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Rheumatology Advance Access originally published online on November 3, 2006
Rheumatology 2007 46(4):657-665; doi:10.1093/rheumatology/kel346
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Distinct expression pattern of IFN-{alpha} and TNF-{alpha} in juvenile idiopathic arthritis synovial tissue

M. Gattorno, L. Chicha1, A. Gregorio, F. Ferlito, F. Rossi, D. Jarrossay1, A. Lanzavecchia1, A. Martini and M. G. Manz1

Second Division of Pediatrics, ‘G. Gaslini’ Institute for Children and University of Genoa, Genoa, Italy and 1Institute of Research in Biomedicine, Bellinzona, Switzerland.

Correspondence to: M. Gattorno, MD, Second Division of Pediatrics, ‘G. Gaslini’ Institute and University of Genoa, Largo G. Gaslini 5, 16147, Genoa, Italy. E-mail: marcogattorno{at}ospedale-gaslini.ge.it


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives. Recent laboratory and clinical data suggest that two prototype autoimmune diseases, systemic lupus erythematosus and rheumatoid arthritis are mainly driven by distinct cytokines, interferon (IFN)-{alpha} and tumour necrosis factor (TNF)-{alpha}, respectively. We here investigated the presence and characteristics of natural type I IFN-producing cells (IPCs), as well as IFN-{alpha} and TNF-{alpha} expression at sites of inflammation in juvenile idiopathic arthritis (JIA).

Methods. Peripheral blood (PB) and synovial fluid (SF) mononuclear cells (MNCs) (n = 25 each) from JIA patients with active disease were studied. IPCs were identified as BCDA-2+CD123+HLA-DR+CD45RA+ cells, and dendritic cells (DCs) as CD11c+CD14–/lowlin– cells by flow cytometry. IPCs and DCs were analysed for Toll-like receptor-7 and -9 mRNA expression by real-time polymerase chain reaction. IFN-{alpha} was measured by enzyme-linked immunosorbent assay in serum, SF and in supernatants of influenza virus-infected, cultured IPCs. Synovial tissues of n = 6 additional JIA patients were analysed by immunohistochemistry using mAbs against CD123, IFN-{alpha}, TNF-{alpha}, CD3, CD19 and CD138.

Results. IPCs were enriched in SF MNCs compared with PB MNCs in all JIA patients. Influenza-induced, but no spontaneous IFN-{alpha} release was detected from SF IPCs, and serum and SF IFN-{alpha} levels were not elevated. Nonetheless, in synovial tissue IFN-{alpha} producing cells accumulated at inflammatory lymph-follicular-like structures, while TNF-{alpha} producing cells were mostly found at the lining and sublining layers.

Conclusions. These data suggest that besides TNF-{alpha}-expressing cells, IFN-{alpha}-producing IPCs are involved in initiation, maintenance or regulation of the inflammatory response in JIA.

KEY WORDS: Interferon-{alpha}, Plasmacytoid cells, Dendritic cells, Juvenile idiopathic arthritis


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Dendritic cells (DCs) are professional antigen-presenting cells, capable of initiating primary adaptive immune responses [1]. Immature DCs continuously sample antigens in tissues, and upon maturation, migrate to local lymphoid organs and present these antigens to lymphocytes [1, 2]. Natural type I interferon (IFN)-producing cells (IPCs) are a distinct small subset of circulating leucocytes, characterized by their typical (plasma cell-like) morphology [3, 4], and by their capacity to produce high amounts of type I IFN ({alpha} and ß) in response to viral infections [5, 6] or bacterial DNA containing unmethylated CG dinucleotide (CpG motifs) [2, 7–9]. IPCs are found in secondary lymphoid organs, express MHC class II molecules, and upon stimulation, mature to antigen-presenting cells, at least in vitro [10]. Therefore, IPCs were included in the DC family and were also termed as plasmacytoid pre-DCs (PDCs) [1].

As major IFN-{alpha} producers, IPCs play an important role in the regulation of immune responses by influencing T-cell activation and functional orientation [7, 11, 12], DC maturation [13], B-cell activation and plasma-cell differentiation [14] (for review see [2]). However, if dysregulated, IFN-{alpha} might lead to autoimmune diseases [15, 16]. Important evidence for this comes from several observations: patients treated with IFN-{alpha} for viral and malignant diseases are at risk for the development of autoimmune disorders [17–20]; elevated levels of IFN-{alpha} were found in sera of systemic lupus erythematosus (SLE) patients during disease flares [21]; cells with morphological and phenotypic characteristics of IPCs were shown to infiltrate skin lesions in SLE [22] and psoriasis [23]; and finally, microarray analysis demonstrated transcription of IFN-inducible genes in blood mononuclear cells (MNCs) from SLE patients [24]. How IPCs are activated to produce IFN-{alpha} in SLE is not fully understood; however, IPCs are thought to respond to immune-complexes formed by autoantibodies and DNA or RNA molecules derived from apoptotic cells [15].

In contrast to SLE, both clinical and laboratory data suggest that in chronic idiopathic arthritis tumour necrosis fator (TNF)-{alpha} is involved in development and maintenance of the disease [25]. Thus, it was proposed that different cytokine expression patterns characterize different autoimmune diseases, with IFN-{alpha} having a prominent role in SLE, and TNF-{alpha} in chronic inflammatory arthritis [16, 26]. However, this dichotomy might not be mutually exclusive as occasional rheumatoid arthritis (RA) onset during IFN-{alpha} treatment was reported [18], and cells with phenotypic and functional characteristics of IPCs were found to infiltrate the inflamed synovial tissue in adult RA [27–29]. Whereas previous studies have characterized the role of DC in adult RA [30, 31] and juvenile idiopathic arthritis (JIA) [32], no information is available on the possible role of IPCs in JIA. Here we analyse IPCs and DCs as well as IFN-{alpha} and TNF-{alpha} expression at the site of inflammation in JIA patients.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Patient and control samples
Parent informed consent was obtained according to institutional guidelines. Peripheral blood (PB) and synovial fluid (SF) samples of 25 consecutive patients with active JIA undergoing therapeutic arthrocentesis for steroid injection (F : M, 20 : 5) were analysed. Thirteen patients had a persistent oligoarticular form, ten an extended oligoarticular form, and two an RF-negative polyarticular form according to ILAR Durban criteria [33]. Clinical characteristics of JIA patients and treatment at study time are presented in Table 1. Synovial tissue samples were obtained from additional six JIA patients (Table 2). PB MNCs from age-matched healthy controls [n = 8; no history of inflammatory or infectious disorders within 4 weeks before examination and normal erythrocyte sedimentaion rate (ESR) and C-reactive protein (CRP)] attending our clinic for routine pre-operative examinations before minor surgery were used after parent informed consent was obtained. IPCs isolated from healthy adult PB MNCs were used as controls as indicated.


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  		TABLE 1. Clinical characteristics of the JIA patients at the moment of the study

 

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  		TABLE 2. Distribution of interferon-{alpha} and tumor necrosis factor-{alpha} producing cells in the synovial tissues of JIA patients

 
Samples were taken from patients and healthy controls, and stored after parental permission in accordance with the informed consent approved by the Ethical Committee of the ‘G. Gaslini’ Institute.

Sample preparation, flow cytometry and cell sorting
PB and SF MNCs were isolated by Ficoll-Hypaque (Sigma, St Louis, MO, USA) density gradient centrifugation. Cells were stored in 90% fetal calf serum (FCS) and 10% dimethylsulfoxide (DMSO) in liquid nitrogen until evaluation. Monoclonal antibodies (mAbs), conjugated with either FITC, PE, CyChrome or APC, against the following antigens were used: BDCA-2 (Ac144, Mylteni, Bergisch Gladbach, Germany), CD11c (3.9, eBioscience, San Diego, CA, USA), CD123 (9F5, BD Pharmingen, Franklin Lakes, NJ, USA), CD56 (B159, BD Pharmingen), CXCR3 (1C6, BD Pharmingen), CCR7 (150503, R&D Systems, Minneapolis, MN, USA), CD3 (S4.1, Caltag, Burlingame, CA, USA), CD19 (sJ25-C1, Caltag), HLA-DR (Immu-357), CD14 (RMO52), CD45RA (2H4), CD80 (MAB 104), CD83 (HB15A), CD86 (HA5-2B7), CD40 (MAB 89), CD62L (DREG 56) (all Immunotech, Marseille, France). PB and SF IPCs were identified as CD123+BCDA-2+ cells. For evaluation of IFN-{alpha} production, PB IPCs were isolated from adult healthy donors and SF IPCs were sorted as CD123+CD3–CD19–CD14–CD56– cells since BDCA-2 ligation on IPCs blocks IFN-{alpha} production [34]. Monocytes were characterized as CD11c+CD14high cells, and DCs as CD11c+CD14–/low CD3–CD19–CD56– cells. Flow cytometric analysis and sorting was performed using a flow activated cell sorting (FACS) Calibur and FACS Vantage SE, respectively (Beckton Dickinson, San Jose, CA, USA). Isotype-matched, irrelevant mAbs were used to determine the level of background staining.

INF-{alpha} production and measurement
To test INF-{alpha} production capacities, cells were plated in U-bottom 96-wells and were infected overnight with 1 : 30 40 HAU (HAU = haemagglutinin units) dilution of influenza virus (strain A/Beijing/353/89 kind gift of I. Julkunen) or CpG 2216 (5 µg/ml; Microsynth, Balgach, Switzerland) as previously reported [9]. INF-{alpha} was measured in culture supernatants by enzyme-linked immunosorbent assay according to the manufacturer instructions (PBL Biomedical Laboratories). Values were normalized to 1000 input cells. To evaluate intracellular IFN-{alpha} content, influenza-stimulated and unstimulated IPCs were spun on slides and were stained for IFN-{alpha} (MMHA-3, PBL laboratories, Piscataway, NJ, USA) and were revealed with alkaline phosphatase anti-alkaline phosphatase complex (APAAP, Dako, Glostrup, Denmark), using fucsin as chromogen.

In addition, IFN-{alpha} levels were measured in sera and SF of 20 JIA patients and in sera of 10 age-matched healthy controls.

Dendritic cell activation
Monocytes (CD11c+CD14high) and DCs (CD11c+CD14–/low) sorted from SF as well as from healthy PB MNC donors were cultured overnight in RPMI 1640 media (Gibco) supplemented with 10% FCS, 100 ng/ml granulocyte macrophage colony stimulating factor (GM-CSF) (Leukomax, Novartis, Switzerland), 20 ng/ml interleukin (IL)-4 (R&D Systems) and 0.1 µg/ml lipopolysaccharide (LPS) (Sigma Chemicals Co.).

Semiquantitative PCR analysis
IPCs and DCs were sorted from SF and RNA was isolated using TRIZOL reagent, according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). RNA samples were treated with DNase I (Invitrogen) to remove contaminating genomic DNA, and first-strand cDNA was synthesized using random primers and Superscript II reverse transcriptase (Invitrogen). Real-time PCR was performed using an ABI PRISM 7700 Sequence Detector (Perkin-Elmer) and TaqMan target mixes (Applied Biosystems). Amplification of endogenous ß-actin (ACTB Hs 99999903 m1) was done for each experimental sample as a control and reference accounting for differences in the amount and quality of total cDNA added to each reaction. Pre-TCR alpha chain was detected using target mix PTCRA (Hs 00300125 m1). For detection of Toll-like receptors (TLRs), the following sequences of primers and probes were used: TLR2 (F: CAGCACTGGTGTCTGGCATG, probe: CTGTGCTCTGTTCCTGCTGATCCTGC, R: GAGCCAGGCCCACATCATT), TLR4 (F: GTTTCCTGCAATGGATCAAGGA, probe: TCGTTCAACTTCCACCAAGAGCTGCCT, R: TGCTTATCTGAAGGTGTTGCACAT) TLR7 (F: TTACCTGGATGGAAACCAGCTACT, probe: AGATACCGCAGGGCCTCCCGC; R: TCAAGGCTGAGAAGCTGTAAGCTA), TLR9 (F: TGAAGACTTCAGGCCCAACTG, probe: AGCACCCTCAACTTCACCTTGGATCTGTC, R: TGCACGGTCACCAGGTTGT).

Immunohistochemistry
Tissue specimens were prepared for immunohistochemical stainings according to standard technique [35]. Briefly, specimens were fixed in 10% formalin for 4 h, dehydrated and embedded in paraffin. Sections of 4 µm were cut and layered on polylysine-coated slides. Slides were deparaffinized in xylene and rehydrated in descending grade (100–70%) of ethanol. Serial tissue sections were incubated overnight at +4°C with mouse Abs against CD123 (9F5, BD Pharmingen), INF-{alpha} (MMHA-3, PBL laboratories, Piscataway, NJ, USA) and for 30 min with Abs against CD3 (polyclonal, Dako), CD20 (L26, Dako), DC-LAMP (104.G4, Immunotech), CD1a (010, Immunotech), CD68 (KP1, Dako), TNF-{alpha} (4C6-H8, MONOSAN-Netherlands), CD138 (MI15, Dako) and CD117 (c-kit, polyclonal, Dako). Secondary labelling was performed for 30 min at room temperature with an anti-mouse Ig antibody conjugated to peroxidase labelled-dextran polymer (EnVision, Dako). The chromogenic diaminobenzidine substrate (DAB, Dako) was applied for 10 min. Slides were washed in PBS and were counterstained with Mayer's haematoxylin. Reactions in the absence of primary antibody and with isotype-matched, irrelevant antibodies (anti-cytomegalovirus, clones DDG9 and CCH2, Dako) were carried out as negative controls.

Statistical analysis
Percentages of PB and SF cell populations, and serum and SF IFN-{alpha} levels were compared by non-parametric Wilcoxon rank test. Differences with normal controls were calculated using Mann–Whitney U-test. Correlations among all the variables considered were evaluated using Spearman rank test.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
IPCs and immature DCs are enriched in SF compared with peripheral blood in JIA patients
Cells with phenotypic characteristics of IPCs (CD123+BDCA-2+) were identified in both PB and SF of all 25 JIA patients (Table 1, Fig. 1A). In 15 JIA patients, PB and SF monocytes (CD11c+CD14high) and DC (DC, CD11c+CD14–/low) were also characterized (Fig. 1B). Notably, when SF CD11c+CD14high and CD11c+CD14–/low cells were sorted and cultured overnight in media supplemented with LPS, GM-CSF and IL-4, the latter population acquired typical DC morphology, supporting the assumption that CD11c+CD14–/low cells were immediate DC precursors or DCs, whereas CD11c+CD14high cells maintained monocyte-like characteristics (Supplementary Fig. 1). In all 25 patients, IPCs were enriched in SF compared with PB (median 1.6%; range 0.8–3 vs 0.35%, range 0.1–1.7% of MNCs, respectively; P < 0.0001) (Fig. 2A). Similarly, in all 15 patients studied, DCs (CD11c+CD14–/low) and monocytes (CD11c+CD14high) were enriched in SF compared with PB (SF DCs median 6.3%, range 1.5–18%; PB DCs median 0.9%, range 0.5–3.4%; P = 0.0002; SF monocytes median 15.9%, range 5.1–38.4%; PB monocytes median 9.4%, range 3.4–28%; P = 0.009) (Fig. 2B and data not shown). No significant differences were found in percentages of PB IPCs and DCs between JIA patients and age-matched healthy controls, respectively (Fig. 2A and B).


Figure 1
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  		FIG. 1. Characterization of type I IPCs, monocytes and DCs in JIA patient PB and SF. Contour plots show (A) CD123+BDCA-2+ IPCs as well as CD11c+CD14highCD3–CD19–CD56– monocytes and (B) CD11c+CD14–/lowCD3–CD19–CD56– DCs in PB and SF of JIA patients. Typical dot plots of (A) n = 25 and (B) n = 15 samples are shown.

 

Figure 2
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  		FIG. 2. IPCs and DCs are enriched in JIA patient SF. Distribution of (A) IFN-{alpha}-producing cells (IPCs, CD123+BDCA2+) and (B) DCs (CD11c+CD14–/low in PB and SF from JIA patients, and in PB from age-matched healthy controls (A: 25 JIA patients; B: 15 JIA patients; A, B: n = 8 healthy age-matched controls). Boxes contain values falling between the 25th and 75th percentiles, whisker lines that extend from the boxes represent the highest and lowest values for each subgroups. Differences between paired PB and SF were evaluated by the Wilcoxon rank test.

 
Percentages of DCs and IPCs in PB and SF did not significantly correlate with parameters of disease activity (number of active joints, physician global score, ERS) or with the disease subtype. Interestingly, however, a trend towards a higher percentage of IPCs in SF of ANA-positive patients (n = 20) compared with ANA-negative patients (n = 5) was observed (median 2.1%, range 0.45–3.2, vs 1.07%, range 0.48–1.9%; P = 0.06, Mann–Whitney U-test).

JIA synovial fluid IPCs display low, while DCs display enhanced levels of activation compared with their respective peripheral blood counterparts
We next studied phenotypical features of PB and SF IPCs, DCs and monocytes. In all three SF populations, CD40 was slightly up-regulated compared with PB (Fig. 3A and B). Costimulatory molecules as CD80 and CD86, as well as CD83, displayed some degree of up-regulation in SF DCs, but were not up-regulated in SF IPCs (Fig. 3 and data not shown). Both chemokine receptors CXCR3 and CCR7 [36] were variably, but not substantially different, expressed in both PB and SF IPCs. In contrast, the homing molecule CD62L (L-selectin), was expressed on circulating IPCs, and was down-regulated on SF IPCs (Fig. 3).


Figure 3
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  		FIG. 3. Phenotypic characterization of monocytes, DCs and IPCs in JIA patient PB and SF. Histograms show indicated marker expression (bold line) and irrelevant isotype controls (dashed lines) on CD11+CD14high monocytes, CD11+CD14–/low DCs, and CD123+BDCA-2+ IPCs in paired (A) peripheral blood and (B) synovial fluid from JIA patients. Histograms depict representative analysis of at least six JIA patients evaluated for each marker.

 
As expected, IPCs isolated from SF expressed mRNA of TLR-7 and -9, but not of TLR-2 and -4, which in contrast were expressed by CD11c+CD14–/low DC (Supplementary Fig. 2).

Thus, IPC isolated from JIA SF display typical phenotypic features of this particular cell subpopulation in terms of surface markers and TLR expression, but in accordance with recent studies in adult spondyloarthropathy (SpA) [27] and RA [28], display little phenotypic activation, while SF monocytes and DCs appear somewhat activated with respect to costimulatory molecule, CD83 and CD40 expression.

JIA synovial fluid IPCs contain but do not spontaneously release IFN-{alpha}
We next evaluated functional activity of JIA patient SF IPCs. Because for both ethical and practical reasons, we were not able to obtain sufficient amounts of PB IPCs from JIA children, PB IPCs from healthy adult blood donors were used as controls in these experiments. Control PB IPCs displayed typical morphology of resting cells and did not contain intracellular IFN-{alpha} (Supplementary Fig. 3A). Upon stimulation with influenza virus or CpG, PB IPC developed DC-like morphology and stained positive for intracellular IFN-{alpha} (Supplementary Fig. 3B). Notably, a number of SF IPCs displayed a DC-like morphology, and some stained positive for IFN-{alpha} (Supplementary Fig. 3C). Although JIA SF IPCs did not spontaneously release IFN-{alpha} in culture supernatants, IFN-{alpha} release upon influenza virus or CpG stimulation from two SF samples tested was comparably efficient as observed from stimulated healthy donor peripheral blood IPCs (Supplementary Fig. 3D) [9]. Thus, compared with circulating healthy donor IPCs, JIA SF IPCs are somewhat activated with respect to DC-like morphology and intracellular IFN-{alpha} content.

In line with lacking spontaneous IFN-{alpha} release from JIA SF IPCs, IFN-{alpha} levels in SF were low (median 92 pg/ml, range 63–149 pg/ml, n = 20), and did not obviously differ from IFN-{alpha} levels in sera of both JIA patients (129 pg/ml, range 67–209 pg/ml, n = 20) and healthy controls (139 pg/ml, 20–184 pg/ml, n = 12).

IFN-{alpha}-expressing IPCs preferentially localize around lymphocyte aggregates while TNF-{alpha}-expressing cells mainly localize at the lining and sublining layer in JIA synovial tissue
Although SF is providing important information on disease activity, the primary inflammatory reaction in JIA occurs in synovial tissues. We thus studied synoviectomy tissues of six additional JIA patients (Table 2). Clusters of CD20 and CD3-positive cells, forming lymph-follicular aggregates were observed in five samples; however, none showed clear germinal centre reactions [37]. One sample displayed a diffuse lymphocytic infiltration with predominant CD3+ T-cell and few scattered CD20+ B-cell infiltrates [37].

IPCs were identified as cells with plasmacytoid morphology and co-localization of CD123+ and IFN-{alpha}+ in serial sections. IPCs were found mainly in areas surrounding T- and B-cell aggregates (Fig. 4A–D), and in some areas in less-organized lymphocytic infiltrates in the sublining layer (Fig. 4E–H). Since CD123 is expressed in synovial tissue on endothelial cells [37] and on mast cells besides on IPCs, [38] those cell types needed to be carefully excluded from the analysis. However, endothelial cells did not stain for IFN-{alpha}+ (Fig. 4H). Furthermore IFN-{alpha} and c-kit (CD117), the receptor for stem cell factor expressed on mast cells, was differently distributed in serial sections (Fig. 4I–K). Interestingly, in all T–B lymphoid aggregates observed (n = 5), IPCs were found in close anatomical relationship with CD138+ plasma cells (Fig. 5A–D). In contrast to IFN-{alpha}, TNF-{alpha} was prominently expressed in the lining and sublining layers either by cells of the monocyte-macrophage lineage (CD68+) and by resident fibroblast-like synoviocytes (CD68–) (Fig. 5E–J and Table 2). Only scattered TNF-{alpha}+ cells were found in the context of the lymphoid aggregates, but with a different localization than IFN-{alpha}-producing cells (Fig. 5E–G). Taken together, in inflammatory JIA tissues IFN-{alpha}-producing IPCs accumulate at lymph-follicular-like structures while TNF-{alpha}-producing cells are mostly found at the lining and sublining layers.


Figure 4
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  		FIG. 4. Co-localization of CD123 and IFN-{alpha} in synovial tissue. (A, B) Co-localization of CD123 and IFN-{alpha} in an area surrounding T- and B-cell aggregates in serial sections in a synovial tissue from a 14-year-old girl with RF-polyarticular JIA (patient 2, Table 2) (20x). (C, D) Details of the same areas (arrows) shown in the box in panels A and B, at higher magnification (40x). (E–F) Different localization of CD123 and IFN-{alpha} in a synovial tissue characterized by a prevalent diffuse perivascular lymphocyte infiltration from a 12-yr-old boy with ANA-positive persistent oligoarticular JIA (patient 1, Table 2) (10x). Details of the same areas are shown in panels G and H, respectively (20x). (I–K) Different distribution of CD123+ IFN-{alpha} + (I, J) cells and mast cells (CD117+) (K) (20x).

 

Figure 5
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  		FIG. 5. IFN-{alpha}-producing cells are localized predominantly around lymph-follicular aggregates while TNF-{alpha}-producing cells are localized mainly at the lining and sublining layer (A–D) T–B aggregate (panel A, CD3 and panel B, CD20) surrounded by IPCs (panel C) and plasma cells (panel D, CD138) in patient 4 (Table 2) (10x). (E–J) Different distribution of IFN-{alpha} and TNF-{alpha} in the context of the synovial membrane from a patient with persistent oligoarticular JIA (patient 6, Table 2). TNF-{alpha} is expressed by cells of monocyte-macrophage lineage (CD68+) and by fibroblast-like synoviocytes (CD68– cells) of lining and sublining layers (black arrows) (panels E and G, 5x). Details of the same areas shown in the box in panels E and G are shown at higher magnification in panels F and H, respectively (20x). Panel I shows the prevalent localization of IFN-{alpha}-producing cells corresponding to lymphoid aggregate (black arrows) (5x). Details of the same area in the box is shown in panel J (20x).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
Based on the findings in paediatric patients affected by systemic onset JIA and SLE it was proposed that two prototype autoimmune diseases, SLE and RA are mainly driven by distinct cytokines, IFN-{alpha} and TNF-{alpha}, respectively [13, 16, 26]. However, it is not clear if these observations on peripheral blood extend to the sites of tissue inflammation. Indeed, the primary initiators and cellular composition of the inflammatory infiltrate in JIA are not fully defined. Here we describe for the first time that IPCs accumulate at the sites of inflammation in JIA and that these IFN-{alpha}-producing cells show a different distribution compared with TNF-{alpha}-producing cells in inflamed synovial tissue. SF IPC numbers were about 5-fold increased compared with PB IPCs; SF IPCs displayed activated, DC-like morphology and contained intracellular IFN-{alpha} at levels comparable with those found in stimulated but not in ex vivo isolated, healthy donor peripheral blood IPCs. However, SF IFN-{alpha} levels were not elevated, and besides morphology and intracellular IFN-{alpha} content, SF IPCs did not show signs of activation as evaluated by costimulatory molecule expression. Furthermore, we show that DCs that display some signs of activation but are not fully matured, are about 7-fold increased in SF compared with PB.

Our findings on SF IPCs in JIA are in line with parallel, recently published studies which demonstrated accumulation of non-activated SF IPC and lack of IFN-{alpha} elevation in adult SpA and RA SF [27, 28]. In these, it was suggested that IPCs are actively recruited to SF but that IFN-{alpha} release was inhibited by so far non-defined soluble factors [28]. Given that the primary site of inflammation in JIA is synovial tissue (ST), SF might only serve as a surrogate for ST disease, and need to be interpreted in this context. We thus evaluated ST: here CD123+ IFN- {alpha}+ cells with plasma cell-like morphology, i.e. IPCs, accumulated at perivascular lymph-follicular-like aggregates [29]. In contrast, TNF-{alpha}-producing cells were mostly found at the lining and sublining layers.

As IPCs are demonstrated to be enriched in diverse autoinflammatory lesions [2, 22, 23, 28], it might be argued that IPC enrichment is a general, unspecific sign of chronic autoimmune disease. However, the peculiar distribution of IFN-{alpha}-producing cells in the context of synovial tissue may indicate an actual role of this cell population in the pathophysiology of JIA.

Elevated SF TNF-{alpha} levels have been demonstrated in JIA [39, 40] and anti-TNF-{alpha} therapy has been proven effective [41, 42]. In line with this, here we found TNF-{alpha} expressed at the level of sublining and lining layer and an accumulation of DCs and monocytes in the SF that likely produce it there. Thus, TNF-{alpha} might critically contribute in the activation of the inflammatory response and determination of tissue damage. With ongoing inflammation, IPCs might be recruited to inflamed synovial tissue together with B and T cells [28], likely via a CD62L-dependant mechanism, and consecutively enter the SF. Indeed, we found this homing molecule down-regulated on SF IPCs.

At inflammatory sites then, multiple cellular cross-regulatory interactions will occur. IPCs might be stimulated to produce IFN-{alpha} by immune complexes formed of anti-nuclear autoantibodies and DNA or RNA molecules via Fc{gamma}RII [15] or apoptotic cell-derived unmethylated CpG-DNA molecules via TLR-9 [15]. These same mechanisms potentially contribute to IPC activation in JIA. In SF, however, monocyte and possibly DC-produced TNF-{alpha} might inhibit major IFN-{alpha} release from IPCs [26–28], while some level of IFN-{alpha} production by IPCs in turn might initiate DC maturation [13], both features fitting with the data demonstrated here. In tissues, however, IFN-{alpha}-producing IPCs might sustain the autoimmune response by induction of plasma-cell differentiation [14] in a paracrine manner. In line with this, we found IPCs in the vicinity of CD138 expressing plasma cells at ST lymph-follicular infiltrates and higher proportion of IPCs in the SF of ANA-positive JIA patients (although not significant at this sample size). In this context, it is particularly intriguing that lymph-follicular aggregate formation in JIA correlates positively with the production of anti-nuclear autoantibodies [43].

On the other hand, CpG-stimulated IPCs have been shown to prime allogeneic CD4+CD25– T cells to differentiate into CD4+CD25+ regulatory T (Treg) cells [44]. These findings together with our recent observation of CD4+CD25+ Treg cell accumulation around the follicular aggregates in synovial tissue in JIA patients [45], might point to a regulatory role of IPCs in the inflamed synovium.

As most clinical data, the current findings on a heterogeneous group of JIA patients are descriptive and do not allow definitive conclusions. To establish the role of IPCs in initiation vs maintenance of disease, it will be helpful to compare primary manifestation with established disease patient groups. However, the study presented here provides the first definitive evidence that IFN-{alpha}-producing IPCs are part of the inflammatory response, suggesting a complementary rather than an alternative contribution of IFN-{alpha} and TNF-{alpha} in the pathogenesis of JIA. Given these findings together with previous data on RA [27–29], the current classification of autoimmune diseases according to a TNF-{alpha} or IFN-{alpha} dichotomy is helpful; however, it might be somewhat too simplistic and likely to be refined in future studies.

Formula


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
This work was partially funded by the Italian Ministry of Health (Ricerca Corrente).

The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 

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Submitted 16 December 2005; revised version accepted 8 September 2006.
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