Limited VH gene usage in B-cell clones established with nurse-like cells from patients with rheumatoid arthritis.

OBJECTIVES: Nurse-like stromal cells (NLC) in synovia and bone marrow of patients with rheumatoid arthritis (RA) can support pseudoemperipolesis, protect from apoptosis and enhance immunoglobulin production of peripheral blood B cells isolated from healthy individuals, suggesting the profound contribution of hyperactivation of B cells in RA. In the course of establishing RA-NLC from RA patients, we observed the growth of B cells in the presence of RA-NLC. METHODS: We cloned B cells from the synovium or bone marrow of RA patients using the limiting dilution technique. For established clones, nucleotide sequences of immunoglobulin and surface antigens were investigated. To investigate the dependence of these clones on NLC, differences in the proliferation and the amount of immunoglobulin produced in the presence or absence of NLC were compared. Immunocytochemical staining of various cells was performed using the antibody these clones produced. RESULTS: Nine B-cell clones established from RA patients showed RA-NLC-dependent growth. These B-cell clones expressed CD19, CD20, CD38, CD39 and CD40, suggesting that the cloned cells were mature and activated. All clones secreted immunoglobulins in culture media, which were specific for intracellular components of various cell lines, including RA-NLC. Interestingly, we found limited usage of immunoglobulin heavy-chain variable regions (VH) among B-cell clones from RA patients. These repertoires were reported to be detected preferentially in fetal livers. CONCLUSION: The present study provides a novel insight into the involvement of RA-NLC in the immunopathogenesis of RA via an autoreactive B cell development and/or activation mechanism.

Comparison of the activities of multinucleated bone-resorbing giant cells derived from CD14-positive cells in the synovial fluids of rheumatoid arthritis and osteoarthritis patients.

OBJECTIVE: To investigate the morphology and function of multinucleated bone-resorbing giant cells derived from CD14-positive cells in the synovial fluids (SF) of patients with rheumatoid arthritis (RA) or osteoarthritis (OA). METHODS: CD14-positive cells were obtained by magnetic-activated cell sorting of primary cultures of mononuclear cells from the SF. Multinucleated bone-resorbing giant cells were induced from the CD14-positive cells in the presence or absence of cytokines. We examined various characteristics, including osteoclast markers, fusion index and bone-resorption activities of the multinucleated giant cells. RESULTS: Multinucleated giant cells were induced from the CD14-positive cells in the SF of the RA and OA patients by the addition of interleukin (IL)-3, IL-5 and IL-7, or granulocyte-macrophage colony-stimulating factor (GM-CSF), respectively. These multinucleated giant cells were positive for tartrate-resistant acid phosphatase (TRAP), carbonic anhydrase II, actin, vitronectin receptor and the calcitonin receptor. However, the average values for the number of nuclei, fusion index and bone-resorption functions of the SF cells from the RA patients were significantly higher than those derived from the OA patients. CONCLUSION: These results suggest that the induction and activities of multinucleated bone-resorbing giant cells may play a pivotal role in bone destruction, and that these processes may be enhanced significantly in RA patients.

DS-Nh as an experimental model of atopic dermatitis induced by Staphylococcus aureus producing staphylococcal enterotoxin C.

DS-Nh mice raised under conventional conditions spontaneously develop dermatitis similar to human atopic dermatitis (AD), which is associated with staphylococcal infection. In the present study, we show that Staphylococcus aureus producing staphylococcus exotoxin C (SEC) was recovered from the culture of the skin lesions of DS-Nh mice with AD-like dermatitis and that the serum levels of anti-SEC antibodies from these mice were elevated. We describe here how to promote experimental AD by epicutaneous injection with SEC-producing S. aureus to DS-Nh mice. In order to assess the role of SEC in the pathogenesis of AD, the mitogenic activity, TCRBV repertoire analysis and the production of IL-4 and IFN-gamma from spleen mononuclear cells (MNC) from DS-Nh stimulated by SEC were compared with those due to SEA, SEB and TSST. The weakest was the mitogenic activity of SEC, and higher IL-4 responses and lower IFN-gamma responses to SEC showed correlation with TCRBV8S2-positive T cells, which were selectively stimulated by SEC. We also demonstrate that SEC-producing S. aureus was able to survive in DS-Nh after intradermal injection. These results suggest a possible role for SEC in the pathogenesis of AD through host-S. aureus relationships.

Differentiation of monocytes into multinucleated giant bone-resorbing cells: two-step differentiation induced by nurse-like cells and cytokines.

Bone resorption in the joints is the characteristic finding in patients with rheumatoid arthritis (RA). Osteoclast-like cells are present in the synovial tissues and invade the bone of patients with RA. The characteristics of these cells are not completely known. In the work reported here, we generated these cells from peripheral-blood monocytes from healthy individuals. The monocytes were co-cultured with nurse-like cells from synovial tissues of patients with RA (RA-NLCs). Within 5 weeks of culture, the monocytes were activated and differentiated into mononuclear cells positive for CD14 and tartrate-resistant acid phosphatase (TRAP). These mononuclear cells then differentiated into multinucleated giant bone-resorbing cells after stimulation with IL-3, IL-5, IL-7, and/or granulocyte-macrophage-colony-stimulating factor. TRAP-positive cells with similar characteristics were found in synovial fluid from patients with RA. These results indicate that multinucleated giant bone-resorbing cells are generated from monocytes in two steps: first, RA-NLCs induce monocytes to differentiate into TRAP-positive mononuclear cells, which are then induced by cytokines to differentiate into multinucleated giant bone-resorbing cells.

A new method for quantitative analysis of the mouse T-cell receptor V region repertoires: comparison of repertoires among strains.

We developed an adaptor ligation PCR-based microplate hybridization assay (MHA) to analyze the repertoires of mouse T-cell receptor (TCR) alpha- and beta-chain variable regions (TCRAV and TCRBV). RNA is transcribed to cDNA and an adaptor is ligated to the 5' end of the cDNA, which is then used as a template for PCR with an adaptor-specific 3' primer and a constant region-specific 5' primer. After hybridization of PCR products with TCRAV-and TCRBV-specific probes on the microplate, quantitative ELISA was carried out. The entire TCRAV or TCRBV repertoires could be analyzed using a single 96-well plate in triplicate and completed in less than 4 h. The assay results demonstrated the high level of specificity and reproducibility of this method. Furthermore, MHA results correlated well with those of fluorescence-activated cell sorting. This method may provide important information about various T-cell-associated diseases including autoimmune disease. The influence of the MHC on mouse TCR repertoires was next studied using the newly developed mouse TCRAV and TCRBV repertoire assay. The analysis in six strains showed no significant correlation between MHC haplotypes and TCRAV and TCRBV repertoires. However, large differences among strains was observed in TCRBV, but not in TCRAV repertoires. There were also large differences within same strain in TCRBV, but not in TCRAV repertoires, indicating differences in individuals independent of genetic factors. These data suggest that TCRBV repertoires are more susceptible than TCRAV repertoires not only to genetic factors but also some environmental factors.

Restricted usage of T-cell receptor alpha-chain variable region (TCRAV) and T-cell receptor beta-chain variable region (TCRBV) repertoires after human allogeneic haematopoietic transplantation.

We analysed T-cell receptor alpha-chain variable region (TCRAV) and T-cell receptor beta-chain variable region (TCRBV) repertoires in peripheral blood mononuclear cells (PBMCs) from 34 recipients of allogeneic bone marrow transplantation (allo-BMT), seven of allogeneic peripheral blood stem cell transplantation and 19 of autologous peripheral blood stem cell transplantation using the quantitative microplate hybridization assay. TCR usage skewed at an early period (6-7 weeks) after BMT. The change was more apparent in allogeneic recipients than in autologous recipients. In particular, a predominant increase was detected in the frequency of VA1-4 (26%, 11 of 41 recipients), VA3-1 (32%) and VB24-1 (28%). Interestingly, acidic amino acid residues frequently followed the arginine residue in complementarity-determining region 3 of BV24S1. We further examined the extent of skew using samples obtained at serial time points after transplantation. The normalization of skewed repertoires occurred over a long period of time (> 8 years). There was a significant difference in the rate of normalization of skewed TCR repertoires between adult and child recipients (P < 0.05). The results suggest that these T cells may have expanded in response to allogeneic antigens, such as miHA (minor histocompatibility antigen), and that altered repertoires are eventually normalized by T-cell regeneration via a thymic-dependent pathway in children.

T-cell activation and induction of antibodies and memory T cells by immunization with inactivated Japanese encephalitis vaccine.

Mouse brain-derived inactivated Japanese encephalitis (JE) vaccine is the only currently internationally accepted vaccine against JE virus. We analyzed cellular and humoral immune responses to the JE vaccine in healthy adults in order to understand the protective immunity induced by this vaccine. Immunization with the JE vaccine induced T-cell activation in vivo, demonstrated by increase in the plasma levels of interleukin (IL)-2 and soluble CD8. JE virus-specific antibodies determined in radioimmunoprecipitation (RIP), hemagglutination inhibition (HI), and neutralization assays were also induced by immunization with the JE vaccine. JE virus-specific memory T cells were detected 60 days after immunization. These results suggest that protective immunity induced by the inactivated JE vaccine includes JE virus-specific T cells as well as antibodies with multiple biological activities.

Establishment of nurse-like stromal cells from bone marrow of patients with rheumatoid arthritis: indication of characteristic bone marrow microenvironment in patients with rheumatoid arthritis.

OBJECTIVE: To investigate the microenvironment of bone marrow (BM) of patients with rheumatoid arthritis (RA). METHODS: Nurse cell-like BM stromal cell lines were established from BM mononuclear cells of patients with RA. We examined the various characteristics of these cell lines, including morphology, pseudoemperipolesis activity, cell surface markers, cytokine production and hyaluronan (HA) production. RESULTS: These RA BM nurse cell-like lines (RA-BMNC) were of mesenchymal origin and positive for CD44, CD54 and HLA-DR. They were defined as nurse cells because of pseudoemperipolesis activity that allowed lymphocytes to migrate underneath. RA-BMNC lines produced HA and multiple cytokines including interleukin (IL)-6, IL-7, IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF). HA production by BM stromal cells was correlated with pseudoemperipolesis activity. RA-BMNC produced significantly higher levels of IL-6, IL-8 and GM-CSF by co-culture with lymphocytes. The cells also produced IL-1beta, G-CSF and tumour necrosis factor only when co-cultured with lymphocytes. The RA-BMNC maintained the growth of CD14+ myeloid cells unique to severe RA. CONCLUSION: The present results both indicate that RA-BMNC are nurse cells and suggest that they may play an important role in the pathogenesis of RA.

Polyclonal expansion of TCRBV2- and TCRBV6-bearing T cells in patients with Kawasaki disease.

We examined T-cell receptor (TCR) usage, cytokine production and antibody responses to superantigens in patients with Kawasaki disease (KD) to facilitate a better understanding of the immunopathogenesis of KD. The mean percentage of VB2- or VB6. 5-bearing T cells in peripheral blood mononuclear cells (PBMC) of patients with acute-phase KD was significantly higher than that of patients in the convalescent phase of KD or in healthy donors. Expansion of VB2- or VB6.5-bearing T cells was polyclonal because DNA sequences in the complementarity determining region 3 of VB2- and VB6.5-positive cDNA clones were all different from each other. The plasma levels of interleukin (IL)-1beta, IL-2, IL-6, IL-8, IL-10, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and granulocyte colony-stimulating factor (G-CSF) were elevated in the acute phase of KD. We previously reported that streptococcal pyrogenic exotoxin C (SPEC) was a potent stimulator of VB2- and VB6.5-positive T cells and, furthermore, serum levels of anti-SPEC antibodies were significantly higher in patients with acute and convalescent KD than in age-matched controls. The results of the present study, together with those of our previous report, suggest that SPEC induces activation and polyclonal expansion of VB2- and VB6.5-positive T cells, and that SPEC-induced activation of T cells may lead to the pathogenesis of KD.

Establishment and characterization of nurse cell-like stromal cell lines from synovial tissues of patients with rheumatoid arthritis.

OBJECTIVE: To investigate the features of synovial stromal cells established from patients with rheumatoid arthritis (RA), and to define these cells as nurse cells. METHODS: Synovial nurse-like stromal cell lines (RA-SNCs) were established from patients with RA. These cell lines were examined for morphology, pseudoemperipolesis activity, cell surface markers, and cytokine production. The interaction between these RA-SNCs and a synovial tissue B cell clone was also examined. RESULTS: RA-SNCs had nurse cell activity. They spontaneously produced interleukin-6 (IL-6), IL-8, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor. Furthermore, they produced IL-1beta and tumor necrosis factor alpha and expressed higher levels of the other cytokines after coculture with the B cell clone. Proliferation and Ig production by the B cell clone were dependent on direct contact with RA-SNCs. CONCLUSION: These results indicate that the RA-SNCs were nurse cells. The findings suggest that RA-SNCs may play an important role in the pathogenesis of RA by producing large amounts of cytokines and maintaining infiltrating lymphocytes.