Essential roles of Toll-like receptor-4 signaling in arthritis induced by type II collagen antibody and LPS

Eun-Kyu Lee1, Sang-Mee Kang1, Doo-Jin Paik1, Jung Mogg Kim2 and Jeehee Youn1

1 Department of Anatomy and Cell Biology, Institute of Biomedical Science and 2 Department of Microbiology, College of Medicine, Hanyang University, Seoul 133-791, Korea

Correspondence to: J. Youn; E-mail: jhyoun{at}hanyang.ac.kr


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Although bacterial LPS has been used to boost the susceptibility to antibody-induced arthritis, the mechanism of the action of LPS remains to be clarified. We investigated whether signals triggered by Toll-like receptor (TLR)-4 mediate the effects of LPS in the context of anti-type II collagen-induced arthritis. The mice defective in the Tlr-4 gene (Tlr-4lps-d) were markedly less susceptible than wild type to arthritis, as manifested in arthritic index, incidence and synovitis. Levels of the pro-inflammatory mediators, tumor necrosis factor-{alpha} and cyclooxygenase-2, in their synovial tissue were also much lower. Serum C3 deposition through the alternative pathway and de novo synthesis of C3 were lower in the Tlr-4lps-d mice in the post-acute phase, pointing to an influence of TLR-4 signals on the turnover rate of complement cascades. T cells from the Tlr-4lps-d mice failed to proliferate in response to an auto-antigen, glucose-6-phosphate isomerase (GPI), unlike those from wild-type mice, and the serum level of GPI-specific IgG antibody was significantly lower than in the wild-type mice. Interestingly, type 2 responses, such as GPI-specific IgG1 and IL-4 production, were up-regulated in the Tlr-4lps-d mice. Taken together, our data suggest that the TLR-4 signaling pathway plays an essential role in the initiation and progression of auto-antibody/LPS-triggered arthritis by inducing pro-inflammatory mediators, C3 deposition, auto-antigen-specific adaptive immune responses and immune deviation between type 1 and type 2 responses.

Keywords: antibody-induced arthritis, LPS, Th1/Th2, Toll-like receptor-4, type II collagen


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease, characterized by anarchic remodeling of joint architecture. Leukocytes that migrate into the joints in conjunction with activated synovial cells produce inflammatory and type 1 cytokines together with degradative enzymes that lead to progressive destruction of cartilage and bone (1). There are also systemic manifestations, such as the production of auto-antibodies. In particular, antibody to type II collagen (CII) is elevated in patients with RA (2, 3). Moreover, mice with certain haplotypes, such as H-2q and H-2r, suffering from collagen-induced arthritis (CIA), generate anti-CII antibodies that can passively transfer arthritis to mice with haplotypes other than H-2q and H-2r, suggesting a role of anti-CII antibodies in the development of arthritis (4). Based on this observation, the anti-CII antibody-induced arthritis (AIA) model was established by injecting mice with purified IgG mAbs to CII (5, 6). However, a huge amount of mAb and/or a combination of different mAb clones were required to achieve full-blown arthritis. Therefore, subsequent injection of bacterial LPS was used to reduce the threshold of the arthritogenic dose of mAb and the required number of mAb clones (710). This model appears to share distal-stage effector mechanisms with RA, as judged by massive recruitment of leukocytes and the involvement of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-{alpha} and IL-1ß (11). Mice lacking the Fc receptor (FcR) {gamma} chain are resistant to anti-CII AIA (12, 13), indicating that FcRs are involved in its causation. In addition, mice deficient in certain complement factors, or treated with anti-complement neutralizing antibody, are less susceptible to CIA than normal mice (1416) suggesting that the complement network also plays a role in AIA development. However, neither immediate effector molecules other than FcRs and complement factors nor the mechanism of the action of LPS has been elucidated.

Toll-like receptors (TLRs), expressed predominantly on innate effector cells such as macrophages and dendritic cells, recognize pathogen-associated molecular patterns and initiate innate inflammatory responses (17). TLRs are phylogenetically conserved members of the type I transmembrane receptor family with an intracellular Toll/IL-1 receptor (TIR) domain (17). An adaptor protein, myeloid differentiation antigen 88 (MyD88), binds to the TIR domain of TLRs through its own TIR motif, while a death domain at its C terminus recruits IL-1R-associated kinase (IRAK) 4 and IRAK1 to the complex. IRAK4 then induces the phosphorylation of IRAK1, allowing TRAF6 to associate with it. TRAF6, once dissociated from the receptor complex, can activate transforming growth factor-ß-activated kinase, which plays a crucial role in the activation of both the nuclear factor-{kappa}B pathway and the mitogen-activated protein kinase cascade. Of the 11 known human TLRs, TLR-4 functions as a signal-transducing receptor for LPS. C3H/HeJ mice are unresponsive to LPS due to a point mutation in the TIR domain of the Tlr-4 gene that prevents downstream signaling (18). TLR-4 also recognizes several other pathogen-associated molecular patterns, including lipoteichoic acids derived from Gram-negative bacteria, and envelope protein from mouse mammary-tumor virus (19, 20). It is also implicated in the recognition of host molecules such as heat shock protein (HSP) 60, HSP 70, fibrinogen and oligosaccharides of hyaluronan (2124). Engagement of TLR-4 with its agonists rapidly activates innate immunity by inducing the production of pro-inflammatory cytokines, such as TNF-{alpha}, IL-1 and IL-18 (25). In addition, stimulation of TLR-4 triggers dendritic cell maturation, which is characterized by increased co-stimulatory and antigen-presenting properties. IL-12 induced by TLR-4 signaling also contributes to the differentiation of activated naive CD4+ T cells into Th1 effector cells, but it is not known whether TLR-4 has any role in the induction of Th2 responses (26). Thus, TLR-4 engagement helps to direct adaptive immune responses to antigens derived from both microbial pathogens and the hosts themselves.

In the present study, to address how LPS synergizes the pathogenic roles of anti-CII antibodies, we tested whether TLR-4 signals influence the susceptibility of mice to anti-CII antibody/LPS-induced arthritis, using mutant mice functionally defective in TLR-4 signaling (Tlr-4lps-d mice). We found that the development of anti-CII AIA was solely dependent on engagement of TLR-4 with its agonist. Moreover, we showed that TLR-4 engagement activates several pathways, each of which has been proposed to play an important role in the pathogenesis of arthritis.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Induction of arthritis
BALB/cJ and BALB/c congenic C.C3 Tlr-4lps-d mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and kept under specific pathogen-free conditions. The mouse work was conducted in accordance with the laboratory animal care guidelines of Hanyang University. The arthritogenic anti-CII mAb cocktail obtained from Chondrex (Seattle, WA, USA) contains four mAbs (F10, A2, D8 and D1) in equal amounts. To induce arthritis, mice were injected intraperitoneally with 2 mg per mouse of anti-CII mAb cocktail and 3 days later with 50 µg per mouse of LPS (Escherichia coli 0111:B4 obtained from Chondrex, or 0127:B8 from Sigma–Aldrich, St. Louis, MO, USA).

Clinical evaluation of arthritis
Starting on day 0 after mAb injection, the mice were inspected blind for disease progression. The clinical severity of disease was scored using the scoring system previously described (1): 0, normal; 1, slight erythema and mild swelling confined to the mid-foot (tarsals) or ankle joint; 2, erythema and mild swelling extending from the ankle to the mid-foot; 3, erythema and moderate swelling extending from the ankle to the metatarsal joints and 4, intensive erythema and severe swelling encompassing ankle, foot and digits. All hind paws were graded, resulting in a maximal clinical score of 8 per mouse, and expressed as the mean arthritic index on a given day. Mice were scored as arthritic if more than one paw had a score >2. The circumference of the ankle of each hind paw was measured with a caliper.

Histopathological assessment
Hind paws were removed postmortem on day 14 after mAb injection, fixed in 10% (w v–1) phosphate-buffered formalin and decalcified in 5.5% EDTA in phosphate-buffered formalin. Decalcified paws were embedded in paraffin, sectioned and stained with hematoxylin and eosin. Arthritic changes in the ankle and foot were scored as previously described (1), where 0 = normal, 1 = weak leukocyte infiltration but no erosion, 2 = modest infiltration and weak erosion, 3 = severe infiltration and invasion of bones and 4 = loss of bone integrity.

Immunohistochemical assays
After de-paraffinization and quenching of endogenous peroxidase, the sections were permeabilized and blocked with 2% BSA and 1.5% normal rabbit serum. They were incubated with either goat anti-cyclooxygenase (COX)-2 antibody (sc-1745, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat anti-TNF-{alpha} antibody (sc-1348, Santa Cruz Biotechnology) at 1 : 50 dilutions, washed and incubated with biotinylated anti-goat IgG antibody (Vector Laboratories, Burlingame, CA, USA). Specific labeling was detected using an avidin–biotin–peroxidase complex (Vector Laboratories) and 3,3'-diaminobenzidine. The sections were counterstained with 1% methylene green solution.

Proliferation assays
When the mice were killed, spleen was excised and teased apart to make a single-cell suspension. Erythrocyte-depleted cells were cultured in 96-well plates at a density of 1 x 106 cells ml–1 (200 µl per well) in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U ml–1 penicillin, 100 µg ml–1 streptomycin and 50 µM 2-mercaptoethanol (Life Technologies, Rockville, MD, USA). The cells were cultured either in medium alone or 50 µg ml–1 glucose-6-phosphate isomerase (GPI) (Sigma–Aldrich) for 72 h, and 1 µCi per well of [3H]thymidine ([3H]TdR) was added for the final 12 h. The cells were harvested on glass fiber filters and [3H]TdR incorporation was measured with a liquid scintillation counter.

Reverse transcription–PCR
The hind paw joints were collected, dissected free of soft tissue and bones, snap frozen in liquid nitrogen and ground into fine pieces to measure the expression of TNF-{alpha} and COX-2. In addition, erythrocyte-depleted splenocytes were assayed for complement factor C3 expression. Total RNA was isolated with the Trizol reagent (Life Technologies), and cDNAs were synthesized by extension of random hexamer primers with 200 units of SuperScript II reverse transcriptase (Life Technologies) in a mixture containing 2 µg of total RNA for 1 h at 37°C. PCR of the cDNA was performed in a final 25 µl containing all four dNTPs, 1.5 mM MgCl2, 1.5 U of Taq polymerase (Takara Shuzo Co., Shiga, Japan) and each primer at 0.4 µM concentration (Bionex Co., Seoul, Korea). The amplification cycles were 94°C for 30 s, 55°C for 45 s and 72°C for 45 s. PCR products were separated on a 1.5% agarose gel after 35 cycles. Primer sequences for the PCR were as follows: COX-2 sense, 5' GCA CTA CAT CCT GAC CCA CT 3'; COX-2 antisense, 5'GAA CCC AGG TCC TCG CTT AT 3'; TNF-{alpha} sense 5' TCT CAT CAG TTC TAT GGC CC 3'; TNF-{alpha} antisense, 5' GGG AGT AGA CAA GGT ACA AC 3'; C3 sense, 5' ACA ACG TAG AGG CCA CAT CC 3'; C3 antisense, 5' GCA TGA TAC ACT GCC ACC AC 3'; ß2 microglobulin (ß2M) sense, 5' TGA CCG GCT TGT ATG CTA TC 3' and ß2M antisense, 5' CAG TGT GAG CCA GGA TAT AG 3'.

Assays of antibodies
After 14 days, sera were collected from AIA-treated mice and analyzed by isotype-specific ELISA to measure levels of GPI-specific antibodies. In brief, ELISA plates (Nunc, Roskilde, Denmark) were incubated overnight at 4°C with 50 µl of 20 µg ml–1 GPI solution. After discarding the coating solution, the plates were blocked with 1% BSA in PBS for 2 h at room temperature and washed. Mouse serum at a 1 : 100 dilution was added to each well. Serum collected from K/BxN mice (27) was serially diluted and used as standard for the GPI-specific total IgG and IgG1 ELISAs. Plates were then kept for 2 h at room temperature, washed and incubated with biotinylated detection antibody (either anti-mouse IgG-biotin or anti-mouse IgG1-biotin) for 1 h, and were also washed and incubated with extravidin–alkaline phosphatase (Sigma–Aldrich). Alkaline phosphatase activity was determined with phosphatase substrate tablets (Sigma–Aldrich) and an ELISA reader at 405 nm. Each sample was tested in triplicate and the mean value was recorded.

Functional assays for the alternative pathway of C3 deposition
An aliquot of mouse serum (10 µl) was added to 90 µl PBS containing 1 x 107 zymosan particles (Sigma–Aldrich), 10 mM ethylene glycol-bis(2-aminoethyl-ether)-N,N,N',N'-tetraacetic acid (EGTA) and 5 mM MgCl2, followed by incubation for 20 min at 37°C. The reaction was stopped with 10 mM EDTA. The zymosan particles were washed with FACS buffer (PBS containing 1% BSA and 0.1% sodium azide), incubated with anti-mouse C3-FITC (ICN Biomedicals, Aurora, OH, USA) and analyzed by FACS.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
LPS requirement for development of AIA
Although the anti-CII AIA model employs co-administration of anti-CII antibodies and LPS, the role of the LPS was ill-defined. To determine whether it is essential or dispensable for the onset and progression of anti-CII AIA in our system, BALB/c mice were injected with anti-CII antibody alone, 50 or 100 µg LPS alone or anti-CII antibody followed later by 50 µg LPS. As shown in Table 1, 50 and 100 µg LPS alone failed to induce arthritis; ~20% of the mice injected with anti-CII antibody alone were slightly arthritic, while co-administration of mice with anti-CII antibody and LPS resulted in severe arthritis in 100% of the mice. Thus, signals triggered by LPS appear to be required for the full-blown development of arthritis induced by a sub-arthritogenic dose of anti-CII antibodies.


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Table 1. Requirement for LPS in anti-CII AIA

 
LPS is an agonist for TLR-4 present on the cell surface. Lymph node and spleen cells obtained from BALB/c congenic mice bearing a loss-of-function mutation of the TLR-4 gene (Tlr-4lps-d) exhibited a dramatically reduced, but not completely abolished, proliferative response to LPS, compared with the wild type (data not shown). Thus, TLR-4 is needed to trigger a major pathway from LPS leading to proliferation. Proliferation in response to PHA was unaffected in the Tlr-4lps-d mice implying that their responses to stimuli other than LPS were intact (data not shown). We used these Tlr-4lps-d mice in subsequent experiments aimed at clarifying the role of TLR-4 in the development of arthritis.

Reduced incidence of arthritis in Tlr-4lps-d mice
We have shown that TLR-4 is needed to transduce LPS signals for growth. To assess whether the LPS dependence of anti-CII AIA (Table 1) is also dependent on TLR-4, mutant and wild-type mice were injected with anti-CII antibody plus LPS, and development of arthritis was observed. As shown in Fig. 1(A), the mutant mice developed only very limited clinical manifestations of arthritis. On day 7, when all wild-type mice were affected with arthritis, only ~30% Tlr-4lps-d mice were early arthritic.



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Fig. 1. Reduced arthritis in TLR-4 mutant mice. A. BALB/c (wt) and TLR-4 mutant (mt) mice were injected with 2 mg anti-CII antibody at day 0 and 50 µg LPS at day 3, followed by clinical scoring. These figures show means ± SEM of three independent sets of experiments (total n = 9 per group; ***P < 0.001, Student's t-test). B. Hind paw sections prepared on day 14 were stained with hematoxylin and eosin. Figures are representative of each group: normal, joints of untreated BALB/c mice; BALB/c wt, joints of AIA-established BALB/c mice and TLR-4 mt, joints of AIA-treated Tlr-4lps-d mice. Original magnification: x40. C. Histopathological index representing the mean histological score per group is shown (***P < 0.001, Student's t-test).

 
The hind paws were subjected to histological examination at the peak of arthritis (day 14). The histopathological index of each hind paw, which was scored according to the severity of leukocyte infiltration and bone invasion, was closely correlated with the clinical data from the individual mice (Fig. 1B). The mean histopathological index of the BALB/c control mice was 3.08 ± 0.23, with severe leukocyte infiltration and bone invasion found in most of the mice. In contrast, the joints of the Tlr-4lps-d mice were essentially normal with only occasional instances of early arthritis, and a much lower mean histopathological index of 1.17 ± 0.11 (Fig. 1C).

Reduced pro-inflammatory mediators in the joints of Tlr-4lps-d mice
COX-2 and TNF-{alpha} are pro-inflammatory mediators with pivotal roles in inflammatory responses. On day 14 after antibody treatment, the level of expression and distribution of COX-2 and TNF-{alpha} in affected joint tissues was assessed by immunohistochemical methods. The level of COX-2 positivity closely paralleled the clinical and histopathological data for individual mice. Joints from control mice with established AIA stained strongly with anti-COX-2 antibody, notably in leukocyte infiltrates and fibroblast-like stromal cells (Fig. 2A). However, the number of COX-2-positive cells was drastically reduced in the joint tissue of the treated Tlr-4lps-d mice and was comparable to that found in normal mice. Similarly, TNF-{alpha}-expressing cells were barely seen in the joints of the Tlr-4lps-d mice, but frequent in those of the wild-type mice with established AIA (Fig. 3A). To assess levels of COX-2 and TNF-{alpha} transcripts in the affected joint tissues, total RNA from the hind paws with soft tissues removed was assayed by reverse transcription–PCR. The levels of COX-2 and TNF-{alpha} transcripts were higher in joints of the control mice than in those of the Tlr-4lps-d mice (Figs 2B and 3B).



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Fig. 2. Deficiency of COX-2-producing cells in joints of Tlr-4lps-d mice. A. Hind paw sections were stained with anti-COX-2 antibody, followed by standard immunohistochemical processing. These figures are representative of each group. a, c, e, joints of AIA-established wild-type mice; b, d, f, joints of AIA-treated Tlr-4 mice and g, normal joint (original magnification: a, b, x40; c, d, g, x100 and e, f, x400). B. On day 14, joints of hind paws were removed and dissected free of soft tissue. Total RNAs were extracted from pooled joints of three mice per group and COX-2 transcripts measured by reverse transcription–PCR. A representative of three independent experiments is shown.

 


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Fig. 3. Deficiency of TNF-{alpha}-producing cells in joints of Tlr-4lps-d mice. A. Hind paw sections were stained with anti-TNF-{alpha} antibody, followed by standard immunohistochemical methods. These figures are representative of each group. a, c, joints of AIA-established wild-type mice; b, d, joints of AIA-treated Tlr-4lps-d mice and e, normal joint (original magnification: a, b, e, x100; c, d, x400). B. On day 14, joints of hind paws were removed and dissected free of soft tissue. Total RNAs were extracted from pooled joints of three mice per group and levels of TNF-{alpha} transcripts determined by reverse transcription–PCR. A representative of three independent experiments is shown.

 
C3 deposition via the alternative pathway was reduced in Tlr-4lps-d mice
Complement pathways, in particular the alternative pathway rather than the classical pathway, participate in the pathogenesis of the arthritis induced by transferring serum from K/BxN mice (28). We examined whether the capacity to deposit C3 induced by the alternative pathway is enhanced in the anti-CII AIA model and whether this process is associated with TLR-4 signals. Aliquots of mouse sera collected during the AIA protocol were incubated with zymosan particles in the presence of EGTA, an inhibitor of the classical pathway of complement cascades, and C3 deposition on zymosan particles was evaluated. Zymosan binding of C3 was increased 1 day after injection of anti-CII antibody, indicating the enhanced serum level of C3. While this acute phase response was the same in wild-type and mutant mice, C3 deposition was sustained or even slightly increased in the wild-type mice by day 8 after antibody injection (i.e. 5 days after LPS injection), whereas it had fallen in the Tlr-4lps-d mice (Fig. 4B). To observe whether this was due to different levels of C3 expression between the strains, we compared the levels of C3 transcripts in spleen cells on day 14 after AIA treatment. These proved to be much higher in the wild-type cells (Fig. 4C). In vitro LPS stimulation led to C3 induction in wild-type cells, confirming that C3 is a target of TLR-4 signaling. Thus, these results demonstrate that, although TLR-4 signaling has no influence on the acute phase of C3 deposition, it plays a role in maintaining AIA by increasing de novo C3 synthesis during the post-acute phase.



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Fig. 4. C3 deposition on zymosan through the alternative pathway. Serum was incubated with zymosan particles in the presence of EGTA and C3 binding to the zymosan particles was analyzed by FACS. A. Representative data for FACS (gray lines, negative controls incubated without serum). B. Percent increase in C3-binding zymosan particles. This figure shows means ± SEMs of three independent sets of experiments (***P < 0.001, total n = 9 per group). C. On day 14 after AIA treatment, spleen cells obtained from TLR-4 wild-type and mutant mice were assayed for C3 transcripts by reverse transcription–PCR before (N) and after exposure to 1 µg ml–1 LPS for 24 h (L).

 
Reduction of auto-antigen-specific T and B cell responses in AIA-subjected Tlr-4lps-d mice
GPI is supposed to serve as an auto-antigen in cases of RA (29) and its animal model K/BxN (27). To determine whether auto-antigen-specific T cells expand during the course of AIA and whether TLR-4 signals play a critical role in this expansion, spleen cells were isolated from the two experimental groups on day 14 after administration of antibody and LPS. Interestingly, in the control group, cells stimulated with GPI protein exhibited about a roughly 3-fold increase in [3H]TdR incorporation over unstimulated cells, whereas the cells from the Tlr-4lps-d mice were refractory to stimulation (Fig. 5A). These results demonstrate that the development of AIA involves the expansion of GPI-specific T cells by a TLR-4-dependent pathway. We also assessed whether B cells cognate to the GPI-specific T cells were activated in vivo by a TLR-4-dependent pathway. Two weeks after induction of AIA, GPI-specific total IgG production was elevated in the sera of wild-type mice but the increase was substantially lower in sera of the Tlr-4lps-d mice (Fig. 5B). Taken together, these data demonstrate that TLR-4 signals play a critical role in the induction of auto-antigen-specific T and B cell responses in anti-CII AIA.



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Fig. 5. Down-regulation of auto-antigen-specific adaptive immune responses in Tlr-4lps-d mice. A. RBC-depleted spleen cells obtained postmortem on day 14 from AIA-treated BALB/c wild-type and Tlr-4lps-d mice were stimulated with GPI protein, and [3H]TdR incorporation was assayed. B. Sera from BALB/c wild-type and Tlr-4lps-d mice were collected before treatment (pre) and at day 14 after AIA treatment (post). Serum levels of GPI-specific total IgG antibodies were measured as described in Methods (AU, arbitrary unit). The data are representative of three independent experiments with similar results (*P < 0.05, ***P < 0.001, Student's t-test).

 
Skewed induction of type 2 responses in AIA-subjected Tlr-4lps-d mice
TLR-4 signals promote the development of Th1, but not Th2 cells, and so skew immune responses toward the Th1 direction. We measured type 1 and type 2 antibody isotypes to GPI in sera from mice subjected to anti-CII AIA to see whether the TLR-4 signals triggered during AIA induction cause an imbalance between Th1 and Th2 responses. Anti-GPI IgG1, a type 2 isotype, was higher in the sera of untreated Tlr-4lps-d mice than in the BALB/c controls, and this difference was more dramatic by day 14 (Fig. 6A). Consistent with this, expression of IL-4, a cytokine representative of type 2 responses, was higher in the joint tissue from AIA-subjected Tlr-4lps-d mice than in the wild-type controls (Fig. 6B). Neither serum IgG2a specific for GPI nor IFN-{gamma} was detectable in the joints of either strain of mice before or after AIA induction (data not shown). These results provide in vivo evidence that TLR-4 signals down-regulate the development of Th2 responses in BALB/c mice.



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Fig. 6. Enhanced Th2 responses in Tlr-4lps-d mice. A. Sera from BALB/c wild-type and Tlr-4lps-d mice were collected before treatment (pre) and on day 14 after AIA treatment (post). Serum levels of GPI-specific IgG1 antibodies were measured as described in Methods (AU, arbitrary unit). B. On day 14, joints of hind paws were removed and dissected free of soft tissue. Total RNAs were extracted from pooled joints of three mice per group and expression levels of IL-4 transcripts determined by reverse transcription–PCR. The data are representative of three independent experiments with similar results (P < 0.02, Student's t-test).

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Previously, it has been shown that in anti-CII AIA models, LPS injection drastically reduced the threshold values of the arthritogenic dose of anti-CII mAb and negated the required multiple epitope specificity of the antibodies (7, 10). However, the molecular mechanism of the action of LPS remains elusive. The present study was performed to investigate the potential roles of TLR-4 signaling in the joint inflammation induced by anti-CII antibodies and LPS. We found that anti-CII antibodies alone without an agonist for TLR-4 were not sufficient to induce arthritis, suggesting an indispensable role of TLR-4 signals in the development of anti-CII AIA. This idea was confirmed by the finding that mice bearing a loss-of-function mutation in TLR-4 failed to develop arthritis in response to anti-CII antibodies and LPS. Importantly, we also found that in response to the attempted induction of AIA, Tlr-4lps-d mice were defective in the production of TNF-{alpha} and COX-2, as well as in the ability to deposit C3 via the alternative pathway, and in GPI-specific T and B cell responses, but gave strong Th2 responses. These results thus suggest that when anti-CII AIA is induced, diverse events occur in a TLR-4-dependent manner, including increased production of pro-inflammatory mediators, sustained activation of the alternative pathway of complement cascades, up-regulation of auto-antigen-specific adaptive immune responses and predominance of Th1 responses relative to Th2 responses.

Evidence of an association between TLRs and RA has accumulated. For instance, expression of TLR-2 and TLR-4 is found in synovial biopsies from patients with active RA (3032), and TLR-2, -4 and -9 transcripts are abundant in synovial tissue of naive mice (30). Moreover, injection of mice with streptococcal cell wall (SCW) fragments readily induced TLR-2, -4 and -9 mRNA (30). In this SCW-induced arthritis model, TLR-2 signaling was important for the onset of joint inflammation, as judged by failure of disease induction in TLR-2- or MyD88-deficient mice, whereas mice lacking TLR-4 developed joint inflammation like wild-type mice, despite being defective in chondrocyte proteoglycan synthesis. This difference appears to reflect the finding that cell wall fragments of certain Gram-positive bacteria such as SCW stimulate a TLR-2- but not a TLR-4-dependent pathway (19). Given that TLR-2 and TLR-4 share a proximal signaling pathway involving the adaptor molecule MyD88, it seems possible that TLR-4 agonists, if present at the onset of joint swelling, could also precipitate the disease. The finding that endogenous products that can be generated by inflammatory processes, such as HSP 60, HSP 70 and hyaluronic acid fragments, serve as TLR-4 agonists further supports this possibility (2124). In fact, it was recently demonstrated that TLR-4 signals are relevant to the pathogenesis of arthritis (33). C3H/HeJ mice injected with serum collected from a K/BxN arthritis model initiated acute paw swelling with similar kinetics to the C3H/HeOU control mice, but the paw swellings subsided more rapidly than in the controls, suggesting that innate immune functions activated via TLR-4 play a role in the maintenance, but not the initiation, of chronic inflammatory processes. These observations conflict with our evidence that TLR-4 signals participate not only in the maintenance of arthritis but also in its onset. This discrepancy presumably derives from the difference in the causative agents used, since we utilized anti-CII IgG2a and IgG2b antibodies plus LPS while the work with the K/BxN arthritis model employed only serum containing mainly anti-GPI IgG1 antibodies. Since different isotypes precipitate distinct outcomes of effector functions, anti-CII AIA and K/BxN serum-transferred arthritis may cause disease by somewhat different mechanisms.

It was demonstrated previously that arthritogenic antibodies, once produced, do not absolutely require T and B cells to provoke disease, since injection of serum from arthritic K/BxN mice into recombination activating gene (RAG)-deficient mice transferred arthritis efficiently (34). However, the observation that B cell-deficient K/BxN transgenic recipients developed more severe and more persistent arthritis than did B cell-deficient non-transgenic recipients raises the possibility that auto-reactive T cells do have an influence as part of a secondary mechanism activated during the late stage of disease. This possibility is in line with our data regarding the development of auto-antigen-specific T and B cells during joint swelling. It should be noted in addition that we also found that the TLR-4 pathway may be essential for inducing auto-antigen-specific T and B cell responses. Thus, even in the context of AIA, in which innate effector mechanisms play major pathogenic roles, auto-antigen-specific adaptive immunity triggered by TLR signaling may contribute to the maintenance of chronic joint inflammation.

While TLR-4 signaling is known to promote Th1-dependent immune responses (26), its roles in Th2 responses remain uncertain. Some investigators found a lack of influence of TLR-4 on Th2 responses (26), whereas others showed that MyD88 knockout mice produced stronger Th2 responses than wild-type mice (35). In our system, we could not detect auto-antigen-specific Th1-dependent IgG2a responses in either the wild-type or mutant mice before or after injection. This is consistent with the absence of IFN-{gamma} production in inflamed joints. The lack of Th1 responses may be due to the Th2-predominant genetic background of BALB/c mice. Alternatively, the small number of GPI proteins present in the soluble phase might tend to induce mainly Th2 responses rather than Th1 responses. We found that the Tlr-4lps-d mice produced higher amounts of auto-antigen-specific Th2-dependent IgG1 responses than wild-type mice even before injection, and this difference became more dramatic after AIA protocol. Thus, these results suggest that, when the genetic and environmental situation leads to Th2 predominance, TLR-4 signals have the regulatory function of preventing Th2 responses from overwhelming Th1 responses.

Taken together, our data show the first evidence that the induction of arthritis by a sub-arthritogenic dose of anti-CII antibodies and LPS is dependent on the TLR-4 pathway. It includes enhanced production of pro-inflammatory mediators, sustained activation of complement cascades, up-regulation of auto-antigen-specific adaptive immune responses and down-regulation of Th2 responses relative to Th1 responses. Although pathogen-associated molecular patterns able to activate the TLR-4 pathway have not yet been identified in inflamed joints, microbial products may not be absolutely required for TLR-4 signaling, since host components enriched in the inflamed joints, such as HSP 60, HSP 70 and fibronectin, have been shown to function as TLR-4 agonists. These findings support our evidence that TLR-4 is instrumental in developing joint inflammation. Thus, targeting of the TLR-4 pathway by interfering with the binding of TLR-4 agonists to their receptor may provide a novel therapy for inflammatory joint diseases such as RA.


    Acknowledgements
 
We thank Seokmann Hong for editing the manuscript, Doo Hyun Chung for providing K/BxN serum and Hee Jung Kang for the valuable discussion. This work was supported by a Korea Research Foundation grant (KRF-2002-041-E00108).


    Abbreviations
 
AIA   antibody-induced arthritis
CIA   collagen-induced arthritis
CII   type II collagen
COX   cyclooxygenase
FcR   Fc receptor
GPI   glucose-6-phosphate isomerase
HSP   heat shock protein
[3H]TdR   [3H]thymidine
IRAK   IL-1R-associated kinase
ß2M   ß2 microglobulin
MyD88   myeloid differentiation antigen 88
RA   rheumatoid arthritis
SCW   streptococcal cell wall
TIR   Toll/IL-1 receptor
TLR   Toll-like receptor
TNF-{alpha}   tumor necrosis factor-{alpha}

    Notes
 
Transmitting editor: K. Yamamoto

Received 24 November 2004, accepted 28 December 2004.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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