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Clin Exp Immunol. Dec 2004; 138(3): 540–546.
PMCID: PMC1809238

Cytokine and chemokine levels in systemic sclerosis: relationship with cutaneous and internal organ involvement

Abstract

Systemic sclerosis (SSc) is a connective tissue disorder characterized by excessive collagen deposition in the skin and internal organs. Several cytokines and chemokines have been implicated in the induction of fibrosis, but a definitive relationship between specific cytokines and organ involvement has not been established yet. Serum samples, PBMC and T cell lines (TCL) obtained from 54 patients affected by SSc and 20 healthy donors (HD) were examined by ELISA for Interferon-γ (IFN-γ), interleukin (IL)-4, IL-6, IL-10, IL-18, Transforming growth factor (TGF)-β1, Tumour necrosis factor (TNF)-α, sCD30, Macrophage derived chemokine (MDC), Monocyte chemoattractant protein (MCP)-1, Macrophage inflammatory protein (MIP)-1α and Regulated on activation normal T-cell expressed and secreted (RANTES). In all the SSc serum samples, we found significantly increased levels of IL6, TNFα and MCP-1 but reduced amounts of γ-IFN and MDC. IL6, IL10, IL18, MIP-1α and TNFα measured in supernatants from PHA-stimulated PBMC and IL6, MCP-1 and RANTES in supernatants from stimulated TCL were also increased in patients. MDC was decreased in all the biological SSc sources studied. TGF-β1, IL10, and sCD30 were produced at a significantly lower level by SSc TCL. Serum IL6 and sCD30 levels were significantly increased in dc-SSc patients compared to lc-SSc as were levels of MCP-1 produced by PBMC and IL10 from TCL. We observed a strict relationship between pulmonary fibrosis and IL10, MCP-1 (both from TCL) and serum IL6. Kidney involvement was related to serum MCP-1 levels and IL18 production from PBMC. Oesophageal involvement correlated with MDC production from PBMC and IL10 synthesis by TCL. We showed that IL-6, IL-10, MDC and MCP-1 are variably associated with internal organ involvement and allow the discrimination between limited and diffuse forms of the disease.

Keywords: IL-6, IL-10, MCP-1, MDC, systemic sclerosis

INTRODUCTION

Systemic sclerosis (scleroderma) is a generalized connective tissue disorder, characterized by a wide spectrum of microvascular and immunological abnormalities, leading to progressive thickening and fibrosis of the skin and other visceral organs, such as the lungs, gastrointestinal tract, heart and kidneys [1,2]. The autoantibodies classically associated with SSc include anticentromere antibodies (ACA) and anti scl-70 (antitopoisomerase I). In addition to these, is the less commonly occurring antinucleolar antibody system (including anti PM-scl, antifibrillarin/anti-U3-ribonucleoprotein, anti Th/To and the anti RNA-polymerase family), that have been associated with poor outcome in SSc [3].

According to American College of Rheumatology (ACR) criteria established in 1988, SSc can be classified into 2 major subsets, on the basis of skin thickening extent: one characterized by diffuse skin fibrosis with more severe internal organ involvement (dc-SSc), the other with limited cutaneous involvement (lc-SSc) [4].

The progressive fibrosis is a consequence of multiple, and only partially known events that lead to increased biosynthesis of matrix proteins by connective tissue cells. A growing body of evidence suggests that a perturbed immunoregulation is involved in the pathogenesis of the disease [510], and dysregulation in production of soluble factors is supposed to play a central role. Among these factors, chemokines and cytokines are important mediators in modulating leucocyte–endothelial interactions. An aberrant recruitment of inflammatory cells into the perivascular dermal matrix of the skin and internal organs is observed in the initial phases of scleroderma and precedes the development of fibrosis [8]. The fibrotic phase might then represent the consequence of cytokines stimulation by inflammatory cells, leading to accumulation of excessive extracellular matrix components synthesized by an activated population of fibroblasts [7,1113]. These profibrotic cytokines include transforming growth factor (TGF)-β[14], tumour necrosis factor (TNF)-α[1518] and interleukin (IL)-6 [9,1822].

Although a number a cytokines and chemokines has been investigated as possible mediators of fibrotic and vascular damage in SSc, specific correlation between cytokines and organ involvement have not been found yet, and a cytokine profile characteristic of SSc is far to be identified.

In this study, we evaluated the expression of representative type 1, type 2, regulatory and inflammatory cytokines, as well as chemokines, in patients affected by SSc using different biological specimens, such as serum and culture supernatants from activated peripheral blood mononuclear cells (PBMC) and T cell lines (TCL). This might allow us to identify patterns of cytokine production in SSc, in distinct conditions, and to possibly correlate them with internal organ involvement and scleroderma subsets.

PATIENTS, MATERIALS AND METHODS

Patients

Fifty-four consecutive patients (50 females and 4 males) and 20 healthy subjects (15 F and 5 M) were included in the study. All scleroderma patients were first evaluated by experienced physicians in our division and met the American College of Rheumatology (formerly, the American Rheumatism Association) criteria for the classification of systemic sclerosis (scleroderma) [23]. Patients were classified as either dc-SSc, showing cutaneous thickening proximal to the elbow, or lc-SSc, characterized by skin sclerosis restricted to the distal extremities. The mean age ± standard deviation (SD) in the total patients group was 54 ± 17 years, the mean age of the controls was 46 ± 11 years.

Clinical assessment

A complete history, physical examination and laboratory evaluation were obtained in each patient. The disease duration was measured from the onset of the first symptom other than Raynaud's phenomenon consistent with SSc (puffy hands, digital pitting scars, sclerodactily with or without proximal scleroderma, dyspnea, dysphagia).

Pulmonary involvement was defined as a forced vital capacity <70% of predicted, as measured by a dry spirometer, or by other confirmatory evidence of fibrosis such as bibasilar fibrosis on chest radiograms and/or amorphous or reticulonodular infiltrates in high resolution computed tomography scans (HRCT). The HRCT was scored as follows: 0 = no modifications, 1 = mild alterations (ground glass and/or linear opacities of 1–2 mm thickness), 2 = moderate alterations (with linear opacities of 2–3 mm thickness), 3 severe alterations (linear opacities of >3 mm and the presence of honeycombing). Pulmonary hypertension (PH) was evaluated indirectly by Doppler echocardiography, according to Denton et al. [24]. Renal involvement was considered as ‘present’ when at least one of the following conditions was documented: hypertensive renal crisis attributable to SSc, or persistent renal impairment (serum creatinine value >2 mg/dl plus a diastolic blood pressure >110 mmHg) or otherwise inexplicable presence of blood and/or protein in the urine. Oesophageal dismotility was diagnosed by manometry. Three degrees of oesophageal motor disorders were identified: normal, moderate (uncoordinated peristalsis) and severe (both aperistalsis and decreased low oesophageal sphincter pressure). Joint involvement was considered as ‘present’ in the presence of symmetric synovitis, flexion contractures and tendon friction rubs.

The study was approved by the Istituto Dermopatico dell’Immacolata (IDI-IRCCS) Institutional review board, and informed consent was obtained from all patients.

Serum samples

Venous blood samples from both patients and healthy donors were centrifuged shortly after clot formation. All samples were stored at −80°C in aliquots and thawed only before the test.

PBMC samples

PBMC from each patients and controls were isolated from venous blood by density gradient sedimentation on Lymphoprep (Pharmacia, Uppsala, Sweden), resuspended in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 2 mm l-glutamine (Gibco), penicillin 100 U/ml and streptomycin 100 µg/ml (Gibco) and 10% v/v foetal calf serum (FCS- Hyclone, Logan, UT, USA) at 106 cells/ml. Activation was carried out by 48- h stimulation with phytohemagglutinin (PHA-P, 1 µg/ml, Murex, Pomezia, Italy) at 37°C IN a 5% CO2 atmosphere. The supernatants were then collected by centrifugation and frozen at −80°C prior to use.

T cell lines samples

Mononuclear cell cultures were transferred to 24-wells plates (Falcon, Oxnard, CA, USA) for six days in presence of complete medium supplemented with 2-ME 50 µm (Sigma Chem. Co., St. Louis, IL, USA) and 20 U/ml of rh-IL-2 (Boehringer Mannheim, Germany) and expanded by weekly splitting and addition of rIL2 supplemented complete medium. Polyclonal TCL obtained from each patients were tested by flow cytometry with CD3, CD19, CD14 and CD56 in order to prove that only T cells were present, and then induced to maximal cytokine production with 10 ng/ml PMA and 1 µg/ml ionomycin (Sigma) at a time of optimal growth (from three to four weeks of culture) in the absence of rhIL-2. Supernatants obtained from 48- h stimulated T cells were filtered and stored in aliquots at −80°C until analysis.

Measurements of Cytokines

Representative Th1-type cytokines (γ-IFN and IL18), Th2-type soluble factors (CD30s and IL4), regulatory (IL-10 and TGF-β1), inflammatory cytokines (IL-6 and TNF-α), and C-C chemokines (MIP-1α, RANTES, MCP-1 and MDC) were measured in serum samples and supernatants from PBMC and TCL with commercially available sandwich ELISA kits (Quantikine, R & D Systems, Minneapolis, MN, USA), following the manufacturer's instructions. The results were interpolated from the standard reference curve provided with each kit. Each sample was tested in duplicate and thawed only once.

Statistical analysis

Data were entered in an electronic database and analysed using the SPSS/PC + statistical package [25]. Socio-demographic and clinical differences between patients with lc-SSc and dc-SSc were investigated using the χ2 test. The levels of the variables of interest in the serum, PBMC, and TCL in the two classes of SSc patients as well as in controls were summarized showing the mean ± SD, in order to provide the most easily interpretable summary measure. However, differences between the levels of these variables among lc-SSc, dc-SSc, and controls were tested using the nonparametric Mann–Whitney U-test.

The association between the cytokine and chemokine levels and the different internal organs involved was studied in univariate and multivariate analysis. In univariate analysis, we used the above mentioned nonparametric test to compare variables in subjects with or without organ involvement.

Finally, to see whether the association between organs involved and different biological variables was present after simultaneously adjusting for the other variables of interest, we performed multiple linear regression analysis for the clinical variables with linear scores (i.e. lungs and oesophagus), and multiple logistic regression for the clinical variables with dichotomous scores (i.e. joints and kidney).

Although we performed multiple pairwise comparisons we chose not to adjust the significance level to control the overall type I error rate, according to the recommendations of Greenland & Rothman [26], Rothman [27] and Savitz & Olshan [28], and therefore we set the significance cut-off level at P < 0·05.

RESULTS

Patients characteristics

Thirty-four patients (33 F and 1 M) had lc-SSc; and 20 (17 F and 3 M) had dc-SSc (Table 1). The disease duration from the onset time to study entry was 11·1 ± 9·9 years (mean ± SD; range 1–37 years). None of the patients had a recent history of infection or other inflammatory diseases, including connective tissue disease. No patients had been treated with disease-modifying therapy for two months. All patients had Raynaud's phenomenon after exposure to low temperature. Antinuclear antibodies, demonstrated by immunofluorescence, were present in the sera of all patients. At the time of the investigation, anticentromere antibodies (ACA) were detected in 14 patients with lc-SSc, while anti-Topoisomerase I antibodies (scl70) were present in 12 with lc-SSc and 18 with dc-SSc (Table 1). The patients included in the study had been evaluated for organ involvement at least 1 month before blood collection. Forty patients had respiratory involvement. Isolated PH, in the presence of a normal forced vital capacity, was found in 18 patients. Kidney involvement was found in 17 patients. Thirty-one patients had oesophageal dismotility. Joint involvement was observed in 32 individuals (Table 1).

Table 1
Clinical and laboratory characteristic of the patients with systemic sclerosis

Cytokine and chemokine levels in serum, activated PBMC and T cell lines

Data obtained in serum and culture supernatants of in vitro stimulated PBMC and TCL are reported in Table 2. We found significantly elevated serum levels of inflammatory cytokines (TNFα and IL6) in SSc patients, with IL6 being higher in dc-SSc compared to lc-SSc. γ-IFN was significantly decreased in all patients in comparison to normal controls. Among the chemokines tested, MCP-1 was found to be increased in patients’ compared to controls’ sera, whereas MDC levels were lower in patients (Table 2).

Table 2
Cytokine and chemokine levels (pg/ml) in serum, PBMC and T cell lines of patients with systemic sclerosis and healthy controls

We next addressed the possibility that PBMC from patients with SSc and/or their subgroups could differ for type and/or amount of cytokine secreted. Therefore we measured the ability of PBMC to produce cytokines and chemokines after in vitro stimulation with PHA. We found that the same inflammatory cytokines (TNFα and IL6) whose serum levels were increased, were also elevated in PBMC supernatants. TNFα correlated with IL6 (P = 0·0251, r: 0·317) and IL10 production (P < 0·0001, r: 0·517). IL18 and IL10 production were also increased, and their levels correlated (P = 0·0184, r: 0·333), although without significant differences between lc-SSc and dc-SSc. MIP1α production was increased and MDC was decreased in patients, whereas dc-SSc showed significantly higher levels of MCP-1 in comparison with lc-SSc (Table 2).

In order to assess the role of T lymphocytes in shaping the cytokine pattern, we then generated a T cell line (CD3+ cells at least ≈ 98% in all samples) from PBMC of each patients and controls, and evaluated their ability to produce chemokines and cytokines after maximal in vitro stimulation. We observed increased levels of IL6 in all patients’ TCL (as also found in serum and PBMC supernatants), thus suggesting that T cells could be an important source of this cytokine in serum and supernatants from PBMC. IL4, sCD30, IL10 and TGF-β1 were significantly reduced and higher levels of MCP-1 and RANTES were found in SSc patients, together with lower levels of MDC. Production of IL6 was significantly correlated with MCP-1 in patients with SSc (r: 0·535, P < 0·0001). IL10 synthesis was higher in dc-SSc than in lc-SSc (Table 2).

Association of cytokines with internal organ involvement

We then classified the patients according to the presence/absence of pulmonary fibrosis, pulmonary hypertension, kidney failure and oesophageal dismotility and evaluated the cytokine measurements described with respect to these clinical parameters (Table 3). We found significantly higher levels of IL10 in TCL supernatants from patients with pulmonary fibrosis (P = 0·021) compared to patients without lung involvement. High levels of serum MCP-1 (P = 0·025) and low levels of IL18 produced by activated PBMC (P = 0·047) were observed in SSc patients with kidney failure. Finally, PBMC of patients with oesophagus dismotility secreted significantly lower amounts of MDC (P = 0·039).To study the relationship of clinical characteristics of SSc with cytokine levels, we undertook a multiple linear regression analysis, while simultaneously adjusting for variables of interest. We found a significant direct correlation between pulmonary fibrosis and serum IL6 (t: 2·097, P = 0·042) and IL10 from T cell lines (t: 2·746; P = 0·04), and an inverse relationship to MCP-1 from T cell lines (t: −2·209, P = 0·032). Furthermore, a direct relationship between IL10 production by T cell lines and oesophageal involvement (t: 2·746; P = 0·01) was observed. Finally, multiple logistic regression analysis revealed a slightly significant correlation between serum MCP-1 levels and kidney involvement (r: 0·168, P = 0·05).

Table 3
Cytokines and chemokines levels in present (yes) or absent (no) organ involvement

DISCUSSION

Several lines of evidences indicate that systemic scleroderma presents deregulated production of cytokines implicated in vascular damage and fibrosis, but their relationship with clinical findings is still unclear. We have used different biological sources to assess the production/amount of immunoregulatory soluble factors, such as type 1 and 2, immunoregulatory and inflammatory cytokines and chemokines, and their possible correlation with lc- and dc- forms of SSc, and with internal organ involvement.

We found very high levels of IL6 in all the biological samples tested (serum, supernatants from activated PBMC and TCL) from SSc patients (Table 2). IL-6 is a pleiotropic cytokine with multiple biological effects on immune regulation, haematopoiesis, inflammation, oncogenesis [29], and it has been implicated in SSc pathogenesis [20,3033]. Several authors have demonstrated increased production of IL6 by fibroblasts in SSc patients [18,21] and furthermore increased production of collagen and glycosaminoglycans, hyaluronic acid and chondroitin-4/6-sulphates from human dermal fibroblasts induced by IL6 [34]. Serum IL6 levels were also able to discriminate between dc-SSc and lc-SSc, and they were higher in the presence of lung involvement (both fibrosis and hypertension), confirming previous results [22,35]. Macrophage derived chemokine (MDC), a chemokine belonging to the CC family, was found to be reduced in all the biological sources from SSc patients (Table 2). MDC is constitutively produced by dendritic cells, B cells, macrophages [36,37] and thymic medullary epithelial cells [38] whereas monocytes, NK cells and CD4+ lymphocytes produce MDC only upon appropriate stimulation [37]. Elevated concentrations of MDC are found in sera of subjects with Th2-dominated disorders, such as mycosis fungoides/Sezary syndrome or atopic dermatitis [39]. MDC was shown to act selectively on chronically activated Th2 lymphocytes by interacting with the CCR4 receptor [37,40]. Contrasting data are available regarding the presence of a Th2 skewed profile in SSc [5,4147]. In our study, we observed high levels of IL18 (a Type 1 proinflammatory cytokine) in PBMC from patients with SSc (Table 2), very low levels of MDC (a Th2 chemoattractant) in all conditions studied, normal values of IL4 and sCD30 in all the biological sources studied (with reduced production of Th2 cytokines from TCL). Therefore, overall data of cytokine and chemokine production in different biological specimens do not indicate a clearly defined pattern in SSc and do not support a type 2 preference.

An intriguing finding was the reduced capability of TCL to secrete regulatory cytokines, such as IL10 and TGF-β1, when compared to TCL from healthy donors (Table 2). It is widely known that regulatory T cells control the reactivity of potentially harmful, self-reactive T cells and prevent autoimmune diseases. Altered functions of regulatory T cells play a role in the breakdown of immunologic self-tolerance, as demonstrated by the development of organ-specific autoimmune disease in animal models [4850]. The reduced amount of these regulatory cytokines produced by TCL is apparently in contrast with the high IL-10 production from PBMC. It is known that the primary sources of IL-10 from PBMC are B cells and monocytes, with variable contribution of T cells [51], and this might explain the discrepancy observed. IL-10 is a cytokine with regulatory functions, affecting the expression of several genes involved in extracellular matrix synthesis and remodelling [52]. It has been demonstrated to up-regulate MCP-1 production in unstimulated UG3 cells [53]. Previous reports have suggested an important role of IL10 in pulmonary involvement and skin fibrosis in patients affected by SSc [20,54]. We observed a correlation between IL10 synthesis by TCL and lung fibrosis (Table 3). We also observed that patients with high levels of IL10 were affected more frequently by kidney involvement and oesophageal dismotility.

TGF-β1, besides its regulatory activity on T cells, has been proposed as one of the most important factors causing fibrosis in SSc, and a number of reports demonstrated an increased expression of TGF-β1 from fibroblast of affected skin in SSc patients [6,55]. However, we found normal values of TGF-β1 in serum and supernatants from PBMC, and reduced production by TCL suggesting that increase of TGF-β1 is not a general feature of SSc [56]. However, It should be taken into consideration that we measured total TGF-β1 levels: it is known that only a small proportion of TGF-β1 is in the biologically active form and differences in the amounts of active vs. latent ligand may play a role in SSc patients [57,58].

We found very high levels of MCP-1 in serum and supernatants from TCL of SSc (Table 2), and a correlation between serum MCP-1 and the presence of kidney involvement (Table 3). MCP-1 levels were clearly higher (around 26×) in long-term T cells cultures from SSc patients indicating that in this condition a long-term stimulation of T-cells induces an over production of this chemokine. Interestingly an up-regulation of MCP-1 production has been demonstrated in animal models of crescentic nephritis [59]. MCP-1, a predominant monocyte chemoattractant and activator of mononuclear cells, has been implicated in a variety of inflammatory and fibrotic diseases. This CC chemokine, in addition to chemoattractant properties, stimulates collagen production by fibroblasts [60]. MCP-1 and RANTES have been implicated in the pathogenesis of SSc [10], with a role in recruiting monocytes from the circulation to the lesional skin of scleroderma [61] and in the development of pulmonary fibrosis [31]. IL6 has been shown to induce MCP-1 in U937 cells [62]. Interestingly, we found a linear correlation between IL6 and MCP-1 levels in supernatants from TCL. This is further reinforced by the recent observation that during inflammation IL-6 induces a shift in chemokine synthesis, from IL8 to MCP-1 production [63] causing the influx of monocytes in place of neutrophils.

In conclusion we identified quantitative and qualitative changes in cytokines and chemokines production in SSc compared to healthy subjects, variably associated with cutaneous or internal organ involvement. It has to be pointed out that our patients had very established disease, therefore it would be interesting to perform longitudinal studies, to evaluate the cytokine profile also in early stages, when the disease is generally much more immunologically active.

Acknowledgments

We thank Monica Bondi and Giulio Pirchio for excellent technical support. This work has been supported by Ministero della Salute and by AIRC (Associazione Italiana per la Ricerca sul Cancro).

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