Seisuke Takemura

Lymphoid Neogenesis in Rheumatoid Synovitis

In rheumatoid arthritis (RA), tissue-infiltrating lymphocytes can be arranged in sophisticated organizations that resemble microstructures usually formed in secondary lymphoid organs. Molecular pathways and host risk factors involved in this process of lymphoid neogenesis remain to be defined. In a series of 64 synovial tissue biopsies, lymphoid follicles with germinal centers (GCs) were found in 23.4% of the patients. Follicular dendritic cells (FDCs) were exclusively present in tissues with GCs, suggesting that the recruitment or in situ maturation of FDCs is a critical factor for GC formation in the synovial membrane. Primary follicles were absent, emphasizing the role of Ag recognition in the generation of inflammation-associated lymphoid organogenesis. Multivariate logistic regression analysis of tissue cytokines and chemokines identified two parameters, in situ transcription of lymphotoxin (LT)-β and of B lymphocyte chemoattractant (BLC; BLC/CXCL13), that were predictors for FDC recruitment and synovial GC formation. LT-β and BLC/CXCL13 were found to be independent variables that could, in part, compensate for each other to facilitate GC formation. Prediction models incorporating in situ transcription of LT-β and BLC/CXCL13 had high negative yet moderate positive predictive values, suggesting that LT-β and BLC/CXCL13 are necessary but not sufficient. LT-β protein was detected on a subset of mantle zone and GC B cells, but also on T cells in follicular structures. BLC/CXCL13 was produced by FDCs in follicular centers, but was predominantly found in endothelial cells and synovial fibroblasts, suggesting heterotypic signaling between cells of the synovial membrane and infiltrating lymphocytes in regulating extranodal lymphoid neogenesis.

T Cell Activation in Rheumatoid Synovium Is B Cell Dependent

Rheumatoid arthritis results from a T cell-driven inflammation in the synovial membrane that is frequently associated with the formation of tertiary lymphoid structures. The significance of this extranodal lymphoid neogenesis is unknown. Microdissection was used to isolate CD4 T cells residing in synovial tissue T cell/B cell follicles. CD4 T cells with identical TCR sequences were represented in independent, nonadjacent follicles, suggesting recognition of the same Ag in different germinal centers. When adoptively transferred into rheumatoid arthritis synovium-SCID mouse chimeras, these CD4 T cell clones enhanced the production of IFN-γ, IL-1β, and TNF-α. In vivo activity of adoptively transferred CD4 T cells required matching of HLA-DRB1 alleles and also the presence of T cell/B cell follicles. HLA-DRB1-matched synovial tissues that were infiltrated by T cells, macrophages, and dendritic cells, but that lacked B cells, did not support the activation of adoptively transferred CD4 T cell clones, raising the possibility that B cells provided a critical function in T cell activation or harbored the relevant Ag. Dependence of T cell activation on B cells was confirmed in B cell depletion studies. Treatment of chimeric mice with anti-CD20 mAb inhibited the production of IFN-γ and IL-1β, indicating that APCs other than B cells could not substitute in maintaining T cell activation. The central role of B cells in synovial inflammation identifies them as excellent targets for immunosuppressive therapy.

BLyS and APRIL in rheumatoid arthritis

The cytokines B lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL) enhance autoimmune disease by sustaining B cell activation. In RA, B cells contribute to the formation of 3 functionally distinct types of lymphoid microarchitectures in the inflamed synovium: ectopic GCs; T cell-B cell aggregates lacking GC reactions; and unorganized, diffuse infiltrates. We examined 72 tissues representing the 3 types of synovitis for BLyS and APRIL production and for expression of APRIL/BLyS receptors. Biologic effects of BLyS and APRIL were explored by treating human synovium-SCID mouse chimeras with the APRIL and BLyS decoy receptor transmembrane activator and CAML interactor:Fc (TACI:Fc). GC+ synovitis had the highest levels of APRIL, produced exclusively by CD83+ DCs. BLyS was present in similar levels in all tissue types and derived exclusively from CD68+ macrophages. In GC+ synovitis, treatment with TACI:Fc resulted in GC destruction and marked inhibition of IFN-gamma and Ig transcription. In contrast, inhibition of APRIL and BLyS in aggregate and diffuse synovitis left Ig levels unaffected and enhanced IFN-gamma production. These differential immunomodulatory effects correlated with the presence of TACI+ T cells in aggregate and diffuse synovitis and their absence in GC+ synovitis. We propose that BLyS and APRIL regulate B cell as well as T cell function and have pro- and antiinflammatory activities in RA.

CD4+,CD28- T cells in rheumatoid arthritis patients combine features of the innate and adaptive immune systems.

OBJECTIVE To determine whether CD4+,CD28- T cells, which are expanded in patients with rheumatoid arthritis (RA), express receptors that typically regulate the function of natural killer (NK) cells. METHODS Expression of the NK cell surface molecules CD158, p70, CD94, CD161, and CD8alpha on T cell subsets was determined by multicolor flow cytometric analysis of peripheral blood mononuclear cells from 36 RA patients. Expression of CD161 on tissue-infiltrating CD4 T cells was determined by 2-color immunohistochemistry analysis of synovial tissue samples. RESULTS Killer cell-inhibitory receptors (KIR) and killer cell-activating receptors (KAR) were exclusively expressed on CD4+,CD28- T cells, with the CD158b molecule being the most frequently detected isoform. A coordinated mechanism inducing KIR/KAR expression was suggested by similarities in the expression of CD158b on CD4 and CD8 T cells. CD4+,CD28- T cells were also positive for CD8-alphaalpha homodimers, another characteristic shared with NK cells. Of the C-type lectin NK cell receptors (NK receptors), CD94 was consistently absent, but CD161 was found on a CD4 T cell population that is significantly expanded in RA patients (P = 0.01). Involvement in disease of NK receptor-expressing CD4 T cells was suggested by the presence of CD4+,CD161+ T cells in follicular microstructures typical of rheumatoid synovitis. CONCLUSION Patients with RA have an expanded and unusual subset of CD4 T cells that infiltrates the tissue lesions and is characterized by a deficiency of CD28, the expression of CD8-alphaalpha homodimers, and the expression of several types of HLA class I-recognizing NK receptors. CD4 T cells bearing NK receptors can bridge functions of the innate and adaptive immune systems, such as responsiveness to specific antigen, rapid release of interferon-gamma, cytotoxicity, independence from classic costimulatory pathways, and integration of multiple activating and inhibitory signals to control effector functions.

Cell-cell interactions in synovitis: Interactions between T cells and B cells in rheumatoid arthritis

In rheumatoid arthritis, T cells and B cells participate in the immune responses evolving in the synovial lesions. Interaction between T cells and B cells is probably antigen specific because complex microstructures typical of secondary lymphoid organs are generated. Differences between patients in forming follicles with germinal centers, T-cell–B-cell aggregates without germinal center reactions, or loosely organized T-cell–B-cell infiltrates might reflect the presence of different antigens or a heterogeneity in host response patterns to immune injury. Tertiary lymphoid microstructures in the rheumatoid lesions can enhance the sensitivity of antigen recognition, optimize the collaboration of immunoregulatory and effector cells, and support the interaction between the tissue site and the aberrant immune response. The molecular basis of lymphoid organogenesis studied in gene-targeted mice will provide clues to why the synovium is a preferred site for tertiary lymphoid tissue. B cells have a critical role in lymphoid organogenesis. Their contribution to synovial inflammation extends beyond antibody secretion and includes the activation and regulation of effector T cells.

Anterior Cruciate Ligament Reconstruction Using a Bone–Patellar Tendon–Bone Autograft to Avoid Harvest-Site Morbidity in Knee Arthroscopy

Although anterior cruciate ligament reconstruction using a bone-patellar tendon-bone (BPTB) autograft has many advantages (e.g., high strength and solid fixation), there are also several complications (e.g., anterior knee pain or kneeling pain) due to harvest-site morbidity associated with the use of this graft type compared with the use of hamstring tendon. Therefore the ultimate goal of anterior cruciate ligament reconstruction using a BPTB graft is to minimize harvest-site morbidity. We have used a technique for harvesting central-third BPTB grafts that involves only a 3-cm-long, longitudinal, curved incision in the medial tibial tuberosity for both graft harvesting and fixation. The purpose of this report is to describe the technique, which can avoid the harvest-site morbidities associated with BPTB autografts during knee arthroscopy. We believe that this less invasive reconstruction may reduce the harvest-site morbidities associated with BPTB grafts because it allows for BPTB graft harvesting without incising the synovial bursa or paratenon and mitigates scarring and adhesion formation.

Participation of substance P distribution in the cytokine production of rheumatoid synovium

Abstract Based on findings which suggested the involvement of the neuropeptide substance P in the pathogenesis of rheumatoid arthritis (RA), we investigated the mechanism of synovial pannus formation in RA, and examined the interaction between the cytokine production of synovial tissues and the concentration of substance P in the cartilage–pannus junction (CPJ). The CPJ and other peripheral synovial tissues were separately obtained from each part of the synovium from the knee joints of seven RA patients. The concentrations of substance P and the cytokines interleukin (IL)-1β and IL-6 in the CPJ and peripheral synovial tissues were determined by enzyme-linked immunosorbent assays. In addition, synovial cells were isolated from the CPJ and peripheral synovial tissues and treated with substance P or neurokinin-1 receptor antagonist to analyze the changes in cytokine production. The substance P levels were 211.2 and 50.5 pg/mg protein in the CPJ and the peripheral synovium, respectively. The IL-1β and IL-6 levels in the CPJ were 24.6 and 12.8 pg/mg protein, respectively. In the peripheral synovium, these levels were 4.3 and 2.5 pg/mg protein, respectively. In the CPJ, the IL-1β and IL-6 levels in tissue containing a high concentration of substance P (>200 pg/mg protein) were 39.4 and 21.6 pg/mg protein, respectively, and those in tissue containing a low concentration of substance P (≤200 pg/mg protein) were 11.6 and 5.1 pg/mg protein, respectively. Synovial cells from the CPJ produced higher levels of IL-1β and IL-6 than those from peripheral tissues. In addition, treatment of the cells with an NK-1 antagonist significantly reduced the production of these cytokines by the synovial cells. The theory that substance P plays a role in the pathogenesis of RA via the upregulation of cytokine production should be considered in further studies on the immunomodulatory properties of substance P in arthritis.

Substance P-induced priming effects on synovial cells

We detected and analyzed an intracellular mechanism of a substance P-induced priming effect on cytokine production using human synovial cells. The synovial tissues were isolated from the knee joints of osteoarthritis patients. After the administration of a low dose of substance P (1 nM) without significant effect alone, the synovial cells were stimulated with substance P (30 μM), phorbol 12-myristate 13-acetate (PMA) (100 nM), and calcium ionophore (A23187), (1 μM). The total interleukin (IL)-1β and IL-6 levels in the supernatant was measured by an enzymelinked immunosorbent assay (ELISA) kit, and the changes in the intracellular calcium concentration ([Ca2+]i) and protein kinase C (PKC) activation were measured by the fura 2-AM fluorescence method and a radioimmunoassay, respectively. The substance P-induced cytokine production was accompanied by an elevation of [Ca2+]i and PKC activation. The amounts of cytokines produced from the substance P (1 nM)-primed synovial cells stimulated with 30 μM substance P were approximately 4 times as much as that observed in non-primed cells. In addition, the priming treatment with 1 nM substance P enhanced not only the subsequent substance P-induced cytokine production, but also the PMA-induced response. However, substance P (1 nM) priming treatment did not affect the A23187-induced response. Furthermore, in substance P-primed cells, substance P (30 μM) induced a significant activation of PKC without changing the [Ca2+]i elevation response. These results suggest that the substance P-priming effect on synovial cells contributed to changes in intracellular mechanisms such as PKC activation.

Relationship Between HLA-DRB1-DQB1 Haplotypes and the Effect of Chicken Cartilage Soup Containing Type II Collagen on Rheumatoid Arthritis

The HLA-DQB1 and HLA-DRB1 genotypes were determined by a PCR-SSO technique in 65 Japanese outpatients with rheumatoid arthritis (RA). Group A consisted of patients having one or two HLA-DRB1*0405-HLA-DQB 1*0401 haplotypes. Group B consisted of patients without the haplotype. Eighteen and 14 patients were given chicken cartilage soup containing type II collagen (32mg/160ml) daily for over 3 weeks in group A and B, respectively (CII group). Nineteen and 14 patients were not treated with the soup in groups A and B, respectively (non-CII group).