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. Author manuscript; available in PMC: 2014 Jan 1.
Published in final edited form as: Arthritis Rheum. 2013 Jan;65(1):236–246. doi: 10.1002/art.37706

IL-13-producing CD8+ T cells mediate dermal fibrosis in patients with systemic sclerosis

Patrizia Fuschiotti 1,*, Adriana T Larregina 1,2,3, Johnan Ho 2, Carol Feghali-Bostwick 4, Thomas A Medsger Jr 5
PMCID: PMC3535505  NIHMSID: NIHMS407622  PMID: 23001877

Abstract

OBJECTIVE

Fibrosis is a major contributor to morbidity and mortality in systemic sclerosis (SSc). T cells are the predominant inflammatory infiltrate in affected tissues and are thought to produce cytokines that drive the synthesis of extracellular matrix proteins by fibroblasts, resulting in excessive fibrosis. We showed that aberrant IL-13 production by peripheral blood effector CD8+ T cells from SSc patients correlates with the extent of skin fibrosis. Here we investigate the role of IL-13 production by CD8+ T cells in dermal fibrosis, an early and specific manifestation of SSc.

METHODS

Extracellular matrix production by normal dermal fibroblasts co-cultured with SSc CD8+ T-cell-supernatants was determined by quantitative PCR and Western blot. Skin-homing receptor expression and IL-13 production by peripheral blood SSc CD8+ T cells were measured by flow cytometry, whereas immunohistochemistry identified IL-13+ and CD8+ cells in sclerotic skin.

RESULTS

IL-13-producing circulating SSc CD8+ T cells express skin-homing receptors and induce a pro-fibrotic phenotype in normal dermal fibroblasts that is inhibited by an anti-IL-13 antibody. High numbers of CD8+ T cells and IL-13+ cells are found in the skin lesions of patients, particularly in the early inflammatory phase of the disease. Thus, IL-13-producing CD8+ T cells are directly involved in modulating dermal fibrosis in SSc.

CONCLUSIONS

We make an important mechanistic contribution to understanding the pathogenesis of dermal fibrosis in SSc by showing that CD8+ T cells homing to the skin early in the disease are associated with accumulation of IL-13 and may represent an important target for future therapeutic intervention.

Systemic sclerosis (SSc or scleroderma) is an idiopathic disorder of connective tissue characterized by vascular abnormalities, immune cell activation and cutaneous and visceral fibrosis 1. Its most characteristic feature is cutaneous fibrosis attributable to excessive deposition of collagen and other connective tissue components by activated dermal fibroblasts 2. Although the pathogenesis is still unclear, this activation is believed to result from fibroblast interaction with immune mediators and other growth factors 2,3.

Microscopic and immunohistochemical studies of skin biopsies from various clinical stages of SSc indicate that vascular injury and endothelial damage are the earliest observable events in pathogenesis 2,4–6, possibly initiated by viruses, autoantibodies, granzymes or oxidative products 2,7. Infiltration of activated lymphocytes and macrophages into the affected skin follows, preceding worsening of vasculopathy and fibrosis 4–6. Interestingly, in situ hybridization studies have demonstrated that collagen synthesizing fibroblasts are located in close proximity to small blood vessels and to the perivascular inflammatory infiltrate 8, thus supporting the hypothesis that inflammatory cells provide important stimuli that drive collagen synthesis in fibroblasts.

Macrophages and T lymphocytes represent the predominant cell type of the inflammatory infiltrates in the dermis of SSc patients 4–6,9. Such infiltrating T cells exhibit increased expression of activation markers and show signs of antigen-driven expansion 10,11. While their antigen specificity is not known, T cell-derived cytokines have been implicated in the induction of fibrosis 12.

We recently found that dysregulated production of the profibrotic cytokine IL-13 by peripheral blood effector CD8+ T cells is associated with more severe skin thickening in SSc 13 and defects in the molecular control of IL-13 production 14. Other studies have suggested that IL-13 plays a role in the pathogenesis of SSc 15–17, however direct evidence of the source and role of IL-13 in SSc patients is still unclear. IL-13 is an immunoregulatory cytokine predominantly secreted by activated Th2 cells, and is involved in the pathogenesis of many fibrotic diseases 18. Although most studies to date have focused on CD4+ T cells because of the strong MHC class II HLA associations in some SSc patient subsets and the presence of distinctive SSc autoantibodies 19, CD8+ T cells are also involved in the pathogenesis of SSc. Increased numbers of CD8+ T cells with elevated production of type 2 cytokines have been found in the bronchoalveolar lavage fluid of SSc patients with lung fibrosis 20, as well as increased numbers of IL-4-producing CD8+ T cells were found in the skin of SSc patients 21. Furthermore, our recent data have shown abnormalities in the number of circulating effector CD8+ T cells in patients with SSc as well as in their cytokine production ability compared to normal individuals 13,22.

In the present study we provide new insight into the pathogenesis of skin fibrosis in SSc by showing that CD8+ T cells and IL-13+ cells are numerous in the skin lesions of patients, particularly in the early stages of the disease. Furthermore, we demonstrate that IL-13 produced by circulating skin-homing CD8+ T cells from SSc patients is able to induce a pro-fibrotic functional phenotype in normal skin fibroblasts. We conclude that IL-13 production by CD8+ T cells is directly involved in modulating dermal fibrosis in SSc and may represent an important target for future therapeutic intervention.

MATERIAL AND METHODS

Patients and samples

Patients

We studied 29 normal individuals and 56 new and return SSc patients seen in our weekly Scleroderma Clinic at the University of Pittsburgh during 2007–2011. These were well-characterized patients in terms of disease type, clinical features and therapy. The age at SSc diagnosis was 47.8±12.9 (mean±SD) and the female to male ratio was 4:1. Thirty-five patients had diffuse (dc) and 21 limited (lc) cutaneous SSc. The disease duration in these two groups was 4.6±3.5 and 6.9±5.3 years, respectively. All demographic, clinical and laboratory data were abstracted on standardized data collection forms and entered into the Pittsburgh Scleroderma Databank time-oriented computer system (MEDLOG) for analysis. All patients fulfilled either the classification criteria for SSc proposed by the American College of Rheumatology 23 or the early SSc diagnostic criteria of LeRoy and Medsger 24. Disease type, severity and duration were assessed according to established criteria 25–27. Twenty-nine% of patients in our cohort were being treated with low doses of corticosteroids, 8% with disease-modifying antirheumatic drugs and 2% with immunosuppressants. We included treated patients since no effects of immunomodulators on IL-13 production by CD8+ T cells have been shown 13.

The 29 healthy controls were either blood donors or employees of the Central Blood Bank of Pittsburgh. The age range was 24–68 years and the female/male ratio was 4 to 6. All participants signed a written consent document. Clinical information and biological specimens were de-identified and coded. Research protocols involving human subjects were approved by the Institutional Review Board of the University of Pittsburgh.

Skin samples

All biopsies were obtained with informed consent and institutional approval. Full-thickness skin biopsy specimens were obtained from the clinically involved skin of 13 scleroderma patients. Patients were classified as having early (duration < 3 years) or late (duration > 6 years) disease as previously described 27. Early scleroderma patients included 4 patients with dcSSc (all female) and 4 patients with localized scleroderma (LSc, 2 females and 2 males). Late disease patients included 5 dcSSc patients (3 female and 2 male). One the late dcSSc skin sample was purchased from the National Disease Research Interchange (Philadelphia, PA). Two patients with early dcSSc were untreated and two were on immunosuppressive drugs. The LSc patients were untreated and 3 of the 5 late dcSSc patients were receiving immunosuppressants.

Skin samples of 3 age- and sex-matched healthy donors were obtained as controls from volunteer non-connective tissue disease patients recruited from the Arthritis and Autoimmunity Center clinics at the University of Pittsburgh Medical Center.

Cell culture

Dermal fibroblast culture

A 3-mm punch biopsy specimen of skin was obtained from 4 normal individuals undergoing cosmetic plastic surgery and with no history of autoimmune diseases. All biopsies were obtained with informed consent and institutional approval. Human skin fibroblasts derived from a fragment of the skin biopsy were prepared as previously described 28. Cells were grown in DMEM medium (GIBCO, Invitrogen) supplemented with 10% FBS (Atlanta Biologicals), glutamine (Biowhittaker), and an antibiotic-antimycotic cocktail (GIBCO Invitrogen) until confluent. All experiments with fibroblasts were performed at passages 3–7.

CD8+ T cell isolation and culture

CD8+ T cells were isolated from PBMC samples by negative selection using the EasySep enrichment kit (StemCell Technologies) as previously described 14. The CD8+ T-cell-enriched fraction contained >90% CD8/CD3-positive cells as determined by flow cytometry 14. All cultures were performed in complete RPMI-1640 medium (GIBCO, Invitrogen) and activated for 5 days with beads coated with anti-CD3 and anti-CD28 mAbs (Dynabeads CD3/CD28 T cell Expander, Dynal Biotech-Invitrogen, bead-to-cell ratio: 1:1) and rhIL-2 (20 U/ml, PeproTech). Tc2 differentiation was achieved by the addition of rhIL-4 (100 U/ml, PeproTech) and anti-IFN-γ mAb (clone B27, 50µg/ml). After 5 days CD8+ T-cell culture supernatants were harvested and IL-13 production by CD8+ T cells was determined by intracellular cytokine staining (ICC).

Co-culture of CD8+ T-cell supernatants with primary dermal fibroblasts

Skin fibroblasts were plated in 48-well plates and grown to near confluence in complete DMEM medium, followed by serum starvation overnight prior to adding aliquots of supernatant from control or SSc CD8+ T cells (1:2 dilution). For IL-13 inhibition experiments, conditioned SSc supernatants were pre-incubated for 1 hour at 37°C with an anti-IL-13 neutralizing antibody (0.1 µg/ml, Peprotech) or an irrelevant mAb (anti-human CD3) or anti-human IL-4 (both from BD Pharmingen, 0.1 µg/ml) and then used for co-culture experiments. Controls included fibroblasts cultured with medium alone or treated with TGF-β1 (2 ng/ml), IL-4 (10 ng/ml) and IL-13 (30 ng/ml), all from Peprotech as previously described 29. Twenty-four hours later fibroblasts and culture supernatants were harvested and quantitative RT-PCR (qPCR) and Western blotting were performed to measure transcription and protein levels of extracellular matrix (ECM) components.

Quantitation of ECM synthesis by dermal fibroblasts

RNA was extracted from fibroblasts using an RNeasy kit, according to the manufacturer’s instructions (Qiagen), and qPCR experiments were performed as described 14. All reagents and primers for human collagen I and III, fibronectin (COL1A1, COL3A1, FN respectively) and GAPDH were purchased from Applied Biosystems. Data analysis was performed with SDS 2.1 software (Applied Biosystems). The differences in the number of mRNA copies in each PCR were corrected by the human GAPDH endogenous control transcript levels.

Fibronectin protein expression by fibroblasts was determined by Western blot. Culture supernatants (sp) and cellular lysates (lys) were obtained according to standard protocols and protein concentration was determined by Bradford protein assay (Bio-Rad Laboratories). 20µg of lys or equal volumes of sp from an equal number of cells were loaded in each lane and were subjected to SDS-PAGE gel electrophoresis and immunoblotting. Membranes were probed with a mouse anti-human FN (EP5) or anti-human GAPDH mAbs (both from Santa Cruz Biotechnology). Signals were detected after incubation with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) and chemiluminescence (SuperSignal-West-Femto, ThermoScientific). Band intensities were quantified using ImageJ (http://rsbweb.nih.gov/ij/).

Flow cytometry

IL-13 production by CD8+ T cells was determined by ICC as previously described 13, using the following mAbs: anti-CD8 PacificBlue®, anti-CD3-APC and anti-human IL-13-FITC (clone PVM13-1) (eBioscience).

For pSTAT-6 determination, fibroblasts were grown to confluence before being serum starved for 24 hours and then co-cultured with CD8+ T-cell supernatants or IL-13 (30 ng/ml). Fibroblasts were harvested with trypsin-EDTA, permeabilized in ice-cold 90% MeOH and then stained with an anti-pSTAT-6 mAb (Cell Signaling), according to the manufacturer’s instructions. A fluorochrome-conjugated irrelevant isotype (mouse IgG) was used as a negative control.

Expression of skin-homing receptors by CD8+ T cells from SSc patients and age-matched normal controls was evaluated by multicolor flow cytometry 13 using a combination of the following antibodies: CD8-PacificBlue®, CD3-AlexaFluor®700, CCR4-PECy7, CCR6-PerCPCy5.5 (eBioscience), CLA-FITC and CCR10-APC (BioLegend).

Labeled cells were analyzed on a 3 laser, 9 detector LSR II instrument (Becton Dickinson) using FlowJo software (Tree Star).

Immunohistochemistry

Sequential paraffin-embedded skin biopsies (5µm) were deparaffinized in xylenes and rehydrated in graded solutions of ethanol. Antigens were retrieved in Tris-EDTA buffer, and endogenous peroxidases quenched with 3% H2O2. Sections were blocked with Protein Blocking Agent (Pierce) and incubated with the following anti-human primary mAbs (1:100 dilution): rat anti-IL-13 (Abcam), rabbit anti-IL-13Rα1 (Sigma) or mouse anti-IL-13Rα2 (Abcam), followed by biotinilated secondary antibodies (Vector Laboratories). For dual staining, sections were stained first with a rabbit anti-CD4 mAb (1:50, Cell Marque) and then with a mouse anti-CD8 mAb (1:50, Abcam). Bound secondary antibodies (Vector Laboratories) were detected using Vectastain ABC kit (Vector Laboratories). Tissue staining was visualized with a DAB substrate (BD Biosciences) and cell nuclei were identified by hematoxylin counterstain. Skin samples were analyzed using an Axiovert PLUS microscope (Carl Zeiss) and images were obtained with an Axiocam (MRC) and analyzed using the Axiovision software. The number of CD4+ and CD8+ T cells infiltrating the skin samples from early, late SSc and localized scleroderma was calculated following quantification of 20 high-power fields (HPF; magnification, X500) and the result was standardized by using a microscope eyepiece grid to determine the cell number/skin mm2.

Statistical analysis

Statistical analyses were performed using InStat (GraphPad Software, Inc.). We used the unpaired two-tailed Student’s t-test or the Mann-Whitney test (non-parametric) to compare two groups. Multiple group comparisons were made using the Kruskal-Wallis test followed by a post-hoc Dunn test. Correlation analysis between levels of IL-13 and COL1A1 mRNA expression was performed by calculating the Spearman rank correlation coefficient. We considered p<0.05 (indicated * in Figures) as significant, p<0.01 (**) as very significant, and p<0.001 (***) as highly significant.

RESULTS

IL-13 produced by CD8+ T cells from SSc patients increases ECM protein production by normal dermal fibroblasts in vitro

We investigated the pro-fibrotic effects of soluble factors produced by SSc CD8+ T cells on normal dermal fibroblasts by co-culturing in vitro with culture supernatants from SSc and normal donor (ND) CD8+ T cells. After 24 hours, ECM protein synthesis (notably COL1A1, COL3A1 and FN) was examined by qPCR and Western blot analysis. Fibroblast gene expression of ECM components shows that CD8+ T cell-supernatants from most of the SSc patients tested cause an increase in mRNA expression of COL1A1, COL3A1 and FN in normal dermal fibroblasts (P=0.002, P=0.008 and P=0.003 respectively, Fig. 1A–C) compared to ND CD8+ culture supernatants. This was confirmed at the protein level by Western blot (Fig. 1D, E). We found that CD8+ T-cell supernatants from SSc patients up-regulate FN protein production by dermal fibroblasts compared to conditioned medium from ND CD8+ T cells. A representative example (Fig. 1D) and the validation in several other patients and NDs (Fig. 1E, P=0.0041) are shown. FN protein was detected both in fibroblast cell lysates (lys) but particularly as secreted protein (sp) in the fibroblast cell cultures. Similar induction was observed in lung fibroblasts (data not shown). As expected 29, the positive controls IL-4, IL-13 and TGF-β induced FN expression by fibroblasts (Fig. 1D). CD8+ T-cell supernatants from patients with dcSSc induce the expression of higher levels of COL1A1 mRNA compared to patients with lcSSc (mean±SD: 1.23±0.58 vs. 0.66±0.45, P=0.013). Most of the patients inducing COL1A1 mRNA expression are high IL-13 producers, as shown in Fig. 1F. To establish whether the observed pro-fibrotic fibroblast activation by SSc CD8+ T cells was mediated by IL-13, we inhibited IL-13 with an anti-IL-13 neutralizing mAb. The stimulatory effect of SSc CD8+ T-cell culture media on COL1A1 mRNA levels was reduced by pre-incubating SSc CD8+ supernatants with the anti-IL-13 antibody (Fig. 2A–B). In contrast, a neutralizing antibody to IL-4 did not inhibit COL1A1 production, suggesting that the pro-fibrotic properties of SSc CD8+ culture supernatants were specific to IL-13. Similar results were obtained for production of COL3A1 and FN (data not shown). Confirmation at the protein level was made by Western blot (Fig. 2C). Altogether, these results demonstrate that IL-13 produced by CD8+ T-cells from SSc patients modulates ECM production by normal dermal fibroblasts in vitro.

Figure 1. CD8+ T-cell-supernatant from SSc patients induces extracellular matrix protein (ECM) production by normal dermal fibroblasts in vitro.

Figure 1

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ECM production was determined in normal dermal fibroblasts co-cultured for 24 hours with CD8+ T-cell supernatants from SSc patients and normal donors (ND). Each symbol represents one patient or ND. Fibroblast mRNA expression of (A) COL1A1, SSc/ND number=16/6, (B) COL3A1, n=16/6, and (C) FN, n=12/6, was quantified by qPCR. Data were normalized by GAPDH. A horizontal line indicates mean response. The Mann-Whitney test was used for significance. (D) Representative Western blot for FN protein expression in fibroblast culture media (sp) and fibroblast lysates (lys) following incubation with CD8+ T-cell supernatants from one SSc patient and one ND (right panel) out of six of each tested. Fibroblasts stimulated with IL-13, IL-4, or TGF-β were used as positive controls (left panel). GAPDH protein was a loading control in lys. (E) Densitometric quantification of secreted FN protein measured as in (D). The difference between the means of 13 SSc CD8+ T-cell supernatants and 12 from NDs is significant (P=0.001; Mann-Whitney test). (F) IL-13 production by SSc CD8+ T cells correlates with COL1A1 mRNA induction in fibroblasts (n=14, Spearman’s rank correlation coefficient, P= 0.002).

Figure 2. An anti-IL-13 antibody inhibits COL1A1 production by CD8+ T-cell-supernatant from SSc patients in normal dermal fibroblasts in vitro.

Figure 2

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COL1A1 mRNA levels were determined in fibroblasts following 24-hour stimulation with CD8+ T cell supernatants from SSc patients with or without pre-incubation with a neutralizing anti-IL-13 antibody or a control anti-IL-4-antibody. The number of transcripts was normalized to human GAPDH. A representative example (A) and multiple CD8+ T-cell supernatants (B, n=13, P=0.0003 by Mann-Whitney test) are shown. (C) Detection of FN in culture supernatants or cell lysates by Western blot in dermal fibroblasts cultured as described in (A). A representative experiment out of five independent experiments from different SSc patients is shown.

SSc CD8+ T-cell supernatants activates STAT-6 in normal dermal fibroblasts in vitro

Signaling via the IL-13 pathway in normal dermal fibroblasts was assessed by measuring levels of pSTAT-6 30. The ability of CD8+ T-cell supernatants from SSc patients and NDs to induce pSTAT-6 was analyzed by flow cytometry. In Fig. 3A we show that IL-13 is a potent activator of STAT-6 in skin fibroblasts, since more cells express pSTAT-6 when treated with IL-13 for 15 min at 37°C than with medium alone (mean±SD: 55.02±11.03% vs. 5.99±2.42%, respectively P<0.001). CD8+ T-cell culture supernatants from NDs were unable to induce signaling through the IL-13 receptor (Fig. 3B), however, signaling could be achieved after skewing CD8+ T cells from NDs in type 2 culture conditions (ND Tc0 4.94±3.75% vs. ND Tc2 40.97±0.11%, P<0.001, Fig. 3B), characterized by production of high levels of type 2 cytokines such as IL-13 and IL-4 14. Co-culture of dermal fibroblasts with CD8+ T-cell supernatants from SSc patients induced strong STAT-6 activation (Fig. 3C) by the increased number of pSTAT-6-positive cells. The incubation of CD8+ T-cell-treated human dermal fibroblasts with an anti-human IL-13 antibody (0.1 µg/ml) suppressed STAT-6 activation, whereas an unrelated antibody used as a control did not affect the levels of pSTAT-6. The experiment was performed in 5 patients with a cumulative reduction of pSTAT-6-positive cell frequency from 53.6±8.9% SSc CD8 T-cell untreated supernatants to 12.4±6.5 after anti-IL-13 antibody supernatant pre-incubation (P<0.001).

Figure 3. STAT-6 phosphorylation (pSTAT-6) is induced in normal dermal fibroblasts after incubation with CD8+ T-cell supernatants from SSc patients.

Figure 3

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Normal dermal fibroblasts, serum starved for 18 hours, were co-cultured for 24 hrs with CD8+ T-cell supernatants from SSc and NDs as described above and pSTAT-6 in the fibroblasts was determined by intracellular staining as described in the Material and Methods section. A representative example out of five independent experiments giving similar results. (A) pSTAT-6 determination in fibroblasts incubated with medium alone or IL-13 (30ng/ml). (B) CD8+ T cells from one normal donor were cultured in Tc0 or Tc2 culture conditions as previously described 14. The supernatants were collected and added to the fibroblasts. pSTAT-6 was determined after 24 hours. (C) Co-culture of one SSc CD8+ T-cell supernatant untreated (black line) or pre-incubated with an anti-IL-13 antibody (grey shade) or an unrelated control antibody (dot line) with skin fibroblasts.

Expression of skin-homing receptors by peripheral blood CD8+ T cells from SSc patients and normal controls

The chemokine receptors (CCRs), CCR4 and CCR10, and the cutaneous lymphocyte antigen (CLA) have each been proposed as critical mediators of skin-specific T lymphocyte homing in mice and humans 31–33. We determined their expression to establish skin homing abilities of peripheral blood CD8+ T cells from SSc patients. We found that SSc patients have a highly significant increase in the frequencies of CD8+CCR10+, whereas circulating CD8+ T cells from normal donors show negligible levels of CCR10 on their surface 34 (Fig. 4A, B). Similarly, we demonstrated a higher proportion of CD8+CLA+ T cells in SSc patients compared to normal controls (Fig. 4A, B). In contrast, no significant differences were found between SSc and ND in the expression of other chemokine receptors that are upregulated in some inflammatory conditions, such as CCR4 and CCR6 31 (Fig. 4B). No correlation was detected between skin homing abilities of SSc CD8+ T cells and disease subtype (mean±SD; CCR10: lcSSc 5.21± 2.25 vs. dcSSc 6.92± 2.92 and CLA: lcSSc 22.01±5.51 vs. dcSSc 23.8±3.38), although this remains to be confirmed in larger numbers of patients. Moreover, we found that the frequency of circulating CLA+CCR10+ cells is increased in CD8+ T cells from SSc patients compared to normal controls (P=0.025 Fig. 4C) as well as the frequency of CD8+CCR4+CCR10+ T cells (P<0.0001, Fig. 4D), defining a strong skin-homing phenotype. We also measured IL-13 expression by CD8+ T cell skin-homing subsets, finding a higher frequency of IL-13+ cells in the CCR10+ subpopulation compared to the CLA+ subset (Fig. 4E, F). Interestingly, this frequency is higher in CD8+CCR4+CCR10+ T cells (P=0.002). The corresponding subsets in NDs contain only small numbers of IL-13+ cells. Taken together, these results show that CD8+ T cells from SSc patients have an increased ability to home to the skin compared to healthy controls, and highlight CCR10 as the most preferentially restricted receptor of the skin-homing CD8+ T cells in SSc.

Figure 4. Skin-homing receptor expression by peripheral blood CD8+ T cells from SSc patients and age-matched NDs.

Figure 4

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Freshly isolated CD8+ T cells were stained by multicolor flow cytometry for skin-homing molecules CLA, CCR4, CCR6 and CCR10. Lymphocyte population was gated according to light scatter characteristics (FSC/SSC) and CD8+ T cells identified for CD8 and CD3 positivity. Each symbol represents one patient or ND. (A) Two-color plots of CCR10 or CLA versus CD8 from one SSc patient and one ND. (B) Frequencies of skin-homing receptor-positive CD8+ T cells show a higher percentage of CD8+CLA+ T cells in SSc patients (n=16) compared to ND (n=9) (P=0.031) and a higher frequency of CCR10+ T cells (P<0.0001). Statistics by the unpaired two-tailed Student’s t test. Values represent the mean percentage of positive cells±SD. A higher proportion of CD8+CCR10+ cells in SSc patients co-express CLA (C) and CCR4 (D) compared to controls (P=0.03 and P<0.0001, respectively: Mann-Whitney test). A horizontal line indicates mean response. (E, F) IL-13 production by skin-homing SSc CD8+ T cell subsets. (E) A representative example, and (F) IL-13 frequency in multiple SSc patients (n=10, P=0.002: Kruskal-Wallis test). IL-13 production was determined by ICC 13. Mean percentage of positive cells±SD is shown.

Increased numbers of CD8+ T cells and IL-13 expression in the sclerotic skin of scleroderma patients

Infiltration of CD8+ T cells and IL-13 secretion were determined by immunohistochemistry in the sclerotic skin of patients with early and late stages of scleroderma. As expected 35, skin from NDs has a defined papillary/epidermal dermal junction, delicate fibroblast and collagen organization and absence of inflammatory infiltrates (Fig. 5Ai). In contrast, affected skin from early SSc patients shows moderate loss of papillary epidermal dermal junction, thickening of collagen fibers (Fig. 5Aii,iii-square) and abundant mononuclear cell infiltrate in the deeper (reticular) dermis and hypodermis 5,6 (Fig. 5Aii,iii). Atrophy of the eccrine sweat glands is also evident. We included in this group 4 patients with early dcSSc and 5 patients with LSc, all with disease duration of less than two years. Although these two forms of scleroderma have very well-defined and dramatic differences in clinical manifestations in both the skin and other organ systems, skin biopsies have similar histological features 35,36. Skin from late stage SSc patients showed no inflammatory lesions, absence of the papillary epidermal/dermal junction (Fig. 5Aiv), thickening of dermal collagen bundles with areas of basophilic degeneration (Fig. 5Aiv-square) and loss of perieccrine adipose cells. A marked accumulation of IL-13-producing cells was detected in the mononuclear cells infiltrating the skin of early scleroderma patients (Fig. 5Bii,iii). The largest numbers of IL-13-positive cells were found in perivascular areas as well as in the dermis. Biopsies from the sclerotic skin of late dcSSc patients showed a lower number of IL-13-positive cells (Fig. 5Biv). No expression of IL-13 was detected in normal skin (Fig. 5Bi). The mononuclear infiltrates in the skin of patients with early disease were composed predominantly by CD8+ T cells compared to CD4+ T cells (Fig. 5Cii,iii and Fig. 5D, P<0.0001). In contrast, skin in late stage SSc showed scant inflammatory mononuclear infiltrates in which the number of CD4+ T cells was significantly higher than CD8+ T cells (Fig. 5Civ and Fig. 5D, P<0.0001). Limited numbers of CD4+ T cells and no CD8+ T cells were found clustered near vessels and cutaneous appendages of normal skin (Fig. 5Ci). Mononuclear cells infiltrating perivascular areas as well as endothelial cells of blood vessels undergoing fibrosis express significant levels of IL-13Rα1 and IL-13Rα2 (Fig. 6A and B). Dermal fibroblasts in areas of active disease in the sclerotic skin express strikingly high levels of IL-13 receptors (Fig. 6A and B). Interestingly, high levels are also expressed by nerves and sebaceous and sweat glands (data not shown). Altogether, these findings establish that IL-13 and CD8+ T cells are numerous in the sclerotic skin of scleroderma patients and strongly suggest that they mediate dermal sclerosis.

Figure 5. Immunohistochemical localization of IL-13 and CD8+ T cells in scleroderma skin biopsies.

Figure 5

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(A) Comparative histological analysis of skin samples from normal donors and early or late scleroderma patients (H&E, 200X). IL-13 expression (B), cell infiltrate characterization (C) in normal skin and samples of patients with early or late scleroderma. (B) is peroxidase anti-peroxidase (brown stain) and (C) is peroxidase and alkaline phosphatase staining identifying CD4+ (black LSc (200X) and brown early and late SSc (400X)) and CD8+ (red) T cells, respectively. Insets 1000X. (D) Quantification of the cellular infiltrate in tissue sections by microscopy is expressed as mean±SD of cell numbers/mm2 skin area. Statistics by Student’s t test.

Figure 6. Immunohistochemical localization of IL-13 receptors in scleroderma skin biopsies.

Figure 6

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IL-13Rα1 (A) and IL-13Rα2 (B) expression in normal skin and samples of patients with early or late scleroderma, using peroxidase anti-peroxidase (brown stain, 200X). Insets 500X.

DISCUSSION

We present evidence that over-expression of IL-13 by CD8+ T cells plays a significant role in dermal fibrosis, one of the earliest and most specific manifestations of scleroderma. Although CD8+ T cells are best known for their involvement in type 1 immune responses and their ability to kill bacteria or viral infected cells, recent advances have revealed a much wider range of functions, including production of type 2 cytokines such as IL-4 and IL-13. These atypical CD8+ T cells can be found in a number of human diseases 37,38. Moreover, effector CD8+ T cells were shown to be a source of IL-13 in a mouse model of airway hyper-responsiveness and inflammation 39, supporting the idea that CD8+ T cells also play important roles in type 2-driven immune responses.

High numbers of CD8+ T cells expressing skin-homing receptors, such as CLA 33 and CCR10 32, are present in the peripheral blood of SSc patients but not in controls, indicating an enhanced ability to migrate to the skin. While we found only a modest increase in the number of CLA+ circulating CD8+ T cells, a highly significant increase in the number of CD8+ CCR10+ T cells was noted. Although CCR10 has been clearly associated with the homing of CD4+ T cells to skin 32, few reports indicate an association with CD8+ T cells 34. Interestingly, expression of CCR10 by specific T-cell subsets has been associated with several T-cell mediated skin diseases 31,32,40. The functional ligand for CCR10, chemokine CCL27, was found highly expressed in the serum and mRNA from sclerotic skin of SSc patients and correlated with some clinical features 41. Most remarkably, we found that circulating CD8+CCR10+ subsets also contain a large percentage of IL-13+ cells, which may contribute to SSc pathogenesis when homed to the skin.

IL-13-producing cells were found at high numbers in the mononuclear cellular infiltrates in the skin of SSc patients, particularly in the early inflammatory phase of the disease, and sequential staining with CD8 and IL-13 antibodies indicates that IL-13 is expressed by CD8+ T cells. Characterization of the inflammatory T cell infiltrate by double staining immunohistochemistry for CD4 and CD8 expression shows that CD8+ T cells are present in higher numbers than CD4+ T cells in the early stages of the disease, while in late stages CD4+ T cells are predominant. These results suggest that CD8+ T cells have an important role in the initiation of the disease, although they may also contribute to disease progression since they remain present in lower numbers in late stage disease. Cytotoxic T lymphocyte-mediated killing using granzyme B represents a source of autoantigens in systemic autoimmune diseases 42 and, interestingly, self protein fragments 43 generated by granzyme B are recognized by autoantibodies in a subset of SSc patients 42. Furthermore, inhibition of endothelial cell growth in SSc by vascular injury appears to be mediated by granzyme B and perforin 43. It is well documented that type 2-cytokine producing CD8+ T cells are cytotoxic 44. Indeed, we found that peripheral blood IL-13-producing effector CD8+ T cells express cytolytic effector molecules such as perforin and granzyme B 22. In addition, immunofluorescence studies of SSc skin biopsies using an anti-granzyme antibody demonstrated the presence of granzyme reactivity, while healthy control tissues were negative, suggesting cytolytic mechanism involvement in the pathogenesis of scleroderma 43. Thus, the activation of cytotoxic cell-mediated pathways may be involved in early vascular damage in SSc and could initiate and propagate the specific autoimmune response in this disease 42,43.

High numbers of IL-13Rα1- and IL-13Rα2-positive cells were found in the perivascular inflammatory infiltrates and in endothelial cells of blood vessels undergoing subintimal thickening. Interestingly, fibroblasts in areas of the dermis and epidermis with increased matrix deposition highly express both receptors, likely upregulated by IL-13 18. In vitro studies demonstrated that IL-13 potently stimulates fibroblast proliferation and ECM protein production 29,45 through IL-13Rα1 signaling 30. Likewise, we show here that CD8+ T-cell supernatants from SSc patients induce STAT-6 activation in normal dermal fibroblasts in vitro as well as synthesis of ECM, both inhibited by a mAb to IL-13, suggesting that IL-13 produced by CD8+ T cells in scleroderma skin may activate IL-13Rα1 signaling in local fibroblasts to promote dermal sclerosis. IL-13Rα2 binds IL-13 with high affinity and specificity 46. However, contrasting functions have been proposed for its effects on fibrosis. On the one hand, lack of signaling activity would provide tight regulation of IL-13 responses by limiting its bioavailability and retarding signaling via IL-13Rα1 47. However, recent studies have also shown that during prolonged experimental inflammatory conditions, its activation leads to TGF-β production by macrophages and, ultimately fibrosis 48. Interestingly, a significant association between IL-13Rα2 gene polymorphisms and susceptibility to SSc has been found in a French cohort of Caucasian patients 49, but a full understanding of its roles in SSc needs to be determined and both functions could coexist.

Tissue fibrosis is a leading cause of morbidity and mortality for a number of human diseases, including SSc. Despite the important impact on human health, inadequate understanding of the mechanisms underlying the initiation and progression of fibrosis limits the development of effective therapeutics for fibroproliferative diseases. Our results make an important mechanistic contribution to understanding the pathogenesis of scleroderma by showing that CD8+ T cells homing to the skin early in the disease are associated with accumulation of IL-13. Furthermore, the activation of cytotoxic cell-mediated pathways may be involved in early vascular damage in scleroderma and potentially in initiating and propagating a specific autoimmune response. These results open avenues for the development of novel therapeutic strategies for SSc aimed at targeting these cells and the cytokines they produce. In addition, such mechanisms are likely to be applicable to other fibrotic disorders.

ACKNOWLEDGEMENTS

We thank Mary Lucas, BSN, MPH, for providing clinical details of patients, and Dana Ivanco, EMT-I, CCMA, CCRC, for recruiting eligible patients to this study (Division of Rheumatology and Clinical Immunology).

Grant support: This work was supported by the Marta Marx Eradication of Scleroderma Award from the Scleroderma Foundation, University of Pittsburgh and Centocor Biotech Inc. to PF; NIH-NIAID R01A1077511 to ATL; and the Scleroderma Research Fund, Taub Fund (Chicago, IL), Zale Foundation (Dallas, TX), Arthritis Foundation Western PA Chapter, Shoemaker Fund (Pittsburgh, PA) to TAM.

Footnotes

The authors declare that no competing financial interests or conflicts exist.

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