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Published online before print March 2, 2004
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in fibroblasts correlates with elevated levels of CXCR3 ligands in septic arthritis synovial fluids
,2
* Laboratories of Molecular Immunology and
Immunobiology, Rega Institute for Medical Research, Leuven, Belgium
1 Correspondence: Laboratory of Molecular Immunology, Rega Institute, K.U.Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium. E-mail: paul.proost{at}rega.kuleuven.ac.be
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(IFN-) and CXCL11/IFN-inducible T cell 
chemoattractant, two chemoattractants for activated T cells and natural killer cells, and ligands for CXC chemokine receptor 3 (CXCR3) were detected in the synovial fluid of septic arthritis compared with osteo- and crystal arthritis patients. In vitro, IFN- and TLR3 ligation by double-stranded RNA (dsRNA) induced the expression of CXCL9 and CXCL11 in leukocytes and skin-muscle fibroblasts, whereas ligation of TLR2, TLR4, TLR5, and TLR9 by peptidoglycan (PGN), lipopolysaccharide (LPS), flagellin, and unmethylated CpG oligonucleotides, respectively, did not. PGN and LPS, but not unmethylated CpG oligonucleotides, even inhibited IFN--induced CXCL9 and CXCL11 expression in leukocytes. In sharp contrast, in fibroblasts, the TLR ligands PGN, dsRNA, LPS, and flagellin synergized with IFN- for the production of CXCL9 and CXCL11. Although TLR ligands stimulate leukocytes to produce CXCL8/interleukin-8 during the early innate defense, they contribute less to the production of CXCR3 ligands, whereas fibroblasts are important sources of CXCR3 ligands. These results illustrate the complex interaction between cytokines and TLR ligands in infection.
Key Words: chemokine • LPS • peptidoglycan • double-stranded RNA • leukocyte
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemokines are classified in subfamilies depending on the position of the NH2-terminal cysteines. The initial chemokine nomenclature was based on the source or target cells of the chemokines. The novel nomenclature uses the abbreviations CC chemokine ligand (CCL), CXCL, XCL, and CX3CL followed by the number of the corresponding gene for CC, CXC, C, and CX3C chemokines, respectively [1 ]. The numbering of chemokine receptors is on a historical basis, and an R (for receptor) replaces the L (for ligand) to indicate a chemokine [5 ]. The CXC chemokines without glutamic acid–leucine–arginine (ELR) motif just in front of the first cysteine attract and activate a broad panel of leukocytes including monocytes, B- and T-lymphocytes, and/or natural killer (NK) cells.
The three CXCR3 ligands, CXCL9/monokine induced by interferon- (Mig), CXCL10/IFN-inducible protein 10, and CXCL11/IFN-inducible T cell chemoattractant (I-TAC), are constitutively expressed in human thymus and are induced by the inflammatory cytokine IFN- in other tissues. They attract and activate NK cells and T helper cell type 1 (Th1) lymphocytes and therefore play a role in thymus lymphopoiesis as well as innate and adaptive immunity [6
7
8
9
10
11
]. CXCR3 is highly up-regulated on Th1 cells, and most of the synovial lymphocytes in rheumatoid arthritis patients are CXCR3+ [12
]. Moreover, administration of anti-CXCR3 antibodies diminishes the recruitment of activated Th1 cells to sites of inflammation [13
]. CXCL11 is a more potent ligand for CXCR3 than CXCL10 and CXCL9 [9
]. At elevated (µM) concentrations, CXCL9, CXCL10, and CXCL11 have been reported to display direct, receptor-independent, defensin-like antimicrobial activity [14
]. CXCL9, which is chemically more basic than CXCL10 and CXCL11 as a result of a number of Arg and Lys residues in its extended COOH-terminal part, has the highest antibacterial potency. Recently, CXCR3 ligands, at elevated concentrations, have been reported as antagonists for CCR3 [15
, 16
]. Thus, CXCL9, CXCL10, and CXCL11 do not only promote Th1 responses by attracting Th1 cells but also inhibit the attraction of CCR3+ Th2 cells. The important role of CXCR3 and its ligands in immunological responses is exemplified in vivo by the implication of CXCR3 and CXCL10 for acute allograft rejection [17
, 18
]. In addition to their role in host defense, CXCR3 ligands have antiangiogenic activity. By inhibiting endothelial cell growth and the angiogenic activity of ELR+–CXC chemokines such as CXCL8/interleukin-8 (IL-8) and CXCL6/granulocyte chemotactic protein-2, CXCR3 ligands delay wound healing and tumor growth and metastasis [19
20
21
22
23
24
].
Recently, we reported that CXCL8 and CXCL10 expression is differentially regulated by lipopolysaccharide (LPS) and peptidoglycan (PGN) in leukocytes and fibroblasts, and we detected enhanced CXCL10 concentrations in synovial fluids from septic arthritis patients [25 ]. In this study, levels of CXCL9 and CXCL11 were evaluated in the synovial fluid of septic arthritis, crystal arthritis, and osteoarthritis patients. The effect of microbial agents that act through Toll-like receptors (TLRs) on the expression of CXCL9 and CXCL11 was investigated in two important cell types of the synovial cavity, i.e., fibroblasts and leukocytes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Reagents
Recombinant human (rh)IFN- and Mig/CXCL9 were purchased from PeproTech (Rocky Hill, NJ). rhI-TAC/CXCL11 was from R&D Systems (Abingdon, UK). Bacterial LPS (Escherichia coli 0111:B4), bacterial PGN (Staphylococcus aureus), the double-stranded (ds)RNA polyriboinosinic:polyribocytidylic acid (polyrI:rC), E.coli flagellin, and the unmethylated CpG oligonucleotide and TLR9 ligand ODN2006 (5'-tcgtcgttttgtcgttttgtcgtt-3') were obtained from Difco Laboratories (Detroit, MI), Fluka Chemie GmbH (Buchs, Switzerland), Sigma Chemical Co. (St. Louis, MO), Inotek Pharmaceuticals Co. (Beverly, MA), and InvivoGen (San Diego, CA), respectively. Endotoxin contamination was limited to 600 pg LPS/µg flagellin, PGN contained 30 pg LPS/mg PGN, and IFN- contained less than 1 pg LPS/µg IFN-, as determined by the limulus amoebocyte lysate assay (BioWhittaker Europe, Verviers, Belgium).
Cells and induction experiments
Human peripheral blood mononuclear cells (PBMC) were isolated from single blood donations (blood transfusion centers of Antwerp and Leuven, Belgium). Erythrocytes were removed by sedimentation in hydroxyethyl starch (Plasmasteril, Fresenius Hemotechnology, Bad Homburg, Germany) for 30 min at 37°C. PBMC were further purified by density gradient centrifugation on Ficoll-sodium diatrizoate (Lymphoprep, Axis-Shield PoC AS, Oslo, Norway) for 30 min at 400 g. PBMC at 2 x 106/ml were seeded in 24-well dishes (1 ml/well) and induced at 37°C in RPMI 1640 with 10% fetal bovine serum (FBS; BioWhittaker Europe) and 50 µg/ml gentamicin (Invitrogen, Groningen, The Netherlands) for 24 h. The cellular concentration and viability of the PBMC were determined after stimulation by trypan blue staining followed by classical cell counting. Human diploid skin muscle-derived fibroblasts were grown in Eagle’s minimum essential medium with Earle’s salts (Invitrogen) containing 10% FBS. Confluent fibroblast monolayers (
0.2x106 cells/1.9 cm2) were treated with varying concentrations of inducers, the conditioned medium was harvested (after 72 h) and centrifuged at 900 g, and the supernatant was stored at –20°C until further analysis. For mRNA extraction, PBMC or fibroblasts were seeded and cultured in 25 cm2 tissue-culture flasks instead of 24-well dishes.
Immunoassays
Sandwich enzyme-linked immunosorbent assays (ELISAs) for human CXCL9 and CXCL11 were developed in our laboratory. Microtiter plates (Maxisorp, Nunc-Immuno Plate, Invitrogen) were coated with 1.67 µg/ml monoclonal mouse anti-human CXCL9 antibody (Ab; R&D Systems) or with 670 ng/ml polyclonal rabbit anti-human CXCL11 Ab (PeproTech) in phosphate-buffered saline (PBS; pH 7.4), containing 0.05% (v/v) Tween-20 (PBS/Tween) and blocked with 0.1% (w/v) casein in PBS/Tween. The CXCL9 or CXCL11 that bound to the capturing Ab was detected with 670 ng/ml polyclonal rabbit anti-human CXCL9 Ab (PeproTech) or 500 ng/ml monoclonal mouse anti-human CXCL11 Ab (R&D Systems). The secondary Ab were detected with peroxidase-conjugated donkey anti-rabbit immunoglobulin G (IgG; CXCL9 ELISA) or with peroxidase-conjugated anti-mouse IgG (CXCL11 ELISA; both diluted 1/5000 in PBS/Tween; Jackson Immunoresearch Laboratories, West Grove, PA). Peroxidase activity was quantified by measuring the conversion of 3,3',5,5',-tetramethylbenzidine (Sigma Chemical Co.) at 450 nm. Both ELISAs did not show cross-reactivity with other chemokines or with the chemokine inducers used. All samples from patients were diluted at least threefold in PBS before analysis.
mRNA isolation and quantitative analysis by TaqMan real-time polymerase chain reaction (PCR)
Total cellular RNA was isolated from stimulated PBMC by the guanidium thiocyanate method (RNeasy mini kit, Qiagen, Westburg, Leusden, the Netherlands). Total mRNA was converted into cDNA with the high-capacity cDNA archive kit, according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). Real-time PCR was performed in 25 µl total volume (50 cycles) on the ABI Prism 7700 sequence detection system. The reaction mixture consisted of 20 µl RNase-free water, 2.5 µl cDNA, 12.5 µl TaqMan Universal PCR master mix (no AmpErase uracil DNA glycosylase), 1.25 µl assay-on-demand for CXCL9 with optimized primers and 6-carboxyfluorescein (6-FAM)-labeled probes, and 1.25 µl 18S rRNA primers and probes (VIC-labeled probes and primer-limiting 18S predeveloped TaqMan assay reagents) as endogenous control (all reagents from Applied Biosystems) in the same reaction vial. Subtraction of the threshold cycle (Ct) for the internal control (18S rRNA) from the Ct for CXCL9 gives the 
Ct of the sample. The relative quantity of CXCL9 mRNA was determined with the Ct method [26
]. The Ct for an induced sample is the difference between the Ct of the induced sample and the Ct of the buffer-treated control sample. The relative quantity of CXCL9 mRNA in the sample equals 2–Ct.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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or microbial TLR ligands was a potent inducer of CXCL9 in PBMC (Fig. 2A
). Stimulation of PBMC with the TLR3 ligand dsRNA resulted in a low but significant induction of CXCL9. All other TLR ligands tested, i.e., the TLR2 ligand PGN, the TLR4 ligand LPS, the TLR5 ligand flagellin, and the TLR9 ligand unmethylated CpG oligonucleotide ODN2006, failed to induce CXCL9 protein production. In addition to CXCL9, CXCL11 was dose-dependently induced with IFN- in PBMC (Fig. 2B) . However, the amount of this most potent CXCR3 ligand produced was approximately tenfold lower compared with the amount of CXCL9. Treatment of PBMC with dsRNA resulted only in a moderate induction of CXCL11. In analogy with CXCL9, CXCL11 protein was not produced in PBMC treated with PGN, LPS, flagellin, or an unmethylated CpG oligonucleotide.
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or TLR ligands resulted in an induction pattern similar to that of PBMC in that only IFN- and dsRNA significantly induced CXCL9 and CXCL11 (Fig. 2C and 2D)
. However, the CXCL11 concentrations reached in fibroblasts were tenfold higher than those in PBMC, and dsRNA was an equally good or better inducer than IFN- of CXCL9 and CXCL11.
Effect of TLR ligands on IFN--induced CXCL9 and CXCL11 production by PBMC
During septic arthritis, TLR ligands and inflammatory cytokines such as IFN- are simultaneously present to activate synovial cells. Therefore, CXCL9 and CXCL11 production was evaluated in PBMC treated with IFN- in combination with PGN, dsRNA, LPS, flagellin, or the CpG oligonucleotide ODN2006. The TLR4 ligand LPS (5 µg/ml) clearly inhibited IFN--induced CXCL9 and CXCL11 protein production in a dose-dependent manner, whereas the TLR2 ligand PGN (10 µg/ml) completely abolished the induction of these CXCR3 ligands by IFN- (Fig. 3
). Real-time quantitative reverse transcriptase-PCR was performed to verify whether the reduction in CXCL9 protein was also detectable at the mRNA level. IFN- (200 ng/ml) treatment of the PBMC resulted, compared with untreated control cells, in about a 100-fold increase of the Mig mRNA. When the PBMC were cotreated with IFN- and LPS or PGN, this resulted in a 14-fold or ninefold decrease of Mig RNA (cf.d., with IFN--treated cells). In contrast to LPS and PGN, the respective TLR3 and TLR5 ligands dsRNA (100 µg/ml) and bacterial flagellin (100 ng/ml) did not significantly alter CXCL9 and CXCL11 protein production in IFN--treated PBMC (Fig. 3)
. In combination with IFN-, the TLR9 ligand ODN2006 synergistically induced CXCL11 but not CXCL9 (Fig. 3)
. In general, the amount of CXCL9 produced was five- to tenfold higher than the amount of CXCL11. Finally, the reduction in CXCL9 and CXCL11 protein production was not caused by an altered viability or proliferation of the PBMC as determined by trypan blue staining and cell counting (data not shown).
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- but not on dsRNA-induced CXCL9 and CXCL11 production by fibroblasts and microbial TLR ligands. Although of the tested TLR ligands, only dsRNA was able to induce CXCL9 and CXCL11 production in fibroblasts, simultaneous stimulation with IFN- and PGN, dsRNA, LPS, or flagellin resulted in a synergistic production of CXCL9 and CXCL11 (Fig. 4
). Indeed, the amounts of CXCL9 and CXCL11 produced by the fibroblasts were ten- to 100-fold higher compared with the amounts produced by IFN- alone. With the exception of the combination of IFN- and flagellin, the detected CXCL9 levels were higher compared with CXCL11 levels. In contrast to other TLR ligands, the TLR9 ligand unmethylated CpG oligonucleotide ODN2006 (1 µM) did not alter the IFN--induced CXCL9 or CXCL11 production in fibroblasts (data not shown). As dsRNA, on its own, is also an efficient inducer in fibroblasts (Fig. 2C
and 2D)
, this TLR3 ligand was combined with other TLR ligands (PGN and LPS). In contrast to simultaneous treatment with IFN- and TLR ligands, combined treatment with dsRNA and PGN or LPS did not result in a synergistic induction of CXCL9 or CXCL11 (Fig. 5
). Thus, IFN- seems to be an essential component to obtain synergistic induction of CXCL9 and CXCL11 in fibroblasts.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in a variety of cells including PBMC and fibroblasts [25
]. However, these three CXCR3 ligands significantly differ in their receptor-binding and -activating properties in that CXCL11 is more potent than CXCL10 and that CXCL10 is more potent than CXCL9 [9
].
In this study, enhanced concentrations of CXCL9 and CXCL11 were detected in septic compared with osteoarthritis and crystal arthritis synovial fluids. The median synovial concentrations of the most potent CXCR3 ligand (CXCL11) were about threefold lower than the concentrations of CXCL9 and of the recently reported CXCL8 and CXCL10 levels in septic arthritis patients [25
, 29
]. Thus, all three CXCR3 ligands likely contribute to the accumulation of lymphocytes and NK cells in the septic synovium. To identify possible inducers and local producer cells for the CXCR3 ligands during microbial infection, fibroblasts and leukocytes were stimulated with IFN- and/or TLR ligands. About fivefold more CXCL9 than CXCL11 was induced in PBMC after stimulation with IFN- alone, whereas fibroblasts produced equal amounts of CXCL9 as PBMC. In contrast to PBMC, fibroblasts produced about equal amounts of CXCL9 and CXCL11. Like CXCL10, but in contrast to the leukocyte chemoattractant CXCL8 [25
], the TLR ligands PGN, LPS, flagellin, and unmethylated CpG oligonucleotides did not induce CXCL9 and CXCL11 expression in leukocytes or fibroblasts. In PBMC, IFN--induced CXCL9, CXCL10, and CXCL11 protein secretion is even inhibited by the bacterial TLR2 and TLR4 ligands, PGN and LPS, but not by unmethylated CpG oligonucleotides. TLR ligands and IFN-, however, synergistically induce CXCL9 and CXCL11 in fibroblasts. This synergistic induction of CXCL9 and CXCL11 resulted in ten- to 100-fold increased levels of both CXCR3 ligands. At such elevated concentrations, not only the chemotactic but also the receptor-independent, defensin-like antimicrobial activity of CXCL9 becomes relevant [14
]. Synergistic induction of CXCR3 ligands in fibroblasts is not restricted to combinations of IFN- with TLR ligands. The synergistic induction of CXCL9 and CXCL10 in fibroblasts with IFN- and TNF- has been reported to depend partially on the cooperation between two transcription factors, i.e., signal transducer and activator of transcription-1 and nuclear factor-
B (NF-B) [30
, 31
]. Moreover, up-regulation of TLR2 expression on synovial fibroblasts by inflammatory cytokines and TLR ligands increases the sensitivity of these cells to PGN [32
, 33
].
These and other previously reported observations can be incorporated into a model for chemokine action in septic arthritis (Fig. 6
). Upon microbial infection, TLR ligands activate synovial macrophages and fibroblasts. Stimulation of TLR on leukocytes directly leads to the induction of CXCL8 and other neutrophil chemotactic cytokines [25
, 34
]. Induction of inflammatory cytokines such as IL-1ß, IFN-, and TNF- in leukocytes by TLR ligands further promotes CXCL8 production. IFN- alone does not induce CXCL8 in leukocytes but acts in synergy with the microbial TLR ligands LPS and PGN to produce additional CXCL8. In comparison with leukocytes, fibroblasts not only are weaker CXCL8-producer cells, but the production of the neutrophil chemoattractants CXCL8 and CXCL6 in fibroblasts is even inhibited by IFN- [25
]. Thus, the production of leukocyte-derived and not fibroblast-derived chemokines is important for neutrophil chemotaxis, leading to protease release and degradation of the ECM components. Furthermore, the secreted and activated protease gelatinase B/MMP-9 activates CXCL8 by clipping its N-terminal domain [35
]. In addition, fragments of the ECM glycosaminoglycan hyaluronan activate TLR4 and together with IFN-, stimulate macrophages to express additional cytokines and chemokines in a NF-B-dependent manner [36
37
38
].
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-induced production of all three CXCR3 ligands in leukocytes (Fig. 3
and ref. [25
]). Although upon stimulation with microbial TLR ligands, fibroblasts only moderately contribute to the production of neutrophil chemoattractants [25
], they are, in contrast to leukocytes, important producers of the CXCR3 ligands CXCL9, CXCL10, and CXCL11 (ref. [25
] and this manuscript). Activation of fibroblasts with TLR ligands in the presence of IFN- results in a synergistic production of all three CXCR3 ligands (Fig. 6)
. The CXCR3 ligands attract lymphocytes and NK cells that produce extra cytokines. Although the amounts of CXCL11 are lower than the amounts of CXCL9 and CXCL10 (Fig. 1
and ref. [25
]), this chemokine may be an important player in the attraction of CXCR3-positive T cells and NK cells as a result of its higher specific activity [9
]. The influx of activated CD26+ CXCR3+ Th1 cells will moreover result in chemokine processing by CD26/dipeptidyl peptidase IV (Fig. 6)
. CXCR3 ligands belong to the most efficiently processed CD26 substrates and become chemotactically inactive after truncation by this protease [22
, 39
]. CD26 will thus extinguish the inflammatory response in a negative-feedback loop. In conclusion, our findings show that although a number of inflammatory chemokines are present in a septic arthritis joint, the production of each chemokine is regulated in a different, inducer- and cell-dependent manner. In vitro, major differences in specific chemokine induction by TLR ligands were observed between leukocytes and fibroblasts, and minor differences were observed among the induction profiles of the three CXCR3 ligands. These data provide novel insights in the complex, inflammatory network that contains TLR ligands, cytokines, chemokines, and proteases.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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Received October 30, 2003; accepted February 3, 2004.
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and leaves RANTES and MCP-2 intact Blood 96,2673-2681 to induce the C-X-C chemokines Mig and interferon-inducible protein-10 in mouse macrophages J. Biol. Chem. 273,35088-35094B activation mediates the cross-talk between extracellular matrix and interferon- (IFN-) leading to enhanced monokine induced by IFN- (MIG) expression in macrophages J. Biol. Chem. 277,43757-43762This article has been cited by other articles:
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), septic arthritis (SA, 
), and crystal arthritis (CA, 
) were collected, and CXCL9 and CXCL11 concentrations were determined by ELISA. The detection limits for the CXCL9 (0.06 ng/ml) and CXCL11 (0.025 ng/ml) ELISA are indicated. Solid and dotted lines represent mean and median values, respectively. Statistical analysis was performed with the Mann-Whitney U-test, and Pvalues are indicated on top of the figures. The scale of the y-axis is logarithmic.
