Interleukin-18 overexpression as a hallmark of the activity of autoimmune inflammatory myopathies
Tucci M
F Dammacco,Dammacco F,Quatraro C,Silvestris F
0
The objective of this study was to explore the role of interleukin (IL)-18 in patients with inflammatory myopathies (IM) such as dermatomyositis (DM) and polymyositis (PM) in relation to the possible predominance of a Th1 immune response in their pathogenesis. Serum concentrations of IL-18, interferon (IFN)-γ, IL-4 and IL-6 were measured in six patients by enzyme-linked immunosorbent assay (ELISA). IL-18 expression was evaluated by in situ hybridization (ISH), whereas CD68, CD8 and CD83 were investigated by immunohistochemistry (IHC) to define the main producers of IL-18. Lastly, the expression of both IL-18 receptor (IL-18R) and monocyte chemoattractant protein (MCP)-1 was also explored by IHC. High serum levels of IL-18 and IFN-γ, and conversely low titres of IL-4 and IL-6, were demonstrated in both diseases. In addition, IL-18 was overexpressed in muscle biopsy specimens from patients with IM. Both macrophages and dendritic cells (DC) surrounding either perivascular and perimysium areas in DM or endomysium in PM were the main producers of IL-18. Endothelial cells (EC), smooth muscle cells (SMC) and CD8+ T cells expressed a high content of IL-18R. Vessel cells overexpressed MCP-1 in parallel with IL-18R. High concentrations of serum IL-18 as well as muscular up-regulation of IL-18 and IL-18R suggest that deregulation of the IL-18/IL-18R pathway is a pathogenetic mechanism in IM. Measurement of IL-18 may thus predict the severity of both DM and PM.
Introduction
Dermatomyositis (DM) and polymyositis (PM) are inflammatory myopathies (IM) characterized by muscular weakness and local accumulation of both T cells and macrophages that surround the perivascular areas and infiltrate the fibres [1,2]. IM are thought to arise from deposition of immune complexes within skeletal muscles [3], whereas other events mediated by functional molecules, including cytokines, integrins and chemokines, promote inflammation, although the extent of their individual participation is not known.
Recent evidence suggests involvement of the T helper (Th)-1 immune response in inducing the high production of both tumour necrosis factor (TNF)-α and interferon (IFN)-γ in the development of muscle inflammation [4], and overexpression of Th1 cytokines has been demonstrated in mononuclear infiltrates surrounding the muscular fibres, in association with up-regulation of adhesion molecules and chemokines [5,6]. In contrast, levels of interleukin (IL)-4 and other Th2 cytokines are not so high in DM and PM.
IL-18 is a Th1 inflammatory cytokine produced mainly by antigen-presenting cells (APC), including macrophages and dendritic cells (DC) [7,8]. It interacts with IL-12 to produce IFN-γ, induces both proliferation and differentiation of naive T cells and exerts intrinsic attraction through its receptor (IL-18R) [9] or the stimulation of several chemokines, including monocyte chemoattractant protein (MCP)-1 [10]. SJL/J mice resembling human inflammatory myositis display muscular accumulation of cytotoxic T lymphocytes (CTL) and high production of IFN-γ, whereas a mild to moderate disease occurs in immunized IL-1 null mice [11]. Further evidence of the Th1 immune response in IM is provided by the demonstration of increased serum IFN-γ and IL-18 levels in patients with autoimmune disorders and those with different clinical presentations of DM and PM [12,13].
To explore the pathogenic role of IL-18, we have investigated its serum levels and muscle expression, and its mainproducers within the inflamed muscles of DM and PM patients.
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Materials and methods
Study population
Six patients with DM and PM, respectively, were enrolled into the study at the Department of Internal Medicine and Clinical Oncology of the University of Bari, together with nine healthy controls. The local ethical committee approved the study and written informed consent was obtained from all subjects. Diagnosis was based on both clinical and laboratory parameters and on muscle biopsy specimens [14,15]. The demographic data were collected on enrolment and a medical history and key laboratory data were recorded. Four male patients aged 41·3 ± 7·4 and two female patients aged 37·6 ± 11·04 were investigated. Laboratory evaluations included creatine kinase, myoglobin and aldolase as well as measurement of both anti-nuclear and anti-Jo1 autoantibodies. Treatment with steroids was addressed after laboratory assessment and biopsy and was continued for 1 year. Control biopsy specimens were provided from a patient suffering from chronic fatigue. Demographics of the study patients are summarized in Table 1.
[Table 1]
Table 1
Demographics of the study subjects.
Enzyme-linked immunosorbent assays (ELISAs)
ELISAs were installed to measure serum IL-18, IFN-γ, IL-4 and IL-6 levels. Samples at 1/10 dilution were tested for the 18 kDa bioactive isoform of IL-18 (Medical and Biological Laboratories, Nagoya, Japan), IFN-γ and IL-4 (Technogenetics, Milan, Italy) using dedicated kits according to the manufacturer's instructions. IL-6 was assessed in our laboratory; 96-well Nunc plates were coated overnight with a purified mouse anti-human IL-6 monoclonal antibody (MoAb; Pharmingen, San Diego, CA, USA) and then with a biotyinylated rabbit anti-IL-6 anti-serum (Pharmingen). Streptavidin–alkaline phosphatase diluted at 1/1000 (Southern Biotechnology Birmingham, AL, USA) completed the test. The reaction was revealed by the o-phenyldiamine chromogen (OPS) solution (Sigma, St Louis, MO, USA) at 405 nm and data were analysed with a dedicated software. Recombinant human IL-6 (Pharmingen) was used for the standard curve.
Immunohistochemistry
Muscle specimens from the patients and controls were studied by immunohistochemistry (IHC) to determine the expression of IL-18 and the α/β subunits of IL-18R (Medical and Biological Laboratories). In addition, CD68 (Vector Laboratories, Burlingame, CA, USA), CD83 (R&D Systems, Minneapolis, MN, USA), CD8 (Vector Laboratories), MCP-1 and von Willebrand factor (vWF; Pharmingen) wereinvestigated with relative MoAbs to identify the cells infiltrating muscle fibres and peri-endothelial areas. Briefly, paraffin-embedded sections were treated in xylene and rehydrated in a gradient of ethanol. After blocking with 2% horse serum, sections were incubated with mouse MoAbs at appropriate concentrations. Binding of the secondary biotinylated horse anti-mouse IgG was detected with the Vectastain ABC system (Vector Laboratories). Endogenous peroxidase activity was blocked with 3% H2O2 for 30 min; 3,3′-diaminobenzidine (DAB) was then added for 10 min and nuclei were counterstained with haematoxylin (DakoCytomation, Milan, Italy). Mouse IgG1 and IgG2a were used as isotypic control. Staining was evaluated by light microscopy.
In situ hybridization (ISH)
ISH was adopted to reveal the expression of IL-18 and its main producers in inflamed muscles. Briefly, after rehydration in gradients of alcohol, paraffin-embedded sections were incubated overnight with 4 µg/ml of the biotinylated long DNA probe for human IL-18 (Maxim Biotech, CA, USA) and the hybridization reaction was developed using the DNA probe hybridization/detection system in situ kit (Maxim Biotech), according to the manufacturer's instructions. Specific staining was then completed with the bromochloroindolyl phosphate/nitroblue tetrazolium (BCIP/NBT) substrate and nuclei were counterstained with nuclear fast red. Further experiments demonstrated the simultaneous expression of CD68, CD83 and CD8 within the muscle areas infiltrated by IL-18+ cells. Thus, after IL-18 detection by ISH, sections were analysed by IHC following the above-mentioned procedure. Positive control was provided by the housekeeping probe of the kit, whereas negative control included both sense oligomers and absence of probe.
Quantification of positive cells and statistical methods
Because muscle sections were heterogeneous and their IL-18-producing cells were located randomly, positive cells were quantified as described previously [16]. Briefly, muscle areas containing the highest density of positive cells were defined as spots, in which positive cells were counted at 50 × magnification in 10 independent 0·3 mm2 fields. Each spot thus corresponded to an approximately 3 mm2 area. The average number of positive cells in two spots was expressed as number of positive cells per mm2. Differences of serum cytokine levels were measured by the Mann–Whitney non-parametric test, and the Spearman's rank correlation test was adopted for comparative analyses.
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Results
Serum IL-18 is elevated in both DM and PM
Figure 1 shows the serum profile of both Th1 (above) and Th2 (below) cytokines from patients and controls. IL-18 levels (Fig. 1a) were significantly higher in patients at diagnosis and their mean value was almost three times that of the controls (mean ± s.d.: 1088·7 ± 131 versus 370 ± 105 pg/ml, P < 0·0001). IL-18 levels correlated with those of creatine kinase (r = 0·3311), myoglobin (r = 0·3834) and aldolase (r = 0·3409, P < 0·05 in all instances). In addition (Fig. 1b), IFN-γ levels paralleled those of IL-18 and were increased in patients compared to controls (905·5 ± 185 versus 205·3 ± 70 pg/ml, P < 0·0001). By contrast, both IL-4 (113·7 ± 90 pg/ml) and IL-6 (227·6 ± 53 pg/ml) levels were only moderately increased in patients. Patients were treated with 1 mg/kg steroids according to a tapering schedule and their serum IL-18 and IFN-γ levels decreased progressively over the course of 1 year (P < 0·05 in both instances). Patients with DM and PM showed a similar trend of increase in their serum levels of cytokines.
[Fig. 1]
Fig. 1
Th1/Th2 cytokine imbalance in inflammatory myopathies (IM). Soluble levels of both Th1 [interleukin (IL)-18 and interferon (IFN)-γ] and Th2 (IL-4 and IL-6) cytokines were measured by enzyme-linked immunosorbent assay (ELISA). At diagnosis (month ...
High IL-18 muscular expression
IHC and ISH evaluated the in situ production of IL-18 in muscle specimens from patients with DM and PM. As shown in Fig. 2a–f, a predominance of IL-18+ cells was demonstrated in representative sections from patients with both DM (27 ± 6 cells/mm2, Fig. 2a,b) and PM (38 ± 9 cells/mm2, Fig. 2c–f), whereas IL-18 was undetectable in the control biopsies (Fig. 2l). However, the distribution of IL-18+ cells was not the same in specimens from DM and PM patients. Those from DM (Fig. 2a,b) showed IL-18+ cells (dark cells) surrounding the perivascular areas of the perimysium and infiltrating the extracellular matrix (Fig. 2a,b), whereas few cells were demonstrated within fibres of the endomysium. By contrast, specimens from PM displayed a prevalent endomysial accumulation of IL-18+ cells (Fig. 2c–f). As shown in Fig. 2c,d, many IL-18+ cells were found within fibres that were extensively infiltrated and damaged (inserts, Fig. 2c,d). In addition, most were located near the small arterioles of the endomysium (Fig. 2e,f), whereas the vessels of the perimysium were intact. Thus, it is conceivable that both diseases are characterized by initial vascular inflammation followed by either perimysial or endomysial migration of inflammatory cells. Further experiments illustrated the molecular expression of IL-18 by ISH. As shown in Fig. 2g,h, DM and PM specimens displayed many IL-18+ cells (dark spots) distributed uniformly around fibres and perivascular areas and containing cytoplasmic accumulations of IL-18/DNA. Figure 2i depicts a representative negative pattern from a control subject.
[Fig. 2]
Fig. 2
Representative pattern of muscle expression of interleukin (IL)-18 in both dermatomyositis (DM) and polymyositis (PM). Prominent expression of IL-18 (dark gray) occurred in all patients with DM (a, b) and PM (c–f). IL-18+ cells in DM were located ...
Infiltrating inflammatory cells express IL-18R
The next experiments investigated the muscle in situ expression of the IL-18R complex formed of the α and β subunits. As shown in Fig. 3a, IL-18Rα was expressed widely in both DM (Fig. 3a,b) and PM (Fig. 3c–f) specimens in parallel with the subunit β (data not shown). A prevalent expression of IL-18Rα+ cells was demonstrated in the extracellular matrix (Fig. 3a) and within the fibres in DM and PM (Fig. 3c,d), respectively. In addition, the majority of cells surrounding the perivascular areas of the perimysium (Fig. 3b) and endomysium (Fig. 3d–f) expressed high receptor levels. Lastly, there were many IL-18R+ cells within the arterial wall (Fig. 3b,e,f), both SMC and EC, whereas they were accumulated predominantly within the fibres of the PM specimens (insert in Fig. 3e).
[Fig. 3]
Fig. 3
Representative pattern of interleukin (IL)-18Rα expression. A dissemination of IL-18R+ within the extracellular matrix of the perymisium was demonstrated in sections from patients with dermatomyositis (DM) (a) in parallel to high accumulation ...
Macrophages and DC infiltrate both perimysium and endomysium
The phenotype of the inflammatory cells infiltrating the muscles was determined by evaluating the in situ expression of CD68 and CD83, namely the markers for macrophages and immature DCs, respectively. Both were detected, although located dissimilarly within the muscles, whereas they were absent in normal muscle. As shown in Fig. 4, CD68+ predominated in the DM specimen (Fig. 4a–c; 25·4 ± 8 mm2) compared to PM (Fig. 4d,e; 11·5 ± 3 mm2, P < 0·05), whereas the number of CD83+ cells (Fig. 4f–i) was slightly higher in PM (9·9 ± 3 mm2 and 6·3 ± 4 mm2, respectively). However, CD68+ cells in DM were located predominantly within the extracellular matrix (Fig. 4a,b) as isolated cells (4·7 ± 1 mm2) surrounding the fibres (Fig. 4c). A similar distribution of CD83+ cells was observed (Fig. 4f). Fewer CD68+ were found in patients with PM (Fig. 4g; 8·1 ± 2 mm2), whereas CD83+ cells (Fig. 4h,i; 10·3 ± 2 mm2) were documented prevalently within the perimysial fibres. In addition, several CD83+ cells displayed a plasmacytoid-like appearance with a remote nucleus in a large cytoplasm (arrows in Fig. 4h) reflecting incomplete maturation, as confirmed by the CD83 expression.
[Fig. 4]
Fig. 4
Macrophages and dendritic cells (DC) infiltrate the inflamed muscles. CD68 expression (dark cells) was prominent in dermatomyositis (DM) (a–c) compared to polymyositis (PM) (d, e) and prevalent in the extracellular matrix in the first and within ...
As a comparative analysis of sections at high magnification showed that CD68 and CD83 positive cells were expressing IL-18, ISH followed by IHC was repeated to identify its principal source. The in situ expression of CD68, CD83, CD8 and CD19 was explored. As shown in Fig. 5, CD68 was detected at high intensity within the cytoplasm of IL-18+ cells (dark spots within nucleus and cytoplasm) in both DM (Fig. 5a) and PM (Fig. 5b) in a fashion similar to CD83 (Fig. 5c,d, respectively). IL-18+ cells were CD8-negative by ISH (Fig. 5f), suggesting that macrophages and DC were the predominant producers of IL-18 in IM, although CD8+/IL18– cells in both diseases were also demonstrated. Other T cells may thus directly exert cytotoxic mechanisms that damage the muscle.
[Fig. 5]
Fig. 5
CD68+/CD83+ cells produce interleukin (IL)-18. As shown, both dermatomyositis (DM) (a, b) and polymyositis (PM) (c, d) were characterized by the presence of CD68+ and CD83+ cells [arrows in (b, d)]. Both subsets displayed a high nuclear and cytoplasmic ...
T lymphocytes infiltrate the muscles and express IL-18R
The next set of experiments identified the phenotype of these infiltrating cells. As shown in Fig. 6 (Fig. 6a,b), many CD8+ cells infiltrated the extracellular matrix of the perimysium in DM, whereas they were fewer in the endomysium (41·8 ± 17 mm2 and 7·3 ± 4 mm2). Conversely, patients with PM (Fig. 6c,d) displayed a homogeneous distribution of CD8+ cells within the perimysial vascular areas (11·3 ± 4 mm2) and endomysium (23·1 ± 5 mm2). In addition, the number of CD8+ cells within the endomysium in PM was double that in DM and modest in vascular sites (P < 0·05 in both instances), thus suggesting different pathogenetic mechanisms, namely early endothelial inflammation in DM and T cell-dependent cytotoxicity in PM. High magnification also revealed that most CD8+ cells expressed IL-18R, thus suggesting that T cells may be attracted within inflamed muscles under the control of IL-18 produced by other inflammatory cells.
[Fig. 6]
Fig. 6
CD8+ infiltrate both vessels and muscles in both dermatomyositis (DM) and polymyositis (PM). Immunostaining of muscle sections with anti-CD8 monoclonal antibody (MoAb) showed T cells infiltrating the extracellular matrix near the arteries in DM (a, b) ...
Vessel cells overexpress MCP-1
The expression of MCP-1 by both ECs and SMCs was evaluated to characterize endothelial inflammation as the primary event for diffusion of inflammatory cells within muscles. As shown in Fig. 7, MCP-1 was strongly expressed. Patients with DM displayed a large accumulation of MCP-1+ cells near and within the vessel wall and both SMC and EC (Fig. 7a–c) overexpressed MCP-1, whereas those with PM expressed MCP-1 to a lower extent (Fig. 7d). In addition, many cells located primarily in the extracellular matrix in DM (insert, Fig. 7c), or within the fibres in PM, expressed MCP-1 thus supporting its role in both attraction and migration of inflammatory cells toward muscle inflammation sites.
[Fig. 7]
Fig. 7
Monocyte chemoattractant protein (MCP)-1 is locally overexpressed in patients with dermatomyositis (DM). Immunohistochemistry (IHC) staining of paraffin-embedded sections with the anti-MCP-1 monoclonal antibody (MoAb) showed many positive cells within ...
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Discussion
Th1 cytokines, adhesion molecules and chemokines promote inflammation and muscle damage in IM [17]. Here, we provide evidence that IL-18, a cytokine driving both Th1 differentiation and IFN-γ production [18], is overproduced during the acute phases of muscle inflammation in patients with DM and PM.
High amounts of IL-18 were found in both macrophages and DC infiltrating the fibres. In addition, cytotoxic CD8+ cells were represented largely within injured fibres and apparently attracted into inflamed muscles under the stimuli of IL-18+ cells through the overexpression of the IL18-R. Lastly, both EC and SMC from patients with DM overexpressed MCP-1 in vascular areas infiltrated by IL-18+ cells, thus emphasizing endothelial inflammation as early pathogenic event in muscle inflammation.
The role of IL-18 in inflammatory disorders has been defined [19] and its serum elevations have been described in patients with lupus nephritis [12,20]. Predominance of Th1+ cells has been reported in patients with IM and both serum IFN-γ and TNF-α have been proposed as effective promoters of muscle inflammation. Here we demonstrated that IL-18 is involved primarily in these disorders and that its serum levels parallel those of IFN-γ and are increased significantly with respect to Th2 cytokines. Accumulation of IL-18+ cells within damaged fibres also suggests that the primary pathogenetic event comprises the Th1 immune response through IFN-γ overproduction, whereas the potential shift towards a Th2 profile with release of IL-4 may theoretically prevent the spread of inflammation [21]. However, the effective role of Th2 cytokines is still controversial. High doses of steroids are used commonly to treat IM. This raises the concern that the Th1/Th2 imbalance may be attributed to therapy [22]. In our study, all patients displayed high Th1 cytokine levels before treatment, followed by a progressive decrement to normal values on clinical remission. Thus, both IL-18 and IFN-γ are effective markers of active disease and may reflect the extent of muscle inflammation.
It has been suggested that serum levels of cytokines may not reflect actual inflammation owing to the interference of the rheumatoid factor and their tendency to bind the soluble receptors [23]. Thus, we demonstrated the in situ expression of IL-18 in biopsy specimens from patients with DM and PM, whereas the excess of IL-18 within inflamed muscles paralleled the serum levels and provided evidence for its pathogenic role. Also, it was evident that the in situ IL-18 expression was correlated with the number of inflammatory cells and the severity of muscle infiltration, thus indicating that its overproduction is involved in progressive muscle impairment.
The variable distribution of IL-18+ within muscles of patients with DM and PM suggests that different pathogenetic mechanisms drive accumulation of the inflammatory cells and Th1 cytokine overproduction. As demonstrated previously, DM is characterized by early activation of complement, leading to the formation and deposition of the membrano-lytic attack complex (MAC) on the endomysial microvasculature and resulting in endothelial damage, hypoperfusion and inflammation followed by perifascicular atrophy [24]. Muscle lesions in PM, on the other hand, are dependent on cytotoxic mechanisms against the myotubules and mediated by autoreactive T-cells recruited by both IFN-γ and TNF-α as well as adhesion molecules [25,26]. Our results parallel these observations and demonstrate that IL-18+ cells are located prevalently in both extracellular matrix and vascular areas, whereas they surround and infiltrate the fibres with a minimal perivascular deposition in DM and PM, respectively. These findings suggest that muscles constitute the principal sites of IL-18 production in IM. However, the prevalent IL-18 accumulation near the endothelial areas or in the context of fibres in patients with DM and PM emphasizes the involvement of different cell populations to drive the progression of muscle injury, either after their extravasation from inflamed endothelium or through a direct cytotoxicity of fibres.
An additional topic of the present study was the source of IL-18 within muscles. A prevalent accumulation of macrophages and DC was demonstrated in perivascular areas and around the fibres in both diseases. These cells produce IL-18 in various diseases [27], including IM [8]. In addition, migration of APC within muscles has been associated with their overexpression of IFN-γ, IL-17 and up-regulation of CCL20/CCR6 complex [8]. Both ISH and IHC suggested that CD68+ macrophages and CD83+ DC express a high IL-18 content and are its main local producers. Comparison of the biopsy specimens showed that most CD83+ cells also expressed IL18-R. Because the CD83 antigen expression indicates incomplete DC maturation, it is conceivable that immature cells migrate within muscles and maturate under the effect of the IL-18/IL18-R pathway activation. However, local DC maturation has been also attributed to up-regulation of both CCL20 and CCR6 chemokines by immature cells, whereas the low IL-4 production and the presence of CCL21, CCL19 and CCR7 chemokines emphasize the muscle migration of mature DC [8]. These observations are consistent with our demonstration of a reduced number of immature CD83+ cells in patients with DM in parallel with the finding of low IL-4 serum levels. Furthermore, IL-18+ inflammatory cells exert chemoattraction of IL-18R+ immature DC and lead to their migration within inflamed tissues and subsequent activation of the immune response [28]. Excess of both cytokines and chemokines by APC may thus concur to their activation in situ, although their individual role in IM is not known.
Muscle cytotoxicity in patients with PM is promoted primarily by autoreactive T cells activated by either IFN-γ or TNF-α[3]. We found that T cells were abundant within infiltrates and expressed both CD8 and the IL-18R, whereas they were almost negative for IL-18 production. Therefore, we reasoned that CD8+ T cells are responsive to IL-18 produced by neighbouring cells through IL-18R overexpression and may be recruited within muscles to induce cytotoxicity, fibre injury and spread of inflammation.
High IL-18R expression by both EC and SMC has been demonstrated in patients with atheromasia and associated with endothelial damage and inflammation operated by IL-18+ cells [29]. Moreover, IL-18 up-regulates both IFN-γ and TNF-α expression as well as different chemokines by vascular cells, molecules that may then facilitate the influx of inflammatory cells. We showed that EC and SMC express the IL-18R and that IL-18+ cells are located prevalently near both perivascular areas and the extracellular matrix in patients with DM. Thus, activation of the IL-18/IL-18R pathway may be a further mechanism promoting endothelial inflammation and the subsequent migration of inflammatory cells within muscles. In this context, it is conceivable that IL-18 exerts a pivotal role in the pathogenesis of IM and, in relation to its cellular producers or peculiar muscular accumulation, activates mechanisms that promote the muscle damage.
IL-18 stimulates the production of different chemokines, including MCP-1 [10]. High MCP-1 production has been associated with an increased cytotoxicity of myocytes as well as with the binding of the MAC to endothelial cells in PM and DM, respectively. Our results show that both EC and SMC strongly express MCP-1, indicating that endothelial inflammation is an early pathogenetic event leading to attraction of inflammatory cells through chemokine overproduction. The parallel expression of IL-18R may be an additional mechanism whereby IL-18 itself increases the extent of inflammation through MCP-1 expression.
In conclusion, our findings suggest that IL-18 has a pathogenetic role in IM by promoting imbalance of the immune system toward a Th1 phenotype. Because its elevations correlate with the severity of muscle injury, its blockade could be of assistance in the management of both DM and PM.
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Acknowledgments
This work was supported by PRIN 2003 and funds from University of Bari.
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Clin Exp Immunol
146(1): 21–31
2006
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1809738/
