Riboflavin in innate and acquired immune responses

Objective and Design: Riboflavin, also known as vitamin B2, is a micronutrient with a key role in maintaining human health. It has also been shown to enhance host resistance to bacterial infections in mice. The aim of this study was to assess the role of vitamin B2 treatment in inflammatory conditions. Subjects and Methods: Three models of inflammatory states were assessed. One of them encompasses neutrophil mediated but T cell/macrophage independent cutaneous inflammation. Another one is delayed type hypersensitivity reaction (DTH), a T cell/macrophage dependent but neutrophil independent inflammatory response. The third one is collagen- induced arthritis, having components from both of the above described reactions. Mice were treated with vitamin B2, administered by peritoneal injections, throughout the course of the experiments. Results: The granulocyte dependent reaction to olive oil was significantly reduced in vitamin B2 treated mice. In contrast, DTH reactivity and collagen II arthritis were not affected by the treatment. Conclusion: Riboflavin administration affects neutrophil migration but does not alter acquired immune responsiveness. Received 20 April 2005; returned for revision 9 June 2005; returned for final revision 29 June 2005; accepted by M. Parnham 5 July 2005     

LIGHT is involved in the pathogenesis of rheumatoid arthritis by inducing the expression of pro-inflammatory cytokines and MMP-9 in macrophages

Macrophages play a crucial role in the perpetuation of inflammation and irreversible cartilage damage during the development of rheumatoid arthritis (RA). LIGHT (TNFSF14) and its receptor TR2 (TNFRSF14) are known to have pro-inflammatory activities in foam cells of atherosclerotic plaques. We tested a hypothesis that LIGHT and TR2 are involved in activation of monocyte/macrophages in RA synovium. Immunohistochemical analysis of RA synovial tissue samples revealed that both LIGHT and TR2 are expressed in CD68 positive macrophages. In contrast, synovial tissue samples from osteoarthritis (OA) patients failed to reveal the expression of LIGHT. Expression of TR2 in RA synovial macrophages was also detected using flow cytometry analysis. To identify the role of LIGHT in the functioning of macrophages in RA, we isolated macrophage enriched cells from RA synovial fluid and stimulated them with LIGHT. LIGHT induced expression of matrix metalloproteinase-9 and pro-inflammatory cytokines such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-8. These data indicate that LIGHT and TR2 expressed in macrophages are involved in the pathogenesis of RA by inducing the expression pro-inflammatory cytokines and matrix degrading enzymes.     

类风湿关节炎患者外周血单个核细胞Sonic Hedgehog信号通路表达的初步研究

目的: 初步探讨类风湿关节炎(RA)患者外周血单个核细胞(PBMCs)和滑膜组织中Sonic Hedgehog(Shh)信号通路相关因子表达及意义。方法: 收集符合1987年美国风湿病学会(ACR)RA分类标准、28个关节疾病活动度评分(DAS28)≥3.2,病情活动RA患者(35例)及年龄、性别相匹配的健康志愿者(35例)外周血2 mL,分离PBMCs,提取总RNA,采用实时荧光定量PCR(real-time PCR)检测Shh信号通路中信号肽Shh、膜受体Ptch1和核转录因子Gli1 mRNA的表达。收集10例病情中度活动(DAS28≥3.2)RA患者的滑膜组织,同时收集5例外伤或半月板损伤(无关节炎)者滑膜组织作为对照组,免疫组化检测Shh、Ptch1和Gli1的蛋白表达情况。结果: RA患者PBMCs中Shh和Gli1 mRNA的相对表达量分别为1.36±1.48和1.15±0.68,对照组上述信号分子的mRNA表达量分别为0.47±0.25和0.49±0.05,2组差异有统计学意义(P<0.05),Ptch1 mRNA表达在2组间的差异无统计学意义(P>0.05);免疫组化示Shh和Gli1蛋白表达的阳性细胞百分率均高于对照组(P<0.05),Ptch1蛋白表达阳性细胞百分率2组无显著差异(P>0.05)。结论: RA患者PBMCs与滑膜组织中检测到Shh通路相关信号分子Shh和Gli1的表达上调,提示RA患者中可能存在Shh信号通路的激活,其在RA发病机制中的作用值得进一步研究。     

Prophylactic simvastatin increased survival during endotoxemia and inhibited granulocyte trafficking in a cell-intrinsic manner

Cross sectional studies have shown that statin-users have improved odds of surviving severe sepsis. Meanwhile controlled clinical trials failed to demonstrate improved sepsis survival with acute statin administration following hospitalization. Here, a lethal murine peritoneal lipopolysaccharide (LPS) endotoxemia model was used to assess the efficacy of chronic versus acute simvastatin on survival. Mirroring clinical observations, chronic but not acute treatment with simvastatin significantly increased survival. At a pre-mortality time point in LPS-treated mice, chronic simvastatin suppressed granulocyte trafficking in to the lungs and peritoneum without otherwise suppressing emergency myelopoiesis, myeloid cells in circulation, or inflammatory cytokines. Chronic simvastatin treatment significantly downregulated inflammatory chemokine gene signature in the lungs of LPS-treated mice. Thus, it was unclear if simvastatin was inhibiting granulocyte chemotaxis in a cell intrinsic or extrinsic manner. Adoptive transfer of fluorescently labeled granulocytes from statin and vehicle treated mice into LPS-treated mice showed that simvastatin inhibited lung-granulocyte trafficking in a cell intrinsic manner. Congruent with this, chemotaxis experiments using in vitro macrophages and ex vivo granulocytes demonstrated that simvastatin inhibited chemotaxis in a cell-intrinsic manner. Collectively, chronic but not acute simvastatin treatment improved survival in murine endotoxemia, and this was associated with cell-intrinsic inhibition of granulocyte chemotaxis.     

CD68-expressing cells can prime T cells and initiate autoimmune arthritis in the absence of reactive oxygen species

It is widely believed that DC, but not macrophages, prime naïve T cells in vivo. Here, we investigated the ability of CD68‐expressing cells (commonly defined as macrophages) in priming autoreactive T cells and initiating collagen‐induced arthritis (CIA) in the mouse. For this purpose, a transgenic mouse was developed (MBQ mouse) where macrophages exclusively expressed the MHC class II H2‐A^q (A^q) on an H2‐A^p (A^p) background. A^q, but not A^p expression mediates susceptibility to CIA through presentation of type II collagen (CII) to T cells. CIA severity is enhanced by a mutation in the Ncf1 gene, impairing reactive oxygen species (ROS) production by the phagocyte NADPH oxidase (NOX2) complex. Expression of functional Ncf1 on macrophages was previously shown to protect from severe CIA. To study the effect of ROS on macrophage‐mediated priming of T cells, the Ncf1 mutation was introduced in the MBQ mouse. Upon CII immunization, Ncf1‐mutated MBQ mice, but not Ncf1 wild‐type MBQ mice nor Ncf1‐mutated A^p mice, activated autoreactive T cells and developed CIA. These findings demonstrate for the first time that macrophages can initiate arthritis and that the process is negatively regulated by ROS produced via the NOX2 complex.     

The role of E- and P-selectin in neutrophil and monocyte migration in adjuvant-induced arthritis in the rat

The role of the endothelial adhesion molecules E- and P-selectin in leukocyte accumulation in arthritis is not known. We investigated this role in rat adjuvant arthritis by employing adhesion function-blocking monoclonal antibodies (mAb) to rat P- and E-selectin. The acute migration (2 h) of radiolabeled rat blood neutrophils and monocytes to joints and skin was determined. Anti-P-selectin mAb significantly reduced accumulation of monocytes (by 50%) and neutrophils (by 40%) in the talar joint, and of neutrophils in tail joints (by 90%). Anti-E-selectin mAb alone did not attenuate leukocyte migration, but when combined with anti-P-selectin mAb, it enhanced inhibition of neutrophil accumulation in the talar and carpal joints. In the same animals, anti-P-selectin mAb significantly inhibited neutrophil and monocyte migration to dermal inflammatory reactions induced by zymosan-activated rat serum (ZAS) containing the chemotactic factor C5a_{des Arg}, endotoxin (LPS), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). In contrast, anti-E-selectin mAb alone had no effect on monocyte or neutrophil accumulation in inflamed skin of arthritic animals, but again enhanced the inhibition when combined with mAb to P-selectin. The addition of anti-L-selectin mAb to anti-P- and E-selectin mAb did not further suppress monocyte or neutrophil migration to inflamed skin or joints. These results demonstrate that optimal leukocyte migration to arthritic joints and inflamed skin is P-selectin dependent, and E-selectin is not essential. However, E-selectin contributes to migration when P-selectin mechanisms are not operative. L-selectin does not play a role in E- and P-selectin-independent leukocyte migration to joints or skin inflammation in arthritic rats. However, it is likely that additional selectin-independent pathways also mediate neutrophil and monocyte migration to joint and skin inflammation.     

Anti-inflammatory activity of the isoquinoline alkaloid,tetrandrine, against established adjuvant arthritis in rats

Two isoquinoline plant alkaloids, tetrandrine (1) and berbamine (2), have been evaluated for anti-inflammatory activity in an acute paw oedema assay and in adjuvant-induced arthritis in rats.1 but not2 suppressed the chronic inflammation in the arthritis model but neither compound was active in the acute inflammation assay. In the adjuvant-induced polyarthritis,1 was not effective when given at the time of inoculation (Day 0), nor just before (Day 7–10) signs of arthritis were evident. However, when given on a therapeutic dose schedule (Days 10–13) or continually (Day –1 to +14) on a prophylactic schedule, signs of arthritis including weight loss due to cachexia were significantly reduced. Given orally,1 was considerably more potent than aspirin but not gastro-irritant and may be a promising lead for the development of a safe and effective treatment of chronic inflammatory diseases.     

低强度脉冲超声干预膝关节炎模型兔软骨细胞Ⅱ型胶原和基质金属蛋白酶13的表达

BACKGROUND: Low intensity pulsed ultrasound is safe, non-invasive, and has strong penetration. In recent years, more and more studies have indicated that low intensity pulsed ultrasound has important role in promoting the repair of damaged articular cartilage.     

Anti-inflammatory effect of all-trans-retinoic acid in inflammatory arthritis

OBJECTIVE: To determine whether all-trans-retinoic acid (ATRA) improves the destruction of joints and the effect of cytokines on DBA/1J mice with collagen-induced arthritis (CIA). METHODS: Starting from the time of type II collagen injection, DBA/1J mice were injected intraperitoneally with PBS or 0.5 mg of ATRA 3 times per week for 35 days. The effects of treatment were monitored by determining arthritis and histological scores and measuring cellular proliferation, production of cytokines (IL-2, IL-10, IL-12, IL-6, IFN-gamma, and TNF-alpha) and IgG, and the expression of mRNAs for inducible nitric oxide synthase (iNOS), monocyte chemoattractant protein-1 (MCP-1), and CXCR3. RESULTS: The arthritis score and incidence of arthritis were lower in the mice treated with ATRA than in those treated with PBS. Histopathologic evidence of joint damage was 34% lower, and the infiltrations of macrophages were reduced in the mice treated with ATRA compared with those treated with PBS. Type II collagen- and ConA-stimulated proliferation of spleen cells, the production of cytokines (IL-6, IL-12, and TNF-alpha), the serum levels of total IgG and IgG1 anti-collagen antibodies, and the expression of mRNAs for MCP-1 were significantly reduced in the mice treated with ATRA than in those treated with PBS. CONCLUSION: ATRA improved the clinical course and reduced the production of inflammatory cytokines, immunoglobulin, and chemokines in murine CIA. These data suggest that ATRA might be also effective for the treatment of inflammatory arthritis like human rheumatoid arthritis.     

The effect of menadione epoxide on the experimental immune arthritis in the rabbit.

It was shown previously that the experimentally induced arthritis in the rabbit can be largely nullified by subsequent treatment with menadione (by gavage). It is now shown that menadione epoxide, as is produced in the vitamin K cycle, also exerts a beneficial effect histologically and biochemically. Such treatment decreased both the glucose 6-phosphate dehydrogenase and the 6-phosphogluconolactonase activities in the synovial lining cells of the challenged joints towards values found in the unchallenged joints; it had only equivocal effects on the 6-phosphogluconate dehydrogenase activity. The results indicated that the epoxide might be interfering primarily with the lactonase activity.     

The effect of lysine acetylsalicylate on joint capsule mechanoreceptors in rats with polyarthritis

Summary Joint capsule mechanoreceptors in arthritic rats are more sensitive to pressure than similar receptors in normal animals. This greater sensitivity was reversed by the intravenous or topical administration of lysine acetylsalicylate in anaesthetised rats in doses of 15 to 50 mgm ASA-equivalent/kg. The reduction in sensitivity began within 5–10 min and reached a minimum mean value of 35% of the control after 35 to 40 min. During this period there was a negative linear or exponential relation between the amplitude of response to a controlled mechanical stimulus and time after administration of lysine acetylsalicylate. Control values of sensitivity were reached about 65–70 min following treatment with lysine acetylsalicylate. The results are interpreted as indicating that the high sensitivity of the arthritic joint capsule receptors is due to locally produced prostaglandins, such as prostacyclin.     

Therapeutic granulocyte and monocyte apheresis (GMA) for treatment refractory sarcoidosis: a pilot study of clinical effects and possible mechanisms of action

Sarcoidosis is a systemic, inflammatory disorder, which in a proportion of patients runs a chronic progressive course despite immunosuppressive treatment. Therapeutic granulocyte and monocyte apheresis (GMA) has been shown to be an effective treatment option for other systemic inflammatory disorders, but has not yet been investigated in sarcoidosis. The aim of this study was to evaluate the response to GMA in sarcoidosis. Seven patients with sarcoidosis refractory to standard immunosuppressive therapy received 10 GMA sessions. All patients underwent chest X-ray, spirometry, a Chronic Respiratory Disease Questionnaire (CRQ-SAS), blood tests and bronchoscopy with bronchoalveolar lavage (BAL) before treatment and at 2–4 weeks and 3 months (except bronchoscopy) after the last treatment session. Bronchoalveolar lavage fluid (BALF) cell differential counts were recorded and T cells from blood and BALF were analysed for markers of activity, differentiation and T regulatory function. Compared to baseline, five of seven patients reported an improvement in dyspnoea score. In BALF there was an increase in the percentage of macrophages and a decrease in the percentage of lymphocytes and CD4⁺/FoxP3⁺ T cells. Furthermore, the decrease in BALF CD4⁺/FoxP3⁺ T cells correlated significantly with an improvement in dyspnoea score. In peripheral blood there was a statistically significant increase in the percentage of CD4⁺/CD27⁻ T cells and a trend towards an initial increase in the percentage of CD4⁺/FoxP3⁺ T cells, followed by a statistically significant decrease. The effects of GMA on regulatory T cells are consistent with those observed in other inflammatory disorders and could potentially translate into a clinical benefit.     

Interaction of circulating immune complexes with granulocyte function in patients with rheumatoid arthritis

Summary The sera and peripheral blood granulocytes of 10 patients with rheumatoid arthritis and of 10 healthy controls were investigated for the presence of soluble immune complexes and for cellular dysfunction. Using the Rajicell-radioimmunoassay, immune complexes were detected in 7 out of 10 rheumatoid sera. In one patient the presence of immune complexes was demonstrated by immunofluorescence. In all rheumatoid patients with circulating immune complexes decreased chemotactic reactivity and diminished bactericidal capacity of the neutrophils were observed. Incubation of rheumatoid granulocytes with pooled AB-serum or pretreatment of neutrophils of healthy controls with immune complexes containing rheumatoid sera resulted in a reduced number of comparable cellular dysfunctions including increased release of lysosomal enzymes, strongly correlated with the presence of intracellular phagocytosed immune complexes. Phagocytosis and increase of oxidative cell metabolism during phagocytosis were not influenced by circulating immune complexes. Based on our in vitro findings we suggest that the described immune complex-dependent granulocyte dysfunctions are possibly responsible for the high risk of local or systemic bacterial diseases in patients with rheumatoid arthritis.     

The effect of LDL apheresis on progression of coronary artery disease in patients with familial hypercholesterolemia

This study investigated the effect of extracorporal lipid-lowering therapy by low-density lipoprotein (LDL) apheresis on coronary artery disease in a population characterized by early development and rapid progression of atherosclerosis. We treated 32 patients aged between 15 and 63 years with drug-refractory familial hypercholesterolemia, treated once a week by immuno-specific LDL apheresis for 3 years in a controlled prospective and non-randomized trial; 25 patients (14 females and 11 males) completed the study. Noninvasive data were obtained by physical examination, 12-lead ECG and exercise testing. Invasive cardiological data were obtained by cardiac catheterization according to a standardized protocol in four cardiological centers. Left ventricular ejection fraction was calculated using planimetry. Coronary stenoses were measured quantitatively in 23 defined coronary segments by a panel of four investigators with an electronic digital caliper. In addition, overall coronary atherosclerosis was visually qualified. Final decisions on a classification into one of three groups (regression, no change, progression) of coronary atherosclerosis were based on panel consensus. Six cardiac events occurred throughout the study: percutaneous transluminal coronary angioplasty in one patient, coronary bypass grafting in three and two deaths. Statistical analysis of exercise testing yielded no significant change for maximum power and work capacity during the study period. Hemodynamic data revealed no significant change; mean ejection fraction was calculated as 65.8 ± 15.9% at study entry and 67.0 ± 12.7% at completion. Quantitative measurement of 111 circumscribed coronary stenoses showed a mean stenosis degree of 45 ± 26% at entry cineangio-film and 43 ± 22% at final cineangio-film demonstrating no significant change. Eight localized stenoses showed a regression of more than 10% and 11 stenoses a progression of more than 10%. Panel consensus decision for overall coronary atherosclerosis resulted in regression in no patients, no change in 16, questionable progression in 3, definite progression in 5, and undecided in one. We conclude that specific LDL-apheresis therapy can be used effectively for primary and secondary prevention of coronary artery disease in patients with familial hypercholesterolemia. Its beneficial effect was the prevention of further progression of coronary artery disease in the majority of the study population.Abbreviations FH familial hypercholesterolemia - LDL low-density lipoprotein     

Anti-inflammatory effect of abciximab-coated stent in a porcine coronary restenosis model

The aim of this study was to examine the anti-inflammatory effect of abciximab-coated stent in a porcine coronary overstretch restenosis model. Ten abciximab-coated stents, ten sirolimus-eluting stents (SES), and ten paclitaxel-eluting stents (PES) were deployed with oversizing (stent/artery ratio 1.3:1) in porcine coronary arteries, and histopathologic analysis was done at 28 days after stenting. There were no significant differences in the neointima area normalized to injury score and inflammation score among the three stent groups (1.58 +/- 0.43 mm(2), 1.57 +/-0.39 mm(2) in abciximab-coated stent group vs. 1.69 +/- 0.57 mm(2), 1.72 +/- 0.49 mm(2) in the SES group vs. 1.92 +/- 0.86 mm(2), 1.79 +/- 0.87 mm(2) in the PES group, respectively). In the neointima, most inflammatory cells were lymphohistiocytes. Significant positive correlations were found between the extent of inflammatory reaction and the neointima area (r=0.567, p<0.001) and percent area stenosis (r=0.587, p<0.001). Significant correlations were found between the injury score and neointimal area (r=0.645, p<0.001), between the injury score and the inflammation score (r=0.837, p<0.001), and between the inflammation score and neointimal area (r=0.536, p=0.001). There was no significant difference in the inflammatory cell counts normalized to injury score among the three stent groups (75.5 +/- 23.1/microL in abciximabcoated stent group vs. 78.8 +/- 33.2/microL in the SES group vs. 130.3 +/- 46.9/microL in the PES group). Abciximab-coated stent showed comparable inhibition of inflammatory cell infiltration and neointimal hyperplasia with other drug-eluting stents in a porcine coronary restenosis model.     

Initiation of an autoimmune response: insights from a transgenic model of rheumatoid arthritis

K/BxN mice develop an inflammatory joint disease with many features characteristic of rheumatoid arthritis. This model is based on a T-cell receptor transgene, KRN, that has been shown to recognize both the foreign antigen bovine RNase, and the ubiquitously expressed self antigen, glucose-6-phosphate isomerase (GPI). We have used this model to investigate the initial events that occur during the autoimmune response to GPI. We and others have identified key mediators in the inflammatory response: Fc receptors (FcRs) (in particular FcRIII), the alternative pathway of complement, neutrophils, and mast cells. Using micro position emission tomography, we demonstrated that anti-GPI Immunoglobulin G (IgG) localizes specifically to the joints where arthritis occurs and that this localization is dependent on mast cells, neutrophils, FcRs, and immune complexes. The trigger of arthritis in this model is the KRN T-cell inducing the production of anti-GPI Ig. By overexpressing the ligand for the KRN T-cells in major histocompatibility complex class II-expressing cells, we demonstrated that KRN T-cells were able to escape tolerance induction in the thymus owing to insufficient levels of antigen in the thymus antigen-presenting cells. This allows the KRN T-cells to exit to the periphery, where they provide help to anti-GPI B-cells, inducing the production of arthritogenic Ig. To understand the joint specificity of the disease, we followed the anti-GPI B-cell response that naturally arises in K/BxN mice and showed that, although the GPI antigen is ubiquitously expressed, the anti-GPI B-cell response is foucused on the lymph nodes draining the affected joints. Together, these studies have given us a greater understanding of how an autoimmune response is initiated at the level of both the adaptive and innate immune systems and demonstrate the versatility of the K/BxN arthritis model for studying human disease.     

Anti-inflammatory effect of tuftsin and its retro-inverso analogue in rat adjuvant arthritis

Tuftsin, an immunostimulating tetrapeptide derived from immunoglobulin heavy chain, and its retro-inverso analogue have been tested in rat adjuvant arthritis. The secondary lesion of rats injected with Freund's adjuvant was significant inhibited by both the natural and retro-inverso tuftsin administered orally. The retro-inverso analogue was at least tenfold more potent than the parent molecule, suggesting an increased stability to degradation, because of its molecular modification. The anti-inflammatory effect of tuftsin may be due to its ability to modulate macrophage function.

     

Anti-inflammatory effect of tuftsin and its retro-inverso analogue in rat adjuvant arthritis

Tuftsin, an immunostimulating tetrapeptide derived from immunoglobulin heavy chain, and its retro-inverso analogue have been tested in rat adjuvant arthritis. The secondary lesion of rats injected with Freund's adjuvant was significant inhibited by both the natural and retro-inverso tuftsin administered orally. The retro-inverso analogue was at least tenfold more potent than the parent molecule, suggesting an increased stability to degradation, because of its molecular modification. The anti-inflammatory effect of tuftsin may be due to its ability to modulate macrophage function.