scholarly journals SAT0301 CIRCULATING FIBROCYTES FROM SYSTEMIC SCLEROSIS PATIENTS AS POSSIBLE TARGET OF CTLA4-IG TREATMENT: AN IN VITRO STUDY

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1096.1-1096
Author(s):  
S. Soldano ◽  
P. Montagna ◽  
S. Paolino ◽  
E. Alessandri ◽  
C. Pizzorni ◽  
...  
Keyword(s):  
Gene Expression ◽  
Systemic Sclerosis ◽  
Costimulatory Molecule ◽  
Skin Fibroblasts ◽  
Type I ◽  
Research Support ◽  
Hla Dr ◽  
Antigen Presenting ◽  
Circulating Fibrocytes

Background:muscle actin (aSMA)+cells involved in the overproduction of extracellular matrix proteins, primarily fibronectin (FN) and type I collagen (COL1) at the level of damaged tissues (1). These cells may originate from different cell types including fibroblasts, endothelial and epithelial cells, and fibrocytes (1). Circulating fibrocytes are bone marrow progenitor cells expressing specific markers of hematopoietic (CD34, CD45, and MHC class II) and stromal cells (COL1 and COL3), chemokine receptors (CCR2, CCR7), and CXCR4 (2). CXCR4 regulates fibrocyte migration into injured tissues allowing their differentiation into fibroblasts/myofibroblasts (2).In vitro, fibrocytes differentiate from circulating CD14+monocytes showing an antigen-presenting capability through the expression of HLA-DR and costimulatory molecule CD86 (2). CTLA4-Ig fusion protein (abatacept) interacts with CD86 on cell surface of antigen presenting cells (APCs), such as macrophages and endothelial cells (3,4).Objectives:To investigate the possible effect of CTLA4-Ig treatment on cultured human fibrocytes and skin fibroblasts isolated from the same systemic sclerosis patients (SSc pts).Methods:Fibrocytes isolated from the peripheral blood mononuclear cells of SSc pts and healthy subjects (HSs) were cultured on fibronectin-coated plates in DMEM at 20% of FBS; for further 8 days (T8) to allow their complete differentiation. Differentiated fibrocytes were maintained in growth medium or treated with CTLA4-Ig at different concentrations (10, 50, 100, and 500μg/ml) for 3 hours. Fibroblasts were isolated from the skin biopsies of the same patients and HSs, cultured until the 3rdpassage in RPMI at 10% FBS and then treated with CTLA4-Ig for 24 and 48 hours. Fibrocytes were characterized as CD45+CXCR4+COL1+cells and the expression of CD86 and HLA-DR was also evaluated. The gene expression of aSMA, COL1, CXCR4, TGFb1 and CD86 was investigated by quantitative real-time polymerase chain reaction in cultured fibrocytes and skin fibroblasts. In cultured skin fibroblasts, COL1 and fibronectin synthesis was evaluated by Western blotting.Results:Treatment with CTLA4-Ig for 3 hours significantly downregulated aSMA and COL1 gene expression in cultured SSc fibrocytes at T8 (p<0.01, p<0.05 vs. untreated fibrocytes), whereas no modulatory effect was observed on the TGFbeta1 and CXCR4 gene expression. In cultured SSc skin fibroblasts, CTLA4-Ig did not induce any significant effect on CD68, TGFb1, COL1 and FN gene expression as well as COL1 and FN protein synthesis, both after 24 and 48 hours. Of note, these cultured SSc skin fibroblasts showed a low expression of CD86.Conclusion:Due to their high expression of CD86, circulating fibrocytes seem to be more responsive to CTLA4-Ig treatment than the skin fibroblasts isolated from the same SSc patient.References:[1]Cutolo M et al. Expert Rev Clin Immunol. 2019;15:753-64.[2]Bucala R. Mol Med.2015;2:S3-5.[3]Cutolo M et al. Clin Exp Rheumatol. 2015;33:250-4.[4]Brizzolara R et al. J Rheumatol. 2013;40:738-40.Disclosure of Interests:Stefano Soldano: None declared, Paola Montagna: None declared, Sabrina Paolino: None declared, Elisa Alessandri: None declared, Carmen Pizzorni: None declared, Greta Pacini: None declared, Federica Goegan: None declared, Alberto Sulli Grant/research support from: Laboratori Baldacci, Carlotta Schenone: None declared, Vanessa Smith Grant/research support from: The affiliated company received grants from Research Foundation - Flanders (FWO), Belgian Fund for Scientific Research in Rheumatic diseases (FWRO), Boehringer Ingelheim Pharma GmbH & Co and Janssen-Cilag NV, Consultant of: Boehringer-Ingelheim Pharma GmbH & Co, Speakers bureau: Actelion Pharmaceuticals Ltd, Boehringer-Ingelheim Pharma GmbH & Co and UCB Biopharma Sprl, Maurizio Cutolo Grant/research support from: Bristol-Myers Squibb, Actelion, Celgene, Consultant of: Bristol-Myers Squibb, Speakers bureau: Sigma-Alpha

scholarly journals POS0330 NINTEDANIB (TYROSINE KINASE INHIBITOR) DOWNREGULATES THE TRANSITION OF CULTURED SYSTEMIC SCLEROSIS FIBROCYTES INTO MYOFIBROBLASTS AND THEIR PRO-FIBROTIC ACTIVITY

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 392.2-392
Author(s):  
S. Soldano ◽  
P. Montagna ◽  
E. Gotelli ◽  
S. Tardito ◽  
S. Paolino ◽  
...  
Keyword(s):  
Gene Expression ◽  
Systemic Sclerosis ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Kinase Inhibitor ◽  
Adherent Cells ◽  
Research Support ◽  
Fibrotic Process ◽  
Circulating Fibrocytes

Background:Fibroblast-to-myofibroblast transition is one of the fundamental steps involved in the fibrotic process that characterise systemic sclerosis (SSc) [1]. Myofibroblasts are α-smooth muscle actin (αSMA) positive cells that contribute to fibrosis through the excessive synthesis and deposition of extracellular matrix (ECM) proteins, primarily fibronectin (FN) and type I collagen (COL1) [2].Among the cells involved in the fibrotic process of SSc, circulating fibrocytes seem to have an emerging role as an important source of fibroblasts and myofibroblasts [3].Nintedanib is a tyrosine kinase inhibitor approved for the treatment of idiopathic pulmonary fibrosis that interferes with the signalling pathways involved in the pathogenesis of fibrosis (4). Nintedanib was recently demonstrated to have a beneficial effect in patients with interstitial lung disease (ILD) associated with SSc (5).Objectives:To investigate nintedanib effect in inhibiting the in vitro transition of circulating SSc fibrocytes into myofibroblasts and their pro-fibrotic activity.Methods:Circulating fibrocytes were obtained from 14 SSc patients (mean age 64±14 years), who fulfilled the 2013 ACR/EULAR criteria for SSc and that underwent complete disease staging in a day-hospital setting at the Rheumatology Division of Genoa University. Five age-matched healthy subjects (HSs) were also analysed. All SSc patients and HSs signed the informed consent and the local EC approved the study. Peripheral blood mononuclear cells were isolated by density gradient centrifugation and plated on FN-coated dishes. After overnight culture, non-adherent cells were removed, and adherent cells were maintained in growth medium for 8 days (T8) to obtain fibrocytes [6]. T8-cultured SSc fibrocytes were maintained in growth medium (untreated cells) or treated with nintedanib 0.1μM and 1μM for 3 and 24 hours. Fibroblast specific protein-1 (S100A4) and αSMA, as markers of fibroblast/myofibroblast phenotype, together with COL1 and FN, were investigated by qRT-PCR and Western blotting. Non-parametric Mann-Whitney and Wilcoxon tests were used for the statistical analysis.Results:Significantly elevated gene and protein expressions of αSMA, S100A4, COL1 and FN were observed in SSc fibrocytes compared to HS fibrocytes (gene: αSMA p<0.001; others p<0.0001; protein: all p<0.05). In accordance with the antibody positivity for Scl70 and the presence or absence of ILD at CT scan, SSc patients were grouped as either Scl70 positive patients with ILD (Scl70+ILD+) or Scl70 negative patients without ILD (Scl70-ILD-). Significant αSMA, S100A4, COL1 and FN gene expressions were found in fibrocytes from Scl70+ILD+ compared to HS fibrocytes (αSMA p<0.001; others p<0.0001). Moreover, fibrocytes from Scl70+ILD+patients showed a more significant gene expression of fibroblasts/myofibroblasts markers compared to Scl70-ILD-patients (p<0.01 for S100A4), whereas no differences were observed for ECM gene expression.Nintedanib reduced the gene and protein expression of αSMA, COL1 and FN in SSc fibrocytes compared to untreated ones with different statistical significance.Noteworthy, nintedanib significantly downregulated αSMA, S100A4, COL1 and FN gene expression (all p<0.05) in Scl70+ILD+fibrocytes, whereas only that of S100A4 and FN was significantly downregulated (p<0.05) in Scl70-ILD- fibrocytes compared to untreated cells.Conclusion:Nintedanib seems to downregulate in vitro the transition of fibrocytes into myofibroblasts and their pro-fibrotic activity, particularly in cells isolated from Scl70+ILD+SSc patients.References:[1]Cutolo M et al. Exp Rev Clin Immunol. 2019;15:753-64.[2]Van Caam A et al. Front. Immunol. 2018;9:2452.doi:10.3389/fimmu.2018.02452.[3]Distler JH et al. Arthritis Rheumatol. 2017;69:257-67.[4]Distler O et al. New Eng J Med. 2019; 380:2518-28.[5]Maher TB et al. Arthritis Rheumatol.2020.doi:10.1002/art.41576.[6]Cutolo M et al. Arthritis Res Ther. 2018;20:157.doi:10.1186/s13075-018-1652-6.Acknowledgements:We thank Stefano-Lutz Willing for the scientific support through the study.Disclosure of Interests:Stefano Soldano: None declared, Paola Montagna: None declared, Emanuele Gotelli: None declared, Samuele Tardito: None declared, Sabrina Paolino: None declared, Claudio Corallo: None declared, Carmen Pizzorni: None declared, Alberto Sulli: None declared, Carlotta Schenone: None declared, Greta Pacini: None declared, Vanessa Smith: None declared, Maurizio Cutolo Grant/research support from: I received grant/research support from Bristol-Myers Squibb, Boehringer, Celgene

scholarly journals AB0168 NINTEDANIB (TYROSINE-KINASE INHIBITOR) INHIBITS THE TRANSITION OF CIRCULATING FIBROCYTES ISOLATED FROM SYSTEMIC SCLEROSIS PATIENTS INTO MYOFIBROBLASTS: AN IN VITROSTUDY

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1384.1-1384
Author(s):  
S. Soldano ◽  
G. Martinelli ◽  
S. Tardito ◽  
S. Paolino ◽  
M. Patanè ◽  
...  
Keyword(s):  
Gene Expression ◽  
Systemic Sclerosis ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Growth Medium ◽  
Mononuclear Cells ◽  
Kinase Inhibitor ◽  
Type I ◽  
Research Support ◽  
Circulating Fibrocytes

Background:Systemic sclerosis (SSc) is a chronic connective disease characterized by microvascular alterations, dysregulated immune response and fibrosis [1,2]. Myofibroblasts are alpha-smooth muscle actin (alphaSMA)+cells and play a crucial role in fibrosis, through the excessive synthesis and deposition of extracellular matrix (ECM) proteins, in particular fibronectin (FN) and type I collagen (COL1) [3]. Despite myofibroblasts primarily derive from resident fibroblasts transition and differentiation, another important source is represented by circulating fibrocytes [4]. Nintedanib is a tyrosine kinase inhibitor approved for the treatment of idiopathic pulmonary fibrosis that interferes with the signalling pathways involved in the pathogenesis of fibrosis [5].Objectives:To investigate the possible effects of nintedanib in contrasting the ability of cultured mature fibrocytes from SSc patients to differentiate into profibrotic myofibroblasts.Methods:Circulating fibrocytes were obtained from peripheral blood mononuclear cells isolated from 5 limited cutaneous SSc patients (mean age 68 +/- 10 years) and then plated on FN-coated tissue culture dishes in growth medium (DMEM at 20% of fetal bovine serum, 1% of penicillin-streptomycin and 1% L-glutamine), to allow the adhesion of fibrocyte precursors. Adherent cells were maintained in growth medium for 8 days in order to allow their differentiation into fibrocytes. Differentiated fibrocytes were treated with nintedanib at the concentrations of 100nM and 1000nM for 3 and 24 hours (hrs) or maintained in growth medium without any treatment. The differentiation of fibrocytes into myofibroblasts was determined evaluating the gene expression of alphaSMA, fibroblast specific protein-1 (S100A4) COL1, FN and CXCR4 by quantitative real-time polymerase chain reaction, and the protein synthesis of alphaSMA, COL1 and FN by western blotting.Results:Nintedanib inhibited alphaSMA and S100A4 gene expression already at the concentration of 100nM in cultured fibrocytes and after 3 hrs of treatment, when compared with untreated cells. Furthermore, both concentrations of nintedanib (100nM and 1000nM) reduced the gene expression of COL1 and FN, whereas only 100nM downregulated the CXCR4 gene expression. At protein level, nintedanib 100nM and 1000nM reduced the synthesis of alphaSMA and COL1 after 24 hrs of treatment, whereas FN synthesis was reduced only by the nintedanib concentration of 1000nM.Conclusion:The preliminary results show that nintedanib may inhibit thein vitrotransition of SSc fibrocytes into myofibroblasts and their profibrotic activity, through the reduction of specific myofibroblast phenotype markers and ECM protein production. The results seem to suggest fibrocytes as further possible target of the antifibrotic action of nintedanib in SSc.References:[1]Cutolo M et al. Expert Rev Clin Immunol. 2019;15:753-64 2. Barsotti S et al. Clin Exp Rheumatol. 2016;34(Suppl.100):S3-S13 3. Wynn TA et al. Nat Med. 2012;18:1028-40. 4.Distler JHW et al. Arthritis Rheumatol. 2017;69:257-67 5.Hilberg F et al. Cancer Res. 2008;68:4774-82.Disclosure of Interests:Stefano Soldano: None declared, Giulia Martinelli: None declared, Samuele Tardito: None declared, Sabrina Paolino: None declared, Massimo Patanè: None declared, Emanuele Gotelli: None declared, Claudio Corallo: None declared, Carmen Pizzorni: None declared, Alberto Sulli Grant/research support from: Laboratori Baldacci, Carlotta Schenone: None declared, Vanessa Smith Grant/research support from: The affiliated company received grants from Research Foundation - Flanders (FWO), Belgian Fund for Scientific Research in Rheumatic diseases (FWRO), Boehringer Ingelheim Pharma GmbH & Co and Janssen-Cilag NV, Consultant of: Boehringer-Ingelheim Pharma GmbH & Co, Speakers bureau: Actelion Pharmaceuticals Ltd, Boehringer-Ingelheim Pharma GmbH & Co and UCB Biopharma Sprl, Maurizio Cutolo Grant/research support from: Bristol-Myers Squibb, Actelion, Celgene, Consultant of: Bristol-Myers Squibb, Speakers bureau: Sigma-Alpha

scholarly journals Effects of CTLA4-Ig treatment on circulating fibrocytes and skin fibroblasts from the same systemic sclerosis patients: an in vitro assay

2018 ◽  
Vol 20 (1) ◽  
Author(s):  
Maurizio Cutolo ◽  
Stefano Soldano ◽  
Paola Montagna ◽  
Amelia Chiara Trombetta ◽  
Paola Contini ◽  
...  
Keyword(s):  
Systemic Sclerosis ◽  
In Vitro Assay ◽  
Skin Fibroblasts ◽  
Vitro Assay ◽  
Circulating Fibrocytes

scholarly journals Nintedanib downregulates the transition of cultured systemic sclerosis fibrocytes into myofibroblasts and their pro-fibrotic activity

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Maurizio Cutolo ◽  
Emanuele Gotelli ◽  
Paola Montagna ◽  
Samuele Tardito ◽  
Sabrina Paolino ◽  
...  
Keyword(s):  
Systemic Sclerosis ◽  
Protein Expression ◽  
Type I Collagen ◽  
Kinase Inhibitor ◽  
Smooth Muscle Actin ◽  
Statistical Significance ◽  
Type I ◽  
Gene And Protein Expression ◽  
Circulating Fibrocytes

Abstract Background Circulating fibrocytes are an important source of fibroblasts and myofibroblasts, which are involved in fibrotic processes, including systemic sclerosis (SSc). The study aimed to investigate the effect of nintedanib (a tyrosine kinase inhibitor) in inhibiting the in vitro transition of circulating SSc fibrocytes into myofibroblasts and their pro-fibrotic activity. Methods Circulating fibrocytes were obtained from 18 SSc patients and 5 healthy subjects (HSs). Cultured SSc fibrocytes were maintained in growth medium (untreated cells) or treated with nintedanib 0.1 and 1 μM for 3 and 24 h. Fibroblast-specific protein-1 (S100A4) and α-smooth muscle actin (αSMA), as markers of fibroblast/myofibroblast phenotype, together with type I collagen (COL1) and fibronectin (FN), were investigated by qRT-PCR and Western blotting. Non-parametric tests were used for statistical analysis. Results Significantly elevated gene and protein expressions of αSMA, S100A4, COL1, and FN were observed in SSc fibrocytes compared to HS fibrocytes (gene: αSMA p < 0.001; others p < 0.0001; protein: all p < 0.05). Interestingly, an increased gene and protein expression of αSMA and S100A4 was found in fibrocytes from SSc patients positive for anti-Scl70 and with interstitial lung disease (ILD) (Scl70+ILD+) compared to Scl70−ILD− patients (S100A4: gene: p < 0.01; protein: p < 0.05), whereas no differences were observed for COL1 and FN. Nintedanib reduced gene and protein expression of αSMA, S100A4, COL1, and FN in SSc fibrocytes compared to untreated ones with different statistical significance. Noteworthy, nintedanib significantly downregulated gene and protein expression of αSMA, S100A4, COL1, and FN in Scl70+ILD+ fibrocytes (all p < 0.05), whereas only that of S100A4 and FN was significantly downregulated (p < 0.05) in Scl70−ILD− fibrocytes compared to the related untreated cells. Conclusions Nintedanib seems to downregulate in vitro the transition of fibrocytes into myofibroblasts and their pro-fibrotic activity, particularly in cells isolated from Scl70+ILD+ SSc patients.

Tissue specificity of type I collagen gene expression is determined at both transcriptional and post-transcriptional levels

1984 ◽  
Vol 4 (9) ◽  
pp. 1843-1852
Author(s):  
R J Focht ◽  
S L Adams
Keyword(s):  
Gene Expression ◽  
Rna Structure ◽  
Type I Collagen ◽  
Translational Efficiency ◽  
Type I ◽  
Collagen Gene ◽  
Differentiated Cells ◽  
Collagen Gene Expression

We analyzed the control of type I collagen synthesis in four kinds of differentiated cells from chicken embryos which synthesize very different amounts of the protein. Tendon, skin, and smooth muscle cells were found to have identical amounts of type I collagen RNAs; however, the RNAs had inherently different translatabilities, which were observed both in vivo and in vitro. Chondrocytes also had substantial amounts of type I collagen RNAs, even though they directed no detectable synthesis of the protein either in vivo or in vitro. Type I collagen RNAs in chondrocytes display altered electrophoretic mobilities, suggesting that in these cells the reduction in translational efficiency may be mediated in part by changes in the RNA structure. These data indicate that control of type I collagen gene expression is a complex process which is exerted at both transcriptional and post-transcriptional levels.

Investigation of the effects of prostaglandin E2 on equine superficial digital flexor tendon fibroblasts in vitro

2010 ◽  
Vol 23 (06) ◽  
pp. 417-423 ◽  
Author(s):  
J. M. Cissell ◽  
S. C. Milton ◽  
L. A. Dahlgren
Keyword(s):  
Gene Expression ◽  
Matrix Protein ◽  
Collagen Type ◽  
Flexor Tendon ◽  
Cell Metabolism ◽  
Collagen Type I ◽  
Type I ◽  
Matrix Metalloproteinase 1 ◽  
Superficial Digital Flexor Tendon

Summary Objectives: To evaluate the effects of pros-taglandin E2 (PGE2) treatment on the metabolism of equine tendon fibroblasts in vitro to aid in investigating the response of tendon fibroblasts to injury and novel therapeutics. Methods: Superficial digital flexor tendon fibroblasts isolated via collagenase digestion from six young adult horses were grown in monolayer in four concentrations of PGE2 (0, 10, 50, 100 ng/ml) for 48 hours. Cells and medium were harvested for gene expression (collagen types I and III, cartilage oligomeric matrix protein [COMP], decorin, and matrix metalloproteinase-1, –3, and –13), biochemical analysis (glycosaminoglycan, DNA, and collagen content), and cytological staining. Results: Gene expression for collagen type I was significantly increased at 100 ng/ml PGE2 compared to 10 and 50 ng/ml. There were not any significant differences detected for gene expression of collagen type III, COMP or dec-orin or for biochemical content and cell morphology. Clinical significance: Under the conditions investigated, exogenous treatment of equine tendon fibroblasts with PGE2 failed to alter cell metabolism in a manner useful as a model of tendon injury. A model that applies cyclic strain to a three dimensional construct seeded with tendon fibroblasts may prove to be a more useful model and merits further investigation for this purpose. The ability to assess cellular responses in an environment where the cells are supported within the extracellular matrix may prove beneficial.

scholarly journals Cell-Free Demineralized Bone Matrix for Mesenchymal Stem Cells Survival and Colonization

Materials ◽  
2019 ◽  
Vol 12 (9) ◽  
pp. 1360 ◽  
Author(s):  
Monica Mattioli-Belmonte ◽  
Francesca Montemurro ◽  
Caterina Licini ◽  
Iolanda Iezzi ◽  
Manuela Dicarlo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Tissue Regeneration ◽  
Demineralized Bone Matrix ◽  
Bone Matrix ◽  
Morphological Observation ◽  
Type I ◽  
Demineralized Bone

Decellularized bone matrix is receiving much attention as biological scaffolds and implantable biomaterials for bone tissue regeneration. Here, we evaluated the efficacy of a cell-free demineralized bone matrix on mesenchymal stem cells (MSCs) survival and differentiation in vitro. The seeding of human umbilical cord-derived MSCs (hUC-SCs) on decellularized bone matrices up to 14 days was exploited, assessing their capability of scaffold colonization and evaluating gene expression of bone markers. Light and Scanning Electron Microscopies were used. The obtained cell-free decalcified structures showed elastic moduli attributable to both topology and biochemical composition. Morphological observation evidenced an almost complete colonization of the scaffolds after 14 days of culture. Moreover, in hUC-SCs cultured on decalcified scaffolds, without the addition of any osteoinductive media, there was an upregulation of Collagen Type I (COL1) and osteonectin (ON) gene expression, especially on day 14. Modifications in the expression of genes engaged in stemness were also detected. In conclusion, the proposed decellularized bone matrix can induce the in vitro hUC-SCs differentiation and has the potential to be tested for in in vivo tissue regeneration.

Alveolar macrophages from systemic sclerosis patients: evidence for IL-4-mediated phenotype changes

2004 ◽  
Vol 286 (6) ◽  
pp. L1202-L1209 ◽  
Author(s):  
Raymond F. Hamilton ◽  
Ed Parsley ◽  
Andrij Holian
Keyword(s):  
Systemic Sclerosis ◽  
Alveolar Macrophages ◽  
Lung Inflammation ◽  
Costimulatory Molecule ◽  
Normal Volunteer ◽  
Macrophage Phenotype ◽  
Histocompatibility Complex ◽  
Ifn Γ ◽  
Lung Lobes

The mechanism of chronic lung inflammation leading to lung fibrosis is unknown and does not have a characteristic inflammatory macrophage phenotype. This study was undertaken to determine whether a change in macrophage phenotype could account for chronic lung inflammation. In this study, human alveolar macrophages (AM) from subjects with systemic sclerosis (SSc) were obtained from bronchoalveolar lavage (BAL) and characterized on the basis of function (response to LPS), phenotype, and relative cell-surface B7 expression. AM from the subjects' disease-involved and noninvolved lung lobes were compared with each other and to AM from normal volunteer BAL. AM from involved SSc lobes produced significantly more interleukin (IL)-1β and PGE2 than AM from uninvolved lobes in response to LPS, but there was no spontaneous production of either mediator. The activator AM phenotype designated by RFD1+ surface epitope was significantly elevated in SSc BAL samples compared with normal BAL, although there were no differences comparing involved vs. noninvolved lobes within SSc subjects. The major histocompatibility complex II costimulatory molecule B7.2 was also significantly elevated in SSc AM compared with normal AM, again with no differences between involved and noninvolved lobes. In an attempt to determine environmental influences on AM phenotypes, normal AM were cultured in vitro with IFN-γ, IL-3, IL-4, IL-10, IL-12, or dexamethasone for 6 days. Of the cytokines examined, only IL-4 induced significant increases in both the activator phenotype RFD1+ and B7.2 expression. Taken together, these results indicate that IL-4 could account for proinflammatory AM phenotype changes and B7 surface-marker shifts, as seen in subjects with SSc.

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