A regulatory cross‐talk between Vγ9Vδ2 T lymphocytes and mesenchymal stem cells

The physiological functions of human TCRVγ9Vδ2⁺ γδ lymphocytes reactive to non‐peptide phosphoantigens contribute to cancer immunosurveillance and immunotherapy. However, their regulation by mesenchymal stem cells (MSC), multipotent and immunomodulatory progenitor cells able to infiltrate tumors, has not been investigated so far. By analyzing freshly isolated TCRVγ9Vδ2⁺ lymphocytes and primary cell lines stimulated with synthetic phosphoantigen or B‐cell lymphoma cell lines in the presence of MSC, we demonstrated that MSC were potent suppressors of γδ‐cell proliferation, cytokine production and cytolytic responses in vitro. This inhibition was mediated by the COX‐2‐dependent production of prostaglandin E2 (PGE₂) and by MSC through EP2 and EP4 inhibitory receptors expressed by Vγ9Vδ2 T lymphocytes. COX‐2 expression and PGE₂ production by MSC were not constitutive, but were induced by IFN‐γ and TNF‐α secreted by activated Vγ9Vδ2 T cells. This regulatory cross‐talk between MSC and Vγ9Vδ2 T lymphocytes involving PGE₂ could be of importance for the antitumor and antimicrobial activities of γδ T cells.     

Mesenchymal stem cells suppress CD8+ T cell‐mediated activation by suppressing natural killer group 2, member D protein receptor expression and secretion of prostaglandin E2, indoleamine 2, 3‐dioxygenase and transforming growth factor‐β

Bone marrow mesenchymal stem cells (BMSCs) inhibit immune cell responsiveness, and especially of T lymphocytes. We showed that BMSCs markedly inhibited the proliferation and cytokine production by CD8⁺ T cells by a cell‐to‐cell contact phenomenon and secretion of soluble factors. BMSCs down‐regulate the expression of natural killer group 2, member D protein (NKG2D) receptors on CD8⁺ T cells when co‐cultured with them. Moreover, CD8⁺ T cells that express low levels of NKG2D had impaired proliferation after triggering by a mitogen. The major histocompatibility complex (MHC) class I chain‐related (MIC) A/B molecule, which is a typical ligand for NKG2D, was expressed on BMSCs, and caused dampening of cell proliferation. Monoclonal antibody blocking experiments targeted to MIC A/B impaired CD8⁺ T cell function, as evaluated by proliferation and cytokine production. In addition, the production of prostaglandin E₂ (PGE₂), indoleamine 2, 3‐dioxygenase (IDO) and transforming growth factor (TGF)‐β1 were increased when BMSCs were co‐cultured with CD8⁺ T cells. The addition of specific inhibitors against PGE₂, IDO and TGF‐β partially restored the proliferation of CD8⁺ T cells. Our results suggest that BMSCs suppress CD8⁺ T cell‐mediated activation by suppressing NKG2D expression and secretion of PGE₂, IDO and TGF‐β. Our observations further confirm the feasibility of BMSCs as a potential adoptive cellular therapy in immune‐mediated diseases such as graft‐versus‐host disease (GVHD).     

The influence of bone marrow‐ and synovium‐derived mesenchymal stromal cells from osteoarthritis patients on regulatory T cells in co‐culture

There is increasing evidence that inflammation in the synovium plays a major role in the progression of osteoarthritis (OA). However, the immunogenic properties of mesenchymal stromal cells (MSCs), which are considered to regulate immunity in various diseases, remain largely unknown in OA. The purpose of this study was to determine the influence of MSCs from OA patients on regulatory T cells (T_regs) in an allogeneic co‐culture model. Bone marrow (BM) and synovial membrane (SM) were harvested from hip joints of OA patients and co‐cultured with lymphocytes enriched in CD4⁺CD25⁺CD127^– regulatory T cells (T_reg⁺LC) from healthy donors. T_reg proportions and MSC markers were assessed by flow cytometry. Cytokine levels were assessed after 2 and 5 days of co‐cultivation. Additionally, T_reg⁺LC cultures were analysed in the presence of interleukin (IL)‐6 and MSC‐supernatant complemented medium. B‐MSCs and S‐MSCs were able to retain the T_reg proportion compared to lymphocyte monocultures. T cell–MSC co‐cultures showed a significant increase of IL‐6 compared to MSC cultures. S‐MSCs produced higher amounts of IL‐6 compared to B‐MSCs, both in single and T cell co‐cultures. The effect of retaining the T_reg percentage could be reproduced partially by IL‐6 addition to the medium, but could only be observed fully when using MSC culture supernatants. Our data demonstrate that retaining the T_reg phenotype in MSC–T cell co‐cultures can be mediated by MSC derived from OA patients. IL‐6 plays an important role in mediating these processes. To our knowledge, this study is the first describing the interaction of MSCs from OA patients and T_regs in an allogeneic co‐culture model.     

Cell contact,prostaglandin E2 and transforming growth factor beta 1 play non‐redundant roles in human mesenchymal stem cell induction of CD4+CD25Highforkhead box P3+ regulatory T cells

Adult human mesenchymal stromal or stem cells (MSC) can differentiate into a variety of cell types and are candidate cellular therapeutics in regenerative medicine. Surprisingly, these cells also display multiple potent immunomodulatory capabilities, including allosuppression, making allogeneic cell therapy a possibility. The exact mechanisms involved in regulatory T cell induction by allogeneic human MSC was examined, using purified CD4⁺ populations and well‐characterized bone marrow‐derived adult human MSC. Allogeneic MSC were shown to induce forkhead box P3 (FoxP3)⁺ and CD25⁺ mRNA and protein expression in CD4⁺ T cells. This phenomenon required direct contact between MSC and purified T cells, although cell contact was not required for MSC induction of FoxP3 expression in an unseparated mononuclear cell population. In addition, through use of antagonists and neutralizing antibodies, MSC‐derived prostaglandins and transforming growth factor (TGF)‐β1 were shown to have a non‐redundant role in the induction of CD4⁺CD25⁺FoxP3⁺ T cells. Purified CD4⁺CD25⁺ T cells induced by MSC co‐culture expressed TGF‐β1 and were able to suppress alloantigen‐driven proliferative responses in mixed lymphocyte reaction. These data clarify the mechanisms of human MSC‐mediated allosuppression, supporting a sequential process of regulatory T cell induction involving direct MSC contact with CD4⁺ cells followed by both prostaglandin E₂ and TGF‐β1 expression. Overall, this study provides a rational basis for ongoing clinical studies involving allogeneic MSC.     

Intrathecal CD4+ and CD8+ T‐cell responses to endogenously synthesized candidate disease‐associated human autoantigens in multiple sclerosis patients

MS pathology is potentially orchestrated by autoreactive T cells, but the antigens recognized remain unknown. A novel APC/T‐cell platform was developed to determine intrathecal CD4⁺ and CD8⁺ T‐cell responses to candidate MS‐associated autoantigens (cMSAg) in clinically isolated syndrome (CIS, n = 7) and MS (n = 6) patients. Human cMSAg encoding open reading frames (n = 8) were cloned into an Epstein–Barr virus (EBV)‐based vector to express cMSAg at high levels in EBV‐transformed B‐cells (BLCLs). Human cMSAg cloned were myelin‐associated and ‐oligodendrocyte glycoprotein, myelin basic protein, proteolipid protein, ATP‐dependent potassium channel ATP‐dependent inwards rectifying potassium channel 4.1, S100 calcium‐binding protein B, contactin‐2, and neurofascin. Transduced BLCLs were used as autologous APC in functional T‐cell assays to determine cMSAg‐specific T‐cell frequencies in cerebrospinal fluid derived T‐cell lines (CSF‐TCLs) by intracellular IFN‐γ flow cytometry. Whereas all CSF‐TCL responded strongly to mitogenic stimulation, no substantial T‐cell reactivity to cMSAg was observed. Contrastingly, measles virus fusion protein‐specific CD4⁺ and CD8⁺ T‐cell clones, used as control of the APC/T‐cell platform, efficiently recognized transduced BLCL expressing their cognate antigen. The inability to detect substantial T‐cell reactivity to eight human endogenously synthesized cMSAg in autologous APC do not support their role as prominent intrathecal T‐cell target antigens in CIS and MS patients early after onset of disease.     

Specific CD8+ T‐lymphocytes control dissemination of measles virus

Measles continues to be an important cause of childhood mortality in developing countries. Measles virus (MV) is lymphotropic and infects high percentages of B‐ and T‐lymphocytes in lymphoid tissues. Cellular immunity is considered crucial for viral clearance; however, MV‐specific T‐lymphocytes generated during primary infection also constitute a potential target for MV infection. We therefore aimed to identify T‐lymphocyte subsets that can clear MV infection without becoming infected. To this end, we infected human EBV transformed B‐lymphoblastic cell lines (B‐LCL) with a recombinant MV strain expressing enhanced GFP, and co‐cultured these with non‐infected B‐LCL resulting in rapid viral spread. MV‐specific CD8⁺ T‐cell clones efficiently suppressed MV dissemination in autologous and HLA‐matched, but not in HLA‐mismatched B‐LCL. In contrast, CD4⁺ T‐cell clones could not control MV dissemination but became a target for MV infection themselves. Furthermore, PBMC collected 6–9 months after acute measles and stimulated with autologous MV‐infected B‐LCL also efficiently suppressed MV dissemination; this was mediated by the fraction containing CD8⁺ T‐lymphocytes. In conclusion, we have developed a powerful tool to study cellular immunity against measles, and demonstrate that control of MV dissemination is mediated by virus‐specific CD8⁺ rather than by CD4⁺ T‐lymphocytes.     

Regulatory effects of IFN‐β on production of osteopontin and IL‐17 by CD4+ T Cells in MS

IFN‐β currently serves as one of the major treatments for MS. Its anti‐inflammatory mechanism has been reported as involving a shift in cytokine balance from Th1 to Th2 in the T‐cell response against elements of the myelin sheath. In addition to the Th1 and Th2 groups, two other important pro‐inflammatory cytokines, IL‐17 and osteopontin (OPN), are believed to play important roles in CNS inflammation in the pathogenesis of MS. In this study, we examined the potential effects of IFN‐β on the regulation of OPN and IL‐17 in MS patients. We found that IFN‐β used in vitro at 0.5–3 ng/mL significantly inhibited the production of OPN in primary T cells derived from PBMC. The inhibition of OPN was determined to occur at the CD4⁺ T‐cell level. In addition, IFN‐β inhibited the production of IL‐17 and IL‐21 in CD4⁺ T cells. It has been described that IFN‐β suppresses IL‐17 production through the inhibition of a monocytic cytokine, the intracellular translational isoform of OPN. Our further investigation demonstrated that IFN‐β also acted directly on the CD4⁺ T cells to regulate OPN and IL‐17 expression through the type I IFN receptor‐mediated activation of STAT1 and suppression of STAT3 activity. Administration of IFN‐β to EAE mice ameliorated the disease severity. Furthermore, spinal cord infiltration of OPN⁺ and IL‐17⁺ cells decreased in IFN‐β‐treated EAE mice along with decreases in serum levels of OPN and IL‐21. Importantly, decreased OPN production by IFN‐β treatment contributes to the reduced migratory activity of T cells. Taken together, the results from both in vitro and in vivo experiments indicate that IFN‐β treatment can down‐regulate the OPN and IL‐17 production in MS. This study provides new insights into the mechanism of action of IFN‐β in the treatment of MS.     

Autologous mixed lymphocyte reaction in man. II. Histamine-induced suppression of the autologous mixed lymphocyte reaction by T-cell subsets defined with monoclonal antibodies

Human peripheral blood mononuclear cells were separted into T and non-T cells by E-rosette formation. The influence of histamine (10^–8–10^–3 M) on the proliferative response of T cells in autologous and allogeneic mixed lymphocyte cultures (MLC) was studied. Pretreatment of responder T cells but not of stimulator non-T cells with histamine for 24 hr resulted in a markedly diminished proliferative response in both autologous and allogeneic MLC. A minimum of 4 hr of incubation of T cells with histamine was required to inhibit autologous MLC. Furthermore, T cells pretreated with histamine followed by mitomycin C treatment, when cocultured with fresh autologous T cells, suppressed their proliferative response in both autologous and allogeneic MLC. Analysis with OKT₄ and OKT₈ monoclonal antibodies revealed that histamine-induced suppressor T cells were contained in OKT ₈ ⁺ -cell subset. Hitamine-treated OKT ₄ ⁺ cells had no suppressive effect on the proliferative responses of autologous T cells. Supernatants of T cells cultured with histamine for 24 hr also suppressed both autologous and allogeneic MLC responses of fresh T cells, suggesting that the suppression could be mediated by a soluble suppressor factor(s). Experiments with H₁ and H₂ agonists and antagonists indicated that histamine-induced activation of suppressor T cells and the production of a soluble suppressor factor(s) were mediated through histamine type 2 receptors.     

High Functional CD70 Expression on α‐Type 1‐Polarized Dendritic Cells from Patients with Chronic Lymphocytic Leukaemia

Antigen‐loaded dendritic cells (DCs) used as anticancer vaccine holds promise for therapy, but needs to be optimized. The most frequently described DC vaccine is being matured with a cocktail containing prostaglandin E₂ (PGE₂DC). However, even though PGE₂DCs express both costimulatory and migratory receptors, their IL‐12p70‐prodcution is low, leading to an insufficient Th1 immune response. As an alternative, α‐type‐1 polarized DCs (αDC1s) have shown a superior production of IL‐12p70 and subsequent activation of effector cells. From chronic lymphocytic leukaemia (CLL) patients, αDC1s can be generated to induce a functional Th1‐immune response. Yet, another costimulatory receptor, CD70, appears to be essential for optimal DC function by promotion of T cell survival and function. So far, PGE₂ is suggested as one of the most important factors for the induction of CD70 expression on DCs. Therefore, we wanted to investigate whether αDC1s have the ability to express functional CD70. We found that CD70 expression on αDC1s could be upregulated in the same manner as PGE₂DCs. In an allogeneic mixed leucocyte reaction, we found that antibody‐blocking of CD70 on αDC1s from controls reduced effector cell proliferation although this could not be found when using CLL αDC1s. Nevertheless, CD70‐blocking of αDC1s from both controls and patients with CLL had a negative influence on the production of both IL‐12p70 and the Th1 cytokine IFN‐γ, while the production of the Th2 cytokine IL‐5 was enhanced. Together, this study further suggests that αDC1s should be considered as a suitable candidate for clinical antitumour vaccine strategies in patients with CLL.     

Tumour Growth Causes Suppression of Autoreactive T-Cell Proliferation by Disrupting Macrophage Responsiveness to Interferon-γ

Normal immune homeostasis is regulated partly by a small population of CD4⁺ T cells that react to autologous major histocompatibility complex class-II molecules on self-cells. Decreased autoreactive T-cell responses are associated with cancer. Tumour growth causes syngeneic macrophages (Mø) to suppress autoreactive T-cell proliferation by decreasing Mø class-II expression and increasing Mø production of the suppressor molecule prostaglandin E₂ (PGE₂). Because interferon-γ (IFN-γ) is a potent Mø activation molecule which regulates both Mø PGE₂ and class-II expression, the effects of IFN-γ on tumour-induced suppression of autoreactive T-cell proliferation were investigated. Exogenous IFN-γ increased normal host (NH) CD4⁺ autoreactive T-cell proliferation stimulated by syngeneic NHMø but decreased proliferation stimulated by tumour-bearing host (TBH) Mø. Antibody (Ab) neutralization of endogenous IFN-γ activity reduced TBH Mø-mediated suppression. Kinetic studies showed that endogenous IFN-γ suppressor activity was not exclusive during T-cell activation. Indomelhacin treatment blocked IFN-γ-induced suppression in TBH Mø-T cell cultures. TBH Mø-T cell cultures contained significantly more PGE₂ than those containing NH Mø. Exogenous IFN-γ increased early PGE₂ production in TBH Mø cultures but decreased production in NHMø cultures. The Ab-mediated neutralization of endogenous transforming growth factor-β or tumour necrosis factor-x reduced TBH Mμ-mediated suppression and blocked IFN-γ-induced suppression. Short-term treatment of Mμ with IFN-γ before their addition to T cells caused TBH Mμ to stimulate T-cell proliferation, which suggests that early suppressor molecule production by TBHMø inhibits synthesis or activity of IFN-γ-induced stimulatory monokines. These results show that tumour growth causes Mø to suppress autoreactive T-cell responses by allowing IFN-γ to induce Mø suppressor molecules, which block production or activity of stimulatory monokines.     

In vitro assessment of mesenchymal stem cells immunosuppressive potential in multiple sclerosis patients

Mesenchymal stem cells (MSC) are promising for multiple sclerosis (MS) treatment. However, clinical results remain controversial, and no criteria are available for predicting the efficiency of MSC therapy. Using an in vitro model of lymphocytes and MSC cocultivation we revealed that the Index of MSC Suppression of myelin-induced memory T cells proliferation was stronger than that of PHA-stimulated proliferation and inversely correlated with patients’ EDSS score. In vitro expression of CD119 (IFNGR1) in mitogen/myelin-stimulated T cells increased in the presence of MSC being inversely correlated with T-lymphocytes proliferation. The Index of MSC Suppression and CD119 expression in T-lymphocytes may be useful when assessing MSC immunosuppressive potential in MS patients.     

The fish oil ingredient,docosahexaenoic acid,activates cytosolic phospholipase A2 via GPR120 receptor to produce prostaglandin E2 and plays an anti‐inflammatory role in macrophages

Docosahexaenoic acid (DHA) is one of the major ingredients of fish oil and has been reported to have anti‐inflammatory properties mediated through the GPR120 receptor. Whether cytosolic phospholipase A₂ (cPLA₂) and lipid mediators produced from cPLA₂ activation are involved in the anti‐inflammatory role of DHA in macrophages has not been reported. We report here that DHA and the GPR120 agonist, GW9508, activate cPLA₂ and cyclooxygenase 2 (COX‐2), and cause prostaglandin E₂ (PGE₂) release in a murine macrophage cell line RAW264.7 and in human primary monocyte‐derived macrophages. DHA and GW9508 activate cPLA₂ via GPR120 receptor, G protein Gαq and scaffold protein β‐arrestin 2. Extracellular signal‐regulated kinase 1/2 activation is involved in DHA‐ and GW9508‐induced cPLA₂ activation, but not p38 mitogen‐activated protein kinase. The anti‐inflammatory role of DHA and GW9508 is in part via activation of cPLA₂, COX‐2 and production of PGE₂ as a cPLA₂ inhibitor or a COX‐2 inhibitor partially reverses the DHA‐ and GW9508‐induced inhibition of lipopolysaccharide‐induced interleukin‐6 secretion. The cPLA₂ product arachidonic acid and PGE₂ also play an anti‐inflammatory role. This effect of PGE₂ is partially through inhibition of the nuclear factor‐κB signalling pathway and through the EP4 receptor of PGE₂ because an EP4 inhibitor or knock‐down of EP4 partially reverses DHA inhibition of lipopolysaccharide‐induced interleukin‐6 secretion. Hence, DHA has an anti‐inflammatory effect partially through induction of PGE₂.     

COX‐2‐Mediated Regulation of VEGF‐C in Association With Lymphangiogenesis and Lymph Node Metastasis in Lung Cancer

The mechanisms underlying the effects of COX‐2 on tumor lymphangiogenesis remain largely undefined. Here, the human lung cancer cell lines A549, 95D, Anip973, and AGZY83‐a with different metastatic capacities were investigated by immunostaining, western blotting, and real‐time RT‐PCR. We observed increased expressions of COX‐2 and VEGF‐C in the three highly metastatic cell lines compared with the less metastatic AGZY83‐a cell line. The COX‐2‐specific inhibitor Celecoxib suppressed VEGF‐C expression whereas the main COX‐2 metabolite PGE₂ elevated VEGF‐C expression in Anip973 and AGZY83‐a cells in positive and negative experiments. To determine the functional link to COX‐2 more specifically and elucidate the mechanistic pathway, we used a siRNA to knock down the high COX‐2 expression in Anip973 cells and transfected a COX‐2 cDNA to enhance the low COX‐2 expression in AGZY83‐a cells, and then treated the cells with EP1/EP4 agonists or antagonists, respectively. The results revealed that the EP1/EP4 agonists significantly increased VEGF‐C production in the COX‐2‐knockdown Anip973 cells. In contrast, the EP1/EP4 antagonists diminished VEGF‐C production in the COX‐2‐overexpressing AGZY83‐a cells. Furthermore, animal models provided evidence that Celecoxib decreased VEGF‐C expression, lymphangiogenesis, and lymph node metastases in Anip973 cells, whereas PGE₂ treatment increased the same factors in the parental AGZY83‐a cells. A positive correlation between COX‐2 and VEGF‐C was also confirmed in vivo. The present data suggest that COX‐2 regulates VEGF‐C expression via the PGE₂ pathway, and that EP1/EP4 receptors are involved in PGE₂‐mediated VEGF‐C production. Thus, COX‐2 may represent a candidate gene for blocking tumor lymphangiogenesis and lymph node metastasis. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Volumetric imaging of myelin in vivo using 3D inversion recovery‐prepared ultrashort echo time cones magnetic resonance imaging

Direct myelin imaging is promising for characterization of multiple sclerosis (MS) brains at diagnosis and in response to therapy. In this study, a 3D inversion recovery‐prepared ultrashort echo time cones (IR‐UTE‐Cones) sequence was used for both morphological and quantitative imaging of myelin on a clinical 3 T scanner. Myelin powder phantoms with different myelin concentrations were imaged with the 3D UTE‐Cones sequence and it showed a strong correlation between concentrations and UTE‐Cones signals, demonstrating the ability of the UTE‐Cones sequence to directly image myelin in the brain. Quantitative myelin imaging with multi‐echo IR‐UTE‐Cones sequences show similar T₂* values for a D₂O‐exchanged myelin phantom (T₂* = 0.33 ± 0.04 ms), ex vivo brain specimens (T₂* = 0.20 ± 0.04 ms) and in vivo healthy volunteers (T₂* = 0.254 ± 0.023 ms), further confirming the feasibility of 3D IR‐UTE‐Cones sequences for direct myelin imaging in vivo. In ex vivo MS brain study, signal loss is observed in MS lesions, which was confirmed with histology. For the in vivo study, the lesions in MS patients also show myelin signal loss using the proposed direct myelin imaging method, demonstrating the clinical potential for MS diagnosis. Furthermore, the measured IR‐UTE‐Cones signal intensities show a significant difference between normal‐appearing white matter in MS patients and normal white matter in volunteers, which cannot be found in clinical used T₂‐FLAIR sequences. Thus, the proposed 3D IR‐UTE‐Cones sequence showed clinical potential for MS diagnosis with the capability of direct myelin detection of the whole brain.     

Activation of suppressor function of human peripheral blood T cells by (+)cyanidanol-3: its application to chronic active liver diseases

(+)Cyanidanol-3, a substance belonging to the flavonoids group, has been used in acute viral hepatitis (AVH) and chronic active liver disease (CALD). Studies were undertaken to determine if (+)cyanidanol-3 could affect a function of mitogen stimulated peripheral blood lymphocytes (PBL) in humans. First, (+)cyanidanol-3 was added directly to pokeweed mitogen (PWM) stimulated PBL or co-culture of B and T cells resulting in severe suppression of immunoglobulin (Ig) production. This suppression was mediated by radiosensitive T cells. Secondly, when normal T cells pre-incubated with 25 micrograms/ml of (+)cyanidanol-3 for 48 h were cultured with freshly prepared autologous or allogeneic normal PBL in the presence of PWM, Ig production was markedly suppressed. Similarly, there was a consistent suppression of blast transformation of concanavalin A (Con A) stimulated autologous or allogeneic responding PBL by (+)cyanidanol-3 pre-treated normal T cells. On the other hand, (+)cyanidanol-3-induced suppressor cell activity of T cells from patients with CALD was significantly decreased (P less than 0.001) when compared with that of normal individuals. These studies may explain the therapeutic effect of (+)cyanidanol-3 on certain types of liver disease.     

IL‐12‐polarized Th1 cells produce GM‐CSF and induce EAE independent of IL‐23

CD4⁺ T‐helper (Th) cells reactive against myelin antigens mediate the mouse model experimental autoimmune encephalomyelitis (EAE) and have been implicated in the pathogenesis of multiple sclerosis (MS). It is currently debated whether encephalitogenic Th cells are heterogeneous or arise from a single lineage. In the current study, we challenge the dogma that stimulation with the monokine IL‐23 is universally required for the acquisition of pathogenic properties by myelin‐reactive T cells. We show that IL‐12‐modulated Th1 cells readily produce IFN‐γ and GM‐CSF in the CNS of mice and induce a severe form of EAE via an IL‐23‐independent pathway. Th1‐mediated EAE is characterized by monocyte‐rich CNS infiltrates, elicits a strong proinflammatory cytokine response in the CNS, and is partially CCR2 dependent. Conversely, IL‐23‐modulated, stable Th17 cells induce EAE with a relatively mild course via an IL‐12‐independent pathway. These data provide definitive evidence that autoimmune disease can be driven by distinct CD4⁺ T‐helper‐cell subsets and polarizing factors.     

IL‐1β/HMGB1 Complexes Promote The PGE2 Biosynthesis Pathway in Synovial Fibroblasts

PGE₂ is a potent lipid mediator of pain and oedema found elevated in RA. Microsomal prostaglandin E synthase‐1 (mPGES‐1) is a terminal enzyme of the PGE₂ pathway inducible by proinflammatory cytokines. mPGES‐1 is markedly upregulated in RA synovial tissue despite antirheumatic treatments, suggesting that multiple inflammatory stimuli contribute to its induction. High‐mobility group box chromosomal protein 1 (HMGB1) is known to induce inflammation both by direct interaction with TLR4 and by enhancement of other proinflammatory molecules signalling, through complex formation. The high expression of extracellular HMGB1 within the inflamed synovium, implies its pro‐arthritogenic role in RA. We aimed to investigate the effects of IL‐1β/HMGB1 complexes on mPGES‐1 and other enzymes of the PGE₂ pathway in synovial fibroblasts (SFs) from patients with arthritis. Furthermore, we studied the effect of COX‐2 inhibition and IL‐1RI antagonism on prostanoid and cytokine production by SFs. Stimulation of SFs with HMGB1 in complex with suboptimal amounts of IL‐1β significantly increased mPGES‐1 and COX‐2 expressions as well as PGE₂ production, as compared to treatment with HMGB1 or IL‐1β alone. Furthermore, NS‐398 reduced the production of IL‐6 and IL‐8, thus indicating that IL‐1β/HMGB1 complexes modulate cytokine production in part through prostanoid synthesis. Treatment with IL‐1RA completely abolished the induced PGE₂ and cytokine production, suggesting an effect mediated through IL‐1RI. IL‐1β/HMGB1 complexes promote the induction of mPGES‐1, COX‐2 and PGE₂ in SF. The amplification of the PGE₂ biosynthesis pathway by HMGB1 might constitute an important pathogenic mechanism perpetuating inflammatory and destructive activities in rheumatoid arthritis.     

Calcium/calmodulin‐dependent protein kinase II regulates cyclooxygenase‐2 expression and prostaglandin E2 production by activating cAMP‐response element‐binding protein in rat peritoneal macrophages

Prostaglandin E₂ (PGE₂) is an important inducer of inflammation, which is also closely linked to the progress of tumours. In macrophages, PGE₂ production is regulated by arachidonic acid release and cyclooxygenase‐2 (COX‐2) expression. In the present study, we found that COX‐2 expression can be achieved by activating Ca²⁺/Calmodulin (CaM)‐dependent protein kinase II (CaMKII) and cAMP‐response element‐binding protein (CREB) in rat peritoneal macrophages. Our results indicated that lipopolysaccharide and PMA could elicit the transient increase of the concentration of intracellular free calcium ions ([Ca²⁺]_i), which induced activation of CaMKs with the presence of CaM. The subtype of CaMKs, CaMKII, then triggered the activation of CREB, which elevated COX‐2 expression and PGE₂ production in a chronological order. These results suggested that Ca²⁺/CaM‐dependent CaMKII plays an important role in mediating COX‐2 expression and PGE₂ production by activating CREB in macrophages. The study also provides more useful information to clarify the mechanism of calcium regulation of PGE₂ production, which plays an essential role in inflammation and cancers.