Increase of Fas-induced apoptosis by inhibition of extracellular phosphorylation of Fas receptor in Jurkat cell line.

Apoptosis signalling through the Fas pathway requires several steps of aggregation of the Fas receptor in the membrane, including aggregation that may occur in the absence of Fas ligand. Association of Fas domains is determinant to signal transmission following Fas ligand binding to a specific domain. The domains involved in Fas aggregation are located in its extracellular region and contain three potential protein kinase C-binding motifs. We therefore studied the possibility that phosphorylation of the extracellular region of Fas might be implicated in the regulation of Fas-mediated apoptosis. Inhibition experiments of extracellular phosphorylation were performed in human Jurkat T leukemia cells with K252b, an impermeant protein-kinase inhibitor. Extracellular phosphorylation of Fas receptor was related to ecto-kinase, as assessed by the [gamma-(32)P] ATP labelling of Fas-116 kDa aggregates, suppressed by K252b inhibitor which significantly increased the sensitivity to Fas-mediated apoptosis. Ecto-PKC involvement was demonstrated by bisindolylmaleimide VIII, a selective inhibitor of protein kinase C which significantly increased both Fas aggregation in the membrane and Fas-mediated apoptosis and by the addition of the PKC pseudo-substrate 19-36 which inhibited the phosphorylation of 116 kDa Fas aggregates. These data support a role for Fas phosphorylation in the decreased sensitivity to apoptosis in the Jurkat T leukemia cell line.

Immunoglobulin D enhances interleukin-6 release from the KU812 human prebasophil cell line.

Despite the role of secreted immunoglobulin D (IgD) remains still largely unknown, previous studies have suggested that secreted IgD could induce basophils degranulation in some allergic asthma patients. In the present study we have searched direct evidence of the action of IgD on KU812 cells, generally classified as an immature basophilic cell line. We analyzed by flow cytometry the capacity of IgD, purified from IgD myeloma sera, to bind KU812 cells. Biotinylated monomeric IgD (mIgD) and biotinylated oligomeric IgD (oIgD) could bind KU812 cells. Blocking experiments with others immunoglobulin isotypes showed that KU812 cells expressed an unspecific receptor for IgD. However, oIgD but not mIgD enhances the release of interleukin-6 (IL-6) from KU812 cells. On the other hand, mIgD and oIgD failed to induce histamine release from KU812 cells or from cord blood derived basophils. Since IL-6 is known to induce basophil differentiation, we proposed that IgD could be implicated in allergic disorders by stimulating IL-6 release by prebasophil cells, then IL-6 could further induce an autocrine maturation of the cells.

Sedimentation field flow fractionation purification of immature neural cells from a human tumor neuroblastoma cell line.

The use of stem cells for therapeutic applications is now an important objective for the future. Stem cell preparation is difficult and time-consuming depending on the origin of cells. Sedimentation field flow fractionation (SdFFF) is an effective tool for cell separation, respecting integrity and viability. We used the human neuroblastic SH-SY5Y clone of the SK-N-SH cell line as a source of immature neural cells. Our results demonstrated that by using SdFFF cell sorter under strictly defined conditions, and immunological cell characterization, we are now able to provide, in less than 15 min, a sterile, viable, usable and purified immature neural cell fraction without inducting cell differentiation.

Multiple myeloma cells are killed by syndecan-1-directed superantigen-activated T cells.

Multiple myeloma (MM) is an incurable plasma cell/plasmablast malignancy with a great need for innovative treatment strategies. Since experimental immunotherapy with targeted superantigens (SAg) proved to be effective in other haematopoietic tumours, we investigated whether this would also hold true for MM. We used the bacterial SAg Staphylococcus enterotoxin A (SEA), a potent activator of T cell cytotoxicity by means of its binding to particular T cell receptor Vbeta sequences on effector cells and MHC class II molecules on target cells. To eliminate potentially unspecific binding via MHC class II, SEA was point mutated (SEAm). In a second step SEAm was genetically fused to protein A (PA), resulting in a fusion protein (PA-SEAm). This fusion protein was used together with four different plasma-cell-specific/associated mAbs to direct T cells towards 10 MM target cell lines. Three of these mAbs were directed against syndecan-1/CD138, known to be highly expressed on MM and plasma cells, but absent on other haematopoietic cells. All MM cell lines proved to be sensitive to SAg-activated T cell killing (15-50% lysis), as measured in a 51Cr-release assay. This effect was clearly mediated via the plasma-cell-reactive antibodies, as control antibodies only conferred a low background lysis. MM therapy based on targeted SAgs could in theory be hampered by dysfunctional T cells in MM patients. However, we show that T cells from MM patients and healthy controls responded equally well to activation by SAg.

Expression of interleukin 13 receptor in glioma and renal cell carcinoma: IL13Ralpha2 as a decoy receptor for IL13.

Glioma and renal cell carcinoma (RCC) cells express high affinity interleukin 13 (IL13) binding sites, but only RCC cell proliferation was inhibited by IL13. Both of these two cell types are IL2-receptor (gamma)c chain-negative. We thus used these cell models to investigate the patterns of expression of IL13Ralpha1, IL13Ralpha2, and IL4Ralpha chains and the role of IL13Ralpha2 in the response to IL13. Using new specific antibodies and flow cytometry, we observed a similar surface expression of IL4Ralpha and IL13Ralpha1 chains in most RCC and glioma cells, whereas IL13Ralpha2 was only present on five of six glioma cell lines. In all glioma cell lines, the amount of IL13Ralpha2 expression was 10 to 30 times higher than that of the two other chains. Although there was no surface or intracellular expression of IL13Ralpha2, its mRNA was detected in three of seven RCC cell lines. The expression on RCC cells of IL13Ralpha2 mRNA and/or that of high-affinity IL13 binding sites is not sufficient to predict IL13Ralpha2 protein expression. Blocking experiments showed that IL4 and IL13 strongly inhibited RCC cell proliferation through a unique receptor composed of IL4Ralpha and IL13Ralpha1 chains. Using RCC cells stably transfected with IL13Ralpha2 cDNA, we showed that the overexpression of IL13Ralpha2 decreased the response to IL13 but not that to IL4. Our results demonstrate that IL13Ralpha2 acts as a decoy receptor for IL13 and that it may exert a tight regulation of IL13 activity without impairing the IL4 response of the same cell target.

Survival prognostic value of serum interleukin-6 level in very old people.

OBJECTIVE: To determine the survival prognostic value of serum interleukin-6 level in very old people. DESIGN: Prospective 12-month clinical survey in a long term ward and in a day care hospital. SETTING: A long-term ward and a day care hospital for very old and dependent people, located in Poitiers (France). PARTICIPANTS: A cohort of 115 old people [Range 64-101], either in a long-term ward (n=71) or in day care hospital (n=44). MEASUREMENTS: Patients were checked for different nutritional parameters and the Interleukin-6 level in the serum at baseline and patients were followed up for one year. RESULTS: A high level of interleukin-6 (Il-6) is associated with increased Prognostic Inflammatory and Nutritional Index (PINI) value, serum CRP thereby demonstrating the inflammatory role of this molecule. It is also associated with an increased risk of death. Using a survival regression model, a high Il-6 serum level observed at the beginning of the study is a bad prognostic indicator, with other biological or nutritional parameters having no significant influence. CONCLUSION: A high level of Interleukin-6 may be a better marker of prognosis than an increased PINI score.

Treatment of B-lymphoproliferative disorder with a monoclonal anti-interleukin-6 antibody in 12 patients: a multicenter phase 1-2 clinical trial.

Severe T-cell immunodeficiency after solid organ or bone marrow transplantation may result in the uncontrolled outgrowth of latently Epstein-Barr virus-infected B cells, leading to B-lymphoproliferative disorder (BLPD). Given the potentially important pathogenic role of IL-6 in BLPD, it was tested whether the in vivo neutralization of IL-6 by a monoclonal anti-IL-6 antibody could contribute to the control of BLPD. Safety and efficacy were assessed in 12 recipients of transplanted organs who had BLPD refractory to the reduction of immunosuppression over 8 days. Five patients received 0.4 mg/kg per day. The next 7 patients received 0.8 mg/kg per day. Treatment was scheduled to last 15 days. It was completed in 10 patients, and in the other 2 patients was discontinued early (days 10 and 13, respectively) because of disease progression. Treatment tolerance was good, and no major side effects were observed. High C-reactive protein levels were found in 9 patients before treatment but were normalized under treatment in all patients, demonstrating efficient IL-6 neutralization. Complete remission (CR) was observed in 5 patients and partial remission (PR) in 3 patients. Relapse was observed in 1 of these 8 patients in whom remission was observed. This relapse was unresponsive to treatment. Disease was stable in 1 patient, but it progressed in 3 patients. Seven patients are alive and well. Two patients died because of disease progression, and 3 patients died while in CR (chronic rejection in 2 patients and BLPD sequelae in 1 patient). These data suggest that the anti-IL-6 antibody is safe and should be further explored in the treatment of BLPD.

Integrin-associated protein (CD47/IAP) contributes to T cell arrest on inflammatory vascular endothelium under flow.

Integrin-associated protein (CD47/IAP) is a pentaspan molecule that regulates integrin functions. We prepared a CD47-deficient Jurkat T cell line to assess its role in the arrest of T cells on inflammatory endothelium. Under flow conditions, constitutive arrest of CD47-deficient cells is strongly decreased as compared to the original cell line, whereas reexpression of CD47 reestablishes their ability to stop. Moreover, cells transfected with a chimera made with the extracellular portion of CD47 and the transmembrane domain of CD7 or several truncated forms of CD47 show that the first transmembrane domain and a short cytoplasmic loop are sufficient for this process. CD47 effect is indirect and depends mainly on the alpha4beta1/VCAM-1 pathway, as shown by blocking antibodies. We detected on endothelium the two CD47 counter receptors known to date: thrombospondin and SIRP1alpha. Blocking experiments show that both are involved. Overall, CD47 participates in the constitutive arrest of T lymphocytes on inflamed vascular endothelium by up-regulating alpha 4beta1 integrins.

[Diminution of the in vitro and in vivo alloreactive response by association of mature dendritic cells and anti-CD4 monoclonal antibodies in the rat]. / Diminution de la réponse alloréactive in vitro et in vivo par association de cellules dendritiques matures du donneur et d'un anticorps monoclonal anti-CD4 chez le rat.

Using a fully mismatched model recipient Lewis-donor Wistar-Furth rats, we showed that the association donor mature (spleen) dendritic cells with a non depleting anti-CD4 monoclonal antibody can induce a more pronounced decrease of alloreactivity in mixed splenocyte reaction than anti-CD4 alone. These results are observed in vitro and 30 days after injection to the Lewis rats. Such data suggest that anti-CD4 monoclonal antibody can guide mature dendritic cells, usually involved in acute rejection, towards tolerance. The mechanisms are still to be resolved.

Interleukine-6 (IL-6) synthesis and gp130 expression by human pericardium.

Growing evidence shows that cytokines of the IL-6 family play an important regulatory role in heart physiology such as inducing cardiomyocyte hypertrophy. The purpose of this study was to see if IL-6 and its soluble receptors (sIL-6R and sgp130) could be detected in pericardial fluids, and to see if they are produced by the pericardium. We report that human pericardial fluid from patients with coronary pathologies contained IL-6, sIL-6R, and sgp130. However, the levels present in sera and pericardial fluid did not correlate, which suggests local production. This observation was confirmed by in vitro studies demonstrating massive IL-6 production by cultured pericardial samples, which could be strongly inhibited by methylprednisolone. RT-PCR studies revealed that IL-6 was weakly expressed in fresh tissues and strongly induced after culture. In situ hybridisation and immunohistochemical analysis showed that IL-6 and gp130 were mainly present in mesothelial cells. sIL-6R and sgp130 were also produced by pericardium in vitro, and their synthesis was upregulated by methylprednisolone. Taken together, these results demonstrate that IL-6 is present in pericardial fluid and that its presence could be due to synthesis by pericardial tissue. In vitro studies suggest that IL-6 production by this tissue could be strongly induced and regulated. A potential paracrine role of these factors in cardiomyocyte functions in normal or pathological conditions is discussed.

In vitro induction of neuronal apoptosis by anti-Fas antibody-containing sera from amyotrophic lateral sclerosis patients.

Sera from 26% of patients with sporadic amyotrophic lateral sclerosis (ALS) induced in vitro apoptosis of a human neuroblastoma cell line, as detected by two methods, and most contained anti-Fas autoantibodies. In contrast, Alzheimer sera (studied as controls) very rarely induced apoptosis and did not contain detectable anti-Fas antibodies. Soluble Fas-ligand levels in ALS sera were not different from those in normal sera, except for slightly higher levels in a single case. In mixed cultures of rat embryonic brain and spinal cord cells, ALS sera (and agonistic anti-Fas monoclonal antibodies and soluble Fas-ligand) induced the apoptosis of a subpopulation of neurons. These neurons were motoneurons on the basis of staining with the monoclonal antibody SMI 32 and Fas expression was restricted to these SMI 32-positive neurons. These data are compatible with the hypothesis of the participation of an autoimmune mechanism possibly related to anti-Fas autoantibodies in certain ALS patients.

Clinical and biologic effects of anti-interleukin-10 monoclonal antibody administration in systemic lupus erythematosus.

OBJECTIVE: To evaluate the safety and clinical efficacy of administering an anti-interleukin-10 (anti-IL-10) monoclonal antibody (mAb) to systemic lupus erythematosus (SLE) patients with active and steroid-dependent disease. In addition, we sought to assess the effects of in vivo IL-10 neutralization on biologic markers of SLE. METHODS: Treatment consisted of 20 mg/day intravenous administration of an anti-IL-10 murine mAb (B-N10) for 21 consecutive days, with a followup period of 6 months. Six patients were studied. RESULTS: Treatment was safe and well tolerated. All patients developed antibodies against B-N10. Cutaneous lesions and joint symptoms improved in all patients beginning during B-N10 administration and continuing to month 6. The SLE Disease Activity Index decreased from a mean +/- SEM of 8.83+/-0.91 on day 1 to 3.67+/-0.67 on day 21 (P = 0.001), 1.50+/-0.84 at month 2, and 1.33+/-0.80 at month 6 (P<0.001). At the end of followup, the disease was clinically inactive in 5 of the 6 patients. Prednisone administration was decreased from a mean +/- SEM of 27.9+/-5.7 mg/day on day 1 to 9.6+/-2.0 mg/day at month 6 (P<0.005). Activity of immune and endothelial cells rapidly decreased, as assessed by the early evolution of several biologic markers. CONCLUSION: This is the first report of IL-10 antagonist administration to humans. The study shows the involvement of IL-10 in the pathogenesis of SLE, and indicates that the use of IL-10 antagonists may be beneficial in the management of refractory SLE.

A combination of anti-interleukin 6 murine monoclonal antibody with dexamethasone and high-dose melphalan induces high complete response rates in advanced multiple myeloma.

To improve the complete response (CR) rate in advanced multiple myeloma (MM) without increasing the toxicity of high-dose therapy, we have used a new conditioning regimen. A combination of BE-8 [an anti-interleukin 6 (IL-6) murine monoclonal antibody] and dexamethasone followed by high-dose melphalan (220 mg/m2) and autologous stem cell transplantation was used to treat a series of 16 patients with advanced multiple myeloma. A strong inhibition of IL-6 activity evaluated by quantification of C-reactive protein was observed in all patients and was correlated with the high CR rate achieved with this combination therapy.

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.

Studies on the interleukin-6-type cytokine signal transducer gp130 reveal a novel mechanism of receptor activation by monoclonal antibodies.

The transmembrane glycoprotein gp130 belongs to the family of hematopoietic cytokine receptors. It represents the common signal transducing receptor component of the so called interleukin-6-type cytokines. For several cytokine receptors including gp130 it has been shown that receptor activation cannot only be achieved by the natural ligand but also by single monoclonal antibodies raised against the receptor ectodomain. These findings have been interpreted in a way that dimerization of cytokine receptors is sufficient for receptor activation. Here we show that the recently described gp130-activating antibody B-S12 actually consists of two different monoclonal antibodies. By subcloning of B-S12 the monoclonal antibodies B-S12-A5 and B-S12-G7 were obtained. The individual antibodies are biologically inactive, in combination they exert B-S12-like activity on hepatoma cells. On Ba/F3 cells stably transfected with gp130 a combination of B-S12-G7 with another monoclonal gp130 antibody, B-P8, is required to stimulate proliferation. Using gp130 deletion mutants we show that all three antibodies map to domains 2 and 3 of gp130 which constitute the cytokine binding module. The individual antibodies inhibit activation of the signal transducer by interleukin-6 and interfere with binding of interleukin-6 to gp130. Interestingly, the combination of B-S12-G7 and a Fab fragment of B-P8 retains biological activity. We conclude from our data that (i) the monoclonal antibodies activate gp130 by mimicking the natural ligand and (ii) enforcement of gp130 dimerization is not sufficient for receptor activation but additional conformational requirements have to be fulfilled.

Agonist anti-gp130 transducer monoclonal antibodies are human myeloma cell survival and growth factors.

We have previously reported obtaining two monoclonal antibodies (mAb) against the human gp130 interleukin-6 (IL-6) transducer which made possible the dimerization of gp130 and the activation of several IL-6-driven functions when used together. We report here that these mAb induce gp130-mediated signaling in human myeloma cells and support the survival and the long-term growth of five IL-6-dependent human myeloma cell lines. Their agonist activity is not affected by neutralizing antibodies to IL-6 or IL-6R. These mAb induce a transient proliferation of primary myeloma cells from most patients with multiple myeloma. Again, IL-6 inhibitors do not affect this agonist activity. By using highly purified primary myeloma cells, we found that these anti-gp130 mAb supported the long-term survival of primary myeloma cells from five patients with primary plasma cell leukemia but failed to induce their long-term growth. For patients with fulminant disease and secondary extramedullary proliferation, the antibodies supported a long-term survival and growth, and anti-gp130 mAb-dependent cell lines were obtained. For patients with medullary involvement only, a co-stimulatory signal is necessary, together with gp130 activation, to trigger cell survival and cycling. Leukemia (2000) 14, 188-197.

Ligand-specific utilization of the extracellular membrane-proximal region of the gp130-related signalling receptors.

The receptor gp130 is used by the interleukin-6 (IL-6)-type cytokines, which include IL-6 and leukaemia-inhibitory factor (LIF). To investigate the role of the three extracellular membrane-proximal fibronectin-type-III-like (FNIII) modules of gp130 and the related receptor for granulocyte colony-stimulating factor (G-CSFR) in cytokine signal transduction we have transfected into murine myeloid M1-UR21 cells the chimaera (GR-FNIII)gp130, which contains the membrane-proximal FNIII modules of the G-CSFR on a gp130 backbone, and its complement, the chimaera (gp130-FNIII)GR. Whereas the binding affinities of (125)I-labelled IL-6 to (GR-FNIII)gp130, or of (125)I-Tyr1,3-G-CSF to (gp130-FNIII)GR, were similar to wild-type gp130 and wild-type G-CSFR, respectively, (125)I-LIF failed to bind with high affinity to (GR-FNIII)gp130. In assays measuring differentiation the (gp130-FNIII)GR cells were fully responsive to G-CSF, whereas the (GR-FNIII)gp130 cells responded fully to the agonistic anti-gp130 monoclonal antibody (mAb) B-S12, but not to IL-6 or LIF. Neutralizing mAbs that recognize the membrane-proximal FNIII modules of gp130 or the G-CSFR differentially interfered with signalling by B-S12, LIF and G-CSF. The data suggest that B-S12 and G-CSF induce the correct orientation or conformation for signalling by the wild-type and chimaeric homodimeric receptors, that the membrane-proximal region of gp130 is important for the correct formation of the signalling IL-6-IL-6 receptor-gp130 complex and that this region is also involved in LIF-dependent receptor heterodimerization and signalling.

Human interleukin-6 is in vivo an autocrine growth factor for human herpesvirus-8-infected malignant B lymphocytes.

Human interleukin-6 (hIL-6) acts as a growth factor in several human B lymphoid cancers. As human herpesvirus-8 (HHV-8) encodes for a viral IL-6 (vIL-6), the viral cytokine may be responsible for several manifestations of HHV-8-related disorders. Using an anti-hIL-6 mAb (B-E8) which does not recognize vIL-6, we investigated the involvement of the human cytokine in the proliferation of HHV-8-positive primary effusion lymphoma (PEL) cells. In vitro, 5/5 PEL cell lines produced hIL-6 (4 to 1,200 pg/ml). The EBV- HHV-8+ cell line (BCBL-1) was adapted to grow in SCID mice. hIL-6 was detected in the serum of mice with grafts, as well as human soluble CD138 (sCD138) and human IL-10 (hIL-10). The serum level of these mediators increased with tumor progression. The effect of treatment with the B-E8 mAb on the tumor progression and survival was evaluated. This treatment significantly slowed down the tumor development: on day 54, there were more mice with low levels of sCD138 and hIL-10 in the treated group than in controls (p = 0.03 and 0.02, respectively); treatment also delayed death (median date of death was day 65 for control mice and day 84 for anti-hIL-6 mAb-treated mice; p < 0.02). Thus, hIL-6 is expressed in addition to vIL-6 in HHV-8-positive malignant B lymphocytes, and the viral cytokine does not totally substitute for human IL-6 in promoting tumor progression.

Circulating concentrations of soluble interleukin 6 receptors gp80 and gp130 in chronic renal failure and effects of renal replacement therapy.

BACKGROUND: Circulating receptors modulate the biological effects of cytokines. Renal insufficiency is known to influence the concentrations of the soluble tumor necrosis factor (TNF) receptors p55 and p75. No data are available on the concentrations of the circulating interleukin 6 (IL-6) receptors gp80 and gp130 during chronic renal insufficiency. METHODS: We compared the serum concentrations of the IL-6 receptors gp80 and gp130 to those of the TNF receptors p55 and p75 in end-stage chronic renal failure, continuous ambulatory peritoneal dialysis, and hemodialysis (HD). RESULTS: In healthy controls the concentrations of gp80, gp130, p55, and p75 in serum were 82.1 +/- 24.3, 87.9 +/- 20.2, 1.1 +/- 0.2, and 1.7 +/- 0.3 ng/ml, respectively. These concentrations were increased to, respectively, 112.2 +/- 18.0, 186.0 +/- 37.7, 10.5 +/- 4.3, and 15.0 +/- 7.5 ng/ml in chronic renal failure, to 138.8 +/- 18.0, 181. 3 +/- 46.1, 25.5 +/- 5.2, and 19.1 +/- 3.4 ng/ml in continuous ambulatory peritoneal dialysis, and to 107.9 +/- 29.4, 146.6 +/- 30. 5, 22.9 +/- 6.3, and 16.8 +/- 6.0 ng/ ml in HD (before dialysis session). The concentrations after HD were higher for p75 only. CONCLUSIONS: The data show that the concentrations of the IL-6 receptors (gp80 and gp130) are elevated in chronic renal insufficiency. The increase is relatively low as compared with the elevation of the TNF receptors in this situation. HD does not result in a consistent change in serum concentrations of the various receptors.