Adriana Delwail

Cutting edge: IL-21 is a switch factor for the production of IgG1 and IgG3 by human B cells

IL-21 is a cytokine that regulates the activation of T and NK cells and promotes the proliferation of B cells activated via CD40. In this study, we show that rIL-21 strongly induces the production of all IgG isotypes by purified CD19+ human spleen or peripheral blood B cells stimulated with anti-CD40 mAb. Moreover, it was found to specifically induce the production of IgG1 and IgG3 by CD40-activated CD19+CD27− naive human B cells. Although stimulation of CD19+ B cells via CD40 alone induced γ1 and γ3 germline transcripts, as well as the expression of activation-induced cytidine deaminase, only stimulation with both anti-CD40 mAb and rIL-21 resulted in the production of Sγ/Sμ switch circular DNA. These results show that IL-21, in addition to promoting growth and differentiation of committed B cells, is a specific switch factor for the production of IgG1 and IgG3.

Role of interleukin-6 in cardiomyocyte/cardiac fibroblast interactions during myocyte hypertrophy and fibroblast proliferation.

The process of cardiac hypertrophy is considered to involve two components: that of cardiac myocyte (CM) enlargement and cardiac fibroblast (CF) proliferation. The interleukin‐6 (IL‐6) family cytokines have been implicated in a variety of cellular and molecular interactions between myocytes and non‐myocytes (NCMs), which in turn have important roles in the development of cardiac hypertrophy. In the study of these interactions, we previously detected very high levels of IL‐6 in supernatants of a “dedifferentiated model” of adult ventricular CMs cultured with CFs. In the present study, we have used this in vitro coculture system to examine how IL‐6 is involved in the interactions between CMs and CFs during CM hypertrophy and CF proliferation. IL‐6 and its signal transducer, 130‐kDa glycoprotein (gp130), were detected by immunostaining cultured CMs and CFs with anti‐IL‐6 or anti‐gp130 antibodies. Addition of anti‐IL‐6 or anti‐gp130 antagonist antibodies into CM/CF cocultures induced a significant decrease in expression of atrial natriuretic peptide (ANP) and β‐myosin heavy chain (β‐MHC) in CMs. The presence of IL‐6 antagonist also resulted in a decrease in the surface area of 12‐day‐old CMs cultured with CFs or in the presence of fibroblast conditioned medium (FCM), and decreased fibroblast proliferation in CM/CF cocultures, particularly in the presence of a gp130 antagonist. The results also show that angiotensin II (AngII) is mainly secreted by CFs and induces IL‐6 secretion in CMs cultured with CFs or with FCM. In addition, the effects of IL‐6 on cardiomyocyte hypertrophy and fibroblast proliferation were inhibited by addition of the AT‐1 receptor antagonist, losartan. These results suggest that IL‐6 contributes significantly to CM hypertrophy by an autocrine pathway and to fibroblast proliferation by a paracrine pathway and that these effects could be mediated by AngII.

Circulating soluble gp130, soluble IL-6R, and IL-6 in patients undergoing cardiac surgery, with or without extracorporeal circulation

OBJECTIVE Soluble forms of interleukin-6 (IL-6) receptors are known to modulate biological activities of IL-6. The purpose of the study was to measure circulating levels of IL-6, sIL-6R and sgp130 in patients undergoing coronary artery bypass grafting with cardiopulmonary bypass (CPB group) or without CPB (non-CPB group). METHODS The CPB group included 19 patients and the non-CPB group 12 patients. Sera levels of IL-6, sIL-6R and sgp130 were measured by specific ELISA at the beginning of the operation (T0, 15 min before skin incision) and 6 h later (T1). RESULTS IL-6 sera levels were respectively 9+/-20 pg/ml (mean+/-SD) and 13+/-19 pg/ml at T0 and reached 340+/-250 pg/ml and 965+/-1060 pg/ml at T1 in CPB and non-CPB groups, indicating a significant increase from T0 to T1, but no differences between the two groups. When compared to T0 values, sgp130 levels decreased in both groups (respectively 105+/-37 and 115+/-35 ng/ml at T0 for CPB and non-CPB groups, and 72+/-25 and 84+/-29 ng/ml at T1) while we are not able to detect differences between the groups. Whatever the group or the time, sIL-6R concentrations remained unchanged. CONCLUSIONS We showed that the increase of IL-6 after artery bypass grafting was similar between patients operated with CPB or without CPB. We conclude that the main inductor of IL-6 release is linked to surgical trauma rather than a reaction to CPB. Since it is known that gp130 inhibits IL-6-biological activities, we suggest that the decrease of sgp130 sera levels could further enhance the inflammatory effects of IL-6 in cardiac surgery.

Specific increase in caspase-1 activity and secretion of IL-1 family cytokines: a putative link between mevalonate kinase deficiency and inflammation

The mevalonate kinase deficiency (MKD), including hyperimmunoglobulinemia D periodic fever syndrome (HIDS) and the more severe mevalonic aciduria are rare, autosomal recessive, autoinflammatory diseases belonging to the hereditary periodic fever (HPF) family. Other members include: familial mediterranean fever (FMF), the cryopyrin-associated periodic syndromes (CAPS) and TNFR-associated periodic syndromes (TRAPS). MKD is caused by mutations in the gene encoding mevalonate kinase (MK), an enzyme of the cholesterol pathway, leading to its inactivation. The molecular mechanisms linking MKD and abnormalities of isoprenoid biosynthesis to cytokine production and inflammation have yet to be fully elucidated. Statins, which are extensively prescribed for lowering cholesterol, are potent inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase, the enzyme directly upstream of MK. In this review, we discuss recent reports demonstrating that in vitro inhibition of the mevalonate pathway by statins specifically increases the production, by activated monocytes, of cytokines of the IL-1 family, by enhancing caspase-1 activity, the enzyme responsible for IL-1beta and IL-18 maturation. The molecular mechanisms involve geranylgeranylation and the enhancement of the activity of G proteins such as Rac-1. Interestingly, activated fibroblasts from MKD patients secrete more IL-1beta than fibroblasts from healthy donors. Taken together, these data highlight the specific enhancement of the IL-1 family of cytokines, the maturation of which is caspase-1-dependent in MKD. Finally, the spectacular decrease in febrile attacks in patients with severe HIDS under IL-1 receptor antagonist (anakinra) treatment, reinforces this hypothesis. Deregulated caspase-1 activation could be responsible for the inflammatory component of MKD, thereby mechanistically linking MKD to FMF and CAPS through cytokines of the IL-1 family.

Increased immunoglobulin A in alcoholic liver cirrhosis: exploring the response of B cells to Toll-like receptor 9 activation

Alcoholic liver cirrhosis (ALC) is characterized by increased circulating levels of immunoglobulins (Igs). ALC patients undergo bacterial translocation evidenced by the presence of bacterial DNA in peripheral blood. Bacterial pathogen‐associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), peptidoglycan (PGN) and unmethylated cytosine‐guanine dinucleotide (CpG) DNA are ligands of Toll‐like receptor (TLR)‐4, TLR‐2 and TLR‐9, respectively. Although TLR activation results generally in the secretion of proinflammatory cytokines, activation of B cells through TLR‐7 or TLR‐9 is involved in their maturation and Ig synthesis. The aim of the present study was to assess Ig synthesis by ALC B cells under PAMP activation in order to evaluate the possible involvement of TLR pathways in the increased Ig levels, and especially the hyper‐IgA observed in ALC. CpG, in combination with interleukin (IL)‐10 or IL‐21, enhanced IgA, IgG and IgM synthesis by healthy donor (HD) PBMCs, but had only a weak effect on ALC PBMCs. Relative CpG‐induced IgA production by purified ALC B cells was less important when compared to HD B cells, in accordance with the lower TLR‐9 expression on ALC B cells compared to HD B cells, but the absolute IgA production by CpG‐activated B cells was enhanced significantly for ALC when compared to HD, in agreement with their intrinsic ability to produce spontaneously more IgA than HD. LPS and PGN had no direct activity on B cells, whereas R848 also enhanced Ig synthesis, as reported recently. Taken together, these results suggest that TLR priming of B cells could account for the hyperimmunoglobulinaemia observed in ALC patients.

Molecular and functional characterization of a new potassium conductance in mouse ventricular fibroblasts

The present work is aimed at identifying and characterizing, at a molecular and functional level, new ionic conductances potentially involved in the excitation-secretion coupling and proliferation of cardiac ventricular fibroblasts. Among potassium channel transcripts which were screened by high-throughput real-time PCR, SUR2 and Kir6.1 mRNAs were found to be the most abundant in ventricular fibroblasts. The corresponding proteins were not detected by western blot following 5 days of cell culture, but had appeared at 7 days, increasing with extended cell culture duration as the fibroblasts differentiated into myofibroblasts. Using the inside-out configuration of the patch-clamp technique, single potassium channels could be recorded. These had properties similar to those reported for SUR2/Kir6.1 channels, i.e. activation by pinacidil, inhibition by glibenclamide and activation by intracellular UDP. As already reported for this molecular signature, they were insensitive to intracellular ATP. In the whole-cell configuration, these channels have been shown to be responsible for a glibenclamide-sensitive macroscopic potassium current which can be activated not only by pinacidil, but also by nanomolar concentrations of the sphingolipid sphingosine-1-phosphate (S1P). The activation of this current resulted in an increase in cell proliferation and a decrease in IL-6 secretion, suggesting it has a functional role in situations where S1P increases. Overall, this work demonstrates for the first time that SUR2/Kir6.1 channels represent a significant potassium conductance in ventricular fibroblasts which may be activated in physio-pathological conditions and which may impact on fibroblast proliferation and function.

Treatment of pediatric Erdheim‐Chester disease with interleukin‐1–targeting drugs

Erdheim-Chester disease (ECD) is a rare nonLangerhans systemic histiocytosis of unknown origin. ECD typically involves bilateral symmetric cortical osteosclerosis of the diaphyseal and metaphyseal regions in the long bones and infiltration of other organs (1). The diagnosis is based on typical histologic findings, with xanthogranulomatous infiltration of tissue by foamy CD68-positive and CD1a-negative histiocytes. Published data on therapeutic approaches, including surgery, corticosteroids, cytotoxic drugs, stem cell transplantation, and others, are limited to case reports and small series and show generally incomplete and/or transient remission and frequent toxicity (2–4). More recently, interferon(IFN ) has been suggested as a treatment option; it has demonstrated variable efficacy and sometimes limited tolerance (5–7). Herein we report on a child with ECD who was treated with anakinra, a recombinant, nonglycosylated homolog of the human interleukin-1 (IL-1) receptor antagonist, and we describe a rationale for this treatment. The patient, a 10-year-old girl, presented with recurrent fever, elevated erythrocyte sedimentation rate (ESR) and increased C-reactive protein (CRP) levels, bone pain, and failure to thrive, and was diagnosed as having ECD. Bone radiography revealed multiple osteolytic and osteosclerotic lesions in the femurs, tibia, and pelvis. Whole-body magnetic resonance imaging (MRI) showed high-intensity signal of the skeletal bone marrow on fat-suppressed T2-weighted images, retroperitoneal infiltration, and diffuse low-intensity signal in the metadiaphyses (Figure 1C). Histologic findings on bone biopsy confirmed the diagnosis of ECD (Figure 2). Treatment with subcutaneous IFN alfa-2a (3 10 units 3 times per week) resulted in normalization of clinical symptoms, ESR and CRP values, and liver and spleen volumes, significant regression of retroperitoneal infiltration, and improvement of bone marrow signal intensity within 4 months. However, after 10 months of treatment the patient experienced relapse, manifested by fever, bone pain, and increased ESR and CRP levels. Treatment with vinblastine (6 mg/m/ week) and prednisone (2 mg/kg/day) for 6 weeks showed no efficacy. The patient was then treated with PEGylated IFN alfa-2a (1.5 g/kg/week) (8), with good efficacy. However, after 12 months of treatment, another relapse occurred. To obtain a rationale for a potential alternative treatment option targeting specific cytokines, IL-1 , IL-6, and tumor necrosis factor (TNF ) levels in unstimulated and lipopolysaccharide (LPS; 1 g/ml)–stimulated peripheral blood mononuclear cells (PBMCs), which had been isolated from whole blood samples with Ficoll-Paque and cultured in RPMI, were measured by enzyme-linked immunosorbent assay. Data on the patient were compared with data from 15 healthy controls. As shown in Figure 1A, the unstimulated PBMCs from the patient displayed increased levels of IL-1 (40.3fold), IL-6 (23.9-fold), and TNF (5.6-fold). Following LPS stimulation, levels of IL-1 , IL-6, and TNF were comparable to those in controls. These findings suggested that high basal levels of IL-1 and IL-6 may participate in the pathophysiology of ECD. Serum levels of IL-1 were within the normal range. We therefore hypothesized that targeting of IL-1 might have a beneficial effect on the disease course, and consequently, treatment with anakinra (2 mg/kg/day) was instituted. Within 1 week, fever and bone pain resolved, and 1 month later, the ESR and CRP normalized (Figure 1B). During the subsequent 10 months the patient gained weight (8 kg) and height (6 cm). However, no significant changes in the bone lesions or retroperitoneal infiltration were seen on MRI 7 months after initiation of this treatment (Figure 1C). During Figure 1. A, Production of cytokines (interleukin-1 [IL-1 ], IL-6, and tumor necrosis factor [TNF ]) by peripheral blood mononuclear cells from the patient and from 15 healthy controls, with or without lipopolysaccharide (LPS) stimulation. B, Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels before and after initiation of treatment with anakinra. IFN2a interferon alfa-2a. C, Comparative magnetic resonance imaging findings before (left) and 6 months after (right) initiation of treatment with anakinra. Frontal view whole-body images (T2-weighted with fat suppression) show no evidence of change in retroperitoneal infiltration (arrows).

Design, synthesis and biological evaluation of new thalidomide analogues as TNF-α and IL-6 production inhibitors.

Several thalidomide analogues were synthesized and compared to thalidomide and its more active analogue, lenalidomide, for their ability to inhibit the production of the pro-inflammatory cytokine tumour necrosis factor (TNF)-α and interleukin (IL)-6 by LPS-activated peripheral blood mononuclear cells (PBMCs). Among these compounds, two analogues containing sulfonyl group displayed interesting downregulation of TNF-α and IL-6 production.

High plasma levels of the pro-inflammatory cytokine IL-22 and the anti-inflammatory cytokines IL-10 and IL-1ra in acute pancreatitis

BACKGROUND/OBJECTIVES Pancreatic acinar cells are major targets of IL-22. Our aim is to study early plasma levels of IL-22, of pro- and anti-inflammatory cytokines in acute pancreatitis, and their association with severity or necrosis infection. METHODS Consecutive patients admitted to the Department of Hepato-Gastroenterology at Poitiers University of Medicine Hospital (France) with a diagnosis of AP were prospectively enrolled. Plasma concentrations of IL-22, IL-6, IL-8, IL-1 α, IL-1β, TNF- α, IFN-γ, IL-17A, IL-10, IL-1ra and IL-4 were assessed by multiple immunoassay at the admission time. A thoracoabdominal contrast-enhanced CT scan was performed at day 2. RESULTS Sixty-two patients were included; 13 patients (21%) had a severe acute pancreatitis, 5 patients (8%) developed necrosis infection and 29 patients (47%) had pleural effusion. Plasma levels of IL-22 were high in AP (135 ± 31 vs 4.2 ± 1.8 pg/ml for controls, p < 0.05), but did not correlate with the severity of the disease, whereas IL-6, IL-10 and IL-1ra where enhanced in patients with severe acute pancreatitis and with pleural effusion. Patients who further developed necrosis infection had higher levels of IL-1ra at admission (p = 0.0004). CONCLUSION In acute pancreatitis, high plasma levels of IL-22 are observed, regardless the severity of the disease. In contrast, severe forms were associated with increased levels of IL-6, IL-10 and IL-1ra. The beneficial or deleterious role of IL-22 in AP remains to be further studied.

High-Fat Diet–Induced IL-17A Exacerbates Psoriasiform Dermatitis in a Mouse Model of Steatohepatitis

Recent studies suggest that psoriasis may be more severe in patients with nonalcoholic fatty liver disease, particularly in those with the inflammatory stage of steatohepatitis [nonalcoholic steatohepatitis (NASH)]. Herein, we investigated the impact of diet-induced steatohepatitis on the severity of imiquimod-induced psoriasiform dermatitis. Mice fed with a high-fat diet developed steatohepatitis reminiscent of human NASH with ballooning hepatocytes and significant liver fibrosis. Mice with steatohepatitis also displayed moderate cutaneous inflammation characterized by erythema, dermal infiltrates of CD45(+) leukocytes, and a local production of IL-17A. Moreover, steatohepatitis was associated with an epidermal activation of caspase-1 and cutaneous overexpression of IL-1β. Imiquimod-induced psoriasiform dermatitis was exacerbated in mice with steatohepatitis as compared to animals fed with a standard diet. Scale formation and acanthosis were aggravated, in correlation with increased IL-17A and IL-22 expression in inflamed skins. Finally, intradermal injection of IL-17A in standard diet-fed mice recapitulated the cutaneous pathology of mice with steatohepatitis. The results show that high-fat diet-induced steatohepatitis aggravates the inflammation in psoriasiform dermatitis, via the cutaneous production of IL-17A. In agreement with clinical data, this description of a novel extrahepatic manifestation of NASH should sensitize dermatologists to the screening and the management of fatty liver in psoriatic patients.