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  • The IL1-like cytokine IL33 and its receptor ST2 are abnormally expressed in the affected skin and visceral organs of patients with systemic sclerosis

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Basic and translational research
Extended report
The IL1-like cytokine IL33 and its receptor ST2 are abnormally expressed in the affected skin and visceral organs of patients with systemic sclerosis
  1. Mirko Manetti1,2,
  2. Lidia Ibba-Manneschi1,
  3. Vasiliki Liakouli3,
  4. Serena Guiducci2,
  5. Anna Franca Milia2,
  6. Gemma Benelli1,
  7. Alessandra Marrelli3,
  8. Maria Letizia Conforti2,
  9. Eloisa Romano2,
  10. Roberto Giacomelli3,
  11. Marco Matucci-Cerinic2,
  12. Paola Cipriani3
  1. 1Department of Anatomy, Histology and Forensic Medicine, University of Florence, Florence, Italy
  2. 2Department of Biomedicine, Division of Rheumatology, AOUC, and Excellence Centre for Research, Transfer and High Education DENOthe, University of Florence, Florence, Italy
  3. 3Department of Internal Medicine and Public Health, Division of Rheumatology, University of L’Aquila, L’Aquila, Italy
  1. Correspondence to Professor L Ibba-Manneschi, Department of Anatomy, Histology and Forensic Medicine, University of Florence, Viale G B Morgagni 85, 50134 Florence, Italy; ibba{at}unifi.it

Abstract

Background Early endothelial cell (EC) activation/damage and profibrotic Th2-associated cytokines play a pivotal role in systemic sclerosis (SSc). Interleukin 33 (IL33) is a novel member of the IL1 family that promotes Th2 responses and inflammation through the ST2 receptor. IL33 is also a chromatin-associated transcriptional regulator in ECs.

Objective To investigate the role of the IL33/ST2 axis in SSc.

Methods Skin biopsies were obtained from 30 patients with SSc (15 early/15 late stage) and 10 healthy subjects. Lung, kidney, heart, oesophagus, stomach, placenta biopsies and bronchoalveolar lavage cells from patients with SSc and controls were also analysed. IL33/ST2 expression was investigated by immunohistology, confocal immunofluorescence microscopy, western blotting and RT-PCR.

Results In skin biopsies from control subjects, constitutive nuclear IL33 protein expression was found in dermal ECs and keratinocytes, while ST2 was weakly expressed in ECs and fibroblasts. In skin biopsies from patients with early SSc, IL33 protein was downregulated or absent in ECs and epidermis while IL33 mRNA was normally expressed or even upregulated. Moreover, ECs, perivascular infiltrating mast cells, CD68-positive macrophages, CD3-positive T cells, CD20-positive B cells and activated fibroblasts/myofibroblasts exhibited strong ST2 expression. In skin biopsies from patients with late SSc, IL33 was constitutively found in most ECs while ST2 immunostaining was weaker. In early SSc, the loss of endothelial IL33 protein and the overexpression of ST2 involved all affected organs. Dermal and pulmonary fibroblasts showed IL33 expression in SSc.

Conclusion IL33 and ST2 are abnormally expressed in SSc. In early SSc, upon EC activation/damage IL33 may be mobilised from ECs to signal through ST2 in key profibrotic players such as inflammatory/immune cells and fibroblasts/myofibroblasts.

https://doi.org/10.1136/ard.2009.119321

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    Introduction

    Although fibrosis is the main pathological hallmark of systemic sclerosis (SSc; scleroderma), endothelial cell (EC) activation/damage, inflammation and widespread small vessel vasculopathy characteristically precede the excessive synthesis and deposition of extracellular matrix that ultimately leads to dysfunction of the skin and several internal organs including the lung, heart, kidney and gastrointestinal tract.1,–,5 In SSc, fibrosis results from a complex interplay among ECs, inflammatory/immune cells and fibroblasts through a number of soluble mediators.1 2 The resulting cellular microenvironment leads to a constitutive activation of fibroblasts which transdifferentiate into contractile myofibroblasts, produce large amounts of collagens, fibronectins and proteoglycans, and secrete growth factors and fibrogenic cytokines that perpetuate the fibrotic process.1 2 Furthermore, an altered balance between T helper type (Th)1 and Th2 cytokines towards a Th2-polarised immune response plays a pivotal role in SSc.6 Indeed, several growth factors, profibrotic Th2-associated cytokines and chemokines have been implicated in the pathogenesis of SSc including transforming growth factor β, platelet-derived growth factor, connective tissue growth factor, interleukin 4 (IL4), IL13, monocyte chemoattractant protein-1 and endothelin-1.1 2 6,–,8

    IL33 (previously known as nuclear factor from high endothelial venules (NF-HEV) and IL1F11) is the most recent addition to the IL1 family of proinflammatory cytokines that also includes IL1α, IL1β and IL18.9 10 Clinical and experimental evidence indicates that IL33 may be a promising therapeutic target for a variety of human diseases including asthma and other allergic conditions, rheumatoid arthritis (RA), atherosclerosis, cardiovascular diseases and liver fibrosis.10,–,15 It has been shown to signal through the IL1 receptor-related protein ST2 (also known as IL1RL1, T1 or IL1R4) and to drive production of proinflammatory and Th2-associated cytokines and chemokines in mast cells, Th2 lymphocytes, basophils, eosinophils, invariant natural killer T cells and natural killer cells.9 16,–,19 IL33 is a chemoattractant for human Th2 cells, and treatment of mice with IL33 resulted in an increase in IL4, IL5 and IL13 production, thus suppressing Th1 cell differentiation and favouring Th2 cell differentiation.9 13 20 Like IL1 signalling, IL33 requires receptor heterodimerisation with IL1 receptor accessory protein (IL1RAcP) to activate nuclear factor (NF)-κB and mitogen-activated protein kinase pathways in target cells expressing its specific receptor ST2.10 17

    A number of studies have indicated that IL33 is constitutively highly expressed in the nucleus of ECs in most healthy human tissues, and that nuclear IL33 has transcriptional regulatory properties and associates with chromatin in vivo.21,–,23 Together, these findings suggest that, similarly to IL1α and chromatin-associated cytokine high mobility group box 1, IL33 is a dual-function protein that may play an important role as both a cytokine and an intracellular nuclear factor.10 Indeed, IL33 has been proposed to act as a transcriptional repressor involved in the control of EC activation, as well as an endogenous “danger signal” or “alarmin” which is rapidly released after EC activation/damage to alert the cells of the immune system.21,–,23 In addition to the endothelium, constitutive nuclear expression of IL33 is found in epithelial cells of tissues exposed to the environment, including skin keratinocytes and epithelial cells of the gastrointestinal tract, as well as in fibroblastic reticular cells of lymphoid tissues which exhibit myofibroblast features.21 Interestingly, IL33 expression was biomechanically induced in rat cardiac fibroblasts during their transdifferentiation into myofibroblasts,14 and was also induced by proinflammatory stimuli in dermal fibroblasts and RA synovial fibroblasts.9 24 Thus, specific expression in activated fibroblasts and myofibroblasts suggests that IL33 may be involved in connective tissue remodelling after injury and fibrosis, processes in which both fibroblast activation and myofibroblast differentiation play critical roles.25

    In view of these findings, we hypothesised that the IL33/ST2 axis could be involved in the complex intercellular cross-talk among ECs, inflammatory/immune cells, fibroblasts and myofibroblasts in SSc. We therefore investigated the expression of IL33 and its receptor ST2 in a wide array of diseased tissues from patients with SSc.

    Methods

    Patients, controls and skin biopsies

    Full-thickness skin biopsies were obtained from the clinically involved skin of one-third of the distal forearm of 30 patients with SSc (26 women). Patients were classified as having limited cutaneous SSc (lcSSc; n = 12) or diffuse cutaneous SSc (dcSSc; n = 18).3 Patients were further classified as being in the early (n = 15, 5 lcSSc, 10 dcSSc) or late (n = 15, 7 lcSSc, 8 dcSSc) phase of SSc according to disease duration (early lcSSc, disease duration <5 years; early dcSSc, disease duration <3 years) and to the clinical and pathological stage of skin involvement.26 Skin samples from the same forearm region of 10 age- and sex-matched healthy donors were obtained as controls. Each skin biopsy was divided into two specimens and processed for immunohistochemistry and biomolecular analyses, respectively. For immunohistochemistry, the specimens were fixed in 10% buffered formalin, dehydrated in graded alcohol series and embedded in paraffin. For protein and RNA extraction, the specimens were immediately immersed in liquid nitrogen and stored at −80°C until use. All patients with SSc underwent a 15-day treatment washout before skin biopsy was performed. During this period only proton pump inhibitors and clebopride were allowed. Patients who could not undergo washout due to severe organ complications were not enrolled in the study. Biopsies were taken with full informed consent and the study was approved by the Institutional Review Board.

    Visceral organ biopsies

    Paraffin-embedded sections from lung, heart, oesophagus and kidney were obtained from full-thickness autopsy samples from two subjects who died from early severe dcSSc with rapidly progressive disease. Full-thickness surgical samples of the gastric wall of four patients with SSc (one late lcSSc, two early dcSSc, one late dcSSc) with severe gastro-oesophageal involvement were also examined. Lung biopsies displayed the typical features of non-specific interstitial pneumonitis and SSc-associated pulmonary fibrosis. The histopathological examination of all organs showed the typical fibrotic changes of SSc. As healthy control tissues, full-thickness samples were obtained from autopsies of patients who underwent surgery for neoplastic pathologies. We carefully selected healthy specimens that appeared to have no inflammatory/neoplastic infiltration according to histopathological examination. Placenta biopsies were obtained after delivery from three women with SSc (two lcSSc, one dcSSc) and four healthy uncomplicated pregnancies matched to SSc cases for gestational age, as described previously.27 All specimens were fixed in formalin and embedded in paraffin. Each subject gave written informed consent to participate in the study.

    Bronchoalveolar lavage cell collection and processing

    Bronchoalveolar lavage (BAL) was performed in the pulmonary lobes that showed interstitial lung disease at high-resolution CT in 10 non-smoking patients with SSc (2 lcSSc, 8 dcSSc) and in 10 non-smoking healthy subjects, as described previously.28 Briefly, BAL fluid was recovered by gentle aspiration and collected into specimen traps, filtered through sterile gauze and centrifuged at 400g for 15 min at 4°C. The pellet was washed with cold phosphate buffered saline (PBS) and slide preparation for cell analysis was made in a Shandon cytocentrifuge (Cytospin II; Shandon Ltd, Runcorn, UK) using 100 µl aliquots of the lavage cell suspension. After fixation in 3.7% buffered paraformaldehyde, cells were processed for immunofluorescence analysis.

    Antibodies

    Mouse monoclonal antibody (mAb) anti-IL33 (clone Nessy-1, 1:50 dilution; Alexis Biochemicals, San Diego, California, USA) and rabbit polyclonal antibody anti-ST2 (IL1RL1/ST2L, 1:100 dilution; Atlas Antibodies, AlbaNova University Center, Stockholm, Sweden) were used as primary antibodies in single and double immunostainings. Mouse mAbs against CD31/pan-EC marker (1:20 dilution; Dako, Hamburg, Germany), podoplanin/lymphatic EC marker (D2-40, 1:50 dilution; Dako), CD45/leucocyte common antigen (1:100 dilution; Dako), CD3/T cell marker (1:10 dilution; AbD Serotec, Oxford, UK), CD20/B cell marker (1:20 dilution; Dako), CD68/macrophage marker (1:50 dilution; Dako), α-smooth muscle actin (α-SMA)/myofibroblast marker (1:50 dilution; Abcam, Cambridge, UK) and rabbit polyclonal antibody anti-CD31 (1:100 dilution; Abcam) were used in double staining experiments.

    Histopathology and immunohistochemistry

    Paraffin sections (5 µm thick) were deparaffinised and either stained with haematoxylin and eosin (H&E) for routine histology or processed for immunohistochemistry. H&amp;E-stained tissue sections were carefully examined by an experienced observer who was blind to sample classification. Immunohistochemistry was performed on serial sections using an indirect immunoperoxidase method as described in the online supplement.

    Confocal laser immunofluorescence microscopy

    Tissue sections were boiled for epitope retrieval in citrate buffer (10 mM, pH 6.0) and blocked for 1 h with 1% bovine serum albumin in PBS (PBS-BSA). Primary antibodies were diluted in PBS-BSA and incubated overnight at 4°C. After extensive washing in PBS, slides were incubated with secondary antibodies for 45 min at room temperature in the dark. The immune reactions were revealed using Alexa Fluor-488-conjugated goat anti-rabbit IgG or Rhodamine Red-X-conjugated goat anti-mouse IgG (1:200 dilution; Molecular Probes, Eugene, Oregon, USA) as secondary antibodies. Double immunostainings with mouse and rabbit reagents were performed by mixing primary antibodies and subsequently mixing fluorochrome-conjugated reagents. Irrelevant isotype- and concentration-matched IgG (Sigma, St Louis, Missouri, USA) were used as negative controls. Cross-reactivity of secondary antibodies was tested in control experiments in which primary antibodies were omitted. Tissue sections and BAL cells were examined with a Leica TCS SP5 confocal laser scanning microscope (Leica Microsystems, Mannheim, Germany) equipped with a Leica PlanApo ×63 oil immersion objective and a HeNe/Argon laser source for fluorescence measurements and differential interference contrast optics for transmission images. Series of optical sections (1024×1024 pixels each) at intervals of 0.4 µm were obtained and superimposed to create a single composite image. Immunostaining was evaluated by an experienced and blinded observer. Slides were examined for cellular immunoreactivity and cell types were distinguished based on their positivity for cell-specific markers and/or their characteristic morphology. For IL33 staining in skin samples, the percentage of positive ECs was calculated semiquantitatively as IL33- and CD31-double positive cells in proportion to all CD31-positive cells. Cell counting was performed on three randomly chosen microscopic fields from three sections per sample by two independent blinded observers and the mean was used for analysis. Densitometric analysis of the intensity of immunofluorescent staining of ST2 was performed on digitised images using the free-share ImageJ software (NIH, Bethesda, Maryland, USA; online at http://rsbweb.nih.gov/ij). Data were analysed using the Student t test assuming equal variances. p Values <0.05 were considered statistically significant. Data are represented as mean±SEM.

    Western blotting

    Protein extraction from skin specimens, western blotting of IL33 and ST2 and statistical analysis of the data were performed as described in the online supplement.

    RT-PCR

    IL33 transcript levels in skin biopsies were determined by RNA purification and semiquantitative RT-PCR analysis as described in the online supplement.

    Results

    IL33 and ST2 expression in skin biopsies from patients with SSc

    Our immunohistological results show that, in healthy skin, epidermal keratinocytes and ECs of dermal vessels constitutively express nuclear IL33 protein (figure 1A,C). In skin specimens from patients with early SSc, IL33 expression was strongly decreased or absent in the nuclei of most ECs in the papillary and reticular dermis, as well as in keratinocytes (figure 1B,D). In contrast, the expression of IL33 was mostly preserved in both keratinocytes and ECs of the few remaining vessels in skin from patients with late-stage SSc (figure 1E). Similar results were found both in lcSSc and dcSSc groups, which were equally balanced between early and late disease. As shown in figure 1F, semiquantitative analysis of IL33 expression on IL33/CD31 double immunostained skin sections showed that about 100% of ECs expressed nuclear IL33 in controls, while the percentage of IL33-immunopositive ECs was significantly decreased in skin from patients with early SSc compared with skin from controls and patients with late SSc (p<0.05). Moreover, in skin from patients with early SSc we observed that IL33 mobilisation from EC nuclei involved mainly microvessels, whereas IL33 expression was preserved in ECs of large vessels (figure 1G). Interestingly, we found that dermal fibroblasts were immunopositive for IL33 in skin from patients with SSc (figure 1H). In contrast, none of the control skin specimens showed IL33 expression in fibroblasts.

    Figure 1
    Figure 1

    Immunohistological and confocal laser immunofluorescence analysis of interleukin 33 (IL33) in the skin of healthy controls and patients with systemic sclerosis (SSc). (A,B) Immunoperoxidase staining of IL33 in skin from (A) controls and (B) patients with SSc. Keratinocytes and dermal endothelial cells (ECs) (inset) constitutively express nuclear IL33 in healthy skin (A). In skin from patients with early SSc, IL33 expression is strongly decreased or absent in the nuclei of most dermal ECs (black arrows in inset) and in keratinocytes (black asterisk) (B). Original magnification ×10 (×40 in insets). (C–E) Representative microphotographs of double immunostaining of IL33 (red) and pan-EC marker CD31 (green) in skin from (C) controls and patients with SSc (D,E). In skin from patients with early SSc, many ECs in the papillary and reticular dermis (white arrows) and keratinocytes (white asterisk) lack nuclear IL33 expression (D). In skin from patients with late SSc, IL33 expression is overall preserved in keratinocytes and ECs of the few remaining vessels (E). Original magnification ×63. (F) Semiquantitative analysis of IL33 expression in skin sections. The percentage of ECs immunopositive for IL33 is significantly decreased in skin from patients with early SSc compared with skin from control subjects and those with late SSc (p<0.05, Student t test); n, number of patients. (G,H) Double immunostaining of IL33 (red) and CD31 (green) in skin from patients with SSc. In skin from patients with early SSc, IL33 expression is preserved in ECs of large vessels (G). Dermal fibroblasts are immunopositive for IL33 in skin from patients with SSc (white arrows, H). Original magnification ×63. (I,J) Total protein extracts of skin from healthy subjects and patients with SSc were analysed by western blotting with anti-IL33 antibodies. Blots were stripped and reprobed with anti-α-tubulin antibodies as a loading control for normalisation. Numbers on the right indicate molecular weight (kDa) (I). Bars represent the intensity of the bands, quantified by densitometry (mean ± SEM) (J). OD, optical density (arbitrary units). *p<0.05 for the comparison with control and late SSc (ANOVA and Tukey's w-test). (K) RT-PCR of IL33 in skin from control subjects and patients with SSc. β-Actin served as the control for normalisation. Numbers on the right represent the size (bp) of each RT-PCR product. The results of a representative experiment are shown.

    Using western blotting, IL33 expression levels were evaluated in crude protein extracts from whole skin samples. The anti-human IL33 antibodies detected a single protein band with the expected molecular weight of 31 kDa (figure 1I).These results paralleled those obtained by immunohistology. Indeed, in skin from patients with early SSc there was a statistically significant decrease in IL33 protein expression levels compared with skin from healthy subjects and those with late SSc (p<0.05, figure 1I,J). Furthermore, IL33 protein expression was decreased in skin from patients with late SSc compared with skin from control subjects (figure 1I,J). On the contrary, RT-PCR analysis revealed that in skin from patients with early SSc, IL33 mRNA levels were similar to or even higher than in skin from controls and patients with late SSc (figure 1K).

    We next examined the expression of ST2 protein. In control skin samples we found that ST2 was weakly expressed in dermal capillary ECs and fibroblasts (figure 2A). In contrast, in skin samples from patients with early SSc we observed a strong ST2 immunopositivity in both dermal ECs and fibroblasts (figure 2B). In addition, infiltrating inflammatory cells and α-SMA-positive myofibroblasts exhibited strong ST2 expression (figure 2B). ST2 immunostaining was weaker in skin from patients with late SSc than in skin from those with early SSc (figure 2C). The results of semiquantitative analysis of ST2 immunofluorescent staining intensity in skin sections showed that ST2 protein expression was significantly increased in the early stage of SSc (p<0.05; figure 2D). Western blotting analysis confirmed these results (figure 2E). As for IL33, no significant difference was found between lcSSc and dcSSc subsets.

    Figure 2
    Figure 2

    ST2 expression in the skin of healthy controls and patients with systemic sclerosis (SSc). (A-C) Representative microphotographs of double immunofluorescence staining of ST2 (green) and α-smooth muscle actin (α-SMA, red) in skin from (A) controls and (B,C) patients with SSc. (A) In skin from control subjects, ST2 is weakly expressed in dermal endothelial cells (ECs) and fibroblasts. (B) In skin from patients with early SSc, a strong immunopositivity for ST2 is evident in dermal ECs lining the capillary vessel lumens, fibroblasts, perivascular infiltrating inflammatory cells and α-SMA-positive stromal and perivascular myofibroblasts (arrows). (C) In skin from patients with late SSc, ST2 immunostaining is weaker than in early SSc. In all skin sections pericytes show α-SMA-positivity. Original magnification ×63. (D) Densitometric analysis of the intensity of ST2 immunofluorescent staining on digitised images of skin sections. Bars represent the mean±SEM optical density (au, arbitrary units) of skin samples from 10 controls, 15 patients with early SSc and 15 with late SSc. *p<0.05 for the comparison with control and late SSc (Student t test). (E) Total protein extracts of skin from control subjects and patients with SSc were analysed by western blotting with anti-ST2 antibodies. Blots were stripped and reprobed with anti-α-tubulin antibodies as a loading control for normalisation. Bars represent the intensity of the bands quantified by densitometry (mean ± SEM). OD, optical density (arbitrary units). *p<0.05 for the comparison with control and late SSc (ANOVA and Tukey's w-test).

    Using double IL33/ST2 immunostaining we observed that, in skin from control subjects, ECs constitutively expressed nuclear IL33 but weakly expressed ST2 while, in skin from patients with early SSc, capillary vessels showing weak or absent IL33 expression displayed strong ST2-immunopositivity (figure 3A,B). Furthermore, in patients with early SSc, ST2 was markedly overexpressed in both blood microvascular (CD31-positive/D2-40-negative) ECs and lymphatic (CD31-positive/D2-40-positive) ECs compared with skin from patients with late SSc and controls (figure 3C–F).

    Figure 3
    Figure 3

    (A,B) Double immunofluorescence analysis of interleukin-33 (IL33) (red) and ST2 (green) in the skin of healthy controls and patients with systemic sclerosis (SSc). In skin from control subjects, endothelial cells (ECs) constitutively express nuclear IL33 but weakly express the ST2 receptor (A). In skin from patients with early SSc, ECs showing weak or absent IL33 expression (arrows) display strong ST2 immunopositivity (B). (C–F) Double immunofluorescent stainings of ST2 (green) and CD31 (red) (C) or D2-40 (red) (D–F) in skin sections. In skin from patients with early SSc a marked colocalisation of ST2 with CD31 (C) and podoplanin/D2-40 (D) gives rise to yellow staining. ST2 is strongly expressed in both blood microvascular (D2-40-negative) ECs (arrowheads, D) and lymphatic (D2-40-positive) ECs (arrow, D) in early SSc. Note the strong ST2 immunopositivity in the perivascular inflammatory infiltrate (asterisk, C,D). The blue stain in (C) and (D) indicates autofluorescent erythrocytes within the lumen of capillary vessels. In skin from patients with late SSc, ST2 is weakly expressed in the few remaining blood (arrowheads, E) and lymphatic (arrow, E) microvessels. Weak immunopositivity for ST2 is also evident in blood ECs (arrowhead, F) and lymphatic ECs (arrows, F) of skin from control subjects. Original magnification ×63.

    In order to further characterise the nature of ST2-expressing cells in skin from patients with early SSc, double immunofluorescence analyses were performed using antibodies against inflammatory/immune cell-specific markers. Strong immunopositivity for ST2 was found in different perivascular infiltrating inflammatory/immune cells, including mast cells, eosinophils, CD68-positive macrophages, CD3-positive T cells and CD20-positive B cells (figure 4).

    Figure 4
    Figure 4

    Expression of ST2 in different perivascular infiltrating inflammatory/immune cells in skin samples from patients with early systemic sclerosis (SSc). Double immunofluorescence analysis was performed using anti-ST2 antibodies (green) and antibodies against mononuclear cell-specific markers (red). ST2 is strongly expressed in CD45-positive leucocytes (A), including eosinophils and mast cells (arrows), CD3-positive T lymphocytes (B), CD68-positive macrophages (C) and also in a CD20-positive B lymphocyte present in the perivascular inflammatory infiltrate (arrow, D). Colocalisation of green- and red-labelled markers gives rise to yellow staining. The blue stain in (C) indicates autofluorescent erythrocytes within the lumen of capillary vessels. Original magnification ×63.

    IL33 and ST2 expression in visceral organs of patients with SSc

    We then investigated the expression of IL33 and ST2 in a wide array of visceral organs which are affected by the fibrotic process in SSc including lung, kidney, heart, gastrointestinal tract and placenta.1 3,–,5 27 Lung, kidney, heart, and oesophageal autopsy samples were obtained from two subjects with early severe dcSSc with rapidly progressive disease.

    Affected lung tissues from patients with SSc-related pulmonary fibrosis had decreased or completely absent nuclear IL33 expression in ECs compared with normal lungs (figure 5A,B). Instead, ST2 expression was markedly increased in ECs, inflammatory cells, alveolar macrophages and fibroblasts in the thickened alveolar septa, which are typical of SSc alveolitis (figure 5B). Similarly, ST2 was overexpressed in inflammatory cells, alveolar macrophages and fibroblasts from the BAL fluid of patients with SSc compared with healthy controls (figure 5C,D). Like the findings in the skin, IL33 expression was observed in stromal fibroblasts in SSc lung (figure 5B).

    Figure 5
    Figure 5

    (A,B) Double immunofluorescence analysis of interleukin 33 (IL33) (red) and ST2 (green) in lung sections from healthy controls and patients with early severe diffuse cutaneous systemic sclerosis (dcSSc) with rapidly progressive disease. In lung tissue from control subjects, IL33 immunopositivity is constitutively present in endothelial cells (ECs) while ST2 is weakly expressed (A). In lung tissue from patients with SSc-related pulmonary fibrosis, IL33 expression is not detected in ECs (arrows, B). In the thickened alveolar septa of lung tissue from patients with SSc, ST2 expression is markedly increased in ECs of capillary vessels, inflammatory cells, alveolar macrophages and fibroblasts (asterisks, B). Some stromal fibroblasts are immunopositive for IL33 in lung tissue from patients with SSc (arrowheads, B). (C,D)ST2 is overexpressed in inflammatory cells, alveolar macrophages and fibroblasts collected from the bronchoalveolar lavage (BAL) fluid of patients with SSc (D) compared with controls (C). The purple stain in (A) indicates autofluorescent erythrocytes. Original magnification ×63.

    In the fibrotic kidney of patients with SSc, nuclear IL33 expression was almost absent in ECs of peritubular capillaries while ST2 was strongly expressed in kidney glomeruli, tubuli and peritubular capillaries (figure 6B). In contrast, control kidney specimens displayed constitutive expression of IL33 in peritubular capillary ECs and weak or no immunopositivity for ST2 (figure 6A). Consistent with previous studies,21 IL33 was not detected in kidney glomeruli in any of the specimens analysed.

    Figure 6
    Figure 6

    Expression of interleukin 33 (IL33) (red) and ST2 (green) in kidney (A,B), heart (C), oesophagus (D), stomach (E,F) and placenta (G,H). Kidney, heart, and oesophageal biopsies were obtained from patients with early severe diffuse cutaneous systemic sclerosis (dcSSc) with rapidly progressive disease. Control kidney specimens show immunopositivity for IL33 in peritubular capillary endothelial cells (ECs) while ST2 immunopositivity is almost undetectable (A). In fibrotic kidney from patients with SSc, IL33 expression is absent in ECs of peritubular capillaries while ST2 is strongly expressed in kidney glomeruli, tubuli and peritubular capillaries (B). In patients with SSc, fibrotic myocardium (C) and oesophagus (D) show strong ST2 expression whereas IL33 is almost undetectable. In gastric wall specimens from control subjects (E), IL33 is constitutively expressed in epithelial cells of the mucosal surface and gastric glands as well as in ECs (inset in E), while ST2 is weakly expressed. In gastric wall specimens from patients with SSc with early severe gastroesophageal involvement (F), ST2 expression is evident in inflammatory infiltrates while IL33 immunopositivity is absent in most ECs and epithelial cells. In contrast, IL33 expression is present in ECs and epithelial cells in gastric wall specimens from patients with SSc with longstanding disease (inset in F). ST2 expression is strongly increased in the trophoblast (asterisk), stromal cells and capillary vessels (arrows) within the chorionic villi of placentas from patients with SSc (H) compared with control subjects (G). The purple/blue stain in (C–H) indicates autofluorescent erythrocytes. Original magnification ×63.

    ST2 was also abundantly expressed in fibrotic myocardium as well as in fibrotic oesophageal submucosa and muscle layers of patients with SSc, while IL33 expression in ECs was almost undetectable in both organs (figure 6C,D). In contrast, constitutive IL33 expression in ECs and weak ST2 immunostaining were found in control heart and oesophagus (data not shown).

    Strong ST2 expression was observed in inflammatory infiltrates of surgical gastric wall specimens from patients with SSc with early severe gastro-oesophageal involvement, whereas IL33 was almost absent in ECs and epithelial cells of the mucosal surface and gastric glands compared with controls (figure 6E,F). Instead, IL33 expression was constitutively found in EC nuclei in the gastric wall of patients with SSc with longstanding disease (figure 6F, inset).

    Finally, ST2 expression was strongly increased in the trophoblast, stromal cells and capillary vessels within the chorionic villi of placentas from women with SSc compared with healthy control placentas (figure 6G,H).

    Discussion

    Our results clearly show that dermal ECs in skin from patients with early SSc have decreased or completely absent nuclear IL33 protein expression. Instead, IL33 mRNA levels were similar to or even higher than in skin from controls which suggests that, although normally synthesised, IL33 protein is mobilised from ECs in early disease stage. IL33, which is constitutively expressed to high levels by resting ECs in most healthy human tissues, has been shown to be rapidly released in the extracellular space upon proinflammatory activation as well as after EC damage or mechanical injury.22 29 Our findings therefore suggest that, in the early stage of SSc, upon EC activation/damage IL33 is released by EC nuclei functioning as an endogenous “danger signal/alarmin”.21 29 Furthermore, a recent study has shown that nuclear IL33 is strongly downregulated in endothelium undergoing tumour or wound healing angiogenesis, and that tumour necrosis factor-α and vascular endothelial growth factor (VEGF) stimulate IL33 release in vivo.22 Despite the lack of evidence of significant angiogenesis, the levels of VEGF are strongly increased in the serum and skin of patients with SSc30 31 and therefore could contribute to IL33 mobilisation from ECs in early disease stage. After release, IL33 might function through the activation of downstream signalling in different ST2-expressing cell types and the recruitment of inflammatory/immune cells. Indeed, in skin from patients with early SSc, ST2 expression is strongly increased in dermal ECs and fibroblasts. Our double immunostaining experiments also show that ST2 is strongly expressed in α-SMA-positive myofibroblasts as well as in perivascular infiltrating cells of both the innate and adaptive immune system including mast cells, eosinophils, macrophages, T and B lymphocytes. It has recently been shown that IL33 acts as a Th2 cell chemoattractant and induces Th2 cell differentiation and production of IL4, IL5 and IL13 profibrotic cytokines.9 20 32 Moreover, IL33 also induces maturation and proinflammatory cytokine production in mast cells and degranulation and survival of eosinophils, which have previously been shown to be activated in SSc.16,–,18 33,–,35

    It is well known that microvessels, more than large vessels, are mainly involved in the early phase of SSc.36 Consistent with these findings we observed that, in skin from patients with early SSc, nuclear IL33 downregulation in ECs involved mainly microvessels whereas IL33 expression was preserved in large vessels. It must be pointed out that constitutive IL33 expression in the few remaining vessels of skin from patients with late SSc could be explained by its possible role in maintaining resting ECs in advanced disease.

    In addition, we found decreased or absent IL33 expression in skin keratinocytes from patients with early SSc as well as in epithelial cells of the gastric mucosa of patients with SSc, confirming that, as recently proposed,21 29 IL33 might also play an important role as epithelial “alarmin” in tissues exposed to the environment.

    Our findings also indicate that the loss of nuclear IL33 expression in microvascular ECs and the overexpression of ST2 in early disease affect not only the skin, but all targeted organs including the lung, kidney, heart, oesophagus, stomach and placenta. In affected lung tissue and in cells from the BAL fluid of patients with SSc with interstitial lung disease, a markedly increased expression of ST2 was detected. Previous studies have shown that ST2 protein induced by proinflammatory cytokines may modulate lung inflammation and fibrosis.37 38 In the bleomycin-induced pulmonary fibrosis mouse model, ST2 expression gradually increases in lung tissue concurrently with several Th2-associated cytokines including transforming growth factor β.38 It has recently been shown that ST2 expression is increased and correlates with collagen expression in mouse and human fibrotic liver.15 In a mouse model of hepatic fibrosis, injection of the fusion protein ST2-Fc was shown to increase Th2 cytokine secretion and to enhance hepatic fibrosis.39 Furthermore, patients with an acute exacerbation of idiopathic pulmonary fibrosis have increased serum levels of soluble ST2.40 Similar results have also been found in the serum of patients with systemic lupus erythematosus, RA, Wegener's granulomatosis and Behcet's disease,41 suggesting a role for the IL33/ST2 system in a wide array of autoimmune diseases.

    In agreement with our findings, previous studies have shown that IL33 expression is restricted to ECs and keratinocytes in healthy human skin.21 22 Interestingly, in the skin of our patients with SSc, dermal fibroblasts—which are known to be activated in SSc—express IL33. Moreover, we observed IL33 expression in pulmonary fibroblasts from patients with SSc. Our findings confirm previous studies showing IL33 expression in fibroblastic cells upon activation, such as activated dermal fibroblasts,9 RA synovial fibroblasts,12 24 rat cardiac fibroblasts differentiating into myofibroblasts14 and activated hepatic stellate cells in chronic liver fibrosis.15 Thus, our observation further supports the possible implication of IL33 in connective tissue remodelling and fibrosis.

    Finally, recent evidence indicates that IL33 may be a substrate for the proapoptotic caspase-329 and that the IL33/ST2 axis promotes angiogenesis and endothelial permeability.42 Since EC apoptosis occurs early in SSc and is followed by impaired angiogenesis,36 43 44 the role of IL33 and ST2 in SSc-associated vasculopathy deserves further investigation.

    This study shows that, in the early stage of SSc, following EC activation/damage, IL33 and its specific receptor ST2 are abnormally expressed in targeted organs. After mobilisation from EC nuclei, IL33 might recruit and stimulate ST2-expressing cells such as inflammatory/immune cells and activated fibroblasts/myofibroblasts, thus modulating both the inflammatory and the fibrotic processes. Further studies on the functional role of the IL33/ST2 axis in the SSc-related fibrotic process are warranted.

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    Footnotes

    • Funding This study was supported by grants from the Ministero dell'Istruzione, dell'Università e della Ricerca (MIUR), the University of Florence (ex60%) and the Associazione per lo studio della Sclerosi Sistemica e delle Malattie Fibrosanti (ASSMaF onlus).

    • Competing interests None declared.

    • Patient consent Obtained.

    • Ethics approval The study was approved by the Institutional Review Board.

    • Provenance and peer review Not commissioned; externally peer reviewed.