Yusuke Chino

Laser microdissection-based analysis of cytokine balance in the kidneys of patients with lupus nephritis

To determine the cytokine balance in patients with lupus nephritis (LN), we analysed kidney‐infiltrating T cells. Renal biopsy samples from 15 systemic lupus erythematosus (SLE) patients were used. In accordance with the classification of International Society of Nephrology/Renal Pathology Society, they were categorized into Class III, Class III+V (Class III‐predominant group, n = 4), Class IV, Class IV+V (Class IV‐predominant group, n = 7) and Class V (n = 4) groups. The single‐cell samples of both the glomelular and interstitial infiltrating cells were captured by laser‐microdissection. The glomerular and interstitial infiltrating T cells produced interleukin (IL)‐2, IL‐4, IL‐10, IL‐13 and IL‐17 cytokines in the Class III‐predominant, Class IV‐predominant and Class V groups. Interferon‐gamma was detected only in the glomeruli of the Class III‐predominant and Class V group samples. The expression level of IL‐17 was correlated closely with clinical parameters such as haematuria, blood urea nitrogen level, SLE Disease Activity Index scores in both glomeruli and interstitium, urine protein level in glomeruli and serum creatinine and creatinine clearance levels in interstitium. This suggests that the glomerular infiltrating T cells might act as T helper type 1 (Th1), Th2 and Th17 cells while the interstitial infiltrating T cells, act as Th2 and Th17 cells in the Class III‐predominant and Class V groups. In contrast, both the glomerular and interstitial infiltrating T cells might act as Th2 and Th17 cells in the Class IV‐predominant group. The cytokine balances may be dependent upon the classification of renal pathology, and IL‐17 might play a critical role in SLE development.

Use of laser microdissection in the analysis of renal-infiltrating T cells in MRL/lpr mice

To clarify the role of T cells in the kidneys of MRL/MpJ-lpr (MRL/lpr) mice, cytokine mRNA expression was analyzed, and tissue localization of T cells was examined by immunohistochemistry. Cells infiltrating the glomeruli, glomerular circumference, and perivascular areas in ten female MRL/lpr mice were captured by laser microdissection (LMD). Nested reverse transcription polymerase chain reaction (RT-PCR) of samples was performed with primers specific for β-actin, T-cell receptor β chain (TCR-Cβ), Thy-1, B220, CD4, CD8, interleukin (IL)-2, IL-4, IL-10, IL-13, IL-17, and interferon (IFN)-γ. Frozen sections of lesions were also stained immunohistochemically. B220, MAC-1, Thy-1, CD4, and CD8 staining was observed in glomeruli and perivascular areas, especially in glomerular circumference areas. T cells infiltrating the glomeruli, glomerular circumference areas, and perivascular areas produce INF-γ, IL-13, and IL-17 predominately. IL-10 positivity was identified in 60% of perivascular T cells but not in a substantial number of glomerular or periglomerular T cells. The results of our study suggest that the pathogenesis of renal lesions in MRL/lpr mice is complex and not due simply to the Th1 and Th2 balance. These findings also support the concept of different molecular mechanisms for glomerulonephritis and vasculitis in these mice.

Significance of antiprothrombin antibodies in patients with systemic lupus erythematosus: clinical evaluation of the antiprothrombin assay and the antiphosphatidylserine/prothrombin assay, and comparison with other antiphospholipid antibody assays

Antibodies against prothrombin are detected by enzyme immunoassays (EIA) in sera of patients with antiphospholipid syndrome (APS). However, there are two methods for antiprothrombin EIA; one that uses high binding plates (aPT-A), and another that utilizes phosphatidylserine bound plates (aPS/PT). We aimed to evaluate and compare aPT-A and aPS/PT in a clinical setting. We performed EIA for anti-PT, anti-PS/PT, IgG, and IgM anticardiolipin antibodies (aCL), and IgG β2-glycoprotein I-dependent aCL (aβ2GPI/CL) with serum samples from 139 systemic lupus erythematosus (SLE) patients (16 with history of at least one thrombotic episode) and 148 controls. We observed that: (1) although titers of anti-PT and anti-PS/PT were significantly related with each other (P < 0.0001, ρ = 0.548), titer of anti-PT and anti-PS/PT differed greatly in some samples; (2) odds ratio and 95% confidence interval for each assay was 3.556 (1.221–10.355) for aPT-A, 4.591 (1.555–15.560) for aPS/PT, 4.204 (1.250–14.148) for IgG aCL, 1.809 (0.354–9.232) for IgM aCL, and 7.246 (2.391–21.966) for aβ2GPI/CL. We conclude that, while all EIA performed in this study except IgM aCL are of potential value in assessing the risk of thrombosis, aPS/PT and aβ2GPI/CL seemed to be highly valuable in clinical practice, and that autoantibodies detected by anti-PT and anti-PS/PT are not completely identical.

Inhibition of transforming growth factor-β signalling attenuates interleukin (IL)-18 plus IL-2-induced interstitial lung disease in mice

Interstitial lung disease (ILD) is an intractable disease induced by various factors in humans. However, there is no universally effective treatment for ILD. In this study, we investigated the role of transforming growth factor (TGF)‐β signalling in the pathogenesis of ILD by using model mice. Injection of interleukin (IL)‐18 plus IL‐2 in C57BL6 (B6) mice resulted in acute ILD by infiltration of natural killer (NK) cells and a significant increase of TGF‐β mRNA in the lung. To examine the pathogenetic role of TGF‐β in ILD mice, we used SB‐431542 (4‐[4‐(1,3‐benzodioxol‐5‐yl)‐5‐(2‐pyridinyl)‐1H‐imidazol‐2‐yl]‐benzamide), which is a potent and selective inhibitor of TGF‐β receptor I (TβRI), also known as activin receptor‐like kinase 5 (ALK5). Treatment of B6‐ILD mice with SB‐431542 resulted in improvement of ILD, delay in mortality, reduction of the expression of interferon (IFN)‐γ and IL‐6 in the lungs. The same treatment also decreased significantly the percentage of natural killer (NK) cells in the lungs (P < 0·05) and mRNA expression levels of certain chemokines such as CCL2, CCL3, CCL4, CCL5 and CXCL10 in B6‐ILD. These findings were confirmed by IL‐18 plus IL‐2 treatment of Smad3‐deficient (Smad3–/–) mice (P < 0·05). Our results showed that inhibition of TGF‐β signalling reduced the percentage of NK cells and the expression of certain chemokines in the lungs, resulting in improvement of ILD. The findings suggest that TGF‐β signalling may play an important role in the pathogenesis of IL‐18 plus IL‐2‐induced ILD in mice.

Clinical features of patients with IgG4-related disease complicated with perivascular lesions

Abstract Objective. To define the clinical features of IgG4-related disease (IgG4-RD) complicated with perivascular lesions. Methods. The clinical features of seven patients with IgG4-RD and perivascular lesions diagnosed at the University of Tsukuba Hospital between October 2008 and October 2013, were analyzed, including clinical background, results of imaging studies, satisfaction of the 2011 comprehensive diagnostic criteria (CDC) for IgG4-RD, laboratory data, distribution of perivascular lesions, involvement of other organs, and response to steroid therapy. Results. We studied six men and one woman with a mean age of 66.9 ± 6.7 years (± SD). Six of seven patients were diagnosed as definite IgG4-RD, while the seventh was considered possible IgG4-RD, based on the CDC for IgG4-RD. Serum IgG4 levels at diagnosis were higher than 135 mg/dl in all seven patients (mean, 933 ± 527). Serum C-reactive protein (CRP) levels were elevated in two only (mean, 1.42 ± 3.56 mg/dl). The perivascular lesions were located in the pulmonary artery (n = 1), thoracic aorta (n = 2), abdominal aorta (n = 6), coronary (n = 1), celiac (n = 1), superior mesenteric (n = 1), renal (n = 2), inferior mesenteric (n = 5), and iliac (n = 3) arteries. In addition to perivascular lesions, six patients showed involvement of other organs. All seven patients were treated with prednisolone (0.6 mg/kg/day), which rapidly improved the perivascular and other organ lesions in six patients (the other one patient have not yet been evaluated due to the short follow-up). Conclusion. Perivascular lesions show wide distribution in patients with IgG4-RD. Serum CRP levels are not necessarily elevated in these patients. Steroid therapy is effective in IgG4-RD and results in resolution of lesions.

Tristetraprolin (TTP) gene polymorphisms in patients with rheumatoid arthritis and healthy individuals

Tristetraprolin (TTP) is an intracellular protein that modulates the production of cytokines, including TNFα, by binding to and destabilizing the mRNAs of these cytokines. Therefore, differences in TTP gene expression may affect the severity of inflammatory diseases, such as rheumatoid arthritis (RA). We searched for polymorphisms in the human TTP gene and for this purpose, we sequenced the entire TTP gene in 20 Japanese individuals (ten with RA and ten healthy volunteers) and found one single nucleotide polymorphism (SNP) in the promoter region. We analyzed this SNP (A/G) by restriction fragment length polymorphism method in 155 RA patients and 100 control subjects. While the frequency of A allele in this SNP was similar in RA patients (74.5%) and controls (76.0%), the disease duration in RA patients with genotype GG was shorter than that of patients with genotypes AA/AG and RA patients with genotype GG had a higher probability of being treated with infliximab. We studied the difference in promoter activity between the two alleles by luciferase assay and found that the promoter activity of TTP promoter region with allele A was around two-fold higher than that with allele G. We conclude that this SNP in the promoter region of the TTP gene mildly affects promoter activity, and thus, may influence the disease activity of inflammatory disorders including RA.

Involvement of NK 1.1–Positive γδT Cells in Interleukin-18 Plus Interleukin-2–Induced Interstitial Lung Disease

Interstitial lung disease (ILD) is induced by various factors in humans. However, the exact mechanism of ILD remains elusive. This study sought to determine the role of natural killer (NK) 1.1(+) γδT cells in ILD. The injection of IL-18 plus IL-2 (IL-18/IL-2) into C57BL6 (B6) mice induced acute ILD that resembled early-stage human ILD. An accumulation of NK1.1(+) γδT cells similar to NK cells was evident in the lungs. The T Cell Receptor (TCR) Vγ and Vδ repertoires of NK1.1(+) γδT cells indicated polyclonal expansion. The expression of IL-2 receptor β (Rβ) and IL-18Rβ in NK1.1(+) γδT cells was higher than in NK1.1(-) γδT cells. IL-18/IL-2 stimulated the proliferation of NK1.1(+) γδT cells, but not NK1.1(-) γδT cells. The IL-18/IL-2-stimulated NK1.1(+) γδT cells produced higher concentrations of IFN-γ than did NK1.1(-) γδT cells. Moreover, NK1.1(+) γδT and NK1.1(-) γδT cells constituted completely different cell populations. The IL-18/IL-2-induced ILD was milder in TCRδ(-/-) and IFN-γ(-/-) mice, compared with B6 mice. Furthermore, cell-transfer experiments demonstrated that NK1.1(+) γδT cells could induce the expansion of NK cells and IFN-γ mRNA in the lung by IL-18/IL-2. Our results suggest that NK1.1(+) γδT cells function as inflammatory mediators in the early phase of IL-18/IL-2-induced ILD.

A typical case of giant cell arteritis with vision loss due to delayed diagnosis

A 75‐year‐old woman lost vision because of delayed recognition of GCA. Early diagnosis and treatment of GCA are important for preventing visual complications. Physicians must remember to evaluate the entire body, not just a single organ/system.

Lateral medullary infarction in a patient with central nervous system lupus.

Dear Editor, A 37-year-old woman with highly active systemic lupus erythematosus (SLE) was referred to our hospital due to a sudden onset of left-sided sensory disturbance and urinary retention followed by dizziness, dysphagia, headache and nausea over a 2-day period. Her previous SLE activities had caused arthritis and nephritis, although no hypertension, diabetes mellitus or dyslipidemia. She had smoked five cigarettes per day and occasionally drank moderate amounts of alcohol. Her baseline medications included prednisolone 12.5 mg/ day, tacrolimus 3 mg/day, mizoribine 150 mg/day, lansoprazole, alfacalcidol and minodronate. On general physical examination, her blood pressure was 156/109 mmHg, heart rate was 92 beats/min and temperature was 38.4°C, with a normal respiratory rate and pulse oximetry oxygen saturation of 99% while breathing ambient air. There were no remarkable observations for the head, neck, chest, abdomen, extremities, skin or joints. On neurological examination, the patient was oriented for time and place, and both pupils were equal, round and reactive to light. The patient showed neither ptosis nor facial weakness, but exhibited anhidrosis on the right side of the face. Her gag reflex was impaired and an indirect laryngeal examination revealed right vocal cord paralysis. The motor functions and reflexes were normal in all extremities. Senses to pain and warm temperature were decreased in the left trunk, left limbs and left side of the face, but proprioception was normal bilaterally. Laboratory data were as follows: blood leukocytes 10 400/lL (normal 4000–9000/lL), C-reactive protein 1.35 mg/dL (< 0.5 mg/dL), erythrocyte sedimentation rate 25 mm/h (3–15 mm/h), hemoglobin A1c 5.9% (4.6–6.2%), low-density lipoprotein cholesterol 127 mg/dL (< 119 mg/dL), total hemolytic complement (CH50) 13.5/mL (25.0–48.0/mL), Complement component 3 (C3) 58 mg/dL (normal 65–135 mg/dL), complement component 4 (C4) 5 mg/dL (13–35 mg/ dL), anti-DNA antibody 81 IU/mL (< 6.0 IU/mL), serum anti-ribosomal P antibody 17 U/mL (< 1.0 (a)