Anti–interleukin‐6 receptor antibody therapy reduces vascular endothelial growth factor production in rheumatoid arthritis

Objective

To investigate whether interleukin‐6 (IL‐6) is a regulator of vascular endothelial growth factor (VEGF) in rheumatoid arthritis (RA).

Methods

Serum VEGF levels in RA patients were assayed before and after 8 weeks or 24 weeks of maintenance therapy with humanized anti–IL‐6 receptor monoclonal antibody (anti–IL‐6R mAb). VEGF secreted by RA synovial fibroblasts cultured in the presence of IL‐6, IL‐1β, and/or tumor necrosis factor α (TNFα) was measured. The inhibitory effect of anti–IL‐6R mAb, recombinant IL‐1 receptor antagonist (IL‐1Ra), and anti‐TNFα mAb on VEGF production was also examined.

Results

Serum VEGF levels in RA patients before anti–IL‐6R mAb therapy were significantly higher than those in healthy controls (P < 0.0005). Treatment of RA patients with anti–IL‐6R mAb normalized serum VEGF levels. In the in vitro study, IL‐6 and IL‐1β each induced a slight amount of VEGF production in synovial cells, but TNFα did not. Although VEGF‐inducing activity of these cytokines was not remarkable when they were added alone, IL‐6 acted synergistically with IL‐1β or TNFα to induce VEGF production. There was no synergistic effect between IL‐1β and TNFα. In the presence of all of these cytokines, anti–IL‐6R mAb eliminated the synergistic effect of IL‐6, IL‐1β, and TNFα, while IL‐1Ra or anti‐TNFα mAb did not.

Conclusion

Anti–IL‐6R mAb therapy reduced VEGF production in RA. IL‐6 is the pivotal cytokine that induces VEGF production in synergy with IL‐1β or TNFα, and this may be the mechanism by which IL‐6 blockade effectively suppresses VEGF production in synovial fibroblasts.

     

Targeting oncogenic interleukin‐7 receptor signalling with N‐acetylcysteine in T cell acute lymphoblastic leukaemia

Activating mutations of the interleukin‐7 receptor (IL7R) occur in approximately 10% of patients with T cell acute lymphoblastic leukaemia (T‐ALL). Most mutations generate a cysteine at the transmembrane domain leading to receptor homodimerization through disulfide bond formation and ligand‐independent activation of STAT5. We hypothesized that the reducing agent N‐acetylcysteine (NAC), a well‐tolerated drug used widely in clinical practice to treat acetaminophen overdose, would reduce disulfide bond formation, and inhibit mutant IL7R‐mediated oncogenic signalling. We found that treatment with NAC disrupted IL7R homodimerization in IL7R‐mutant DND‐41 cells as assessed by non‐reducing Western blot, as well as in a luciferase complementation assay. NAC led to STAT5 dephosphorylation and cell apoptosis at clinically achievable concentrations in DND‐41 cells, and Ba/F3 cells transformed by an IL7R‐mutant construct containing a cysteine insertion. The apoptotic effects of NAC could be rescued in part by a constitutively active allele of STAT5. Despite using doses lower than those tolerated in humans, NAC treatment significantly inhibited the progression of human DND‐41 cells engrafted in immunodeficient mice. Thus, targeting leukaemogenic IL7R homodimerization with NAC offers a potentially effective and feasible therapeutic strategy that warrants testing in patients with T‐ALL.     

Interleukin‐6 receptor shedding is enhanced by interleukin‐1β and tumor necrosis factor α and is partially mediated by tumor necrosis factor α–converting enzyme in osteoblast‐like cells

Objective

Interleukin‐6 (IL‐6) and soluble IL‐6 receptor (sIL‐6R) activation of gp130 represents an alternative pathway for osteoclast development in inflammatory conditions. The goal of the present study was to investigate changes in sIL‐6R levels in response to the inflammatory cytokines IL‐1β and tumor necrosis factor α (TNFα) and to determine the role of TNFα‐converting enzyme (TACE) in this process.

Methods

Levels of sIL‐6R in the culture media of MG63 and SAOS‐2 osteoblast‐like cell lines after exposure to various agents were determined by immunoassay. TACE protein levels were measured by Western immunoblotting. Cells were transfected with small interfering RNA (siRNA) or with an expression plasmid for IL‐6R and TACE to determine the potential involvement of TACE in IL‐6R shedding.

Results

IL‐1β and TNFα increased the levels of sIL‐6R in the culture media of MG63 osteoblast‐like cells. This effect was not influenced by cycloheximide or 5,6‐dichlorobenzimidazole riboside but was markedly inhibited by the calcium chelator EGTA and by the TACE and matrix metalloproteinase inhibitor hydroxamate (Ru36156). IL‐1β and TNFα had no influence on the alternatively spliced form of IL‐6R RNA. Levels of sIL‐6R were reduced when MG63 cells were transiently transfected with TACE siRNA. Transfection of SAOS‐2 cells with expression plasmids for IL‐6R and TACE produced a dose‐dependent increase in sIL‐6R levels.

Conclusion

IL‐1β‐ and TNFα‐mediated induction of IL‐6R shedding in osteoblast‐like cells is at least partly dependent on TACE activation.

     

Tumor necrosis factor α and interleukin‐1β modulate calcium and nitric oxide signaling in mechanically stimulated osteocytes

Objective

Inflammatory diseases often coincide with reduced bone mass. Mechanoresponsive osteocytes regulate bone mass by maintaining the balance between bone formation and resorption. Despite its biologic significance, the effect of inflammation on osteocyte mechanoresponsiveness is not understood. To fill this gap, we investigated whether the inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin‐1β (IL‐1β) modulate the osteocyte response to mechanical loading.

Methods

MLO‐Y4 osteocytes were incubated with TNFα (0.5–30 ng/ml) or IL‐1β (0.1–10 ng/ml) for 30 minutes or 24 hours, or with calcium inhibitors for 30 minutes. Cells were subjected to mechanical loading by pulsatile fluid flow (mean ± amplitude 0.7 ± 0.3 Pa, 5 Hz), and the response was quantified by measuring nitric oxide (NO) production using Griess reagent and by measuring intracellular calcium concentration ([Ca²⁺]_i) using Fluo‐4/AM. Focal adhesions and filamentous actin (F‐actin) were visualized by immunostaining, and apoptosis was quantified by measuring caspase 3/7 activity. Cell‐generated tractions were quantified using traction force microscopy, and cytoskeletal stiffness was quantified using optical magnetic twisting cytometry.

Results

Pulsatile fluid flow increased [Ca²⁺]_i within seconds (in 13% of cells) and NO production within 5 minutes (4.7‐fold). TNFα and IL‐1β inhibited these responses. Calcium inhibitors decreased pulsatile fluid flow–induced NO production. TNFα and IL‐1β affected cytoskeletal stiffness, likely because 24 hours of incubation with TNFα and IL‐1β decreased the amount of F‐actin. Incubation with IL‐1β for 24 hours stimulated osteocyte apoptosis.

Conclusion

Our results suggest that TNFα and IL‐1β inhibit mechanical loading–induced NO production by osteocytes via abrogation of pulsatile fluid flow–stimulated [Ca²⁺]_i, and that IL‐1β stimulates osteocyte apoptosis. Since both NO and osteocyte apoptosis affect osteoclasts, these findings provide a mechanism by which inflammatory cytokines might contribute to bone loss and consequently affect bone mass in rheumatoid arthritis.

     

Chondrocyte AMP‐activated protein kinase activity suppresses matrix degradation responses to proinflammatory cytokines interleukin‐1β and tumor necrosis factor α

Objective

Interleukin‐1β (IL‐1β) and tumor necrosis factor α (TNFα) stimulate chondrocyte matrix catabolic responses, thereby compromising cartilage homeostasis in osteoarthritis (OA). AMP‐activated protein kinase (AMPK), which regulates energy homeostasis and cellular metabolism, also exerts antiinflammatory effects in multiple tissues. This study was undertaken to test the hypothesis that AMPK activity limits chondrocyte matrix catabolic responses to IL‐1β and TNFα.

Methods

Expression of AMPK subunits was examined, and AMPKα activity was ascertained by the phosphorylation status of AMPKα Thr¹⁷² in human knee articular chondrocytes and cartilage by Western blotting and immunohistochemistry, respectively. Procatabolic responses to IL‐1β and TNFα, such as release of glycosaminoglycan, nitric oxide, and matrix metalloproteinases 3 and 13 were determined by dimethylmethylene blue assay, Griess reaction, and Western blotting, respectively, in cartilage explants and chondrocytes with and without knockdown of AMPKα by small interfering RNA.

Results

Normal human knee articular chondrocytes expressed AMPKα1, α2, β1, β2, and γ1 subunits. AMPK activity was constitutively present in normal articular chondrocytes and cartilage, but decreased in OA articular chondrocytes and cartilage and in normal chondrocytes treated with IL‐1β and TNFα. Knockdown of AMPKα resulted in enhanced catabolic responses to IL‐1β and TNFα in chondrocytes. Moreover, AMPK activators suppressed cartilage/chondrocyte procatabolic responses to IL‐1β and TNFα and the capacity of TNFα and CXCL8 (IL‐8) to induce type X collagen expression.

Conclusion

Our findings indicate that AMPK activity is reduced in OA cartilage and in chondrocytes following treatment with IL‐1β or TNFα. AMPK activators attenuate dephosphorylation of AMPKα and procatabolic responses in chondrocytes induced by these cytokines. These observations suggest that maintenance of AMPK activity supports cartilage homeostasis by protecting cartilage matrix from inflammation‐induced degradation.

     

Both the lymphotoxin and tumor necrosis factor pathways are involved in experimental murine models of colitis

Background & Aims: Membrane lymphotoxin (LT) α/β, a member of the tumor necrosis factor (TNF) family of immune regulatory molecules, is involved both in the development of secondary lymphoid tissues and the maintenance of organized lymphoid tissues in the adult. Defects observed in the mucosal immune system in animals with a genetically disrupted LTα/β pathway coupled with the expression of LTα/β in activated T cells motivated an examination of the importance of this pathway in experimental colitis. Methods: Soluble LTβ receptor (LTβR) immunoglobulin fusion protein was used to inhibit the LTα/β/light axis in two independent rodent models of colitis: CD45RB^hi CD4⁺–reconstituted SCID mice and bone marrow–transplanted tgϵ26 mice (BM → tgϵ26). Results: Treatment with LTβR immunoglobulin attenuated the development of both the clinical and histological manifestations of the disease in these two murine models of colitis. Given the success of TNF inhibitors in the treatment of human Crohn's disease, the effects of LTβR immunoglobulin have been compared with antibody to TNF in the BM → tgϵ26 model, and both treatments were equally efficacious. Conclusions: The LT pathway plays a role in the development of colitis as important as that of the TNF system and, therefore, represents a potential novel intervention point for the treatment of inflammatory bowel disease.GASTROENTEROLOGY 1998;115:1464-1475     

Mechanical injury potentiates proteoglycan catabolism induced by interleukin‐6 with soluble interleukin‐6 receptor and tumor necrosis factor α in immature bovine and adult human articular cartilage

Objective

Traumatic joint injury can damage cartilage and release inflammatory cytokines from adjacent joint tissue. The present study was undertaken to study the combined effects of compression injury, tumor necrosis factor α (TNFα), and interleukin‐6 (IL‐6) and its soluble receptor (sIL‐6R) on immature bovine and adult human knee and ankle cartilage, using an in vitro model, and to test the hypothesis that endogenous IL‐6 plays a role in proteoglycan loss caused by a combination of injury and TNFα.

Methods

Injured or uninjured cartilage disks were incubated with or without TNFα and/or IL‐6/sIL‐6R. Additional samples were preincubated with an IL‐6–blocking antibody Fab fragment and subjected to injury and TNFα treatment. Treatment effects were assessed by histologic analysis, measurement of glycosaminoglycan (GAG) loss, Western blot to determine proteoglycan degradation, zymography, radiolabeling to determine chondrocyte biosynthesis, and Western blot and enzyme‐linked immunosorbent assay to determine chondrocyte production of IL‐6.

Results

In bovine cartilage samples, injury combined with TNFα and IL‐6/sIL‐6R exposure caused the most severe GAG loss. Findings in human knee and ankle cartilage were strikingly similar to those in bovine samples, although in human ankle tissue, the GAG loss was less severe than that observed in human knee tissue. Without exogenous IL‐6/sIL‐6R, injury plus TNFα exposure up‐regulated chondrocyte production of IL‐6, but incubation with the IL‐6–blocking Fab significantly reduced proteoglycan degradation.

Conclusion

Our findings indicate that mechanical injury potentiates the catabolic effects of TNFα and IL‐6/sIL‐6R in causing proteoglycan degradation in human and bovine cartilage. The temporal and spatial evolution of degradation suggests the importance of transport of biomolecules, which may be altered by overload injury. The catabolic effects of injury plus TNFα appeared partly due to endogenous IL‐6, since GAG loss was partially abrogated by an IL‐6–blocking Fab.

     

The inflammatory circuitry of miR-149 as a pathological mechanism in osteoarthritis

Osteoarthritis (OA) is a multifactorial degenerative pathology, whose progression is exacerbated by pro-inflammatory cytokines signaling. Among the changes triggered in chondrocytes during inflammation, modified expression of tiny epigenetic regulators as microRNAs was shown having deleterious implications for articular cartilage. Aim of the present study was to identify differentially expressed microRNAs in human OA cartilage and to determine their relevance to pathological progression. An OA model based on inflammatory stimulation of a chondrocytic human cell line was used to analyze microRNAs deregulation, and results revealed miR-149 severely down-regulated by IL1β and TNFα. Real-time PCR analysis of miR-149 was exerted also in human primary chondrocytes isolated from cartilage of OA donors and postmortem from subjects with no known history of OA, confirming down-regulation in osteoarthritis. Moving on a functional study, miR-149 regulatory effect on tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL1β) and interleukin 6 (IL6) 3′UTRs was evaluated by luciferase assays, and chondrocytes production of TNFα upon miR-149 transfection was measured by enzyme-linked immuno sorbent assay. We found that miR-149 is down-regulated in OA chondrocytes, and this decrease seems to be correlated to increased expression of pro-inflammatory cytokines such as TNFα, IL1β and IL6. OA is a multifactorial disease and we think that our results give new insights for understanding the complex mechanisms of osteoarthritic pathogenesis.     

Stress‐induced cartilage degradation does not depend on the NLRP3 inflammasome in human osteoarthritis and mouse models

Objective

The main feature of osteoarthritis (OA) is degradation and loss of articular cartilage. Interleukin‐1β (IL‐1β) is thought to have a prominent role in shifting the metabolic balance toward degradation. IL‐1β is first synthesized as an inactive precursor that is cleaved to the secreted active form mainly in the “inflammasome,” a complex of initiators (including NLRP3), adaptor molecule ASC, and caspase 1. The aim of this study was to clarify the roles of IL‐1β and the inflammasome in cartilage breakdown.

Methods

We assessed IL‐1β release by cartilage explants from 18 patients with OA. We also evaluated the lipopolysaccharide (LPS)–, IL‐1α–, and tumor necrosis factor α (TNFα)–induced activity of matrix metalloproteinase 3 (MMP‐3), MMP‐9, and MMP‐13 in NLRP3‐knockout mice and wild‐type mice and the inhibition of caspase 1 with Z‐YVAD‐FMK and the blockade of IL‐1β with IL‐1 receptor antagonist (IL‐1Ra). Cartilage explants from NLRP3‐knockout mice and IL‐1R type I (IL‐1RI)–knockout mice were subjected to excessive dynamic compression (0.5 Hz, 1 MPa) to trigger degradation, followed by assessment of load‐induced glycosaminoglycan (GAG) release and MMP enzymatic activity.

Results

Despite the expression of NLRP3, ASC, and caspase 1, OA cartilage was not able to produce active IL‐1β. LPS, IL‐1α, and TNFα dose‐dependently increased MMP‐3, MMP‐9, and MMP‐13 activity in cultured chondrocytes and in NLRP3^−/− chondrocytes, and this effect was not changed by inhibiting caspase 1 or IL‐1β. The load‐induced increase in GAG release and MMP activity was not affected by knockout of NLRP3 or IL‐1RI in cartilage explants.

Conclusion

OA cartilage may be degraded independently of any inflammasome activity, which may explain, at least in part, the lack of effect of IL‐1β inhibitors observed in previous trials.

     

Delayed addition of tumor necrosis factor (TNF) antagonists inhibits the generation of CD11c+ dendritic cells derived from CD34+ cells exposed to TNF-α

We have developed a method that cells exhibiting typical dendritic cell (DC) characteristics are generated from human CD34⁺ cells and phagocytose cogenerating erythroid progenitor cells in the presence of tumor necrosis factor-α (TNF-α), interleukin-3, stem cell factor and erythropoietin. Using this system, we titrated the effects of TNF antagonists, etanercept and infliximab, on TNF-α activity. We found that 1 μg/ml etanercept dramatically inhibited the generation of CD11c⁺ cells accompanying with a complete recovery of the generation of erythroid progenitors. Infliximab at 200 μg/ml exhibited a similar effect to that observed for etanercept. The delayed addition of etanercept to this culture system at day five resulted in significant inhibitory effects on the generation of CD11c⁺, CD4⁺ and CD86⁺ cells. These results indicate that TNF antagonists administered at a concentration that is achievable in vivo, neutralize the biologic effects of TNF-α in generating CD11c⁺ cells and that a delay in the administration of these antagonists for as long as 5 days partially inhibits the biologic activity of TNF-α. These findings may contribute to a great understanding of anti-TNF therapy in patients with an overproduction of cytokines such as hemophagocytic syndromes.     

Phenotypic characterization of malignant progenitor cells in patients with idiopathic myelofibrosis

Objective/BackgroundIdiopathic myelofibrosis (IM) is a clonal hematological malignancy originating from pluripotent hematopoietic stem cells (HSC). HSC are very rare potent cells that reside in the bone marrow (BM) and at a lower level in peripheral blood (PB). Previous studies showed that IM PB CD34⁺ cells contain not only BM repopulating cells belonging to the malignant clone but also residual normal HSC.MethodsIn the current study, we separated the subpopulations of IM PB CD34⁺ cells using IL-3Rα/CD123 labeling and further characterized them by genetic and functional analyses.ResultsWe differentiated IM PB CD34⁺ cells into three subpopulations (IL-3Rα^high, IL-3Rα^low, and IL-3Rα^negative). IL-3Rα^high CD34⁺ cell subgroup represents a small population in IM PB CD34⁺ cells which was not seen in normal G-CSF mobilized CD34⁺ cells. IM IL-3Rα^high CD34⁺ cells contained significant higher percentage of cells bearing marker chromosome detected by fluorescence in situ hybridization (FISH) analysis. In the absence of growth factors, IM IL-3Rα^high CD34⁺ cells exhibited abnormal colony forming ability and carried greater percentage of JAK2V617F mutant allele compared with IL-3Rα^low and IL-3Rα^negative CD34⁺ cells.ConclusionThese data indicate that IL-3Rα^high CD34⁺ cells from IM enriched for the malignant progenitor cells and IL-3Rα/CD123 may be a potential biomarker and therapeutic target for IM. Our findings will be further validated in future studies with a larger sample size and serial transplant in murine models.     

Association of tumor necrosis factor receptor type II polymorphism 196R with systemic lupus erythematosus in the Japanese: Molecular and functional analysis

Objective

To investigate whether a polymorphism(s) or mutation(s) in the tumor necrosis factor receptor II (TNFRII) gene is involved in the pathogenesis of systemic lupus erythematosus (SLE).

Methods

All 10 exons of the TNFRII gene were analyzed by exon‐specific polymerase chain reaction–single‐strand conformation polymorphism, followed by nucleotide sequencing of exons that displayed aberrant bands. To analyze the function of the TNFRII polymorphisms, the full‐length TNFRII complementary DNA of each allele was transfected in HeLa cells and then studied for specific binding of ¹²⁵I‐TNFα, as well as interleukin‐6 (IL‐6) production and cytotoxic activity after treatment with recombinant human TNFα.

Results

We identified 4 polymorphisms, at codons 56, 181, 196, and 232. The latter 2 had amino acid substitutions M196R and E232K, respectively. Only the 196R allele was significantly associated with SLE in our 105 Japanese SLE patients, with an allele frequency of 20.5%, compared with 12.6% in 99 healthy controls (P = 0.0335). More importantly, using TNFRII‐transfected HeLa cells, we demonstrated significantly increased IL‐6 production by 196R TNFRII compared with 196M TNFRII. The cytotoxic activity induced by 196R TNFRII was also increased compared with that of 196M TNFRII. This increase was achieved without affecting the binding affinity of TNFα to TNFRII, as demonstrated by the finding that specific TNFα binding to the HeLa transfectants of 196R and 196M TNFRII was similar, with K_d values of 3.12 × 10⁻¹⁰M and 4.34 × 10⁻¹⁰M, respectively.

Conclusion

These results suggest that 196R TNFRII, which transduces the signals of TNFα more effectively than does 196M TNFRII, is involved in the pathogenesis of SLE.

     

The role of fibroblasts and fibroblast‐derived factors in periprosthetic osteolysis

Objective

This study was undertaken to investigate how fibroblasts respond to stimulation with particulate wear debris and/or conditioned media obtained from pathologic tissue, and whether these activated fibroblasts express compounds that are involved in bone resorption.

Methods

Conditioned media from explant cultures of synovial tissue, periprosthetic soft tissue (interface membranes), titanium particles, and proinflammatory cytokines were used to stimulate fibroblasts. RNase protection assay was used to measure altered gene expression, and enzyme‐linked immunosorbent assay, Western blot hybridization, and flow cytometry were used to determine fibroblast protein expression. Tartrate‐resistant acid phosphatase staining was used to identify multinucleated osteoclast‐like cells.

Results

The most dominant compounds measured in the conditioned media from interface membranes were tumor necrosis factor α (TNFα), monocyte chemoattractant protein 1 (MCP‐1), interleukin‐1β (IL‐1β), IL‐6, IL‐8, and vascular endothelial growth factor. Fibroblasts phagocytosed particulate wear debris and responded to cytokine/chemokine stimulation. The most prominent up‐regulated genes and proteins secreted by fibroblasts in response to stimulation were matrix metalloproteinase 1, MCP‐1, IL‐1β, IL‐6, IL‐8, cyclooxygenase 1 (COX‐1), COX‐2, leukemia inhibitory factor 1, transforming growth factor β1 (TGFβ1), and TGFβ receptor type I. In addition, interface membrane fibroblasts expressed RANKL and osteoprotegerin in response to stimulation with conditioned media, TNFα, or IL‐1β. Stimulated fibroblasts cocultured with bone marrow cells in the presence of macrophage colony‐stimulating factor induced osteoclastogenesis.

Conclusion

Interface membrane fibroblasts respond directly to particulate wear debris, possibly via phagocytosis, expressing proinflammatory cytokines and RANKL. Thus, these cells may be actively involved in osteoclastogenesis and pathologic (periprosthetic) bone resorption.

     

Sequential expression of CD39 regulates late developmental T helper type 17 plasticity imparting a regulatory cell phenotype

BackgroundCD39 is an immune cell phenotype marker that exhibits ectonucleotidase activity, converting extracellular nucleotides to nucleosides. Although CD39 has been associated with regulatory T cells (Treg), the ectoenzyme is also expressed by a subpopulation of memory T cells (CD4^mem) with effector functions. We postulate that CD39 imparts plasticity to effector T helper type 17 (Th17) as well as co-ordinating Treg cellular programmes of differentiation.MethodsExperiments were performed with peripheral blood mononuclear cells obtained from healthy blood donors. Sorted CD4⁺CD45RO⁺ memory cells (CD4^mem) were exposed to interleukin (IL) 6 plus IL1β plus recombinant transforming growth factor β1 (rTGF) or IL6 plus IL1β plus IL23, or IL6 plus IL1β plus rTGF β1 plus IL23 to induce Th17 polarisation. Cells at Th17 stage were then treated with high-dose IL2 plus anti-CD3/anti-CD28 to favour Treg differentiation and then re-exposed to Th17 differentiating conditions to induce putative reverse or suppressive Th17 (rev/regTh17) cell. Impacts of purinergic mediators on cell effector phenotype and functions were assessed.FindingsCD4^mem could be differentiated sequentially to Th17, Treg, and rev/regTh17. In contrast to the inflammatory properties associated with prototypic Th17 cells, rev/regTh17 exhibited a suppressive phenotype (ie, CD39^high, CD73^high, FOXP3⁺) and were able to control CD4⁺CD25^? cell proliferation and pro-inflammatory cytokine (IFNγ, IL17) production. rev/regTh17 did not upregulate CD39, CD73, and FOXP3 and did not undergo increase in their suppressive function after culture with adenosine.InterpretationDifferential levels of expression of CD39 designate early Th17 cells from later Treg/revTh17 cell plasticity. The potential for Treg to revert to the inflammatory Th17 phenotype is mitigated by expression of CD39, as indicated by enhancements of suppressive function in vitro.FundingUK Medical Research Council.     

Cytokine profile in prediction of acute lung injury in patients with acute pancreatitis

Objectives

To study the role of cytokines in prediction of acute lung injury (ALI) in acute pancreatitis.

Comparative analysis of the pathological events involved in immune and non‐immune TRALI models

Background and Objectives Transfusion‐related acute lung injury (TRALI) is characterized by leukocyte transmigration and alveolar capillary leakage shortly after transfusion. TRALI pathogenesis has not been fully elucidated. In some cases, the infusion of alloantibodies (immune model), whereas in others the combination of neutrophil priming by proinflammatory molecules with the subsequent infusion of biological response modifiers (BRMs) in the hemocomponent (non‐immune model) have been implicated. Our aim was to compare the pathological events involved in TRALI induced by antibodies or BRMs using murine models. Materials and Methods In the immune model, human HNA‐2⁺ neutrophils were incubated in vitro with a monoclonal antibody (anti‐CD177, clone 7D8) directed against the HNA‐2 antigen and injected i.v. in NOD/SCID mice. In the non‐immune model, BALB/c mice were treated with low doses of lipopolysaccharide (LPS) followed by platelet‐activating factor (PAF) infusion 2 h later. Forty minutes after PAF administration, or 6 h after neutrophil injection, lungs were isolated and histological analysis, determination of a variety of cytokines and chemokines including keratinocyte‐derived chemokine (KC), MIP‐2, the interleukins IL‐1β, IL‐6, IL‐8 as well as TNFα, cell influx and alveolar capillary leakage were performed. Results In both models, characteristic histological findings of TRALI and an increase in KC and MIP‐2 levels were detected. In contrast to the immune model, in the non‐immune model, there was a dramatic increase in IL‐1β and TNFα. However, capillary leakage was only detected if PAF was administrated. Conclusions Regardless of the triggering event(s), KC, MIP‐2 and integrins participate in TRALI pathogenesis, whereas PAF is essential for capillary leakage when two events are involved.     

Role of exogenous interleukin 4 in the dynamics of acute liver injury

Background

Mortality in acute liver failure is very high, and the only reliable treatment is transplantation. Interleukin 4 (IL4) has pleiotropic effects on a network of cells directing repair, regeneration, and fibrosis. Pretreatment with subcutaneous IL4 complexed with anti-IL4 antibody (IL4c) promotes repair of carbon tetrachloride-mediated murine liver injury by stimulating hepatocyte proliferation. We hypothesised that IL4c could be administered therapeutically and act by a variety of mechanisms beyond engagement of hepatocytes.