A Rac1 inhibitory peptide suppresses antibody production and paw swelling in the murine collagen-induced arthritis model of rheumatoid arthritis

Introduction

The Rho family GTPase Rac1 regulates cytoskeletal rearrangements crucial for the recruitment, extravasation and activation of leukocytes at sites of inflammation. Rac1 signaling also promotes the activation and survival of lymphocytes and osteoclasts. Therefore, we assessed the ability of a cell-permeable Rac1 carboxy-terminal inhibitory peptide to modulate disease in mice with collagen-induced arthritis (CIA).

Methods

CIA was induced in DBA/1 mice, and in either early or chronic disease, mice were treated three times per week by intraperitoneal injection with control peptide or Rac1 inhibitory peptide. Effects on disease progression were assessed by measurement of paw swelling. Inflammation and joint destruction were examined by histology and radiology. Serum levels of anti-collagen type II antibodies were measured by enzyme-linked immunosorbent assay. T-cell phenotypes and activation were assessed by fluorescence-activated cell sorting analysis. Results were analyzed using Mann-Whitney U and unpaired Student t tests.

Results

Treatment of mice with Rac1 inhibitory peptide resulted in a decrease in paw swelling in early disease and to a lesser extent in more chronic arthritis. Of interest, while joint destruction was unaffected by Rac1 inhibitory peptide, anti-collagen type II antibody production was significantly diminished in treated mice, in both early and chronic arthritis. Ex vivo, Rac1 inhibitory peptide suppressed T-cell receptor/CD28-dependent production of tumor necrosis factor α, interferon γ and interleukin-17 by T cells from collagen-primed mice, and reduced induction of ICOS and CD154, T-cell costimulatory proteins important for B-cell help.

Conclusions

The data suggest that targeting of Rac1 with the Rac1 carboxy-terminal inhibitory peptide may suppress T-cell activation and autoantibody production in autoimmune disease. Whether this could translate into clinically meaningful improvement remains to be shown.     

IL-10 signaling in CD4+ T cells is critical for the pathogenesis of collagen-induced arthritis

Introduction

IL-10 is a very important anti-inflammatory cytokine. However, the role of this cytokine in T cells in the pathogenesis of collagen-induced arthritis is unclear. The purpose of this study was to define the role of IL-10 signaling in T cells in the pathogenesis of collagen-induced arthritis.

Methods

IL-10 receptor dominant-negative transgenic (Tg) and control mice were immunized with bovine type II collagen to induce arthritis. The severity of arthritis was monitored and examined histologically. T-cell activation and cytokine production were analyzed using flow cytometry. T-cell proliferation was examined by [³H]thymidine incorporation. Antigen-specific antibodies in serum were measured by ELISA. Foxp3 expression in CD4⁺ regulatory T cells (Tregs) was determined by intracellular staining or Foxp3-RFP reporter mice. The suppressive function of Foxp3⁺CD4⁺ Tregs was determined in vitro by performing a T-cell proliferation assay. The level of IL-17 mRNA in joints was measured by real-time PCR. A two-tailed nonparametric paired test (Wilcoxon signed-rank test) was used to calculate the arthritis and histological scores. Student's paired or unpaired t-test was used for all other statistical analyses (InStat version 2.03 software; GraphPad Software, San Diego, CA, USA).

Results

Blocking IL-10 signaling in T cells rendered mice, especially female mice, highly susceptible to collagen-induced arthritis. T-cell activation and proliferation were enhanced and produced more IFN-γ. The suppressive function of CD4⁺Foxp3⁺ regulatory T cells was significantly impaired in Tg mice because of the reduced ability of Tregs from Tg mice to maintain their levels of Foxp3. This was further confirmed by transferring Foxp3-RFP cells from Tg or wild-type (Wt) mice into a congenic Wt host. The higher level of IL-17 mRNA was detected in inflammatory joints of Tg mice, probably due to the recruitment of IL-17⁺γδ T cells into the arthritic joints.

Conclusion

IL-10 signaling in T cells is critical for dampening the pathogenesis of collagen-induced arthritis by maintaining the function of Tregs and the recruitment of IL-17⁺γδ T cells.     

Altered peptide ligands inhibit arthritis induced by glucose-6-phosphate isomerase peptide

Introduction

Immunosuppressants, including anti-TNFα antibodies, have remarkable effects in rheumatoid arthritis; however, they increase infectious events. The present study was designed to examine the effects and immunological change of action of altered peptide ligands (APLs) on glucose-6-phosphate isomerase (GPI) peptide-induced arthritis.

Methods

DBA/1 mice were immunized with hGPI_325-339, and cells of draining lymph node (DLN) were stimulated with hGPI_325-339 to investigate the T-cell receptor (TCR) repertoire of antigen-specific CD4⁺ T cells by flow cytometry. Twenty types of APLs with one amino acid substitution at a TCR contact site of hGPI_325-339 were synthesized. CD4⁺ T cells primed with human GPI and antigen-presenting cells were co-cultured with each APL and cytokine production was measured by ELISA to identify antagonistic APLs. Antagonistic APLs were co-immunized with hGPI_325-339 to investigate whether arthritis could be antigen-specifically inhibited by APL. After co-immunization, DLN cells were stimulated with hGPI_325-339 or APL to investigate Th17 and regulatory T-cell population by flow cytometry, and anti-mouse GPI antibodies were measured by ELISA.

Results

Human GPI_325-339-specific Th17 cells showed predominant usage of TCRVβ8.1 8.2. Among the 20 synthesized APLs, four (APL 6; N329S, APL 7; N329T, APL 12; G332A, APL 13; G332V) significantly reduced IL-17 production by CD4⁺ T cells in the presence of hGPI_325-339. Co-immunization with each antagonistic APL markedly prevented the development of arthritis, especially APL 13 (G332V). Although co-immunization with APL did not affect the population of Th17 and regulatory T cells, the titers of anti-mouse GPI antibodies in mice co-immunized with APL were significantly lower than in those without APL.

Conclusions

We prepared antagonistic APLs that antigen-specifically inhibited the development of experimental arthritis. Understanding the inhibitory mechanisms of APLs may pave the way for the development of novel therapies for arthritis induced by autoimmune responses to ubiquitous antigens.     

Indoleamine 2,3-dioxygenase-expressing dendritic cells are involved in the generation of CD4+CD25+ regulatory T cells in Peyer's patches in an orally tolerized, collagen-induced arthritis mouse model

Introduction

The present study was devised to understand the role of systemic indoleamine 2,3-dioxygenase (IDO) in the tolerance induction for orally tolerized mice in collagen-induced arthritis (CIA). We examined whether IDO-expressing dendritic cells (DCs) are involved in the generation of CD4⁺CD25⁺ regulatory T cells during the induction of oral tolerance in a murine CIA model.

Methods

Type II collagen was fed six times to DBA/1 mice beginning 2 weeks before immunization, and the effect on arthritis was assessed. To examine the IDO expression, the DCs of messenger RNA and protein were analyzed by RT-PCR and Flow cytometry. In addition, a proliferative response assay was also carried out to determine the suppressive effects of DCs through IDO. The ability of DCs expressing IDO to induce CD4⁺CD25⁺ T regulatory cells was examined.

Results

CD11c⁺ DCs in Peyer's patches from orally tolerized mice expressed a higher level of IDO than DCs from nontolerized CIA mice. IDO-expressing CD11c⁺ DCs were involved in the suppression of type II collagen-specific T-cell proliferation and in the downregulation of proinflammatory T helper 1 cytokine production. The suppressive effect of IDO-expressing CD11c⁺ DCs was mediated by Foxp3⁺CD4⁺CD25⁺ regulatory T cells.

Conclusion

Our data suggest that tolerogenic CD11c⁺ DCs are closely linked with the induction of oral tolerance through an IDO-dependent mechanism and that this pathway may provide a new therapeutic modality to treat autoimmune arthritis.     

Circulating mediators of bone remodeling in psoriatic arthritis: implications for disordered osteoclastogenesis and bone erosion

Introduction

Rheumatoid arthritis (RA) is a systemic disease manifested by chronic inflammation in multiple articular joints, including the knees and small joints of the hands and feet. We have developed a unique modification to a clinically accepted method for delivering therapies directly to the synovium. Our therapy is based on our previous discovery of an analog peptide (A9) with amino acid substitutions made at positions 260 (I to A), 261 (A to B), and 263 (F to N) that could profoundly suppress immunity to type II collagen (CII) and arthritis in the collagen-induced arthritis model (CIA).

Methods

We engineered an adenoviral vector to contain the CB11 portion of recombinant type II collagen and used PCR to introduce point mutations at three sites within (CII_{124-402, 260A, 261B, 263D}), (rCB11-A9) so that the resulting molecule contained the A9 sequence at the exact site of the wild-type sequence.

Results

We used this construct to target intra-articular tissues of mice and utilized the collagen-induced arthritis model to show that this treatment strategy provided a sustained, local therapy for individual arthritic joints, effective whether given to prevent arthritis or as a treatment. We also developed a novel system for in vivo bioimaging, using the firefly luciferase reporter gene to allow serial bioluminescence imaging to show that luciferase can be detected as late as 18 days post injection into the joint.

Conclusions

Our therapy is unique in that we target synovial cells to ultimately shut down T cell-mediated inflammation. Its effectiveness is based on its ability to transform potential inflammatory T cells and/or bystander T cells into therapeutic (regulatory-like) T cells which secrete interleukin (IL)-4. We believe this approach has potential to effectively suppress RA with minimal side effects.     

Toll-like receptor homolog RP105 modulates the antigen-presenting cell function and regulates the development of collagen-induced arthritis

Introduction

RP105 is a Toll-like receptor homolog expressed on B cells, dendritic cells (DCs), and macrophages. We investigated the role of RP105 in the development of collagen-induced arthritis (CIA).

Methods

CIA was induced in RP105-deficient DBA/1 mice and the incidence and arthritis index were analyzed. The cytokine production by spleen cells was determined. The functions of the DCs and regulatory T cells (Tregs) from RP105-deficient or control mice were determined by adding these cells to the lymph node cell culture. Arthritis was also induced by incomplete Freund's adjuvant (IFA) plus collagen or by injecting anti-collagen antibody and lipopolysaccharide.

Results

RP105-deficient mice showed accelerated onset of arthritis and increased severity. Interferon-gamma (IFN-γ) and tumor necrosis factor-alpha production by spleen cells from RP105-deficient mice was increased in comparison with that from wild-type mice. The DCs from RP105-deficient mice induced more IFN-γ production, whereas Tregs from those mice showed less inhibitory effect against IFN-γ production. RP105-deficient mice also showed more severe arthritis induced by collagen with IFA.

Conclusions

These results indicate that RP105 regulates the antigen-presenting cell function and Treg development, which induced the attenuation of the cell-mediated immune responses and, as a result, suppressed the development of CIA.     

Age-related changes in arthritis susceptibility and severity in a murine model of rheumatoid arthritis

Background

Rheumatoid arthritis (RA) most often begins in females in the fourth-fifth decade of their life, suggesting that the aging of the immune system (immunosenescence) has a major role in this disease. Therefore, in the present study, we sought to investigate the effect of age on arthritis susceptibility in BALB/c mice using the proteoglycan (PG)-induced arthritis (PGIA) model of RA.

Results

We have found that young, 1-month-old female BALB/c mice are resistant to the induction of PGIA, but with aging they become susceptible. PG-induced T cell responses decline with age, whereas there is a shift toward Th1 cytokines. An age-dependent decrease in T cell number is associated with an increased ratio of the memory phenotype, and lower CD28 expression. Antigen-presenting cells shifted from macrophages and myeloid dendritic cells in young mice toward B cells in older mice. The regulatory/activated T cell ratio decreases in older mice after PG injections indicating impaired regulation of the immune response.

Conclusion

We conclude that immunosenescence could alter arthritis susceptibility in a very complex manner including both adaptive and innate immunities, and it cannot be determined by a single trait. Cumulative alterations in immunoregulatory functions closely resemble human disease, which makes this systemic autoimmune arthritis model of RA even more valuable.     

TriVax-HPV: an improved peptide-based therapeutic vaccination strategy against human papillomavirus-induced cancers

Background

Therapeutic vaccines for cancer are an attractive alternative to conventional therapies, since the later result in serious adverse effects and in most cases are not effective against advanced disease. Human papillomavirus (HPV) is responsible for several malignancies such as cervical carcinoma. Vaccines targeting oncogenic viral proteins like HPV16-E6 and HPV16-E7 are ideal candidates to elicit strong immune responses without generating autoimmunity because: (1) these products are not expressed in normal cells and (2) their expression is required to maintain the malignant phenotype. Our group has developed peptide vaccination strategy called TriVax, which is effective in generating vast numbers of antigen-specific T cells in mice capable of persisting for long time periods.

Materials and methods

We have used two HPV-induced mouse cancer models (TC-1 and C3.43) to evaluate the immunogenicity and therapeutic efficacy of TriVax prepared with the immunodominant CD8 T-cell epitope HPV16-E7_49-57, mixed with poly-IC adjuvant and costimulatory anti-CD40 antibodies.

Results

TriVax using HPV16-E7_49-57 induced large and persistent T-cell responses that were therapeutically effective against established HPV16-E7 expressing tumors. In most cases, TriVax was successful in attaining complete rejections of 6–11-day established tumors. In addition, TriVax induced long-term immunological memory, which prevented tumor recurrences. The anti-tumor effects of TriVax were independent of NK and CD4 T cells and, surprisingly, did not rely to a great extent on type-I or type-II interferon.

Conclusions

These findings indicate that the TriVax strategy is an appealing immunotherapeutic approach for the treatment of established viral-induced tumors. We believe that these studies may help to launch more effective and less invasive therapeutic vaccines for HPV-mediated malignancies.     

Anti-T cell immunoglobulin and mucin domain-2 monoclonal antibody exacerbates collagen-induced arthritis by stimulating B cells

Introduction

T cell immunoglobulin and mucin domain-2 (TIM-2) has been shown to regulate CD4 T cell activation. However, the role of TIM-2 in the autoimmune disease models has not been clarified yet. In this study, we investigated the effects of anti-TIM-2 monoclonal antibodies (mAbs) in collagen-induced arthritis (CIA) to determine whether TIM-2 contributes to the development of T helper (Th) 1 or Th17 cells and joint inflammation.

Methods

DBA/1 mice were treated with anti-TIM-2 mAbs during the early or late phase of CIA. Type II collagen (CII)-specific CD4 T-cell proliferative response and cytokine production were assessed from lymph node cell culture. The serum levels of CII-specific antibody were measured by ELISA. The expression of TIM-2 on CD4 T cells or B cells was determined by flow cytometric analysis.

Results

Administration of anti-TIM-2 mAbs in early phase, but not late phase, significantly exacerbated the development of CIA. Although anti-TIM-2 mAbs treatment did not affect the development of Th1 or Th17 cells in the draining lymph node, the serum levels of anti-CII antibodies were significantly increased in the anti-TIM-2-treated mice. TIM-2 expression was found on splenic B cells and further up-regulated by anti-immunoglobulin (Ig)M, anti-CD40, and interleukin(IL)-4 stimulation. In contrast, CD4 T cells did not express TIM-2 even when stimulated with both anti-CD3 and anti-CD28 mAbs. Interestingly, anti-TIM-2 mAbs enhanced proliferation and antibody production of activated B cells in vitro.

Conclusions

TIM-2 signaling influences both proliferation and antibody production of B cells during the early phase of CIA, but not induction of Th1 or Th17 cells.     

Three dimensional structure directs T-cell epitope dominance associated with allergy

Background

CD4+ T-cell epitope immunodominance is not adequately explained by peptide selectivity in class II major histocompatibility proteins, but it has been correlated with adjacent segments of conformational flexibility in several antigens.

Methods

The published T-cell responses to two venom allergens and two aeroallergens were used to construct profiles of epitope dominance, which were correlated with the distribution of conformational flexibility, as measured by crystallographic B factors, solvent-accessible surface, COREX residue stability, and sequence entropy.

Results

Epitopes associated with allergy tended to be excluded from and lie adjacent to flexible segments of the allergen.

Conclusion

During the initiation of allergy, the N- and/or C-terminal ends of proteolytic processing intermediates were preferentially loaded into antigen presenting proteins for the priming of CD4+ T cells.     

Comparative analysis of collagen type II-specific immune responses during development of collagen-induced arthritis in two B10 mouse strains

Introduction

Immune responses against collagen type II (CII) are crucial for the development of collagen-induced arthritis (CIA). The aim of the present study was to evaluate and compare the CII-directed T cell and antibody specificity at different time points in the course of CIA using two mouse strains on the B10 genetic background - B10.Q, expressing A^q MHC class II molecules, and B10.DR4.Ncf1^*/*, expressing human rheumatoid arthritis-associated MHC II DR4 molecules (DRA*0101/DRB*0401).

Methods

B10.Q and B10.DR4.Ncf1^*/* mice were immunized with CII emulsified in adjuvant and development of CIA was assessed. T cells from draining lymph nodes were restimulated in vitro with CII peptides and interferon-gamma (IFN-γ) levels in culture supernatants were evaluated by ELISA. CII-specific antibody levels in serum samples were measured by ELISA.

Results

At four different CIA time points we analyzed T cell specificity to the immunodominant CII epitope 259-273 (CII259-273) and several posttranslationally modified forms of CII259-273 as well as antibody responses to three B cell immunodominant epitopes on CII (C1, U1, J1). Our data show that CII-specific T and B cell responses increase dramatically after disease onset in both strains and are sustained during the disease course. Concerning anti-CII antibody fine specificity, during all investigated stages of CIA the B10.Q mice responded predominantly to the C1 epitope, whereas the B10.DR4.Ncf1^*/* mice also recognized the U1 epitope. In the established disease phase, T cell reactivity toward the galactosylated CII259-273 peptide was similar between the DR4- and the A^q-expressing strains whereas the response to the non-modified CII peptide was dramatically enhanced in the DR4 mice compared with the B10.Q. In addition, we show that the difference in the transgenic DR4-restricted T cell specificity to CII259-273 is not dependent on the degree of glycosylation of the collagen used for immunization.

Conclusions

The present study provides important evaluation of CII-specific immune responses at different phases during CIA development as well as a comparative analysis between two CIA mouse models. We indicate significant differences in CII T cell and antibody specificities between the two strains and highlight a need for improved humanized B10.DR4 mouse model for rheumatoid arthritis.     

Plasmid-encoded NP73-102 modulates atrial natriuretic peptide receptor signaling and plays a critical role in inducing tolerogenic dendritic cells

Background

Atrial natriuretic peptide (ANP) is an important endogenous hormone that controls inflammation and immunity by acting on dendritic cells (DCs); however, the mechanism remains unclear.

Objective

We analyzed the downstream signaling events resulting from the binding of ANP to its receptor, NPRA, and sought to determine what aspects of this signaling modulate DC function.

Methods

We utilized the inhibitory peptide, NP73-102, to block NPRA signaling in human monocyte-derived DCs (hmDCs) and examined the effect on DC maturation and induced immune responses. The potential downstream molecules and interactions among these molecules involved in NPRA signaling were identified by immunoprecipitation and immunoblotting. Changes in T cell phenotype and function were determined by flow cytometry and BrdU proliferation ELISA. To determine if adoptively transferred DCs could alter the in vivo immune response, bone marrow-derived DCs from wild-type C57BL/6 mice were incubated with ovalbumin (OVA) and injected i.v. into C57BL/6 NPRA-/- knockout mice sensitized and challenged with OVA. Lung sections were stained and examined for inflammation and cytokines were measured in bronchoalveolar lavage fluid collected from parallel groups of mice.

Results

Inhibition of NPRA signaling in DCs primes them to induce regulatory T cells. Adoptive transfer of wild type DCs into NPRA-/- mice reverses the attenuation of lung inflammation seen in the NPRA-knockout model. NPRA is associated with TLR-2, SOCS3 and STAT3, and inhibiting NPRA alters expression of IL-6, IL-10 and TGF-β, but not IL-12.

Conclusions

Modulation of NPRA signaling in DCs leads to immune tolerance and TLR2 and SOCS3 are involved in this induction.     

Resting CD4+ effector memory T cells are precursors of bystander-activated effectors: a surrogate model of rheumatoid arthritis synovial T-cell function

Background

Previously we described a system whereby human peripheral blood T cells stimulated for 8 days in a cytokine cocktail acquired effector function for contact-dependent induction of proinflammatory cytokines from monocytes. We termed these cells cytokine-activated (Tck) cells and found that the signalling pathways elicited in the responding monocytes were identical whether they were placed in contact with Tck cells or with T cells isolated from rheumatoid arthritis (RA) synovial tissue.

Methods

Here, using magnetic beads and fluorescence-activated cell sorting, we extensively phenotype the Tck effector cells and conclude that effector function resides within the CD4⁺CD45RO⁺, CCR7^-, CD49d^high population, and that these cells are derived from the effector memory CD4⁺ T cells in resting blood.

Results

After stimulation in culture, these cells produce a wide range of T-cell cytokines, undergo proliferation and differentiate to acquire an extensively activated phenotype resembling RA synovial T cells. Blocking antibodies against CD69, CD18, or CD49d resulted in a reduction of tumour necrosis factor-α production from monocytes stimulated with CD4⁺CD45RO⁺ Tck cells in the co-culture assay. Moreover, blockade of these ligands also resulted in inhibition of spontaneous tumour necrosis factor-α production in RA synovial mononuclear cell cultures.

Conclusion

Taken together, these data strengthen our understanding of T-cell effector function, highlight the multiple involvement of different cell surface ligands in cell-cell contact and, provide novel insights into the pathogenesis of inflammatory RA disease.     

Functional consequences of DECTIN-1 early stop codon polymorphism Y238X in rheumatoid arthritis

Introduction

We have previously demonstrated that ex vivo inhibition of costimulatory molecules on antigen-pulsed dendritic cells (DCs) can be useful for induction of antigen-specific immune deviation and suppression of autoimmune arthritis in the collagen induced arthritis (CIA) model. The current study evaluated a practical method of immune modulation through temporary systemic inhibition of the costimulatory molecule CD40.

Methods

Mice with collagen II (CII)-induced arthritis (CIA) were administered siRNA targeting the CD40 molecule. Therapeutic effects were evaluated by clinical symptoms, histopathology, Ag-specific T cell and B cell immune responses.

Results

Systemic administration of CD40-targeting siRNA can inhibit antigen-specific T cell response to collagen II, as well as prevent pathogenesis of disease in both a pre- and post-immunization manner in the CIA model. Disease amelioration was associated with suppression of Th1 cytokines, attenuation of antibody production, and upregulation of T regulatory cells.

Conclusions

These studies support the feasibility of transient gene silencing at a systemic level as a mechanism of resetting autoreactive immunity.     

Semi-allogeneic dendritic cells can induce antigen-specific T-cell activation, which is not enhanced by concurrent alloreactivity

Background

Alloreactive T-cell responses are known to result in the production of large amounts of proinflammatory cytokines capable of activating and maturing dendritic cells (DC). However, it is unclear whether these allogeneic responses could also act as an adjuvant for concurrent antigen-specific responses.

Objective

To examine effects of simultaneous alloreactive and antigen-specific T-cell responses induced by semi-allogeneic DC.

Methods

Semi-allogeneic DC were generated from the F₁ progeny of inbred strains of mice (C57BL/6 and C3H, or C57BL/6 and DBA). We directly primed antigen-specific CD8⁺ and CD4⁺ T-cells from OT-I and OT-II mice, respectively, in the absence of allogeneic responses, in vitro, and in the presence or absence of alloreactivity in vivo.

Results

In vitro, semi-allogeneic DC cross-presented ovalbumin (OVA) to naïve CD8⁺ OT-I transgenic T-cells, primed naïve CD4⁺ OT-II transgenic T-cells and could stimulate strong alloreactive T-cell proliferation in a primary mixed lymphocyte reaction (MLR). In vivo, semi-allogeneic DC migrated efficiently to regional lymph nodes but did not survive there as long as autologous DC. In addition, they were not able to induce cytotoxic T-lymphocyte (CTL) activity to a target peptide, and only weakly stimulated adoptively transferred OT-II cells. The CD4⁺ response was unchanged in allo-tolerized mice, indicating that alloreactive T-cell responses could not provide help for concurrently activated antigen-specific responses. In an EL4 tumour-treatment model, vaccination with semi-allogeneic DC/EL4 fusion hybrids, but not allogeneic DC/EL4 hybrids, significantly increased mouse survival.

Conclusion

Expression of self-Major histocompatibility complex (MHC) by semi-allogeneic DC can cause the induction of antigen-specific immunity, however, concurrently activated allogeneic bystander responses do not provide helper or adjuvant effects.     

Low CD4/CD8 T-cell ratio associated with inflammatory arthropathy in human T-cell leukemia virus type I Tax transgenic mice

Background

Human T-cell leukemia virus type I (HTLV-1) can cause an aggressive malignancy known as adult T-cell leukemia/lymphoma (ATL) as well as inflammatory diseases such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). A transgenic mouse that expresses HTLV-1 Tax also develops T-cell leukemia/lymphoma and an inflammatory arthropathy that resembles rheumatoid arthritis. The aim of this study was to identify the primary T-cell subsets involved in the development of arthropathy in Tax transgenic mice.

Principal Findings

By 24 months of age, Tax transgenic mice developed severe arthropathy with a cumulative incidence of 22.8%. The pathological findings of arthropathy in Tax transgenic mice were similar to those seen in human rheumatoid arthritis or mouse models of rheumatoid arthritis, with synovial proliferation and a positive rheumatoid factor. Before the onset of spontaneous arthropathy, young and old Tax transgenic mice were not sensitive to collagen and did not develop arthritis after immunization with type II collagen. The arthropathic Tax transgenic mice showed a significantly decreased proportion of splenic CD4⁺ T cells, whereas the proportion of splenic CD8⁺ T cells was increased. Regulatory T cells (CD4⁺CD25⁺Foxp3⁺) were significantly decreased and CD8⁺ T cells that expressed the chemokine receptor CCR4 (CD8⁺CCR4⁺) were significantly increased in arthropathic Tax transgenic mice. The expression of tax mRNA was strong in the spleen and joints of arthropathic mice, with a 40-fold increase compared with healthy transgenic mice.

Conclusions

Our findings reveal that Tax transgenic mice develop rheumatoid-like arthritis with proliferating synovial cells in the joints; however, the proportion of different splenic T-cell subsets in these mice was completely different from other commonly used animal models of rheumatoid arthritis. The crucial T-cell subsets in arthropathic Tax transgenic mice appear to resemble those in HAM/TSP patients rather than those in rheumatoid arthritis patients.     

Detection of autoantibodies to citrullinated BiP in rheumatoid arthritis patients and pro-inflammatory role of citrullinated BiP in collagen-induced arthritis

Introduction

Anti-citrullinated protein/peptide antibodies (ACPAs) are highly specific to rheumatoid arthritis (RA) patients and are thought to have a close relationship with the pathogenesis of arthritis. Several proteins, including fibrinogen, vimentin, and alpha-enolase, were reported as ACPA-target antigens, and their importance in RA pathogenesis was widely proposed. We identified citrullinated immunoglobulin binding protein (citBiP) as another ACPA target in RA patients and examined its pro-inflammatory role in arthritis.

Methods

We measured the levels of anti-citBiP, anti-BiP, and anti-cyclic citrullinated peptide (CCP) antibodies in the serum of RA patients (n = 100), systemic lupus erythematosus (SLE) patients (n = 60), and healthy controls (n = 30) using ELISA and immunoblotting. Epitope mapping was performed using 27 citBiP-derived peptides. In the mouse study, after DBA/1J mice were immunized with BiP or citBiP, serum titers of ACPAs were measured by ELISA and immunohistochemistry. The development of collagen-induced arthritis (CIA) was observed in BiP- or citBiP-pre-immunized mice.

Results

The serum levels of anti-BiP and anti-citBiP antibodies were significantly increased in RA patients, although only anti-BiP antibodies were slightly increased in SLE patients. Interestingly, anti-citBiP antibody levels were higher than anti-BiP antibody levels in 72% of RA patients, whereas no significant increase in anti-citBiP antibody levels was detected in SLE patients and healthy controls. The serum levels of anti-CCP antibodies were correlated with those of anti-citBiP antibodies in RA patients (R² = 0.41). Several citrulline residues of citBiP were determined to be major epitopes of anti-citBiP antibodies, one of which showed cross-reactivity with CCP. Immunization of DBA/1J mice with citBiP induced several kinds of ACPAs, including anti-CCP and anti-citrullinated fibrinogen antibodies. Pre-immunization with citBiP exacerbated CIA, and anti-CCP antibody levels were increased in citBiP-pre-immunized CIA mice.

Conclusions

CitBiP is a newly described ACPA target that may play a pro-inflammatory role in arthritis.