assessm assessments ents of disease severity (scales ranging from 0 to 10) [38] [38].. Predictors of disease severity have been shown to include skin involvement, severity of Raynaud's phenomenon, shortness of breath, gastrointestinal symptoms and pai pain, n, num numberof berof �nge ngertipulcers rtipulcers,, ten tender derand andswo swolle llen n joi joints nts,, cre creati atinin nine, e, and fatigue [38] fatigue [38].. 2.3. Cell isolatio isolation n and culture culture Full thi Full thickn cknessskin essskin bio biopsi psies es wer were e col collec lected ted at bed bedsid side e and pla placed ced int into o Falcon tubes containing 10 ml Dulbecco's modi �ed Eagle medium (DMEM) (Gibco BRL, Grand Island, NY, USA). To separate epidermis from dermis 0.5% dispase (Invitrogen, (Invitrogen, Burlington, Canada) was added and the skin biopsies were incubated at 37 °C, in 5% CO 2 for 2 h. Cells were isolated by incubation of dermal samples overnight at 37 °C under gentle mixing in a solution of 0.2% collagenase H (Sigma, St Louis, MO, USA) in DMEM, and 100 U/ml penicillin, and 100 μ g/ml g/ml streptomycin strept omycin (Schering (Schering Inc., Pointe Claire, Canada). DMEM with 5% fetal calf serum (FCS) was added to neutralize the activity of collagenase. The mixture was centrifuged for 5 min at 500 g . The cell pellet was resuspended with 1 ml DMEM, and cell viability was assessed by Trypan Blue exclusion. exclusion. Cells were then plated in T-75 culture � asks (Becton Dickinson, Franklin Lakes, NJ, USA) and maintained in 12 ml DMEM supplemented with 5% FCS and antibiotics at 37 °C in a 5% CO2/95% air atmosphere. Culture media were changed every 3 days until unt il cel cells ls rea reache ched d 90% con �uen uency. cy. At thi thiss poi point nt the cells were sub-cultured at a ratio of 1:3 by trypsinization. It is expected that these cell preparations would be enriched in �broblasts, but they could also contain any other cell types capable of adherence to tissue culture plastic. 2.4. Stimulatio Stimulation n of skin cells by cytokines cytokines and growth growth factors factors Skin cel Skin cells ls wer were e plat plated ed in T-2 T-25 5 cul cultur ture e �as asksat ksat 4 × 105 cells/�askand were cultured to near con�uence in DMEM supplemented with 5% FCS and ant antibi ibioti otics, cs, fol follow lowed ed by 24 h of inc incubat ubation ion in ser serumum-fre free e con condi dition tionss before stimulation stimulation with growth factors or cytokines. Cells were either left untreated or treated with 10 ng/ml recombinant interleukin-1 β (IL-1β), interl interleukin eukin-6 -6 (IL-6) (IL-6),, inter interleukin leukin-17 -17 (IL-1 (IL-17), 7), oncost oncostatin atin M (OSM),, transf (OSM) transforming orming growth factor factor--β (TGFβ) or platel platelet-deri et-derived ved growth factor (PDGF) (R&D Systems, Minneapolis, MN, USA) in 3.5 ml DMEM for 48 h. Conditioned media were collec collected ted and secr secreted eted proteins were analyzed by SDS-PAGE followed by western blotting. 2.5. Soybean Soybean trypsin trypsin inhibitor inhibitor (SBTI) biotinyla biotinylation tion Soybean Soyb eantry trypsi psin n inh inhibi ibitor tor(SBT (SBTI, I, MW 24 kDa kDa)) was wasuse used d as a con controlfor trolfor the ef �ciency of acetone precipitation of cell culture media prior to SDS-PAGE. SDS-PAG E. The protein was biotinylated with sulfosucc sulfosuccinimidyl inimidyl-6-6(biotin-amido) (biotin-a mido) hexanoat hexanoate e (Sulfo-N (Sulfo-NHS-LC-Bi HS-LC-Biotin; otin; Pierce, Rockford, IL, USA), following the manufacturer's instructions, instructions, to allow detection in western blots with the streptavidin–biotin system used for detection of Chi3L1. Excess Sulfo-NHS-LC-Biotin was removed by overnight dialysis (Nominal MWCO: 12,000–14,000; Fisher Scienti�c, Ottawa, Canada) into 10 l of 100 mM Tris, pH 7.6 at 4 °C. Ten ng biotinylated SBTI was suf �cient for use as a control for detecti detection on by wester western n blotting.
4
Y.Y. Ho et al. / BBA Clinic Clinical al 1 (2014 (2014)) 2 –11
2.6. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting
3. Results
3.1. Patient Patient characterist characteristics ics Cell conditioned media (300 μ l) l) supplemented with 10 ng biotinylated SBTI were precipitated with 600 μ l cold acetone overnight at − 20 °C. The pellet was washed twice with 1 ml cold acetone, dried and resuspended in 24 μ l H2O. Sample buffer containing reducing agent was added to the reconstituted pellet sample, and incubated at 70 °C for 10 min. Proteins were then separated on NuPAGE Bis –Tris 4–12%, 1 mm gels, at 180 V for 1 h. The proteins proteins were were then transferr transferred ed to nitrocellulose membrane (0.45 μ m) m) (Bio-Rad, Hercules, Hercules, CA, USA) at 30 V for 90 min. After transfer, membranes were blocked for 1 h with 5% dried skimmed milk in Tris-buffered saline –Tween 20 (TBS-T). The membranes were probed overnight at 4 °C with primary antibody: rabbit anti-human anti-human Chi3L1 polyclonal polyclonal antibo antibody dy raise raised d again against st the C-terminal C-term inal peptid peptide e CGTNA CGTNAIFDAL IFDALAAT AAT conjug conjugated ated to ovalbu ovalbumin. min. The membra mem brane ness wer were e was washedfour hedfour tim times,with es,with TBS TBS-T -T fol follow lowed ed by inc incubat ubation ion with donkey anti-rabbit, biotinylated species-speci �c antibody (1:5000 dilution dilu tion;; GE Heal Health th Care Care,, Litt Little le Chal Chalfont font,, UK) for 60 min. The membranes were again washed with TBS-T, then incubated with a streptavidin –biotinylated horseradish peroxidase complex (1:750 dilution; GE Health Care, Little Chalfont, UK) for 60 min. After washing the membranes with TBS-T, the protein bands were visualized using ECL reagents (GE Health Care, Pittsburgh, PA, USA). Puri �ed Chi3L1, used as a standard, was isolated from human chondrocyte cultures [39] cultures [39].. 2.7. Immuno Immuno �uorescence analysis of cell phenotype Fibroblasts were subcultured in 6-well plates on microscope coverslips at a density of 2 × 10 5 cells per well. Cells were cultured to near con�uen uence ce in DME DMEM M sup supple plemen mented ted wit with h 5% FCS and ant antibi ibioti otics, cs, fol follow low-ed by 24 h in serum-free conditions before stimulation with growth factors or cytokines. Cells were either left untreated or treated with 10 ng/ml OSM or TGF β in 3.5 ml DMEM for 48 h, then rinsed with phosphate-buffe phosphat e-buffered red saline (PBS) and � xed with 4% paraformaldehyde paraformaldehyde at room temperature for 15 min. After three PBS washes, the cells were permeabilized with PBT (PBS plus 1% Triton X-100) for 30 min prior to blocking with 0.1% bovine serum albumin (BSA), 5% goat serum in PBS for 1 h at room temperature. Primary antibodies diluted in blocking solution solution were then added and incubated overnight overnight at 4 °C. Primary antibodies: rabbit anti-human Chi3L1 polyclonal antibody at 1:200 1:20 0 dilu dilution tion,, mous mouse e anti anti-hum -human an smoo smooth th mus muscle cle acti actin n (αSMA) monoclonal antibody at 1:1000 dilution (Invitrogen, Camarillo, CA, USA), mouse anti-human nestin monoclonal antibody at 1:200 dilution, mouse anti-human CD73 monoclonal antibody at 1:200 dilution, mouse anti-human anti-hum an STRO-1 monoclo monoclonal nal antibody at 1:200 dilution, mouse antihuman TIE-2 monoclonal antibody at 1:200 dilution, mouse anti-human anti-human CD45 monoclonal antibody at 1:200 dilution, mouse anti-human CD34 monoclonal antibody at 1:200 dilution (Abcam, Cambridge, MA, USA), mouse anti-human LSP-1 antibody at 1:200 dilution (Abnova Cooperation, Taipei, Taiwan), and rabbit NG2 chondroitin sulfate proteoglycan polyclonal antibody at 1:200 dilution (Millipore, Temecula, CA, USA). Mouse IgG1 at 1:200 dilution (Abcam, Cambridge, MA, USA) or normal rabbit IgG at 10 μ g/ml g/ml were used as negative controls. The cells were then the n was washed hedthr three ee tim times es in PBS PBSbef before oreinc incubat ubation ionfor for1 1 h at roo room m tem temper per-ature atur e withsecon withsecondaryantibo daryantibodie dies: s: goatanti-r goatanti-rabb abbit it Ale Alexa xa 488and goa goatt anti anti-mouse Alexa 594. Following three PBS washes, the coverslips were mounted inverted on glass slides with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA) and sealed with nail polish. The labeled cells were evaluated by �uorescence microscopy, and quanti�ed by cell counting using 20 repres representativ entative e �elds. 2.8. Statistica Statisticall analysis analysis Data were analyzed using the Mann–Whitney U test.
Sixty-one Sixtyone ski skin n cel celll cul cultur tures es fro from m 41 SSc pat patien ients ts (35 wom women, en, 6 men men;; mean age 57 years, range 39 –77 years) were used in this work. The mean disease duration was 10.2 years, ranging from 1.3 to 48.9 years. The features of the patients studied are summarized summarized in Table 1. 1. For 20 patients, skin cells derived from biopsies taken from both the arm and abdomen were available and analyzed with respect to Chi3L1 production, allowing comparison of the behavior of cells from the same patient, patien t, isolated from these two different different sites (Table (Table 2); 2); for 9 patients, patients, only cells derived from the arm were available ( Table 3) 3) and for for 12 patients, only cells derived from the abdomen were available ( Table 3). 3). Modi�ed Rodnan scores and skin scores from the sites where the biopsies were taken are indicated in Table in Table 4. 4. The control group (Table ( Table 5) 5) consisted of 9 females and 1 male, with a mean age of 53 years, range 44–61 years. Biopsies from both the arm and abdomen were taken from 5 members of this group, the remainder provided arm biopsies only. 3.2. Chi3L1 Chi3L1 secretion secretion by SSc cells Chi3L1 production and its regulation by cytokines or growth factors thought to be associat associated ed with SSc disease pathology pathology were analyzed in 15 cell lines from the skin of 10 control individuals and 61 cell lines from skin biopsies of 41 SSc patients. Cells were stimulated with IL-1 β, IL-6, IL-17, OSM, TGF β or PDGF and Chi3L1 levels in the culture media were analyzed by SDS-PAGE and western blotting. Chi3L1 secretion was not detectable in any of the 15 unstimulated healthy control skin cell ce ll li line ness an anal alyz yzed ed,, no norr wasit in indu duce ced d by an any y of th the e cy cyto toki kine ness or gr grow owth th ors tested in this study (Fig. ( Fig. 1A). 1A). In contrast, endogenous Chi3L1 factors fact secretion was observed in 22 of the 61 SSc skin cell lines analyzed. The level of secreted Chi3L1 was increased when these 22 SSc skin cell lines were stimulated with IL-1 β or OSM, with OSM having the more prominent effect (Fig. (Fig. 1B), 1B), while IL-6, IL-17, TGFβ and PDGF had no effect. All cell preparations from SSc patients which produced Chi3L1 always responded to both IL-1β and OSM, and there was no synergism betw be twee een n the these se cy cyto toki kine nes. s. Fo Forr th thos ose e 39 SS SScc ce cell ll li line ness wh whic ich h di did d no nott sh show ow endoge end ogenous nous Chi Chi3L1 3L1 sec secre retion tion,, the pro protei tein n was detec detected ted in 19 of the cel celll lines lin es aft after er sti stimul mulati ation on wit with h ILIL-1 1 or OSM OSM.. Non None e of theothercytok theothercytokine iness and growth factors tested affected Chi3L1 secretion. Since the greatest stimulation of Chi3L1 production was induced by OSM, this cytokine was used in further studies. SSc skin � broblasts have been reported to possess unstable phenotypes with respect to ECM production [40] production [40].. This could be due to the sampling sampli ng site of the biopsy or to variati variations ons in the stability of the � broblast pheno phenotype type from diffe different rent indiv individuals iduals.. To ascer ascertain tain that diffe difference rencess in the secretion of Chi3L1 between the different SSc cell lines were not due to instability of the � broblas broblastt phenotype, 3 cell lines that endogenouslysecre nou slysecretedChi3L tedChi3L1 1 wer were e con contin tinue ued d in cul cultur ture e fro from m pas passag sage e 7 to pas pas-sage 10. Endogenous Chi3L1 secretion was maintained and remained inducible by OSM at all passages though absolute levels did vary (Fig. 2). 2). Moreover, cell lines that did not express Chi3L1 never attained Table 1 Summary Summar y features of patients studied studied (n = 41).
Variables
Value
% or range
Age (years) [mean] Female (n) Disease duration (years) [mean] Type of systemic sclerosis sclerosis Limited (n) Early limited (n) Diffuse (n) Early diffuse (n)
57 35 10.2
39–77 85% 1.3–48.9
23 9 18 2
56% 22% 44% 5%
Y.Y. Ho et al. / BBA Clinical 1 (2014) 2 –11
5
Table 2 Chi3L1 secretion secretion pattern of skin cells isolated from paired biopsy sites of SSc patients.
Patient characteristics Pati Pa tien entt ID
1 2 3 4 S 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Gend Ge nder er
Female Male Female Female Female Female Female Female Female Male Female Female Female Female Female Female Female Male Female Female
Biopsy site Age Ag e (y (yea ears rs))
53 69 58 52 55 63 60 46 78 63 49 64 66 51 70 61 69 77 77 46 55
Dise Di seas ase e du dura rati tion on (years)
SSc subtype
2.5 1.7 1.5 1.3 2.0 1.3 23.5 15.6 11.0 8.8 2.9 14.8 5.6 13.6 28.5 10.3 48.9 14.5 6.9 12.6
Limited Limited Diffuse Limited Limited Diffuse Limited Diffuse Diffuse Diffuse Limited Limited Limited Diffuse Limited Limited Limited Limited Diffuse Diffuse
Forearm
Abdomen
Clinically involved
Endogenous Chi3L1 secretion
Induction by OSM
Clinically involved
Endogenous Chi3L1 secretion
Induction by OSM
No No No Yes No No No No No No Yes No No No No No Yes Yes No No No Yes Yes
+ + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + +
No No Yes No No No No No No No No No No Yes Yes No No No No Yes
+ + + + + +
+ + + + + +
−
−
−
−
−
−
−
−
− − − − − − − −
−
−
−
−
+ + + +
−
−
−
−
−
−
−
−
−
−
− −
Cells wer Cells were e isol isolate ated d fromskin biop biopsie siess as indi indicat cated ed andcultur andcultured ed in the abs absenceor enceor pres presenceof enceof OSM.Chi3L1secre OSM.Chi3L1secretionwas tionwas dete determi rminedby nedby wes westernblott ternblotting.Chi3L1secre ing.Chi3L1secretionwas tionwas indu inducedby cedby OSM in cell cellss isol isolatedfrom atedfrom forearm forearm biopsies biopsies in all 20 pat patient ients. s. + indi indicate catess detection detection of endo endogeno genous us Chi3 Chi3L1 L1 or an incr increaseover easeover the endo endogeno genous us leve levell induced induced by OSM, but does not re�ect absolute levels.
this property in culture (data not shown). These observations indicate that Chi3L1 is not secreted by normal skin cells nor is its production induced by in �ammatory or �brogenic cytokines in these cells. In contrast it is produced in an inducible and stable fashion by a majority of SSc skin cell preparations and is likely to re �ect an in vivo situation. Three Thr ee dif differ ferent ent patt pattern ernss of Chi Chi3L1 3L1 sec secret retion ion and reg regula ulatio tion n wer were e observed in the SSc skin cell preparations used for this study ( Fig. 3). 3). Group 1 represents the skin cells that constitutively secreted Chi3L1 and this process was stimulated by OSM. Group 2 represents the skin cells that did not endogenously secrete Chi3L1, but for which Chi3L1 secretion could be induced to various levels by OSM. Group 3 skin cells cel ls beh behave aved d sim simila ilarlyto rlyto nor normalcontr malcontrols ols,, as the they y did not end endoge ogenous nously ly
secrete secret e Chi Chi3L1 3L1 and Chi Chi3L1 3L1 sec secre retio tion n was not ind induci ucible ble wit with h OSM OSM.. Of the SSc skin cell lines studied, 22 (36%) belonged to Group 1, 19 (31%) to Group 2, and 20 (33%) to Group 3. Absolute levels of endogenous and/or inducible Chi3L1 varied considerably within each group (Fig. (Fig. 3) 3) ranging from barely detectable to high levels. 3.3. Compa Comparison rison of respo response nse patte patterns rns of perip peripheral heral and abdom abdominal inal skin cells From the data summarized in Tables in Tables 2 and 3 it can be seen that Chi3L1 secretion was inducible by OSM in all 29 cell cultures prepared from the peripheral skin (arm biopsies) of SSc patients. Endogenous secretion secretio n was observed in 16 (55%) of these preparati preparations. ons. In contrast,
Table 3 Clinical parameters and Chi3L1 secretion from cells of SSc patients obtained from either the forearm or the abdomen.
Patient characteristics
Chi3L1 secretion
Pattie Pa ient nt ID
Gend nder er
Age Ag e
Dise Di seaase duratio ion n (y (yea ears rs))
SScc su SS subb-ttyp ype e
Biop opssy si sitte
Biops psy y cl clin inic icaall lly y in invo volv lved ed
Endo En doge geno nou us
Indu duce ced d by OS OSM M
25 27 28 30 31 33 36 37 38 21 22 23 24 26 29 32 34 35 39 40 41
Female Female Female Female Male Female Female Female Male Female Female Female Male Female Female Female Female Female Female Female Female
70 62 46 63 53 63 45 57 43 49 59 55 70 43 56 39 56 59 62 67 63
17.5 4.7 7.1 28.9 6.3 5.8 5.6 5.5 4.9 10.1 1 2.3 6.9 10.8 10 18.7 5.5 8.8 9.7 7.9 1.3 12.1 2.6
Diffuse Limited Diffuse Limited Limited Limited Diffuse Diffuse Limited Diffuse Diffuse Limited Diffuse Limited Diffuse Diffuse Limited Diffuse Limited Limited Limited
Arm Arm Arm Arm Arm Arm Arm Arm Arm Abdomen Abdomen Abdomen Abdomen Abdomen Abdomen Abdomen Abdomen Abdomen Abdomen Abdomen Abdomen
No Yes No No Yes No Yes No No Y es Ye No No No No No No No No No No No No No No No No No No No No
+ +
+ +
+ + + + + + + + +
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
− −
+ − −
−
−
+
+
−
−
+
+
Cells were were isolated isolated from from skin biopsies obtainedfrom the indicat indicated ed sites and cultured cultured in the absence or presence of OSM. Chi3L1 secretio secretion n was determ determined ined by western blottin blotting g of cultur culture e media. med ia. Chi3 Chi3L1 L1 sec secret retion ion wasinduce wasinduced d by OSM in cell cellss isol isolatedfrom atedfrom all for forear earm m bio biopsie psies. s. + indi indicat cates es det detecti ection on of endo endogeno genous us Chi3 Chi3L1 L1 or an incr increaseover easeover the endo endogeno genous us leve levell indu induced ced by OSM, but does not re�ect absolute levels.
6
Y.Y. Ho et al. / BBA Clinic Clinical al 1 (2014 (2014)) 2 –11
Table 4 Patient skin scores.
Patient Pati ent ID Gend Gender er Age (ye (years) ars) mRS mRSS S Skin sco score re (ar (arm) m) Skin scor score e (ab (abdom domen) en) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 25 27 28 30 31 33 36 37 38 21 22 23 24 26 29 32 34 35 39 40 41
Female Male Female Female Female Female Female Female Female Male Female Female Female Female Female Female Female Male Female Female Female Female Female Female Male Female Female Female Male Female Female Female M ale Ma Female Female Female Female Female Female Female Female
53 69 58 52 55 63 60 46 78 63 49 64 66 51 70 61 69 77 46 55 70 62 46 63 53 63 45 57 43 49 59 55 70 43 56 39 56 59 62 67 63
2 4 33 2 9 13 1 17 10 10 1 2 4 31 3 2 2 2 0 29 10 10 10 36 7 7 9 39 6 2 2 29 2 2 30 7 29 12 4 12 11
0 0 4 0 0 0 0 4 0 0 0 0 0 4 0 0 0 0 0 2 2 1 0 5 0 1 0 4 0
0 0 2 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 0 2
Fig. 1. Comparison of Chi3L1 secretion by normal (A) and SSc (B) skin cells. Results are shown for representative samples from 15 normal skin cells and 22 SSc skin cell lines. Skin cells were grown in monolayer culture and stimulated for 48 h with cytokines or growth factors (IL-1 β, IL-6, IL-17, OSM, TGF- β and PDGF). Chi3L1 levels in cultur culture e media were wer e mea measur sured ed by SDSSDS-PAG PAGE E and imm immunob unoblot lottin ting. g. Skincell line liness usedin thisexperi thisexperiment ment were in passage 5. The left-ha left-hand nd lane of the gel shows shows the migrat migration ion of molecula molecularr weight weight markers, and the right-hand lane shows the migration of puri�ed Chi3L1. Each medium sample sam ple was spik spiked ed wit with h equa equall amou amounts nts of biot biotinyl inylate ated d SBTIto mon monitoraceton itoracetone e prec precipit ipitaation ef �ciency.
0 0 1 0 0 0 0 1 0 0 0 0
abdominal biopsi abdominal biopsies es from from 32 patie patients nts yield yielded ed less respons responsive ive cell cell preparations; induction of Chi3L1 secretion by OSM was observed in 12 samples (38%), and 8 of these produced the protein endogenously, while the remaining 20 samples behaved like cells from healthy
Table 5 Chi3L1 secretion from skin cells obtained from healthy control individuals.
Cont Co ntro roll ID
1 2 3 4 5 6 7 8 9 10
Gend nder er
Male Female Female Female Female Female Female Female Female Female
Age Ag e
60 58 5 8 50 50 47 4 7 51 51 57 5 7 44 44 50 50 49 4 9 61
Bio Bi ops psy y si sitte
Arm Arm Abdomen Arm Abdomen Arm Arm Abdomen Arm Arm Arm Abdomen Arm Arm Abdomen
individuals, i.e. no Chi3L1 production individuals, production was detectab detectable le under any of the experi exp erimen mental tal con condit dition ions. s. It sho should uld be note noted d tha thatt all ce cell ll lin lines es whe where re endogenou doge nouss sec secret retion ion of Chi Chi3L1 3L1 was obs observ erved ed res respond ponded ed to OSM OSM,, independent of the biopsy site (arm or abdomen). Matching biopsies taken from both the arm and abdomen were available availabl e from 20 of the SSc patients used in this study, allowing comparison of the capacity to secrete Chi3L1 relative to the biopsy site in individual individ ual patient patientss (Table 2). 2). In 6 patients, unstimulated cells from both sites endogenously produced Chi3L1 and its secretion was increased by OSM, and in a further 4 patients Chi3L1 production was not detectable in unstimulated cells from both sites, but was induced following addition of OSM. No Chi3L1 production either in the absence
Chi3 Ch i3LL1 sec ecre rettio ion n Basa Ba sall
Indu In duce ced d by OS OSM M
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
Cells were isolated from skin biopsies obtained from healthy individuals and cultured in the absence or presence of OSM. Culture media were analyzed for the presence of Chi3L1 by western blotting. Paired biopsies were obtained from 5 individu individuals. als. No Chi3L1 was detectable detectable in any of the 15 samples either in unstimulated cultures cultures or following exposure to OSM.
Fig. 2. Comparison of Chi3L1 secretion in response to OSM at differ different ent cell passage passages. s. One representative represent ative sample of 3 SSc skin cell lines that endogenously secreted Chi3L1 and were wer e upup-reg regulat ulated ed upo upon n stim stimulat ulationby ionby OSM is show shown. n. Skincellswere gro grown wn in mono monolaylayer culture with or without OSM stimulation for 48 h. Chi3L1 secretion into the culture mediumwas med iumwas mea measur sured ed by SDSSDS-PAG PAGE E andimmuno andimmunoblo blottin tting. g. P7–P10 P10:: cellpassa cellpassage ge numb number; er; C: unstimulated cells; O: OSM-induced cells. The left-hand lane of the gel shows the migration of molecular weight markers, markers, and the right-hand lane shows the migrati migration on of puri�ed Chi3L1. Biotinylated Biotinylated SBTI SBTI was added to each medium sample to monitor monitor acetone precipitation ef �ciency.
Y.Y. Ho et al. / BBA Clinical 1 (2014) 2 –11
7
of sti stimul mulati ation on or fol follow lowing ing add additi ition on of OSM was obs observ erved ed in abd abdomi ominal nal cell preparat preparations ions from 10 patien patients, ts, while while peripheral peripheral cells from these these individuals always produced Chi3L1. This is illustrated in Fig. 4, 4, which shows the response of cells from arm and abdominal skin biopsies taken from two representative SSc patients at the same time and analyzed at the same cell passage level. Skin cells from both the arm and abdomen of patient 14 (Fig. ( Fig. 4A) 4A) did not endogenously secrete Chi3L1, but Chi3L1 secretion was inducible by IL-1 and OSM in the cell ce llss fr frombothsit ombothsites es.. Sk Skincel incells ls fr fromthe omthe ar arm m ofpati ofpatien entt 8 (Fi Fig. g. 4B) endog endog-enously secreted Chi3L1 and Chi3L1 secretion was upregulated by IL-1 andOSM, butabdomi butabdominalskin nalskin cel cells ls fro from m thesame pat patien ientt didnot end endogogenously secrete Chi3L1 nor was the protein inducible by IL-1 or OSM. Thus while peripheral skin cells from SSc patients always contain a cell population which is primed to allow upregulation of Chi3L1 by in�ammatory cytokines, this is not always the case for abdominal skin cells. 3.4. Variation Variation in Chi3L1 Chi3L1 secretion secretion pattern pattern with disease disease duration duration
OSM-inducible Fig. 3. Classi�cation of SSc skin cell phenotype based on endogenous and OSM-inducible Chi3L1 secretion. secretion. Skin cells were grown in monolaye monolayerr culture culture with or without without OSM stimulation for 48 h. Chi3L1 secretion into the culture medium was then measured by SDSPAGE and immunoblotting. immunoblotting. Skin cell lines used in this experiment were in passage 5. Eight representative representative samples of 61 cell preparations from 41 SSc patients are shown. Group 1: SSc cells that endogenously secrete Chi3L1 and for which OSM stimulation upregulates Chi3L1 secretion. Group 2: SSc cells that do not endogenously secrete Chi3L1, but the protein is inducible by OSM. Group 3: SSc cells that do not endogenously secrete Chi3L1 and its secretion is not inducible with OSM. The left-hand lane of each gel shows the migration of molecular weight markers, and the right-hand lane shows the migration of puri�ed Chi3L1. Biotinylated SBTI was added to each medium sample to monitor acetone precipitation ef �ciency.
Chi3L1 Chi 3L1 sec secre retio tion n pat patter terns ns did not app appear ear to cor correl relate ate withany of the typical features associated with SSc. Endogenous secretion and/or inducibi duc ibilit lity y by OSM was obs observ erved ed in pat patien ients ts diag diagnos nosed ed wit with h eit eithe herr lim limitited or diffuse SSc, and in biopsies taken from both clinically involved (�brotic lesions) and non-involved sites (Tables ( Tables 2 and 3). 3). Also, Chi3L1 secret sec retionby ionby ski skin n cel cells ls of SScpatie SScpatientswas ntswas notcorre notcorrelat lated ed wit with h ski skin n sco scores res (Table 4) 4) or with clinical outcomes, such as digital ulcers, interstitial lung disease and pulmonary hypertension, disease activity and disease severi sev erity ty (dat (dataa not sho shown) wn).. How Howeve ever, r, it was note noted d tha thatt pati patient entss exhibiting endogenous secretion of Chi3L1 in cells from both biopsy si-
Fig. 4. Com Fig. Compar parisonof isonof Chi3 Chi3L1 L1 sec secreti retion on by skincellsfrom armand abd abdomi ominalbiopsie nalbiopsies. s. Resu Resultsfrom ltsfrom twoSSc pat patient ientss areshown.Skin cell cellss wer were e grow grown n in mon monolay olayer er cult cultureand ureand stim stimulat ulated ed for 48 h with a variety of cytokines and growth factors (IL-1 β, IL-6, OSM, TGF-β and PDGF). Skin cell lines used in this experiment were in passage 5. Chi3L1 secretion into the culture medium was measur measured ed by SDS-PAGE SDS-PAGE and immunoblo immunoblotting. tting. The left-ha left-hand nd lane of the gel shows shows the migrat migration ion of molecula molecularr weight weight markers markers,, andthe right-h right-hand and lane shows the migrati migration on of puri�ed Chi3 Chi3L1.Biotin L1.Biotinylat ylated ed SBTIwas adde added d to eachmediu eachmedium m sam sampleto pleto moni monitoraceton toracetone e prec precipit ipitatio ation n ef �cien ciency.Phenoty cy.Phenotype pe det detailsof ailsof pat patient ientss 14 (A)and 8 (B)are pres presente ented d in inTab Table le 2.
8
Y.Y. Ho et al. / BBA Clinic Clinical al 1 (2014) 2 –11
tes were in the early phase of disease, with a disease duration of b 2.5 year yearss (Tab Table le 2). The sho shorte rtest st dise disease ase dur duratio ation n for pati patients ents exhibiting other secretion patterns was 2.9 years with a median of 12.6 12. 6 year years. s. Mea Mean n dis diseas ease e dur durati ation on wassigni�cantl cantly y reduc reduced ed for patien patients ts showing Chi3L1 expression in cells from both sites (P = 0.0005) and at only the forearm site (P = 0.026). This suggests that the capacity for endogenous production of Chi3L1 may be related to pathological processes active at early disease stages.
3.5. Characteriza Characterization tion of Chi3L1-e Chi3L1-expres xpressing sing cells cells Six dermal cell lines from SSc SSc patients previously shown to endogenously produce Chi3L1 in cells derived from both arm and abdomen were analyzed in this study. A heterogeneous cell population was observed, with Chi3L1 endogenous expression only being detected in 5.4–10.7% of the cells, which were often seen in clusters ( Fig. 5A). 5A). Thus Chi3L1 production is not a feature of the general �broblast
Fig. 5. Identi�cation of cells in SSc dermal cell cultur culture e that endogenously endogenously express Chi3L1. Chi3L1. Cells were identi identi�ed for Chi3 Chi3L1 L1 and αSMA, a marker of mature mature myo�broblasts, using immunouoresce scence nce mic microsc roscopy.One opy.One repr represen esentat tative ive of 6 cult culturelines urelines is show shown n forcells in pass passage6. age6. (A)The Chi3 Chi3L1 L1 expr expressi essing ng cell cellss arepresen arepresentt in clus cluster terss andrepres andrepresentapprox entapproximat imately5 ely5–10 10% % of �uore the cellpopula cellpopulation tion.. (B)Unstim (B)Unstimulat ulated ed Chi3 Chi3L1L1-exp expres ressingcells singcells do notco-loc notco-localiz alize e wit with h myo�brob broblas lasts. ts. (C)OSM sti stimul mulatio ation n near nearly ly doub doubles les the num numberof berof Chi3 Chi3L1L1-expr expressi essing ng cell cellss butdoes not affect myo�broblasts. (D) TGF-β depletes the expression of Chi3L1 but increas increases es the abunda abundance nce of myo�broblasts. Bars represent 100 μ m. m.
Y.Y. Ho et al. / BBA Clinical 1 (2014) 2 –11
population. Some myo�broblasts with characteristic actin stress � bers are evident in the cell preparations, but they do not co-localize with the Chi3L1 expressing cells (Fig. ( Fig. 5B). 5B). Upon OSM stimulation, the number of Chi3L1 expressing cells nearly doubled to 10.2 –19.6% of the total tot al cel celll pop populat ulation ion,, wit with h the cells still bei being ng unr unrelat elated ed to the myo�broblasts (Fig. (Fig. 5C). 5C). In contra contrast, st, upon TGF-β stimulation, when many man y of the SSc skin cells cells are transfor transformed med to αSMA-expressing myo�brobla broblasts, sts, Chi3L1 expre expression ssion is absent (Fig. ( Fig. 5D). 5D). This suggests that Chi3L1 expression in SSc dermal cells is up-regulated by proin�ammatory stimulation but down-regulated by pro-�brotic stimulation, and that cells other than typical � broblasts and myo�broblasts are responsible for its production. In recent years, it has been demonstrated that the dermis of SSc patients may contain a multiplic multiplicity ity of cell types other than � broblasts and myo�brobl broblasts. asts.Thes These e cell celltypes typesincl include ude bone marr marrow-de ow-derive rived d mesenchymal stem cells [41] cells [41],, pericytes [42] pericytes [42],, endothelial progenitor cells [43],, and circulating progenitor cells, such as �brocytes and monocytes [43] [41,, 44, 45] [41 45].. TheSSc cel celll lin lines es wer were e the theref refore ore ana analyz lyzed ed for theexpre theexpressi ssion on of the markers speci �c for these other cell types to determine whether they could be the source of Chi3L1 expression. Immuno �uorescence dual localization revealed revealed that the SSc dermal cells that endogenously expressed Chi3L1 also co-expressed the progenitor/stem cell marker, nestin (Fig. (Fig. 6A). 6A). These cells also expressed STRO-1, CD73, TIE2, LSP-1 and NG2, but not CD34 and CD45 (results not shown). Cell counting showed that the nestin-positive nestin-positive cells constitute constituted d 4.9 –9.9% of the total cell population. Dermal cells from healthy controls showed no expression of the aforementioned cell markers (results not shown). Thus
9
endogenous Chi3L1 production in the SSc dermis appears to originate endogenous from cells with stem cell-like characteristics. It is, however, not clear if these cells represent a single cell type or are of multiple cell lineages. While Chi3L1 expressing cells nearly doubled upon OSM stimulation, the number of cells expressing nestin (or STRO-1, CD73, TIE2, LSP-1 and NG2) remained relatively constant constant at 5.2–10.1% of the total cell population from patients with endogen endogenous ous Chi3L1 expression. Interestingly, upon OSM stimulation, some of the cells that expressed Chi3L1 Chi 3L1 mos mostt exu exuber berant antly ly did not exp expres resss the ste stem m cel celll mar marker kers. s. How Howevever, these cells are observed to be closely associated with those that express both Chi3L1 and nestin (Fig. (Fig. 6B). 6B). In addition, � ve SSc dermal cell lines that did not endogenously express Chi3L1 but in which the protein was inducible by OSM were also studied. Immuno�uorescence for Chi3L1 con �rmed that there was no endogenous Chi3L1 expression, and indicated that upon OSM stimulation Chi3L1 expression was induced in approximately 4.1 –7.9% of cells. Unlike cells that endogenously express Chi3L1, this group of cells were not stained for nesti nestin n (Fig. ( Fig. 6C) 6C) or any of the other progenitor/ stem cell markers tested. 4. Discussion
In this study we investigated the capacity of skin cells from SSc patients and from healthy individuals to secret secrete e Chi3L1 and we assessed the regulation of this process by growth factors and cytokines shown to be associ associated ated with the patholo pathology gy of SSc. The results demonstrate demonstrate that Chi3L1 is not secret secreted ed endogenously endogenously by normal skin cells nor is it
Co-localizatio lization n of Chi3L1Chi3L1-expressi expressing ng cells and nestin by �uore uorescen scence ce mic micros roscopy copy.. (A)SSc derm dermal al celllinesthat endo endogeno genouslyexpre uslyexpress ss Chi3 Chi3L1 L1 als also o exp expressnestin ressnestin.. Wit With h bothmark bothmarkers ers Fig. 6. Co-loca Fig. approximately 5–10% of the cells showed positive staining. One representative of 6 culture lines is shown for cells in passage 6. (B) Upon OSM stimulation of SSc dermal cell lines that endogenously endogenous ly expressed Chi3L1, the number of Chi3L1-expressing Chi3L1-expressing cells doubled while the number of cells expressing nestin remained unchanged. The Chi3L1 expressing cells that do not express nestin are in close proximity to those that express both nestin and Chi3L1. One representative of 6 culture lines is shown for cells in passage 6. (C) SSc dermal cell lines that do not endogenous endogenously ly express Chi3L1 also do not express nestin. Approximately Approximately 4–8% of these cells can be induced to express Chi3L1 upon stimulation stimulation of OSM. One representative representative of 3 culture lines is shown for cells in passage 6. Bar represen represents ts 100 μ m. m.
10
Y.Y. Ho et al. / BBA Clinic Clinical al 1 (2014 (2014)) 2 –11
induced induce d by in�amm ammato atory ry cyt cytoki okinessuch nessuch as OSMand ILIL-1. 1. In con contra trast,all st,all cell preparations obtained from peripheral skin biopsies of SSc patients responded respon ded to OSM with incr increased eased Chi3L1 secretion secretion into the culture medium, and endogenous production was clearly detectable in a proportion proporti on of the cell prepar preparations ations.. This property was indepe independent ndent of clinical status, but cells from both arm and abdomen of subjects with early disease were more likely to demonstrate endogenous secretion of Chi3L1 than those with more advanced disease. The cells responsible respon sible for endogenous production production of Chi3L1 did not appear to be typical � broblasts or myo�broblasts, but consisted of about 5 –10% of the SSc skin cell population that had stem cell-like features. The increased production of Chi3L1 in response to OSM, however, involved additional cells. Thus the cells involved in �brosis and contrac contracture ture appear to be distinct from those responsible for Chi3L1 production. The lack of Chi3L1 production by normal skin �broblasts in the absenc abs ence e or pre presen sence ce of gro growth wth fac factor torss or cyt cytoki okine ness is con consis sisten tentt wit with h lit lit-erature reports [28, reports [28, 39, 46, 47]. 47] . Thus the detection of Chi3L1 in culture media from SSc skin cell preparations was unexpected, indicating the presence of a cell population with an altered phenotype compared to healthy healt hy skin.Identi�cat cationof ionof Chi Chi3L1 3L1 pro produc ducingcell ingcellss by imm immuno uno�uorescence microscopy suggested that a relatively small proportion of the cells cel ls act actual ually ly pro produc duced ed Chi Chi3L1 3L1 eve even n aft after er exp exposu osure re to OSM OSM.. The cel cells ls responsible for endogenous production production of Chi3L1 expressed expressed a variety of progenitor/stem cell markers, namely nestin, STRO-1, CD73, TIE2, LSP1 and NG2. Nestin is a class VI intermediate � lament protein reported to be expressed in neural tube-associated neural stem cells [48] [48],, but is also al so us used ed as a ge gene nera rall ma mark rker er fo forr pr prog ogen enit itor or ce cell lls, s, in incl clud udin ing g skin-derived progenitor cells [49] cells [49] and and endothelial progenitor cells [50] cells [50].. STRO-1 and CD73 are cell surface proteins commonly used as markers for bone marr marrow-de ow-derive rived d mese mesenchy nchymal mal stem cell cellss [51] [51].. TI TIE2is E2is anend anendoothelia the liall tyr tyrosi osine ne ki kinas nase e rec recept eptor or tha thatt has bee been n pro propos posed ed to be the ear earlie liest st mammalian endothelial cell lineage marker [52] marker [52].. LSP-1 is a marker for �brocytes — circulating bone marrow-derived mononuclear cells that have the capacity to deposit extracellular matrix in wound healing and numerous �broti broticc disor disorders ders,, incl including udingSSc SSc [53] [53].. NG NG2 2 isa ce cellsurf llsurfac ace e pr prooteoglycan teogl ycan widely expre expressed ssed in both vasculogenic vasculogenic and angio angiogenic genic neovas neo vascu culat lature ure,, andis acc accept epted ed as a mar marke kerr forperic forpericyte ytess in mic microv rovess essels els [54].. The cell [54] cellss that express express Chi3L Chi3L1 1 did not stain for CD34 and CD45 (re(results not shown), suggesting that the cells do not have hematopoietic potenti pote ntial. al. At pre presen sent, t, the ori origin gin of the cel cells ls coco-exp expres ressin sing g Chi Chi3L1 3L1 and pro pro-genitor/stem cell markers is not known, but bone marrow appears to be thecommo thecom mon n so sour urce ce of su suchcell chcells. s. It is al also so no nott cl clea earr if th the e ab abil ilityof ityof th the e pro pro-geni ge nitor tor/s /ste tem m ce cell llss to re reac actt wi with th a di dive vers rsityof ityof ma mark rker erss is a re�ect ection ion of dif dif-ferent fer ent tran transie sient nt sta stages ges with within in a sin single gle cel celll dif differ ferent entiati iation on path pathway way or is an indication of different subpopulations. It is interesting to note that a recent ce nt st study udy ha hass sh show own n tha thatt Ch Chi3 i3L1 L1 ex expr pres essi sion on is a pro prope perty rty of bot both h un undi diffferentiated and differentiated mesenchymal stem cells [55] cells [55].. The present study showed that OSM stimulation of SSc dermal cell cultures that endogenously express both Chi3L1 and progenitor/s progenitor/stem tem cell markers nearly doubled the number of Chi3L1 expressing cells but not cell cellss that bear bearprogen progenitor/ itor/stem stem cell cellmark markers. ers. Howev However, er, the induc induction tion of Chi Chi3L1upreg 3L1upregulat ulationis ionis obs observ erved ed to occ occur ur in cel cells ls in ver very y clo close se pro proxim xim-ity to the cel cells ls tha thatt coco-exp expres resss Chi Chi3L1 3L1 and prog progeni enitor tor/st /stem em cel celll mar marker kers. s. These Thes e OSM-r OSM-respons esponsive ive cell cellss appea appearr to be activa activated ted �brobl broblasts asts sincethey also co-express S100A4 (�broblast-speci�c protein, FSP-1) (results not shown), and it is possible that they could represent the differentiated product pr oduct of the progenitor/stem cells. OSM was also observed to induce expression of Chi3L1 in some SSc dermal cell cultures that did not show endogenous Chi3L1 expression and contained no cells with progenitor/stem cell markers. The cells that were induced by OSM to express Chi3L1 could also represent activated � broblas broblasts ts as they also coexpress S100A4 (results (results not shown). As the patients without endogenous no us Ch Chi3 i3L1 L1 ex expr pres essi sion on te tend nd to ha have ve a lo long nger er di dise seas ase e du durat ratio ion n it is po posssible that all progeni progenitor/stem tor/stem cells that were present earlier have now differentiated. differenti ated. Unlike normal � broblasts, these cells are primed to respond to in�ammatory cytokines with increased Chi3L1 production.
While peripheral skin cells from SSc patients always produced Chi3L1 following OSM stimulation, abdominal cells behaved in a more sporadic fashion. This is not unexpected, as peripheral skin is more likely to be clinic clinically ally involved in SSc than the abdomen. However, the present � ndings suggest that even when the skin on the forearm appears clinically normal, a pathophysiologic process characterized by the pre presen sence ce of cel cells ls cap capabl able e of Chi Chi3L1 3L1 sec secret retion ion is ongo ongoing ing.. The �nding thatt in mat tha matche ched d sam sample pless end endoge ogenou nouss Chi Chi3L1 3L1 sec secret retion ion ten tends ds to be mor more e consistently consis tently upregulated upregulated in cells from both sites in SSc patients with a short disease duration, suggests that with time the widespread pathological process that is re �ected by generali generalized zed endogenous Chi3L1 secretion diminishes and is replaced by more focal pathology. If Chi3L1 secretion does indeed re �ect an active process, there is no a priori reason for a clear relationship between clinical skin thickening and Chi3L1 secretion. It is possible that Chi3L1 production is not re �ected in clinically detectable skin thickening associated with � brosis because it is either too early for this feature to present, or because turnover of ECM is initially able to keep pace with the increased ECM production associated assoc iated with �br bros osis is.. It is al also so un uncl clea earr wh whet ethe herr Ch Chi3 i3L1 L1 ha hass anydire anydirect ct effect on collagen production, though it may do so indirectly by stimu � broblast proliferation [29] lation of � proliferation [29].. Thepresent Theprese nt stu study dy sho showed wed tha thatt end endoge ogenou nouss sec secre retionof tionof Chi Chi3L1 3L1 and and// or inducibility by OSM did not distinguish patients with limited or diffuse SSc. Also, Chi3L1 secretion by SSc skin cells could not be related to cli clinic nical al fea featur tures es,, suc such h as ski skin n sco score, re, dig digital ital ulc ulcers ers,, int inters erstit titial ial lungdisease, pulmonary hypertension and disease activity. However, other groups gro ups hav have e rep report orted ed tha thatt SSc pat patien ients ts wit with h hig higher her Chi Chi3L1 3L1 ser serum um lev levels els exhibited exhibi ted an elevated incidence incidence of pulmonar pulmonary y � brosis, greater disease severity sever ity and shorter survival time [23] [23].. An enhanced Chi3L1 serum level has also been reported to be associated with joint involvement in SSc patients [22, patients [22, 24]. 24]. However, the cellular source of serum Chi3L1 is not known, but is likely to re �ect variable levels of secretion from many sources. sources. As such, it is not possible to relate the level of serum Chi3L1 to the level in any given tissue. Only direct analysis of speci �c tissues, tissu es, as perfor performed med in this this study, will will permit permit this issue issue to be resol resolved. ved. While the biological functions of Chi3L1 have not been determined in SSc, the protein could be involved in modulating the in �ammatory response. Previous work has shown that Chi3L1 can modulate the response spo nse of con connec nectiv tive e tis tissue sue cel cells ls to in�ammato ammatory ry cytok cytokines ines by reduc reducing ing the response of human chondrocytes to IL-1 or TNF- α [32] [32].. This suggests ges ts tha thatt Chi Chi3L1 3L1 is pro produc duced ed in res respons ponse e to pro pro-in -in�amma ammatory tory stimu stimulalation, but once it is secreted, secreted, it can exert a pro �brotic effect by reducing the turnover of extracellular matrix components. Thus, the induction and up-regulation of Chi3L1 by IL-1 and/or OSM in some SSc skin cells could occur during in�ammation, where it may limit tissue damage. Con�ict of intere interest st
Author declares that there are no con �ict of interest. Acknowledgem Acknow ledgements ents
Thiss pro Thi projec jectt was funded by the Can Canadi adian an Ins Institu titutes tes of Hea Health lth Research, the Scleroderma Society of Canada, and the Shriners of North America. We thank Solene Tatibouet for the statistical analyses. References [1] C.G. Helmick, Helmick, D.T. Felson, R.C. Lawrence, S. Gabriel, R. Hirsch, C.K. Kwoh, M.H. Liang, H.M. Kremers, M.D. Mayes, P.A. Merkel, S.R. Pillemer, J.D. Reveille, J.H. Stone, Estimates of the prevalence of arthritis and other rheumatic conditions in the United States. Part I, Arthritis Rheum. 58 (2008) 15–25 25.. [2] A.J. Geirsson, K. Steinsson, S. Guthmundsson, V. Sigurthsson, Systemic sclerosis sclerosis in Iceland. A nationwide epidemiological study, Ann. Rheum. Dis. 53 (1994) 502 –505 505.. [3] T.R. Katsumoto, M.L. Whit�eld, M.K. Connolly, The pathogenesis of systemic sclerosis, Annu. Rev. Pathol. 6 (2011) 509 –537 537..
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