John Wijdenes

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 plasmocyte selective monoclonal antibody (B-B4) recognizes syndecan-1.

We developed a new monoclonal antibody, B‐B4, which specifically identifies human plasma cells. It strongly reacts with all multiple myeloma cell lines and with malignant plasma cells of all tumour samples of the multiple myeloma patients tested. B‐B4 does not react with any peripheral blood, bone marrow or tonsil cells. Cloning of the B‐B4 antigen reveals that the monoclonal antibody recognizes syndecan‐1. It appears that the monoclonal antibody B‐B4 is a suitable marker for human plasmocyte identification among haemopoietic cells and a useful probe for the diagnosis of haematological malignancies. Furthermore, this monoclonal antibody can be used for depletions prior to CD34 grafting.

Role of interleukin-6 in the paraneoplastic inflammatory syndrome associated with renal-cell carcinoma

We investigated the possible causative role of interleukin 6 (IL‐6) in the paraneoplastic inflammatory syndrome and in paraneoplastic cholestasis (Stauffer syndrome) associated with renal‐cell carcinoma in a series of 119 patients with metastases. IL‐6 levels were found significantly higher in patients with paraneoplastic fever and weight loss. Patients with detectable serum IL‐6 (n = 90, 76%) had significantly higher serum CRP, haptoglobin, and serum alkaline‐phosphatase and gammaglutamyl‐transferase levels. Platelets, polymorphonuclear neutrophil (PMN) and monocyte counts were also significantly higher in patients with detectable serum IL‐6; in contrast, hemoglobin levels were significantly lower in patients with serum IL‐6 over 80 pg/ml. Three of these patients were included in a phase‐II trial of an anti‐IL‐6 monoclonal antibody given daily during 21 days. Reductions of CRP, haptoglobin and serum alkalin phosphatases were observed in all 3 patients during anti‐IL‐6 administration, with a subsequent increase up to or above pre‐treatment levels after the end of anti‐IL‐6. Decrease of platelets, PMN and monocyte counts were also observed in the 3 patients during anti‐IL‐6 administration, with a normalization of cell counts in a patient with increased platelets, PMN and monocyte counts. Hemoglobin concentration, serum albumin concentration and lymphocyte counts remained stable in the 3 patients during and after anti‐IL‐6 administration. Serum IL‐6, as evaluated by IRMA, decreased in the 3 patients during anti‐IL‐6 administration, but increased above pre‐treatment levels after the end of anti‐IL‐6 administration. These results demonstrate that IL‐6 is involved in the physiopathology of paraneoplastic syndromes observed in patients with metastatic renal‐cell carcinoma, in particular CRP and haptoglobin increase, paraneoplastic cholestasis, also paraneoplastic thrombocytosis, neutrophilia and monocytosis. Int. J. Cancer 72:424–430, 1997.

The myeloma cell antigen syndecan-1 is lost by apoptotic myeloma cells

Syndecan‐1 is a cell membrane proteoglycan that binds extracellular matrix components and various growth factors. It is expressed only on malignant plasma cells in bone marrow samples from patients with multiple myeloma (MM). Several reports have suggested that syndecan‐1 was present only on a part of the myeloma cells. By using either IL‐6‐dependent myeloma cell lines or primary myeloma cells stained by annexin V, we report here that syndecan‐1 was rapidly lost by myeloma cells undergoing apoptosis. In the same experimental conditions, expression of other cell membrane antigens such as CD38, HLA class‐I or CD49d on apoptotic myeloma cells was not affected. In addition, we show that syndecan‐1 loss was independent of activation of the gp130 IL‐6 transducer. Dexamethasone induced a strong apoptosis of myeloma cells associated with the loss of syndecan‐1. Finally, by using freshly‐explanted tumoural samples, we show that syndecan‐1 rapidly disappeared from myeloma cells in association with induction of apoptosis. In conclusion we showed that syndecan‐1 is a marker for viable myeloma cells which is rapidly lost by apoptotic cells. These results emphasize the usefulness of anti‐syndecan‐1 antibodies to purge tumoural cells from haemopoietic grafts or to purify these cells for further manipulations for immuno or gene therapies.

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.

Expression of Interleukin 13 Receptor in Glioma and Renal Cell Carcinoma: IL13Rα2 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 γc chain-negative. We thus used these cell models to investigate the patterns of expression of IL13Rα1, IL13Rα2, and IL4Rα chains and the role of IL13Rα2 in the response to IL13. Using new specific antibodies and flow cytometry, we observed a similar surface expression of IL4Rα and IL13Rα1 chains in most RCC and glioma cells, whereas IL13Rα2 was only present on five of six glioma cell lines. In all glioma cell lines, the amount of IL13Rα2 expression was 10 to 30 times higher than that of the two other chains. Although there was no surface or intracellular expression of IL13Rα2, its mRNA was detected in three of seven RCC cell lines. The expression on RCC cells of IL13Rα2 mRNA and/or that of high-affinity IL13 binding sites is not sufficient to predict IL13Rα2 protein expression. Blocking experiments showed that IL4 and IL13 strongly inhibited RCC cell proliferation through a unique receptor composed of IL4Rα and IL13Rα1 chains. Using RCC cells stably transfected with IL13Rα2 cDNA, we showed that the overexpression of IL13Rα2 decreased the response to IL13 but not that to IL4. Our results demonstrate that IL13Rα2 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.

Human cardiomyocyte hypertrophy induced in vitro by gp130 stimulation

OBJECTIVES Recent in vivo and in vitro studies in animals have demonstrated that cytokines of the IL-6 family are involved in cardiac hypertrophy and in protection of cardiomyocytes against apoptosis. The present study aims to analyse the capacity of human atrial cardiac cells (i.e., cardiomyocytes and fibroblasts) to display the gp130 receptor subunit, and to evaluate its functionality. METHODS Twenty human atrial biopsies were used for immunohistochemistry, in situ hybridisation, and western blot analysis or dissociated for isolation and primary culture of cardiac cells. RESULTS Fibroblasts present in tissue or maintained in primary culture clearly express gp130 whereas the signal in cardiomyocytes is weaker. Culture of cardiac cells with a gp130 agonist antibody enhances atrial natriuretic peptide (ANP), beta myosin heavy chain (beta-MHC) expression in cardiomyocytes, and significantly increases the cell surface area microm(2)). This process could involve STAT3 (signal transducer and activator of transcription 3) phosphorylation. CONCLUSIONS These results demonstrate that gp130 activation in human cardiac cells leads to cardiomyocyte hypertrophy. We discuss several hypotheses on the role of IL-6-type cytokines on cardiomyocyte functions.

Epitope mapping of recombinant human gamma interferon using monoclonal antibodies

Five monoclonal antibodies (MAbs B22, B27, 3-6, 32 and 35) specific for human recombinant IFN-gamma were characterized. These MAbs were used to set up quantitative sandwich ELISAs which allowed the detection of 1.25 ng/ml of IFN-gamma when diluted in normal human serum. Epitope mapping of the IFN-gamma molecule using these MAbs demonstrated that antibodies 3-6 and 32 which did not inhibit the biological activity of IFN-gamma recognized an epitope localized on the 15 C-terminal amino acids, suggesting that this portion of the molecule was not implicated in the biological activity of IFN-gamma. Sandwich ELISAs were performed using various pairs of MAbs. The level of reactivity obtained when antibodies B22 and B27 were used simultaneously as catcher and tracer was similar to the result obtained with two antibodies recognizing different epitopes. These results confirm that the IFN-gamma molecule is a dimer in solution and indicate that the two sites of the IFN-gamma dimeric molecule which are associated with the biological activity (epitope B22/B27) are fully exposed. In contrast, the C-terminus is only partially accessible to the antibodies 3-6/32, suggesting that the dimerization of IFN-gamma molecule results in the interaction of regions of the monomers that are homologous and adjacent to the C-terminus.

Identification and location on syndecan-1 core protein of the epitopes of B-B2 and B-B4 monoclonal antibodies

Using a phage display peptide library, we characterized the epitope of two monoclonal antibodies reacting with syndecan‐1: B‐B2 and B‐B4. The identified epitopes QDIT, for B‐B2, and LPEV, for B‐B4, were found to align with residues 36–39 and 90–93 of the mature protein, respectively. In contrast to B‐B4, the B‐B2 epitope is close to a potential glycosaminoglycan attachment site. Since syndecan‐1 is heavily glycosylated and post‐translational modifications are cell type specific, these results might explain the differences observed in the reactivity pattern of B‐B2 and B‐B4 and suggest that these monoclonal antibodies are useful probes to study cell surface exposed syndecan‐1.

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.