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- Materials and methods
Plasma cytokines play an important role in the pathogenesis of Sjögren's syndrome (SS) by initiating and perpetuating various cellular and humoural autoimmune processes. The aim of the present study was to describe a broad spectrum of T-cell and B-cell cytokines, growth factors, chemokines and molecules that could contribute to cell death in SS. A novel protein array system was utilized to measure simultaneously the levels of 25 plasma cytokines of patients with primary SS and healthy individuals. Furthermore, we correlated these plasma cytokine levels with potential laboratory and clinical parameters related to disease activity in SS. A subset of plasma cytokines [e.g. interleukin-1β (IL-1β), IL-6, CXCL8 (IL-8), IL-12 p40, IL-15, tumour necrosis factor-α (TNF-α), epidermal growth factor, CCL4 (MIP-1β), CCL2 (MCP-1), CCL11 (Eotaxin), CCL5 (RANTES), TNF-RI and TNF-RII] was found to significantly differ between patients and controls. Also, distinct populations of cytokines were found to differentiate between patients with normal versus elevated ESR or IgG levels and patients with the presence or absence of extra-glandular manifestations (EGMs). Our results support the assumption that the multiplex cytokine array system can be successfully utilized in the diagnosis and disease management of SS. Furthermore, it may provide a powerful tool in the design of individualized anticytokine therapies.
- Top of page
- Materials and methods
Sjögren's syndrome (SS) is a chronic, slowly progressive, systemic autoimmune disease, which predominantly affects middle-aged women, although it can be seen in patients of all ages, including children . It is characterized by lymphocytic infiltration and destruction of the exocrine glands, resulting in xerostomia and keratoconjunctivitis sicca and the presence of other exocrinopathic symptoms . SS can develop alone (primary SS) or in association with other autoimmune diseases (secondary SS) such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), polymyositis (PM), systemic sclerosis (sS) or thyroiditis .
Cytokines are small soluble peptides used by the immune system to communicate and influence cellular function. They are released by cells and their function can be autocrine or paracrine or endocrine. The key role of a few circulating cytokines in the pathogenesis of SS by triggering the perpetuation of the cellular and humoral autoimmune processes has been described [2, 3]. In previous studies, mostly serum Th1/Th2 cytokines were selectively reported in SS [3–8]. However, no extensive studies have been carried out to describe a broad variety of cytokines and their relation to clinical and/or laboratory features of the disease.
In RA and in SLE, disease activity/severity indices have been established to evaluate the patient's disease status. In SS, a similar disease activity assessment has not been fully established yet [9, 10]. Besides clinical and general immunological tests and since circulating cytokines may correspond to the activation status of immunocompetent cells, the evaluation of these cytokines might be a good indicator of disease activity. Although it is noteworthy that several aspects, including kinetics of expression, mode of induction, regulation of receptor expression and competition for occupancy and the stage of the disease (acute versus chronic), are critical to the cytokine net effect, all of these aspects need to be understood if we are to define a cytokine's whole nature .
The aim of this preliminary study was to simultaneously describe a broad spectrum of T-cell and B-cell cytokines, growth factors, chemokines and molecules that could contribute to cell death in SS by utilizing a multiplex cytokine array system. Furthermore, we correlated these plasma cytokine levels with potential laboratory and clinical parameters for disease activity assessment in SS.
Application of a multiplex cytokine array system might become a powerful tool to extensively describe the SS cytokine profile and to assess the role of these molecules in the pathogenesis of the disease. Furthermore, it ought to improve the design of special, individualized, cytokine-targeted therapy for these patients.
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- Materials and methods
The aim of the present study was to describe subpopulations of circulating cytokines likely to drive a broad spectrum of autoimmune processes in SS. We could identify a subset of cytokines that significantly differ between controls and SS patients as well as differentiate between various subsets of patients with SS (Table 2). Cytokines were grouped into the following categories: ‘cytotoxic cellular cytokines’, ‘humoural cytokines’, ‘growth factors’, ‘chemokines’ and ‘death receptors’. We could characterize each population of patients by their cytokine profile, compare these disease groups and pinpoint special subsets of cytokines that are attributed to the particular disease subset.
Table 2. Plasma cytokines that significantly differ between controls and patients as well as various subsets of patients with Sjögren's syndrome (SS)
|Plasma cytokines that significantly differ between healthy controls and patients|
|Categorized group||Significant cytokines||Healthy controls (pg/ml) (median)||SS patients (pg/ml) (median)||P value|
| ||CCL4 (MIP-1β)||59.26||21.99||0.056|
| ||CCL2 (MCP-1)||343.5||56.57||<0.0001|
| ||CCL11 (Eotaxin)||109.6||5.67||<0.0001|
| ||CCL5 (RANTES)||7903.2||1304.01||<0.0001|
|Plasma cytokines that significantly differ between subsets of SS patients|
|Normal versus elevated ESR|
|Categorized group||Significant cytokines||Normal ESR (pg/ml) (median)||Elevated ESR (pg/ml) (median)||P value|
| ||CCL5 (RANTES)||790.1||1574||0.048|
|Presence versus absence of EGMs|
|Categorized group||Significant cytokines||Without EGM (pg/ml) (median)||With EGM (pg/ml) (median)||P value|
The key cytokines that discriminate between SS and healthy individuals were IL-1β, IL-12p40, IL-15 and TNF-α of the ‘cytotoxic cellular cytokines’ group; IL-6 of the ‘humoral cytokines’; EGF of the ‘growth factors’; CXCL8 (IL-8), CCL4 (MIP-1β), CCL2 (MCP-1), CCL11 (Eotaxin) and CCL5 (RANTES) of the ‘chemokines’ and TNF-RI, TNF-RII of the ‘death receptors’ group. These findings support the hypothesis that a broad spectrum of impaired immune functions are involved in the pathogenesis of SS, affecting cellular, humoural immune responses, leucocyte function, cell proliferation/growth and cell death. Although a few of the cytokines related to SS have been described previously [13–17], most of these significant cytokines have not been related to SS before. With the novel, advanced multiplex cytokine array system, a distinct SS-related plasma cytokine pattern has been revealed that discriminated between patients and healthy individuals.
Additionally, we analysed the correlation between the expression of these cytokines and various laboratory and clinical parameters to characterize the disease status of SS patients (e.g. ESR, serum IgG levels, the presence of anti-SSA and/or anti-SSB autoantibodies, FC values of minor salivary gland histology and the presence of EGMs besides glandular features).
The most prominent cytokines, likely to differentiate between SS patients with normal or elevated ESR levels were IL-2, EGF, CCL3 (MIP-1α), CCL5 (RANTES) and TNF-RI. Our results indicate that behind the elevated ESR in SS lies a complex disturbance of lymphocyte and leucocyte function, characterized by inappropriate regulation of cell-proliferation/growth and cell death.
The presence or absence of EGMs, the most essential ‘clinical candidate’ of disease activity evaluation in SS, was correlated with the circulating levels of 25 cytokines simultaneously. From the ‘cytotoxic cytokines’ group, IL-12p40 was found to significantly differ between SS patients with or without EGMs.
Interestingly, the peripheral plasma cytokine levels did not correlate significantly with the presence or absence of hypergammaglobulinaemia in SS or with the disease-specific autoantibody levels. It is more likely that factors other than peripheral plasma cytokines drive the humoural autoimmune response in SS alternatively, an indirect association between these cytokines and immunoglobulin production exists.
Furthermore, the cytokine comparison in patients with low and high salivary gland FC values did not show significant differences between the two populations raising the possibility that the development of sialadenitis in SS might be driven by a local network of pro-inflammatory cytokines. Further investigations of salivary gland tissue cytokines, utilizing the multiplex cytokine assay, may be used to describe the in situ cytokine milieu in SS salivary glands.
Our results imply that a complex disorder of secreted plasma cytokine levels driving various immunocompetent cell types can be found in SS patients. Also, different patterns of circulating cytokines are specific to diverse types of the disease. We assume that the utilization of the multiplex cytokine array system in SS provides a powerful tool to subcategorize the disease and along with common clinical and laboratory parameters help to evaluate disease activity.
In this study, we focused on individual time points to describe the cytokine profile of the patients, although fluctuations in cytokine levels sometimes accompanies disease flare or remissions. However, in separate ongoing studies utilizing the multiplex cytokine assay, serial serum cytokine level measurements did not seem to fluctuate signficantly (unpublished results).
In this study, cytokine levels with known indicators of disease flare used in the clinical management of SS (e.g. ESR, serum IgG levels, the presence of anti-SSA and/or anti-SSB autoantibodies, FS values of minor salivary gland histology or the presence of EGMs besides glandular features) were assessed in order to describe the characteristic cytokine pattern accompanying a flare as a measure of ‘disease activity’. Moreover, to exclude modulations in the circulating cytokine levels, we selected patients in this study, who at the time of the analysis were not taking immunomodulatory medications, which would seriously affect the cytokine levels.
One of the interesting future applications of this technique is on patients with secondary SS. As this is a preliminary study, we wanted to describe the effective utilization of the multiplex cytokine assay in a clearly defined disease population and avoid complex overlap syndromes, or associated diseases, like secondary SS. As the multiplex cytokine assay is a recently developed, unique technique, we believe that primarily it is important to gain information from relatively well-defined diseases, and furthermore, on the basis of such knowledge apply the technique on more complex diseases. Our further goal is to apply this technology on other systemic autoimmune diseases, also to describe complex disease entities (e.g. secondary SS). A future important application of this technology will be to evaluate the circulating cytokine pattern and thereby help subcategorizing patients with SS and develop this technology to become a powerful diagnostic tool in the future. By the simultaneous monitoring of general laboratory values and serum multiplex cytokine levels, individual therapy planning could be achieved. Additionally, this assay could be used to evaluate the efficacy of an ongoing therapy. Instead of long, empirical therapies, one can optimize combination therapies by individual pro-inflammatory cytokine targeting and also plan advanced cost-benefit strategies. Although this strategy is relatively expensive (100$ per sample for reagents alone), the amount of information obtained from the multiplex cytokine assay is considerable and gives a near-complete description of the pathogenic cytokines and is therefore both powerful and represents a good cost-benefit. We assume that the utilization of the multiplex cytokine array system in the diagnosis and therapy design in SS will provide an advanced disease management in the future.