The spontaneous remission of juvenile idiopathic arthritis is characterized by CD30+ T cells directed to human heat-shock protein 60 capable of producing the regulatory cytokine interleukin-10

OBJECTIVE: To test the hypothesis that T cell reactivity to self heat-shock protein 60 (Hsp60) in patients with remitting juvenile idiopathic arthritis (JIA) is part of an antiinflammatory, regulatory mechanism. METHODS: Using peripheral blood-derived mononuclear cells (PBMCs) and synovial fluid-derived mononuclear cells (SFMCs) obtained from patients with JIA, we analyzed the expression of CD30 and the induction of regulatory cytokines in response to human and mycobacterial Hsp60. RESULTS: In oligoarticular JIA patients, in vitro activation of PBMCs and SFMCs with Hsp60 induced a high expression of CD30 on CD4+, activated (HLA-DR-positive), memory (CD45RO+) T cells. The expression of CD30 induced by human Hsp60 was much higher than that induced by mycobacterial Hsp60. In oligoarticular JIA patients with active disease, the expression of CD30 in response to human Hsp60 was paralleled by a high interleukin-10 (IL-10):interferon-gamma (IFNgamma) ratio. In addition, restimulated human Hsp60-specific T cell lines from oligoarticular JIA patients showed a high production of IL-10 and a low production of IFNgamma. In contrast, PBMCs and SFMCs from polyarticular JIA patients responded to human Hsp60 with virtually no expression of CD30 and a low IL-10:IFNgamma ratio. CONCLUSION: The results show that T cells responding to human Hsp60 in oligoarticular JIA patients express CD30, and during active phases of the disease, these T cells have a cytokine profile with a high IL-10:IFNgamma ratio. These findings suggest that in oligoarticular JIA patients, human Hsp60-specific CD4+ cells have a regulatory function and contribute to disease remission.     

Serum heat shock protein 60 can predict remission of flare-up in juvenile idiopathic arthritis

Heat shock protein (Hsp) 60 has been implicated in the pathogenesis of various inflammatory and autoimmune diseases. This study aimed to investigate synovial fluid and serum concentrations of Hsp60 and anti-Hsp60 and their relationship with juvenile idiopathic arthritis (JIA). Forty-eight patients with JIA, including 22 oligo-articular, 19 poly-articular, and 7 systemic diseases, and 33 normal controls were enrolled in this study. Synovial fluid and serum Hsp60 and anti-Hsp60 concentrations were measured via ELISA. Serum concentrations of Hsp60 of active and inactive oligo- and poly-articular JIA were significantly higher than those of normal controls. Serum concentration of anti-Hsp60 in active oligo-articular JIA was higher than that of normal controls (49.25 vs. 35.76 ng/mL, p = 0.059). Similarly, serum concentration of anti-Hsp60 in active poly-articular JIA was significantly higher than that of inactive samples (65.05 vs. 26.54 ng/mL, p = 0.008). In addition, serum concentration of Hsp60 correlated with the time required for remission from flare-ups in patients with JIA. Serum concentration of Hsp60 correlated well with time required for remission from flare-ups in patients with JIA, representing a potential disease marker to monitor disease activity.     

Inhibition of adjuvant‐induced arthritis by interleukin‐10‐driven regulatory cells induced via nasal administration of a peptide analog of an arthritis‐related heat‐shock protein 60 T cell epitope

Objective

To prevent and treat experimental arthritis via nasal administration of an altered peptide ligand (APL) from the major arthritogenic epitope in adjuvant‐induced arthritis (AIA) and to explore the mechanisms involved.

Methods

Peptides were administered nasally before and after induction of arthritis. Splenocytes and lymph node cells draining both the site of inflammation and the site of tolerance induction were used for cell transfer and were studied for antigen‐specific T cell characteristics. In addition, attempts were made to stop T cell tolerance in vitro, using anticytokine antibodies.

Results

Nasal administration of a modulatory APL of the heat‐shock protein 60 (Hsp60) 180–188 T cell epitope, alanine 183, had a suppressive effect in AIA that far exceeded that of the wild‐type epitope. In addition to its effectiveness in preventing AIA, alanine 183 may be effective in the treatment of ongoing AIA. The protective effect of alanine 183 can be passively transferred using activated splenocytes. Nasal administration of alanine 183 did not lead to detectable T cell proliferation or interleukin‐2 (IL‐2) production in mandibular lymph node cells, while transforming growth factor β (TGFβ), IL‐10, and IL‐4 were readily produced. Likewise, after nasally induced tolerance, followed by induction of arthritis, inguinal lymph node cells produced IL‐4, TGFβ, and IL‐10. After neutralizing in vitro the individual cytokines with anticytokine antibodies, only blocking of IL‐10 production led to reversal of tolerance, at the site of tolerance induction and the site of inflammation.

Conclusion

Nasal administration of an APL of Hsp60 180–188 induces highly effective protection against AIA through generation of regulatory cells that produce IL‐4, TGFβ, and IL‐10, whereas the induced tolerance is driven mainly by production of IL‐10.

     

Escherichia coli heat‐labile enterotoxin B subunit prevents autoimmune arthritis through induction of regulatory CD4+ T cells

Objective

The receptor‐binding B subunit of Escherichia coli heat‐labile enterotoxin (EtxB) is a highly stable, nontoxic protein that is capable of modulating immune responses. This study was conducted to determine whether mucosal administration of EtxB can block collagen‐induced arthritis (CIA) and to investigate the mechanisms involved.

Methods

Clinical arthritis in DBA/1 mice was monitored following mucosal administration of EtxB on 4 occasions. The dependence of disease prevention on receptor binding by EtxB and the associated alterations to the immune response to type II collagen (CII) were assessed. Adoptive transfer experiments and lymph node cell cocultures were used to investigate the underlying mechanisms.

Results

Both intranasal and intragastric delivery of EtxB were effective in preventing CIA; a 1‐μg dose of EtxB was protective after intranasal administration. A non–receptor‐binding mutant of EtxB failed to prevent disease. Intranasal EtxB lowered both the incidence and severity of arthritis when given either at the time of disease induction or 25 days later. EtxB markedly reduced levels of anti‐CII IgG2a antibodies and interferon‐γ (IFNγ) production while not affecting levels of IgG1, interleukin‐4 (IL‐4), or IL‐10. Disease protection could be transferred by CD4+ T cells from treated mice, an effect that was abrogated upon depletion of the CD25+ population. In addition, CD4+CD25+ T cells from treated mice were able to suppress anti‐CII IFNγ production by CII‐primed lymph node cells.

Conclusion

Mucosal administration of EtxB can be used to prevent or treat CIA. Modulation of the anti‐CII immune response by EtxB is associated with a reduction in Th1 cell reactivity without a concomitant shift toward Th2. Instead, EtxB mediates its effects through enhancing the activity of a population of CD4+ regulatory T cells.

     

Quantitative alterations of CD8+ T cells in juvenile idiopathic arthritis patients in remission

The study was aimed to investigate whether quantities of CD8⁺ T cell subsets are normal in juvenile idiopathic arthritis (JIA) patients with disease remission compared to age-matched healthy donors (HD) and whether chronological age may have an impact on proportions of naive CD8⁺ T cells. CD8⁺ T cell subsets were analyzed in 17 JIA patients and 32 age-matched HD by flow cytometry. JIA patients showed lower CD3⁺CD8⁺ T cells compared to HD. Total counts of CD8⁺CD28⁺ and CD8⁺CD28⁺CD45RA⁺ T cells were inversely correlated to chronological age in JIA patients and HD. In JIA patients, percentages of CD8⁺CD28⁺CD45RA⁺ T cells and of CD62L-expressing CD8⁺CD28⁺CD45RA⁺ T cells showed a negative correlation with age. The trend to lower CD28⁺CD45RA⁺ T cell proportions in aged JIA patients in remission may reflect a disturbed T cell homeostasis independently of disease activity and may be due to an intrinsic effect in reconstitution of the peripheral T cells.     

Inhibition of adjuvant‐induced arthritis by DNA vaccination with the 70‐kd or the 90‐kd human heat‐shock protein: Immune cross‐regulation with the 60‐kd heat‐shock protein

Objective

Adjuvant arthritis can be induced in Lewis rats by immunization with Mycobacterium tuberculosis (Mt). The mycobacterial 65‐kd heat‐shock protein (Hsp65) is targeted by arthritogenic T cells. However, Hsp65 and the mycobacterial 71‐kd heat‐shock protein are also recognized by T cells that can down‐regulate adjuvant‐induced arthritis (AIA). We have recently demonstrated that vaccination with human Hsp60 DNA inhibits AIA. The present study was undertaken to analyze the role of the T cell responses to self HSP molecules other than Hsp60 in the control of AIA.

Methods

Lewis rats were immunized with DNA vaccines coding for human Hsp70 or Hsp90 (Hsp70 plasmid [pHsp70] or pHsp90), and AIA was induced. The T cell response to Mt, Hsp60, Hsp70, and Hsp90 (proliferation and cytokine release) was studied, and the T cell response to Hsp60 was mapped with overlapping peptides.

Results

The Hsp70 or Hsp90 DNA vaccines shifted the arthritogenic T cell response from a Th1 to a Th2/3 phenotype and inhibited AIA. We detected immune crosstalk between Hsp70/90 and Hsp60: both the Hsp70 and Hsp90 DNA vaccines induced Hsp60‐specific T cell responses. Similarly, DNA vaccination with Hsp60 induced Hsp70‐specific T cell immunity. Epitope mapping studies revealed that Hsp60‐specific T cells induced by pHsp70 vaccination reacted with known regulatory Hsp60 epitopes.

Conclusion

T cell immunity to Hsp70 and to Hsp90, like Hsp60‐specific immunity, can modulate the arthritogenic response in AIA. In addition, our results suggest that the regulatory mechanisms induced by Hsp60, Hsp70, and Hsp90 are reinforced by an immune network that connects their reactivities.

     

Treatment of murine collagen‐induced arthritis by the stress protein BiP via interleukin‐4–producing regulatory T cells: A novel function for an ancient protein

Objective

Following the demonstration that the stress protein, BiP, prevented induction of collagen‐induced arthritis (CIA) in HLA–DRB*0101^+/+ (HLA–DR1^+/+) mice, we investigated the immunotherapeutic ability of BiP to suppress disease during the active phase of CIA in HLA–DR1^+/+ and DBA/1 mice.

Methods

BiP was administered either subcutaneously or intravenously to DBA/1, HLA–DR1^+/+, or interleukin‐4 (IL‐4)–knockout mice at the onset of arthritis. Immune cells were used in adoptive transfer studies or were restimulated in culture with BiP or type II collagen (CII). Proliferation and cytokine release were measured. In addition, serum anti‐CII antibodies were measured by enzyme‐linked immunosorbent assay. Disease progression was scored using a visual analog scale.

Results

BiP was successful in suppressing established CIA in HLA–DR1^+/+ and DBA/1 mice. Serum levels of anticollagen IgG antibodies were reduced in BiP‐treated mice. T cells from BiP‐immunized mice produced Th2 cytokines, in particular, IL‐4. Treatment with BiP was also shown to increase the production of CII‐specific IL‐5, IL‐10, and interferon‐γ at the termination of the study. Development of severe CIA was prevented by the intravenous transfer of BiP‐specific cells at the time of CIA induction in HLA–DR1^+/+ mice or by transferring BiP‐specific cells to DBA/1 mice at the onset of disease. BiP failed to ameliorate the development of CIA in IL‐4^−/−, HLA–DR1^+/+ mice.

Conclusion

These novel results show that BiP can suppress active CIA by the induction of regulatory cells that act predominantly via IL‐4. Thus, BiP is a potential immunotherapeutic agent for the treatment of patients with rheumatoid arthritis.

     

Impaired suppression of synovial fluid CD4+CD25− T cells from patients with juvenile idiopathic arthritis by CD4+CD25+ Treg cells

Objective

Natural CD4+CD25+FoxP3+ Treg cells play a crucial role in maintaining immune homeostasis and controlling autoimmunity. In patients with juvenile idiopathic arthritis (JIA), inflammation occurs despite the increased total numbers of Treg cells in the synovial fluid (SF) compared to the peripheral blood (PB). This study was undertaken to investigate the phenotype of CD4+ T cells in PB and SF from JIA patients, the function of synovial Treg cells, and the sensitivity of PB and SF CD4+CD25− effector T cells to the immunoregulatory properties of Treg cells, and to study the suppression of cytokine secretion from SF effector T cells by Treg cells.

Methods

The phenotypes of effector T cells and Treg cells of PB and SF from JIA patients and healthy donors were determined by flow cytometry. The functionality of isolated Treg cells and effector T cells was quantified in ³H‐thymidine proliferation assays. Cytokine levels were analyzed using Bio‐Plex Pro assay.

Results

Compared to PB, SF showed significantly elevated numbers of activated and differentiated CD4+CD45RO+ T cells. Sensitivity of SF effector T cells to the suppressive effects of Treg cells from both PB and SF was impaired, correlating inversely with the expression of CD69 and HLA–DR. However, SF effector T cell cytokine secretion was partly suppressed by SF Treg cells.

Conclusion

Our findings indicate that regulation is impaired in the SF of patients with JIA, as shown by the resistance of effector T cells to immunoregulation by functional Treg cells. This resistance of the SF effector T cells might be due to their activated phenotype.

     

A novel heat‐shock protein coinducer boosts stress protein Hsp70 to activate T cell regulation of inflammation in autoimmune arthritis

Objective

Stress proteins, such as members of the heat‐shock protein (HSP) family, are up‐regulated by cells in inflamed tissue and can be viewed functionally as “biomarkers” for the immune system to monitor inflammation. Exogenous administration of stress proteins has induced immunoregulation in various models of inflammation and has also been shown to be effective in clinical trials in humans. This study was undertaken to test the hypothesis that boosting of endogenous HSP expression can restore effective immunoregulation through T cells specific for stress proteins.

Methods

Stress protein expression was manipulated in vivo and in vitro with a food component (carvacrol), and immune recognition of stress proteins was studied.

Results

Carvacrol, a major compound in the oil of many Origanum species, had a notable capacity to coinduce cellular Hsp70 expression in vitro and, upon intragastric administration, in Peyer's patches of mice in vivo. As a consequence, carvacrol specifically promoted T cell recognition of endogenous Hsp70, as demonstrated in vitro by the activation of an Hsp70‐specific T cell hybridoma and in vivo by amplified T cell responses to Hsp70. Carvacrol administration also increased the number of CD4+CD25+FoxP3+ T cells, systemically in the spleen and locally in the joint, and almost completely suppressed proteoglycan‐induced experimental arthritis. Furthermore, protection against arthritis could be transferred with T cells isolated from carvacrol‐fed mice.

Conclusion

These findings illustrate that a food component can boost protective T cell responses to a self stress protein and down‐regulate inflammatory disease, i.e., that the immune system can respond to diet.

     

Effective treatment of collagen‐induced arthritis by adoptive transfer of CD25+ regulatory T cells

Objective

Regulatory T cells play an important role in the prevention of autoimmunity and have been shown to be effective in the treatment of experimental colitis, a T cell–mediated and organ‐specific disease. We previously demonstrated that intrinsic CD25+ regulatory T cells modulate the severity of collagen‐induced arthritis (CIA), which, in contrast to colitis, is a systemic antibody‐mediated disease and an accepted model of rheumatoid arthritis. We undertook this study to determine whether regulatory T cells have the potential to be used therapeutically in arthritis.

Methods

We transferred CD4+,CD25+ T cells into mice exhibiting arthritis symptoms, both immunocompetent mice and mice subjected to lethal irradiation and rescued with syngeneic bone marrow transplantation.

Results

A single transfer of regulatory T cells markedly slowed disease progression, which could not be attributed to losses of systemic type II collagen–specific T and B cell responses, since these remained unchanged after adoptive transfer. However, regulatory T cells could be found in the inflamed synovium soon after transfer, indicating that regulation may occur locally in the joint.

Conclusion

Our data indicate that CD25+ regulatory T cells can be used for the treatment of systemic, antibody‐mediated autoimmune diseases, such as CIA.

     

Acute CD4+ T lymphocyte–dependent interleukin‐1–driven arthritis selectively requires interleukin‐2 and interleukin‐4, joint macrophages,granulocyte–macrophage colony‐stimulating factor,interleukin‐6, and leukemia inhibitory factor

Objective

To further investigate the effects of interleukin‐1 (IL‐1) in immune‐mediated joint inflammation, we examined the role of IL‐2, Th1 interferon‐γ (IFNγ), and Th2 (IL‐4) cytokines, joint macrophages, and macrophage‐derived cytokines (IL‐12 p40, IL‐6, leukemia inhibitory factor [LIF], oncostatin M [OSM], and granulocyte–macrophage colony‐stimulating factor [GM‐CSF]) in a CD4+ T lymphocyte–dependent model of acute arthritis.

Methods

Methylated bovine serum albumin (mBSA)/IL‐1–induced arthritis was elicited in wild‐type, gene‐knockout, and monoclonal antibody–treated mice. Synovial lining macrophages were selectively depleted by intraarticular injection of clodronate liposomes prior to disease induction. The severity of arthritis was assessed histologically.

Results

Mice deficient in IL‐2 were almost completely protected from arthritis, and neutralization of IL‐4 reduced the severity of disease. In contrast, arthritis severity and resolution appeared to be independent of IFNγ. Synovial lining macrophage depletion markedly reduced arthritis severity. IL‐6 or LIF deficiency was only modestly protective, although as previously reported, GM‐CSF deficiency conferred profound disease resistance. IL‐12 p40–deficient mice (which lack IL‐12 and IL‐23) and OSM receptor–deficient mice were susceptible to mBSA/IL‐1–induced arthritis.

Conclusion

Acute mBSA/IL‐1–induced arthritis is dependent on IL‐2 and IL‐4, but not IFNγ. In vivo, the Th1/Th2 paradigm may be distorted by the presence of macrophage‐derived cytokines such as IL‐1. Synovial lining macrophages are essential in mBSA/IL‐1–induced arthritis. However, the requirement for macrophage‐derived cytokines is selective; that is, IL‐6, LIF, and especially GM‐CSF are necessary, but IL‐12, IL‐23, and OSM are dispensable. IL‐1 may therefore influence both adaptive and innate immune mechanisms in acute inflammatory arthritis.

     

Vasoactive intestinal peptide induces CD4+,CD25+ T regulatory cells with therapeutic effect in collagen‐induced arthritis

Objective

CD4+,CD25+ T regulatory cells (Treg) control the immune response to a variety of antigens, including self antigens, and may offer opportunities to intervene in the course of autoimmune diseases. Several models support the idea of the peripheral generation of CD4+,CD25+ Treg from CD4+,CD25− T cells, but little is known about the endogenous factors and mechanisms controlling the peripheral expansion of CD4+,CD25+ Treg. We undertook this study to investigate the capacity of the vasoactive intestinal peptide (VIP), an immunosuppressive antiarthritic neuropeptide, to induce functional Treg in vivo during the development of collagen‐induced arthritis (CIA).

Methods

We measured the number of CD4+,CD25+ Treg following VIP administration to CIA mice, and we characterized their phenotype and their ability to suppress activation of autoreactive T cells. We determined the capacity of VIP to induce Treg in vitro as well as the use of Treg in the treatment of CIA, measuring the clinical evolution and the inflammatory and autoimmune components of the disease.

Results

The administration of VIP to arthritic mice resulted in the expansion of CD4+,CD25+,Foxp3+ Treg in the periphery and joints, which inhibited autoreactive T cell activation/expansion. VIP induced more efficient suppressors on a per‐cell basis. The VIP‐generated CD4+,CD25+ Treg transfer suppressed and significantly ameliorated the progression of the disease.

Conclusion

These results demonstrate the involvement of the generation of Treg in the therapeutic effect of VIP on CIA. The generation of highly efficient Treg by VIP ex vivo could be used as an attractive therapeutic tool in the future, avoiding the administration of the peptide to patients with rheumatoid arthritis.

     

Increased intraarticular interleukin‐7 in rheumatoid arthritis patients stimulates cell contact–dependent activation of CD4+ T cells and macrophages

Objective

To determine the level of intraarticular expression of interleukin‐7 (IL‐7) in patients with rheumatoid arthritis (RA) and to investigate the mechanisms by which IL‐7 facilitates activation of CD4+ T cells and monocyte/macrophages in RA.

Methods

IL‐7 levels were measured in synovial fluid obtained from patients with RA and patients with osteoarthritis (OA). Immunohistologic analysis was used to assess the expression of IL‐7 in synovial tissue from patients with RA. Proliferation and activation markers were determined in order to measure the effect of IL‐7 on mononuclear cells, isolated CD4+ T cells, and monocyte/macrophages from the peripheral blood and synovial fluid. Cocultures of CD4+ T cells and monocytic cells in the absence or presence of a semipermeable membrane were performed to assess the extent to which IL‐7 induces its effects, either contact dependently or via soluble mediators.

Results

IL‐7 levels were increased in synovial fluid from patients with RA compared with the levels in synovial fluid from patients with OA. Macrophages, fibroblasts, and endothelial cells in the joint lining tissue expressed abundant IL‐7. In vitro, synovial fluid CD4+ T cells and macrophages were hyperresponsive to IL‐7 when compared with peripheral blood cells. Furthermore, IL‐7 enhanced cell contact–dependent activation of CD4+ T cells and monocyte/macrophages.

Conclusion

The abundant intraarticular expression of IL‐7 and the stimulation by IL‐7 of contact‐dependent activation of CD4+ T cells and monocytic cells indicate that this cytokine plays an important proinflammatory role in RA synovitis. Further identification of IL‐7–induced pathways may improve understanding of the important interactive role of CD4+ T cells and monocytic cells in RA.

     

日本血吸虫热休克蛋白60通过诱导CD4+CD25+Treg 细胞表达IL?10和TGF?β增强其免疫抑制功能

目的 探讨日本血吸虫热休克蛋白60（SjHSP60）增强小鼠CD4+CD25+调节性T细胞（Treg）免疫抑制功能的可能机制。 方法 采用体外研究探索SjHSP60对Treg细胞功能的影响,通过体外细胞增殖实验检测SjHSP60促进Treg细胞免疫抑制功能增强的途径,利用流式细胞术检测Treg细胞表达IL?10和TGF?β的水平以及Treg细胞Foxp3和CTLA?4表达水平。 结果 体外实验证实,SjHSP60可增强Treg细胞的免疫抑制功能（t = 6.466,P = 0.006）;体外细胞增殖实验表明,SjHSP60刺激后的Treg细胞可通过分泌可溶性细胞因子IL?10（t = 7.363,P = 0.002）和TGF?β（t = 11.262,P = 0）发挥免疫抑制功能;流式细胞术检测显示,SjHSP60刺激后的Treg细胞IL?10（t = 12.367,P = 0）、TGF?β（t = 8.431,P = 0.001）、Foxp3（比例：t = 5.225,P = 0.006;平均荧光强度：t = 8.319,P = 0.001）和CTLA?4（比例：t = 3.518,P = 0.024;平均荧光强度：t = 4.164,P = 0.014）表达水平显著增加。 结论 SjHSP60可通过促进Treg细胞表达IL?10和TGF?β增强其免疫抑制功能,并可能与SjHSP60诱导Treg细胞表达Foxp3和CTLA?4有关。     

The diagnostic significance of soluble CD163 and soluble interleukin‐2 receptor α‐chain in macrophage activation syndrome and untreated new‐onset systemic juvenile idiopathic arthritis

Objective

Macrophage activation syndrome is characterized by an overwhelming inflammatory reaction driven by excessive expansion of T cells and hemophagocytic macrophages. Levels of soluble interleukin‐2 receptor α (sIL‐2Rα) and soluble CD163 (sCD163) may reflect the degree of activation and expansion of T cells and macrophages, respectively. This study was undertaken to assess the value of serum sIL‐2Rα and sCD163 in diagnosing acute macrophage activation syndrome complicating systemic juvenile idiopathic arthritis (JIA).

Methods

Enzyme‐linked immunosorbent assay was used to assess sIL‐2Rα and sCD163 levels in sera from 7 patients with acute macrophage activation syndrome complicating systemic JIA and 16 patients with untreated new‐onset systemic JIA. The results were correlated with clinical features of established macrophage activation syndrome, including ferritin levels.

Results

The median level of sIL‐2Rα in the patients with macrophage activation syndrome was 19,646 pg/ml (interquartile range [IQR] 18,128), compared with 3,787 pg/ml (IQR 3,762) in patients with systemic JIA (P = 0.003). Similarly, the median level of sCD163 in patients with macrophage activation syndrome was 23,000 ng/ml (IQR 14,191), compared with 5,480 ng/ml (IQR 2,635) in patients with systemic JIA (P = 0.017). In 5 of 16 patients with systemic JIA, serum levels of sIL‐2Rα or sCD163 were comparable with those in patients with acute macrophage activation syndrome. These patients had high inflammatory activity associated with a trend toward lower hemoglobin levels (P = 0.11), lower platelet counts, and significantly higher ferritin levels (P = 0.02). Two of these 5 patients developed overt macrophage activation syndrome several months later.

Conclusion

Levels of sIL‐2Rα and sCD163 are promising diagnostic markers for macrophage activation syndrome. They may also help identify patients with subclinical macrophage activation syndrome.

     

Reactivity of γ/δ T cells to human 60‐kd heat‐shock protein and their cytotoxicity to aortic endothelial cells in Takayasu arteritis

Objective

Increased numbers of circulating γ/δ T cells with a restricted T cell receptor repertoire, as well as colocalization of the expression of heat‐shock protein Hsp60/65 and γ/δ T cells in the arterial lesions of patients with Takayasu arteritis (TA), indicate that γ/δ T cells may react to Hsp60 and cause damage to the arterial endothelium. In this study we investigated the proliferative responses of γ/δ T cells to human Hsp60 and their cytotoxicity to human aortic endothelial cells (ECs) in patients with TA.

Methods

Blood samples were obtained from 12 patients with TA, 8 patients with systemic lupus erythematosus (SLE) (as disease controls), and 10 healthy control subjects. Proliferative responses of circulating γ/δ T cells to human Hsp60 were detected by flow cytometry–based bromodeoxyuridine incorporation assay. Cytotoxicity of the γ/δ T cells to human aortic ECs was analyzed by colorimetric lactate dehydrogenase release assay.

Results

The γ/δ T cells of 11 of 12 patients with TA exhibited reactivity to Hsp60, whereas none of the γ/δ T cells from patients with SLE or healthy controls showed reactivity (both P < 0.001). The mean ± SD proliferative response of γ/δ T cells in patients with TA was 21.4 ± 11.3%, compared with 4.2 ± 1.2% in patients with SLE and 4.01 ± 1.82% in healthy controls (both P < 0.001). In addition, compared with the control groups, the γ/δ T cells of patients with TA had increased spontaneous cytotoxicity to aortic ECs (22.1 ± 15.0% versus 9.6 ± 2.13% in SLE patients and 8.1 ± 4.7% in healthy controls; both P < 0.005), which was further enhanced following stimulation of γ/δ T cells with Hsp60. The cytotoxicity of the γ/δ T cells was significantly inhibited by treatment of these cells with concanamycin A and anti–Fas ligand–blocking antibodies.

Conclusion

The results show that γ/δ T cells in patients with TA are reactive to Hsp60 and exhibit cytotoxicity to aortic ECs, suggesting a key role of Hsp60 and γ/δ T cells in the pathogenesis of TA.

     

Identification of immunodominant CD4+ T cell epitopes in patients with Yersinia‐induced reactive arthritis by cytometric cytokine secretion assay

Objective

In reactive arthritis (ReA), a bacteria‐specific T cell response to the triggering microbe is detected in synovial fluid. So far, direct characterization of bacteria‐specific T cells and identification of the immunodominant fine specificities remain difficult due to the lack of appropriate techniques. The aim of the present study was to directly determine the fine specificity of CD4+ T cells specific to ReA‐associated bacteria‐derived protein.

Methods

In 2 patients with Yersinia‐induced ReA, live Yersinia Hsp60–specific CD4+ T cells were directly isolated from synovial fluid after stimulation with Yersinia‐derived protein Hsp60 using a cytometric cytokine secretion assay. Generated short‐term T cell lines were then tested in vitro for their peptide epitope specificity. Also, direct cross‐reactivity of one line with Chlamydia‐ and human‐derived Hsp60 was assessed.

Results

Generated short‐term CD4+ T cell lines were highly antigen‐specific and revealed single immunodominant peptide epitopes that were confirmed by direct testing with single peptides in both peripheral blood and synovial fluid cells. Yersinia Hsp60–specific T cells of one patient cross‐reacted directly with human Hsp60.

Conclusion

Our results demonstrate the feasibility of direct assessment of live, potentially pathogenic, antigen‐specific interferon‐γ+ CD4+ T cells taken from inflammatory lesions of patients with rheumatic diseases such as ReA. This might have implications not only regarding pathogenesis, but also in the design of new immunotherapies.