Modulation of inflammation and response to dexamethasone by Annexin 1 in antigen-induced arthritis

OBJECTIVE: Annexin 1 (Anx-1) is a putative mediator of the antiinflammatory actions of glucocorticoids (GCs). This study investigated the role of Anx-1 in experimental arthritis and in GC-mediated inhibition of inflammation, using antigen-induced arthritis (AIA) in Anx-1 knockout (Anx-1(-/-)) mice. METHODS: Arthritis was induced by intraarticular injection of methylated BSA (mBSA) in mice preimmunized with mBSA. Disease was assessed after 7 days by histologic examination of the knee joints. Serum levels of anti-mBSA IgG were determined by enzyme-linked immunosorbent assay. Cytokine messenger RNA (mRNA) expression was detected by real-time polymerase chain reaction. RESULTS: A significant exacerbation of arthritis was observed in the Anx-1(-/-) mice compared with wild-type (WT) mice. This was associated with increased mRNA expression of synovial interleukin-1 beta, tumor necrosis factor alpha, interleukin-6, and macrophage migration inhibitory factor. Dexamethasone significantly reduced the histologic severity of synovitis and bone damage in the WT mice, but exerted no inhibitory effects in the Anx-1(-/-) mice, and also significantly reduced the serum levels of anti-mBSA IgG and the numbers of peripheral blood neutrophils and lymphocytes in WT mice, but had no such effect in Anx-1(-/-) mice. CONCLUSION: Anx-1 exerts endogenous antiinflammatory effects on AIA via the regulation of cytokine gene expression, and also mediates the antiinflammatory actions of dexamethasone in AIA.     

Adjuvant arthritis as a model of inflammatory cachexia

Objective. To determine whether adjuvant arthritis (AA) leads to changes in body composition and cytokine production similar to those seen in patients with rheumatoid arthritis. Methods. AA was induced in Lewis rats using Freund's complete adjuvant. Body cell mass was measured by determining the concentration of total exchangeable potassium using ⁴²K gavage. Splenocyte production of interleukin-1 (IL-1) and tumor necrosis factor α (TNFα) was measured by bioassay. Weight and food intake were also measured. Results. Animals that developed AA lost 6% of their body weight by the onset of clinically evident arthritis (day 14; P < 0.01) and lost 20% by the end of the inflammatory phase of AA (day 28; P < 0.0001). Body cell mass fell 24.7 ± 8.6% (mean ± SEM) in animals with AA, but did not change significantly in controls (increase of 6.3 ± 7.9%) (P < 0.03). Pair-fed animals lost one-fourth of the weight lost by the animals with AA (P < 0.01), indicating that anorexia alone does not explain inflammatory cachexia. Weight loss was correlated with TNFα production by spleen mononuclear cells (r = 0.68, P < 0.007), and a weaker correlation was seen with IL-1 production (r = 0.45, P < 0.04). Conclusion. AA in rats is a useful model of inflammatory cachexia that mimics the human pathophysiology in important ways, and is consistent with cytokine-driven cachexia in chronic inflammatory arthritis.     

DNA analysis for the kininogen gene of patients with kininogen deficiency in Japan

The kininogen (KGN) gene status was examined in 4 families with both high molecular weight (HMW) and low molecular weight (LMW) KGM deficiency and one family with only HMW-KGN deficiency reported in Japan. No abnormal HMW-KGN or LMW-KGN was detected in those with these deficiencies by immunoblot analysis using monoclonal antibodies (HKG-H12, HKG-L7, HKG-L17) for human HMW-KGN. HMW-DNA prepared from peripheral blood leucocytes was digested with endonuclease, EcoRI, Bam HI, Hind III, Sca I, Bg1II, Xba I, Msp I, Pst I, Hpa I, PvuII, HaeIII, Rsa I, Alu I, or Taq I, and studied by Southern blot analysis with human LMW prekininogen cDNA (phKG 36) as a probe. A gross deletion or insertion of the KGN gene was not detected in those with both HMW- and LMW-KGN deficiencies. On the other hand, partial defect in intron 7 (G) was found in those with only HMW-KGN deficiency, suggesting that this defect might be related to abnormality of the alternative RNA splicing events for HMW-prekininogen mRNA.     

Nerves in inflammatory synovium: immunohistochemical observations on the adjuvant arthritis rat model.

Previous evidence has been presented that neurogenic input may influence adjuvant induced arthritis (AA) in rats. We now present evidence of alterations in synovial nerves in AA. Nerves were studied in well perfused and fixed rats, using immunohistochemistry with the sensitive avidin-biotin peroxidase complex (ABC) method and heterologous antisera to cytoskeletal protein gene product 9.5 (PGP) and the neuropeptides substance P and calcitonin gene related peptide (CGRP). The innervation of synovium was compared in normal rats and rats with AA. Observations concordant with what has been reported for neuropeptide nerves in the synovium of patients with rheumatoid arthritis (RA) are presented. It has been suggested that neural peptide substances are reduced in nerves of synovium from patients with RA. In the AA rat a specific reduction of lining zone and sublining nerves in the synovium was noted. The AA rat model is very suitable for studying the involvement of synovial nerves in arthritis, permitting optimal preservation of immunoreactive neural epitopes.     

Modulation of inflammation and response to dexamethasone by Annexin 1 in antigen‐induced arthritis

Objective

Annexin 1 (Anx‐1) is a putative mediator of the antiinflammatory actions of glucocorticoids (GCs). This study investigated the role of Anx‐1 in experimental arthritis and in GC‐mediated inhibition of inflammation, using antigen‐induced arthritis (AIA) in Anx‐1 knockout (Anx‐1^−/−) mice.

Methods

Arthritis was induced by intraarticular injection of methylated BSA (mBSA) in mice preimmunized with mBSA. Disease was assessed after 7 days by histologic examination of the knee joints. Serum levels of anti‐mBSA IgG were determined by enzyme‐linked immunosorbent assay. Cytokine messenger RNA (mRNA) expression was detected by real‐time polymerase chain reaction.

Results

A significant exacerbation of arthritis was observed in the Anx‐1^−/− mice compared with wild‐type (WT) mice. This was associated with increased mRNA expression of synovial interleukin‐1β, tumor necrosis factor α, interleukin‐6, and macrophage migration inhibitory factor. Dexamethasone significantly reduced the histologic severity of synovitis and bone damage in the WT mice, but exerted no inhibitory effects in the Anx‐1^−/− mice, and also significantly reduced the serum levels of anti‐mBSA IgG and the numbers of peripheral blood neutrophils and lymphocytes in WT mice, but had no such effect in Anx‐1^−/− mice.

Conclusion

Anx‐1 exerts endogenous antiinflammatory effects on AIA via the regulation of cytokine gene expression, and also mediates the antiinflammatory actions of dexamethasone in AIA.

     

Systemic inflammation and neurodegeneration in a mouse model of multiple sulfatase deficiency

Sulfatases are involved in several biological functions such as degradation of macromolecules in the lysosomes. In patients with multiple sulfatase deficiency, mutations in the SUMF1 gene cause a reduction of sulfatase activities because of a posttranslational modification defect. We have generated a mouse line carrying a null mutation in the Sumf1 gene. Sulfatase activities are completely absent in Sumf1(-/-) mice, indicating that Sumf1 is indispensable for sulfatase activation and that mammals, differently from bacteria, have a single sulfatase modification system. Similarly to multiple sulfatase deficiency patients, Sumf1(-/-) mice display frequent early mortality, congenital growth retardation, skeletal abnormalities, and neurological defects. All examined tissues showed progressive cell vacuolization and significant lysosomal storage of glycosaminoglycans. Sumf1(-/-) mice showed a generalized inflammatory process characterized by a massive presence of highly vacuolated macrophages, which are the main site of lysosomal storage. Activated microglia were detected in the cerebellum and brain cortex associated with remarkable astroglyosis and neuronal cell loss. Between 4 and 6 months of age, we detected a strong increase in the expression levels of inflammatory cytokines and of apoptotic markers in both the CNS and liver, demonstrating that inflammation and apoptosis occur at the late stage of disease and suggesting that they play an important role in both the systemic and CNS phenotypes observed in lysosomal disorders. This mouse model, in which the function of an entire protein family has been silenced, offers a unique opportunity to study sulfatase function and the mechanisms underlying lysosomal storage diseases.     

Molecular characterization of total kininogen deficiency in Japanese patients

Kininogens are multifunctional plasma glycoproteins. There are two forms of human kininogen: low molecular weight kininogen (LK) and high molecular weight kininogen (HK). Both are derived from the same gene by alternative splicing. Some patients with kininogen deficiency have been reported to be deficient only in HK while others are deficient in both HK and LK (total kininogen deficiency). We analyzed three Japanese patients with total kininogen deficiency by the Csp45I digestion study of exon 5 as previously reported in Williams trait and found that two had the same point mutation of C to T at base 22 of exon 5, resulting in a transition of CGA (Arg) codon to TGA (Stop) codon. This is the first report of molecular characterization of total kininogen deficiency in the Japanese population.     

The analgesic effect of electroacupuncture on inflammatory pain in the rat model of collagenase-induced arthritis: mediation by opioidergic receptors

Electroacupuncture (EA) is widely practiced for the treatment of osteoarthritic (OA) pain, but its therapeutic mechanisms have not yet been fully studied, especially in the experimental OA rat model. In order to induce collagenase-induced arthritis (CIA) in rats, male Sprague–Dawley rats were intra-articularly injected with 0.05 ml of 4 mg/ml collagenase solution in the left knee of the hind limb, followed by a booster injection 4 days later. Maximal gross, histopathological features and biomarker activity changes consistent with human OA characteristics were observed four weeks after the first collagenase injection. In the exploratory preliminary study of EA stimulation parameters, low-frequency train pulse EA stimulation (2 Hz, 0.07 mA, 0.3 ms) delivered to the Zusanli (ST36) acupoint exerted an antinociceptive effect with acupoint specificity in a rat model of CIA. The antinociceptive effect of Zusanli EA was blocked by intraperitoneal pretreatment with naloxone (μ-opioid receptor antagonist, 2 mg/kg) and naltrindole (δ-opioid receptor antagonist, 1 mg/kg), but not with norbinaltophimine (κ-opioid receptor antagonist, 20 mg/kg). The synergistic antinociceptive effects of Zusanli EA were achieved with statistical significance by i.p. pretreatment with DAMGO (μ-opioid receptor agonist, 1 mg/kg) and with [D-Ala2]-Deltorphin II (δ-opioid receptor agonist, 6 mg/kg), but not with (±)-U-50488 (κ-opioid receptor agonist, 3 mg/kg). These results suggest that the 2-Hz EA can attenuate the osteoarthritic pain in CIA, and the analgesic effects of EA can be mediated by μ-opioid and δ-opioid, but not by κ-opioid receptors.     

A new case of high-molecular-weight kininogen inherited deficiency

A preoperative hemostasis study discovered a prolonged activated partial thromboplastin time in a 23-year-old Portuguese Caucasian woman without personal or past family history of hemorrhage or thrombosis. This was corrected by pooled plasma that excluded circulating anticoagulant. Activated partial thromboplastin time was prolonged whatever the activator, particularly ellagic acid, and was not corrected by prolonged kaolin incubation. Levels of factors VIII and XII were normal; factor XI and prekallikrein levels were either moderately low or normal according to activators and defective reagents used. High-molecular-weight kininogen (HMWK) level assessed by coagulation and immunological method was virtually nil. Fibrinolysis activity was normal before and after venous occlusion. The programmed operation was performed without any particular preparation and no complication arose. Family investigation found heterozygous HMWK deficiency in the proposita's father and three of her siblings.     

Exploring the reciprocal relationship between immunity and inflammation in chronic inflammatory arthritis

Experimental models seeking to explore how susceptible individuals develop rheumatoid arthritis (RA) propose that genetic and environmental factors shape a complex series of molecular and cellular interactions leading to a chronic inflammatory response. T lymphocytes and MHC class II genes have featured prominently in these models. More recent studies have suggested that perpetuation of inflammation in a disease-susceptible host might occur through failure to down-regulate the inflammatory process. One prediction from this model is that effective mechanisms of immunoregulation might be most easily investigated in non-susceptible individuals. However, this has been difficult to study in man. Based on the observation that extended MHC haplotypes are strongly associated with RA in different ethnic groups, I have explored the function of human MHC-encoded genes in transgenic mice using two different experimental approaches. First, by comparing the molecular interactions between disease-associated or non-associated HLA-DR4 molecules and CD4+ T lymphocytes, it has been possible to gain insight into how immune responses in non-susceptible individuals might differ from T-cell responses observed in a susceptible host. This has been achieved using transgenic mice expressing RA disease-associated and non-associated human HLA class II molecules. Secondly, the effects of prolonged exposure of T cells to the proinflammatory cytokine tumour necrosis factor alpha (TNF) have been studied in vitro and in vivo, focusing on T-cell receptor (TCR) signalling and effector responses. In studies of HLA class II transgenic mice, the major differences between disease-associated and non-associated alleles in terms of T-cell responses occur at the level of presentation of antigenic peptides, and the sustained expression of inflammatory cytokines such as TNF. Chronic exposure of T cells to inflammatory cytokines such as TNF induces a phenotype which resembles RA synovial T cells, including the induction of non-deletional and reversible hyporesponsiveness to TCR ligation and uncoupling of proximal TCR signal transduction pathways. The experimental findings are consistent with a model in which HLA class II-driven inflammatory cytokine expression uncouples TCR signalling pathways in the susceptible host in such a way as to profoundly suppress proliferative and immunoregulatory cytokine responses, while at the same time promoting cell survival and effector responses.     

Bim deficiency leads to exacerbation and prolongation of joint inflammation in experimental arthritis

OBJECTIVE:: Rheumatoid arthritis (RA) is characterized by hyperplasia of the synovial lining, inflammation, and destruction of cartilage and bone. Since there are only a few detectable cells undergoing apoptosis in the joint, it is possible that a defect in apoptosis may contribute to synovial hyperplasia. This study sought to identify and characterize the direct role of apoptotic regulators in a mouse model of inflammatory arthritis. METHODS: Using a serum transfer model, experimental arthritis was induced in mice lacking the proapoptotic Bcl-2 family genes Bak (Bak-/-), Bax (Bax-/-), or Bim (Bim-/-), as compared with wild-type (WT) control mice. Physical examination for edema of the ankles and histopathologic analysis of ankle sections were used to determine the severity of arthritis. The serum and ankles were examined for production of chemokines and cytokines using enzyme-linked immunosorbent or Luminex-based assays. RESULTS: Bim-/- mice displayed increased severity and prolongation of arthritis. In contrast, Bak-/- and Bax-/- mice showed no difference in the severity of arthritis as compared with WT mice. In addition, Bim-/- mice had elevated levels of proinflammatory chemokines and cytokines, decreased joint and serum production of antiinflammatory cytokines, fewer TUNEL-positive cells, and reduced levels of active caspase 3 as compared with WT mice. CONCLUSION: These studies are the first to demonstrate a role for the proapoptotic Bcl-2 protein Bim in the effector phase of RA. The findings indicate that Bim potentially functions to repress the effector phase of arthritis by regulating the milieu of the joint and serum, and by inducing apoptosis.     

Partial chondroprotective effect of zoledronate in a rabbit model of inflammatory arthritis.

OBJECTIVE: To test the hypothesis that bisphosphonate treatment has a chondroprotective effect in the carrageenan model of inflammatory arthritis (IA). METHODS: Experimental IA was induced in rabbits by intraarticular injections of carrageenan. One group also received subcutaneous injections of zoledronate, a new bisphosphonate. After 4 weeks, the joints were harvested. Articular cartilage degradation and the degree of synovitis were assessed by light microscope using qualitative grading scores. Articular cartilage and subchondral and cancellous bone were evaluated histomorphometrically. Bone microhardness was measured. RESULTS: Carrageenan injected knees showed changes of inflammatory arthritis with cartilage erosion. Zoledronate treatment partially protected the articular cartilage from degradation. This effect was unlikely due to an antiinflammatory effect of the drug as the carrageenan induced synovitis was unaffected by zoledronate treatment. The treatment preserved subchondral bone thickness and cancellous bone volume and prevented focal breaks in the osteochondral barrier. The subchondral bone hardness was also maintained. CONCLUSION: Zoledronate had a partial effect in a rabbit model of inflammatory arthritis. The chondroprotection may be due to the prevention of bone resorption. By maintaining an intact subchondral bone, normal joint loading may have been maintained and contact between the bone marrow and the articular cartilage averted.     

免疫刺激DNA序列与过敏原联用对哮喘小鼠模型气道过敏性炎症的作用

目的　观察免疫刺激DNA序列 (ISS DNA)单用和与过敏原卵白蛋白 (OVA)合用 ,对支气管哮喘 (简称哮喘 )小鼠模型气道过敏性炎症的作用及作用维持时间。方法　BALB/c小鼠 36只 ,分ISS组 (A组 ) 12只、ISS +OVA组 (B组 ) 12只、OVA组 (C组 ) 6只和生理盐水 (NS)组 (D组 ) 6只。A、B、C组用OVA致敏及激发。A组和B组根据注射次数再分A1、B11次注射组 (1次腹腔注射ISS DNA 10 0μg或ISS DNA 10 0 μg +OVA 10 μg)和A2 、B2 2次注射组 (2次腹腔注射ISS DNA或ISS DNA +OVA)。各组在第 1次激发后 6周中 ,每周采血 1次测特异性IgE ,最后处死小鼠测支气管肺泡灌洗液 (BALF)中细胞计数及分类 ,肺组织病理检查 ,检测脾细胞分泌γ干扰素 (IFN γ)的水平。结果　BALF中嗜酸细胞 (EOS)数A1组为 (2 39± 0 81)× 10 4/ml;A2 组为 (2 .6 2± 0 .77)× 10 4/ml;B1组为 (1.80± 0 .12 )× 10 4/ml;B2 组为 (1.84± 0 .6 7)× 10 4/ml,与C组 [(12 .4 3± 2 .13)× 10 4/ml]比较差异有显著性 (P <0 .0 5 )。脾细胞分泌的IFN γA1组为 (5 10± 10 2 )pg/ml;A2 组为 (492± 98)pg/ml;B1组为 (5 32± 12 0 )pg/ml;B2组为 (46 9± 132 )pg/ml,与C组 [(194± 80 )pg/ml]比较差异有显著性 (P <0 .0 5 )。血清IgE前 4周B组与C组比较     

PTEN在支气管哮喘大鼠气道炎症中的作用

目的 探讨PTEN在支气管哮喘(简称哮喘)大鼠气道炎症中的作用.方法 20只SPF级雄性SD大鼠随机分为2组,对照组和哮喘组.以卵清白蛋白致敏激发法复制大鼠哮喘模型,每只大鼠左肺留取肺组织,右肺进行支气管肺泡灌洗并留取支气管肺泡灌洗液(BALF).对BALF进行细胞分类计数;应用双抗体夹心酶联免疫吸附试验(Sandwich ELISA)法测定BALF中IL-4、IL-12浓度;采用免疫组织化学法和Western blot法测定PTEN蛋白的表达和量的变化.结果 ①BALF IL-4的浓度哮喘组显著高于对照组,BALF中IL-12的浓度哮喘组显著低于对照组(P值均＜0.01);②免疫组织化学显示PTEN蛋白主要表达细胞是支气管上皮细胞,Western blot法显示哮喘组支气管PTEN蛋白的表达显著低于对照组(P＜0.01);③支气管上皮细胞PTEN蛋白表达量分别与BALF中的IL-4浓度呈显著负相关,与BALF中的IL-12浓度呈显著正相关.结论 哮喘大鼠PTEN蛋白表达明显减少,它能减少Th2因子的表达,可能与哮喘大鼠气道炎症中Th1/Th2平衡密切相关.     

Rituximab specifically depletes short-lived autoreactive plasma cells in a mouse model of inflammatory arthritis

There is increasing appreciation of the important role of B cells in many autoimmune diseases and consequently, increasing interest in treating these disorders through B cell-depletion therapy with rituximab, an anti-CD20 monoclonal antibody. Yet, precisely how this and related drugs exert their therapeutic effects remains controversial. In particular, it is unclear how, in a number of contexts, rituximab can greatly reduce the titer of serum autoantibodies without substantially altering the overall antibody titer. We have studied the action of this drug in the K/BxN mouse model of inflammatory arthritis after first crossing in a human CD20 transgene. Rituximab treatment of these mice led to a decrease in the titer of serum antibodies targeting glucose-6-phosphate isomerase, the relevant autoantigen, but not in the total antibody titer. Glucose-6-phosphate isomerase-specific plasma cells did not reside primarily in the bone marrow as expected but rather in the spleen and lymph nodes, where they had short lives, expressed CD20, and were rapidly depleted by rituximab. These data support a model whereby autoreactive plasma cells (at least certain specificities thereof) are intrinsically different from protective antimicrobial plasma cells in their differentiation, migration, and survival properties. Rituximab targets the former and spares the latter.     

PTEN在支气管哮喘大鼠气道炎症中的作用

目的探讨PTEN在支气管哮喘（简称哮喘）人鼠气道炎症中的作用。方法20只SPF级雄性SD大鼠随机分为2组。对照组和哮喘组。以卵清白蛋白致敏激发法复制大鼠哮喘模型,每只大鼠左肺留取肺组织,右肺进行支气管肺泡灌洗歼留取支气管肺泡灌洗液（BALF）。对BALF进行细胞分类计数;应用双抗体夹心酶联免疫吸附试验（Sandwich ELISA）法测定BALF中IL-4、IL-12浓度;采用免疫组织化学法和Western blot法测定PTEN蛋白的表达和量的变化。结果①BALF IL-4的浓度哮喘组显著高于对照组,BALF中IL-12的浓度哮喘组屁著低于对照组（P值均〈0．01）;②免疫组织化学显示PTEN蛋白主要表达细胞是支气管上皮细胞,Western blot法显示哮喘组支气管PTEN蛋白的表达显著低于对照组（P〈0．01）;③支气管上皮细胞PTEN蛋白表达量分别与BALF中的IL-4浓度呈显著负相关,与BALF中的IL—12浓度呈显著正相关。结论哮喘大鼠PTEN蛋白表达明显减少,它能减少Th2因子的表达,可能与哮喘大鼠气道炎症中Th1／Th2平衡密切相关。