Fluoxetine-induced change in rat brain expression of brain-derived neurotrophic factor varies depending on length of treatment.

Recent studies indicate that brain-derived neurotrophic factor (BDNF) may be implicated in the clinical action of antidepressant drugs. Repeated (2-3 weeks) administration of antidepressant drugs increases BDNF gene expression. The onset of this response as well as concomitant effects on the corresponding BDNF protein is however, unclear. The present study investigated the effects of acute and chronic administration of the selective serotonin reuptake inhibitor, fluoxetine (10mg/kg p.o.), upon regional rat brain levels of BDNF mRNA and protein expression. To improve the clinical significance of the study, fluoxetine was administered orally and mRNA and protein levels were determined ex vivo using the techniques of in situ hybridisation histochemistry and immunocytochemistry respectively. Direct measurement of BDNF protein was also carried out using enzyme-linked immunosorbent assay (ELISA). Four days of once daily oral administration of fluoxetine induced decreases in BDNF mRNA (hippocampus, medial habenular and paraventricular thalamic nuclei). Whilst 7 days of treatment showed a non-significant increase in BDNF mRNA, there were marked and region-specific increases following 14 days of treatment. BDNF protein levels remained unaltered until 21 days of fluoxetine treatment, when the numbers of BDNF immunoreactive cells were increased, reaching significance in the pyramidal cell layer of CA1 and CA3 regions of Ammon's horn (CA1 and CA3) but not in the other sub-regions of the hippocampus. Indicative of the highly regional change within the hippocampus, the ELISA method failed to demonstrate significant up-regulation at 21 days, measuring levels of BDNF protein in the whole hippocampus. In contrast to the detected time dependent and biphasic response of the BDNF gene, activity-regulated, cytoskeletal-associated protein (Arc) mRNA showed a gradual increase during the 14-day course of treatment. The results presented here show that BDNF is expressed differentially depending on length of fluoxetine administration, which could contribute in explaining the slow onset of antidepressant activity observed with selective serotonin reuptake inhibitors.

Optimizing therapeutic strategies to inhibit circulating soluble target molecules with monoclonal antibodies: example of the soluble IL-6 receptors.

Therapeutic targeting of soluble molecules such as cytokines can be achieved with monoclonal antibodies (mAb). Anti-IL-6 mAb have been shown to form circulating complexes, resulting in the increase of the half-life of the cytokine in vivo. In IL-6-related diseases, the soluble human IL-6 receptors (shIL-6R), which have been shown to possess strong agonist activity, circulate in the plasma at a high concentration and must be neutralized. Their clearance was studied in mice that had been made to express circulating shIL-6R after i.p. grafting of mouse thymoma cells transfected with a gene coding for shIL-6R, treated with various anti-shIL-6R mAb recognizing different epitopes of the molecule. Injection of one anti-hIL-6R mAb stabilized the short-lived hIL-6R and led to their accumulation. The same result was observed when two mAb directed against two different epitopes of the hIL-6R were used. Clearance of the receptors was only achieved when three mAb specific for three different epitopes were injected. A permanent clearing of the hIL-6R could be obtained by repeated injections of the clearing mixture. No correlation was found between the ability of the mAb to clear the sIL-6R and to immunoprecipitate them in agarose gel. The F(ab')2 fragments lost the clearing ability of the intact mAb. These results clearly show that therapeutic clearance of sIL-6R by mAb need at least three mAb directed against three different epitopes of the molecule, a conclusion which is likely to apply for clearing any soluble target molecule.

Gamma-interferon induces apoptosis of the B lymphoma WEHI-279 cell line through a CD95/CD95L-independent mechanism.

Gamma-interferon (IFN-gamma) a cytokine produced by CD4+ T helper type 1 cells, CD8+ T cells and natural killer (NK) cells, plays a central role in the development of humoral and cell-mediated immunity. IFN-gamma participates in the maturation and differentiation of B cells, but it has been previously reported that IFN-gamma may inhibit the early stages of B cell activation. We report that the inhibition of the B lymphoma cell WEHI-279-proliferation induced by IFN-gamma, involves the induction of typical features of apoptosis (nuclear chromatin condensation and fragmentation, cell shrinkage, phosphatidyl-serine (PS) exposure and mitochondrial membrane potential (delta psim) loss). IFN-gamma-mediated B cell apoptosis was decreased by the addition of the T helper type 2 cytokine, IL-4. WEHI-279 cells express CD95 and undergo apoptosis after treatment with either an agonistic anti-CD95 Ab or with a soluble recombinant CD95L. However, incubation with CD95-Fc or TRAIL-R1-Fc fusion proteins, did not prevent IFN-gamma-mediated apoptosis, suggesting that IFN-gamma-mediated apoptosis occurs independently of CD95/CD95L and TRAIL-R/TRAIL interactions. IFN-gamma-mediated apoptosis is associated with caspase-3 activation that can be prevented by the addition of the broad caspase inhibitor zVAD-fmk. These data indicate that IFN-gamma may play a major role in the regulation of B cell apoptosis, and suggest the involvement of an alternative pathway which is independent of the death receptors.

Identification of centerin: a novel human germinal center B cell-restricted serpin.

For naive B cells to mature in response to antigen triggering and become either plasma cells or memory B cells, a complex array of events takes place within germinal centers (GC) of secondary lymphoid organs. With the long-term objective of defining and characterizing molecules that control the generation of GC, we have subtracted RNA messages derived from highly purified B cells at the follicular mantle stage of differentiation from GC B cells. Using this approach, we have identified a novel molecule, centerin, belonging to the family of serine-protease inhibitors or serpins. Transcription of centerin is highly restricted to GC B cells and their malignant counterparts, Burkitt's lymphoma lines. The putative centerin protein shares the highest sequence identity with thyroxine-binding globulin and possesses arginine/serine at its P1/P1' active site, suggesting that it interacts with a trypsin-like protease(s). In addition, several other sequence features of centerin also indicate that it serves as a bonafide protease inhibitor. Finally, we demonstrate differentially up-regulated transcription of this novel gene by resting, naive B cells stimulated in vitro via CD40 signaling, while Staphylococcus aureus Cowan strain-mediated B cell activation fails to generate this reponse. Because CD40 signaling is required for naive B cells to enter the GC reaction and for GC B cells to survive, it is likely that centerin plays a role in the development and/or sustaining of GC.

Interleukin-6 receptor signaling. I. gp80 and gp130 receptor interaction in the absence of interleukin-6.

Interleukin-6 (IL-6) is used as a growth factor by various tumor cells. It binds to a gp80 specific receptor (IL-6R) and then to a gp130 transducing chain. Both receptor chains are released as soluble functional proteins which circulate in biological fluids. To study the physiological role of these soluble receptors, both proteins were purified from human plasma and the kinetic constants of equilibria between IL-6 and its natural soluble IL-6R (sIL-6R) and gp130 receptor (sgp130) were measured using surface plasmon resonance analysis. Unexpectedly, natural sIL-6R and natural sgp130 were found to interact (Kd = 2.8 nM) in the absence of IL-6. No interaction was seen between the recombinant soluble receptors or between either natural soluble receptor and its recombinant partner. This binary complex was not due to copurification of IL-6 and was detected in human plasma of healthy donors. It results from either direct interaction between the two natural soluble receptors or indirect binding mediated by a yet unidentified copurified plasma molecule playing the role of an IL-6 antagonist. Once formed, the binary complex was found to be unable to bind IL-6. Soluble gp130 had already been shown to inhibit IL-6 signaling by inactivating the IL-6/IL-6R complex. In addition we show that, in the absence of IL-6, circulating natural sgp130 is able to inhibit directly the circulating sIL-6R that is a strong synergic molecule of IL-6 signaling.

Plasmablastic morphology--an independent prognostic factor with clinical and laboratory correlates: Eastern Cooperative Oncology Group (ECOG) myeloma trial E9486 report by the ECOG Myeloma Laboratory Group.

We studied the prognostic significance of plasmablastic (PB) multiple myeloma (MM) in Eastern Cooperative Oncology Group Phase III trial E9486. Two reviewers independently reviewed 453 cases. They agreed on 37 PB (8.2%) cases and 416 non-PB cases, achieving an 85% concordance (P < .0001). These PB cases had significantly lower hemoglobin and serum albumin levels, higher calcium and beta 2-microglobuin levels, and higher percentage BM plasma cells (PC) by immunofluorescence. They had higher bone marrow PC labeling indices, higher serum soluble interleukin-6 receptor (sIL-6R) levels, and a higher probability of ras mutations. Three treatment regimens were used: vincristine, bis-chloro-ethyl nitrosourea (BCNU) melphalan, cyclophosphamide, and prednisone (VBMCP) alone; VBMCP with added cyclophosphamide (HiCy); or recombinant interferon alpha 2 (rIFNalpha2). Although the numbers are low, patients with PB had a significantly lower response rate versus non-PB MM when treated with VBMCP (treated, 47.1% v nontreated, 66.5% [P = .015]). Patients with nonresponding PB had a significantly higher progression rate than non-PB cases (30.6% v 11.8% [P < .0001]), especially with VBMCP alone (35.3% v 15.8% [P = .002]), and with added HiCy (37.5% v 9.8% [P < .0001]), but not with added rIFNalpha2. Event-free and overall survival of PB MM was shorter (median years, 1.1 v 2.7 and 1.9 v 3.7, respectively [P < .0001 for both]). In multivariate analysis, PB classification was also highly prognostic. There is no survival difference between the patients who were classified as PB by both reviewers versus patients classified as PB by only one reviewer. We conclude that PB MM is a discrete entity associated with more aggressive disease and shortened survival. Tumor cell ras mutations and increased sIL-6R may contribute to a higher proliferation rate and reduced survival. There were significant improvements in response and progression with the addition of HiCy and rIFNalpha2 to VBMCP, but the numbers were small and improved survival could not be shown.

Dimerization and activation of the common transducing chain (gp130) of the cytokines of the IL-6 family by mAb.

The objective of our research was to study the mechanisms of activation of mAb against the gp130 transducer chain common to the IL-6 cytokine family. It has been found that among the 56 anti-gp130 available worldwide, none was able to activate the growth of IL-6-dependent myeloma cell lines. When certain of them were associated in pairs they allowed the cells to grow; alone, they were inhibitory. The same activation was also obtained by cross-linking certain anti-gp130 mAb on the cell membrane with a goat anti-mouse Ig antiserum. A bispecific mAb was prepared by the somatic fusion of two hybridomas secreting two mAb whose association was able to activate gp130 signaling; the bispecific mAb was inactive. The activating mAb were able to support long-term proliferation of the IL-6-dependent myeloma cell lines, which indicates that they are potential valuable growth factors of tumor cells and hematopoietic stem cells. When they were injected into SCID mice, they allowed human IL-6-dependent myeloma cell lines to grow, develop tumors and metastasize. By studying the functional epitopes of the cell membrane gp130 receptors, it was shown that the activating mAb induced gp130 dimerization and STAT3 activation, as did IL-6.

Functional interaction of the gp80 and gp130 IL-6 receptors in human B cell malignancies.

IL-6, or cytokines of the IL-6 family using gp130 as transducer chain receptor, have been suggested to play a role in certain B lymphoid neoplasia. The presence of cell membrane gp80 and gp130 IL-6 receptors was studied in 98 patients with various leukaemia and non-Hodgkin's malignant lymphoma using flow cytofluorometry and immunohistology. Except neoplasia of immature B cells which expressed neither of the receptors, the majority of B cell tumours expressed one or both of them, mantle cell lymphoma being found to express the highest density of receptors. Using IL-6-dependent XG myeloma cell lines and mAb recognizing various gp80 and gp130 functional epitopes, it has been shown that IL-6 activation leads to a modified expression of some epitopes. In particular, the decrease or the disappearance of a gp130 epitope called A1 signed gp130 dimerization which is the first step of the gp130 activation pathway. Gp80 and gp130 epitope analysis was achieved in 17 of the patients. In four, an epitope phenotype compatible with a cytokine-induced activation was found. The cells of five B-CLL patients which expressed both gp80 and gp130 receptors were incubated with IL-6 to induce activation. In three of the cases they were found to rearrange their receptors in activated forms but not in the two others, showing that cells able to be activated or not can be found. These results confirm that gp130 signalling might play an important role in the pathogenesis of certain B cell neoplasia.

Specific inhibition of IL-6 signalling with monoclonal antibodies against the gp130 receptor.

A family of cytokines [IL-6, IL-11, oncostatin M (OM), leukaemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF) and cardiotrophin-1] involved in various inflammatory or tumoral diseases share the same gp130 signal transducer chain. The complex formed with their specific receptors associates with a common transducing gp130 membrane protein (gp130) resulting in the formation of high avidity receptor and activation of tyrosine kinases. With the view of identifying gp130 domains specifically involved in IL-6 signalling, the authors prepared 37 new anti-gp130 mAb and analysed the structure-function relationship of the molecule. By cross-competition ELISA, the mAb were classified in 10 subgroups called A to J. By ELISA and BIAcore analysis, the mAb were found to recognize at least 18 antigenic specificities of the gp130 chain. The mAb reacted against the soluble and the membrane forms of gp130 as well. Their ability to inhibit the proliferation of the human myeloma cell line XG-4 of which the growth is strictly dependent on the presence of either exogenous IL-6, or LIF, or OM, or CNTF was studied. Besides mAb with no evident neutralizing effect (G and H) and mAb which neutralized equally well the activity of all tested cytokines (all mAb of groups A, I and J), some showed a selective effect. Those of group F inhibited also the proliferation induced by the 4 cytokines, but more specifically that dependent on the CNTF. mAb of groups B and E specifically inhibited the growth induced by IL-6, whereas those of group C inhibited that induced by LIF and OM. These results show the presence of different gp130 epitopes specifically involved in the signaling induced by the cytokines of the gp130 family. In ELISA, only mAb of group B and E were found to inhibit the binding of the IL-6-IL-6R complex to gp130, showing that they identified one or two domains of gp130 involved in its interaction with the IL-6-IL-6R complex. Precise identification of this(ese) epitope(s) would be useful to better understand the mechanisms of the IL-6 signalling.

Activation of the gp130 signaling pathway by monoclonal antibodies directed against the gp130 molecule.

Six cytokines of the interleukin (IL)-6 family involved in various inflammatory or tumoral diseases share the same gp130 signal transducer chain. We made a panel of anti-gp130 monoclonal antibodies (mAb) to study the structure and function of the gp130 molecule. These mAb recognized different epitopes of the gp130 that we called A to J. Most of the mAb were found to be inhibitors and we studied whether some of them could also induce gp130 activation. When used alone, none of them was able to initiate the proliferation of IL-6-dependent cell lines. However, some particular associations of the mAb were able to induce a proliferative response. mAb B1 could activate the lines in association with F1 or with I2 but not with I1, which in ELISA was similar to I2. In contrast mAb B2, which in ELISA appeared to be very similar to B1, was able to activate the cells in association with I1 but not with F1 or I2. Two other mAb belonging to specificities A and C were found to be activators either in association with I1 only, or with I1 or B2, respectively. These associations of mAb appeared to be nearly as potent activators as IL-6 itself. Although we still have no precise idea of the mechanisms involved, they are interesting tools to study the molecular interactions leading to gp130 activation and, from a practical point of view, valuable growth factors of hematopoietic stem cells.

Major role of the soluble interleukin-6/interleukin-6 receptor complex for the proliferation of interleukin-6-dependent human myeloma cell lines.

Interleukin-6 (IL-6) is a proinflammatory cytokine which possesses a central growth factor activity for certain tumor cells such as plasma cells in multiple myeloma (MM). Upon binding of IL-6, soluble IL-6 receptor (sIL-6R) has been shown to retain its affinity for IL-6 and to associate with the signal-transducing gp130 chain. Therefore, contrary to the majority of soluble cytokine receptors, it plays an agonist role in IL-6 signaling. In order to test its physiological importance as compared to that of its membrane counterpart, we studied cells from two myeloma cell lines which need exogenous IL-6 to proliferate and release sIL-6R into their culture supernatant. Using a new culture system where the supernatant recirculated permanently through an anti-IL-6R affinity column, all sIL-6R was removed from the culture medium throughout the culture period. Under these conditions IL-6-dependent cells were unable to grow in the presence of physiological concentrations of IL-6, showing the major role of the sIL-6R for sustaining the proliferation of these cell lines. Increasing IL-6 concentrations well over the physiological values allowed the cells to proliferate again. No effect was seen when sIL-6R was removed from the supernatant of an IL-6-independent myeloma cell line. These results show that the levels of circulating sIL-6R (and thus those of IL-6/sIL-6R complex) are worth looking at in pathologies involving IL-6 hyperactivity.

Identification of a novel antigenic structure of the human receptor for interleukin-6 involved in the interaction with the glycoprotein 130 chain.

The receptor for interleukin-6 (IL-6) is characterized by a ligand-binding glycoprotein 80 (gp80) transmembrane chain (IL-6R) which associates with a signal-transducer gp130 chain. We previously raised a series of monoclonal antibodies (mAb) recognizing different epitopes of the human IL-6R and interfering with the function of the receptor. One of them, M182, was able to diminish the proliferation of IL-6-dependent plasmacytoma cell lines although it was found unable to inhibit the binding of IL-6 to its receptor. Using an enzyme-linked immunosorbent assay for measuring the binding of IL-6 IL-6R to the gp130 chain, we showed that M182 was directed against a structure directly involved in the IL-6R gp130 interaction. M182 was able to potentiate the inhibitor effect of anti-IL-6R mAB which interfere with the binding of IL-6, leading to complete inhibition of the proliferation of IL-6-dependent cell lines. M182 was also found to synergize with inhibitory anti-IL-6 mAb. Therefore this structure appears to be an important regulatory domain of the IL-6R and a valuable target for inhibiting IL-6 signalling.

Detection of membrane and soluble interleukin-6 receptor in lymphoid malignancies.

We studied the membrane expression of the gp80 chain of IL-6 receptor (IL-6R) by quantitative flow cytometry in chronic lymphocytic leukaemia (CLL) and leukaemic centrocytic lymphoma using a panel of seven monoclonal antibodies. IL-6R was detected in 18/26 CLL cases and 4/7 lymphoma cases, with a mean antigen density < 3000 molecules/cell. Multiple labelling experiments confirmed the IL-6R expression by neoplastic cells. Specific mRNA was found by RT-PCR in neoplastic cells. A specific ELISA test was designed using two anti-IL-6 receptor MAbs to measure the serum soluble IL-6R (sIL-6R) in CLL (n = 48). B-cell non-Hodgkin's lymphoma (NHL; n = 40), and monoclonal gammopathy (MG; n = 32). SIL-6R was higher in CLL (170 +/- 12.6 ng/ml) in NHL (160 +/- 12 ng/ml) and MG patients (183 +/- 23 ng/ml) than in age-matched controls (100 +/- 5.6 ng/ml; P < 0.001) and higher in high-grade than low-grade NHL. No correlation was noted with a previous treatment. Among CLL cases the patients classified as stage B according to the Binet's staging of the disease had the highest sIL-6R values, thus suggesting a link with tumour cell mass.

Immunomodulating IL-6 activity by murine monoclonal antibodies.

The human anti-mouse immunoglobulin antibody (HAMA) response, which occurs frequently after injection of murine monoclonal antibodies (MAb) directed against cellular targets, has been reported extensively in several studies. We analysed here HAMA in 12 patients (six with multiple myeloma, MM, and six with metastatic renal cell carcinoma, MRCC) who were treated with B-E8, an IgG1 MAb against interleukin-6 (IL-6). Efficiency of the treatment was evidenced by the drop in the serum levels of C reactive protein (CRP), of which the in vivo production is under the control of IL-6. Three patients with MM and the six patients with MRCC became immunized to the injected MAb. HAMA appeared between days 7 and 15 after the beginning of the treatment. The nine patients made IgG antibodies; four also made IgM. All of immunized patients made anti-idiotype antibodies specific to B-E8. Two of them also developed HAMA directed to murine IgG1 isotype; in these two patients B-E8 MAb cleared rapidly from the circulation with loss of treatment efficiency. In the patients who developed only anti-idiotype antibodies, serum levels of B-E8 remained unchanged and CRP production remained inhibited, indicating that treatment efficiency was not affected by the presence of HAMA. Circulating B-E8 MAb were still able to bind to IL-6 and to inhibit IL-6-independent proliferation despite the presence of anti-idiotypic HAMA. Therefore, in contrast to HAMA against MAb directed against cellular targets, HAMA against anti-IL-6 MAb idiotopes led neither to clearance nor to functional inactivation of the injected MAb.(ABSTRACT TRUNCATED AT 250 WORDS)

Human anti-mouse antibody response to the injection of murine monoclonal antibodies against IL-6.

We analysed human anti-mouse antibodies (HAMA) in 12 patients (six with multiple myeloma (MM) and six with metastatic renal cell carcinoma (MRCC) who were treated with B-E8, an IgG1 MoAb against IL-6. Efficiency of the treatment was evidenced by the drop in the serum levels of C-reactive protein (CRP), the in vivo production of which is under the control of IL-6. Three patients with MM and the six patients with MRCC became immunized to the injected MoAb. HAMA appeared between days 7 and 15 after the beginning of the treatment. The nine patients made IgG antibodies; four also made IgM. All immunized patients made anti-idiotype antibodies specific to B-E8. Two of them also developed HAMA directed to murine IgG1 isotype; in these two patients B-E8 MoAb cleared rapidly from the circulation with loss of treatment efficiency. In the patients who developed only anti-idiotype antibodies, serum levels of B-E8 remained unchanged and CRP production remained inhibited, indicating that treatment remained efficient in the presence of HAMA. Circulating B-E8 MoAbs were still able to bind to IL-6 and to inhibit IL-6-dependent proliferation despite the presence of anti-idiotypic HAMA. Therefore, in contrast to HAMA produced against MoAb directed against cellular targets, HAMA against anti-IL-6 MoAb idiotopes led neither to clearance nor to functional inactivation of the injected MoAb. This was further shown by resuming the B-E8 treatment with success in a patient who still had anti-idiotypic HAMA.

Growth factor activity of IL-6 in the synovial fluid of patients with rheumatoid arthritis.

OBJECTIVE: We set out to determine whether the ability of synovial fluids (SF) in patients with rheumatoid arthritis (RA) to facilitate the proliferation of synovial tissue-derived fibroblastic cell lines was related to the presence of growth factors and/or cytokines. METHODS: The growth factor activity of 20 RA SF was measured by their ability to induce anchorage-independent growth of the rat NRK-49F (49F) fibroblastic strain. The presence of transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) was also assessed using neutralising anti-TGF-beta or anti-PDGF-AB mAbs. Cytokines were measured by functional assays or ELISA: RESULTS: We observed a correlation between growth factor activity and the IL-6 levels in SF. Both were correlated to the erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels in SF and serum. IL-6 (at concentrations above 10(4) U/ml), synergized with growth factors in the induction of the anchorage independent (AI) growth of 49F cells. Pretreatment of SF with a neutralising anti-IL-6 mAb substantially reduced the capacity of these SF to induce AI growth of 49F cells, confirming the growth factor activity of IL-6 in this test. In contrast, IL-6 alone or in association with PDGF, epidermal growth factor (EGF) or TGF-beta had no effect on the anchored growth of synovial tissue-derived fibroblasts, and treatment of SF with a neutralising anti-IL-6 mAb did not affect their ability to increase the growth rate of synovial tissue-derived fibroblasts. CONCLUSIONS: These results strongly suggest that IL-6 is responsible for the observed correlation between the growth factor activity of SF and inflammatory indexes such as ESR and CRP. However, neither IL-6 nor PDGF were responsible for the observed positive effect of SF on synovial fibroblastic cell lines.

IL-6-induced changes in synthesis of alpha 1-acid glycoprotein in human hepatoma Hep3B cells are distinctively regulated by monoclonal antibodies directed against different epitopes of IL-6 receptor (gp80).

The synthesis of the human acute-phase alpha 1-acid glycoprotein (AGP) is primarily controlled by IL-6 and IL-1 in liver cells. In the present study, monoclonal antibodies against human gp80 interleukin-6 receptor (IL-6R) were utilized to study the role of the IL-6R in the control of the IL-6-induced AGP synthesis in the human hepatoma Hep3B cell line. Two of the 4 MAbs used in this study, M164 and M195, identified 2 different epitopes involved in IL-6 binding and two others, M91 and M182, recognized epitopes not involved in IL-6 binding. Dose-response experiments indicated that up to 55% of AGP synthesis was inhibited by 10(5) ng/ml of MAbs 164 or 195 when Hep3B cells were treated by IL-6 for 48h. Kinetics of the inhibition of AGP synthesis after addition of anti-IL-6R indicated that the decrease of the IL-6-induced AGP synthesis by Hep3B cells was obtained immediately after the addition of the anti-IL-6R MAbs. Of the two MAbs not involved in IL-6 binding, M91 was unable to interfere with the IL-6-induced AGP synthesis whereas, surprisingly, M182 decreased it by about 25%. Since M182 was also able to interfere with the proliferative response of an IL-6 dependent plasma cell line, our results suggested that M182 may be directed to a structure involved in the IL-6/IL-6R gp130 complex formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration of an anti-interleukin-6 monoclonal antibody to patients with acquired immunodeficiency syndrome and lymphoma: effect on lymphoma growth and on B clinical symptoms.

Increased interleukin-6 (IL-6) production and expression by malignant cells of the IL-6 receptor has been evidenced in a subgroup of non-Hodgkin's lymphomas, suggesting that this cytokine plays a role in lymphoma growth and in B clinical symptoms. In this study, the effect of the administration of an anti-IL-6 monoclonal antibody (MoAb) was analyzed in 11 patients seropositive for human immunodeficiency virus-1 and suffering from an immunoblastic or a polymorphic large-cell lymphoma. The antibody (BE-8, 10 to 40 mg/day) was administered for 21 days. Neutralization of in vivo IL-6 effect was assessed by monitoring C-reactive protein levels in the serum. In 5 patients, the lymphoma progressed during treatment. Among them were the 2 patients in whom endogenous IL-6 effect was not neutralized. Five patients experienced a stabilization, and 1 a partial remission. This effect on lymphoma growth lasted for 8 to 28 weeks. The anti-IL-6 MoAb had a clear effect on lymphoma-associated fever and cachexia. The mean body weight increase was 1.4 +/- 0.5 kg between day 1 and day 21, and reached 12 kg in 120 days in 1 patient who received three courses of treatment. Side effects were a consistent but moderate thrombocytopenia, and an occasional and moderate decrease of neutrophil counts. Immunization against the MoAb was observed in only 2 patients. These results indicate that in some cases of lymphomas growth of malignant cells may be partially IL-6-dependent and that neutralizing endogenous effect of IL-6 completely abrogates B clinical symptoms.

Reproducible obtaining of human myeloma cell lines as a model for tumor stem cell study in human multiple myeloma.

We report a novel, reproducible methodology which enabled 10 human myeloma cell lines (HMCL) to be obtained from each of 10 tumor samples harvested from 9 patients with extramedullary proliferation. Fresh samples were cultured with interleukin 6 (IL-6) and granulocyte macrophage-colony stimulating factor (GM-CSF) at a high cell density and resulting HMCL growth became progressively dependent on IL-6 alone, no longer requiring GM-CSF. These HMCL, which had the same immunoglobulin gene rearrangements as the patients' original myeloma cells, were designated XG-1 to XG-9. XG HMCL had a plasma cell morphology, expressed plasma cell antigen (Ag), namely cytoplasmic immunoglobulins, CD38, B-B4 Ag, and CD77, and lacked the usual B-cell Ag. They also expressed activation antigens such as CD28 with coexpression of CD28 and its ligand, B7 Ag, in four HMCL. Six HMCL expressed CD40, 4 CD23, and 5 its ligand, CD21. The XG HMCL bore adhesion molecules VLA-4 and CD44 (all 10 HMCL), VLA-5 (7 HMCL), and CD56 (4 HMCL). Finally, cytogenetic study of 8 HMCL indicated a 14q+ chromosome, and t(11,14) translocation was found in 6 of 8 and 5 of 8 HMCL, respectively. The possibility of obtaining malignant plasma cell lines reproducibly from each patient with extramedullary proliferation offers a unique tool for studying the phenotype and abnormalities of the still unidentified tumor stem cell in this disease.

Epitope analysis of human IL-6 receptor gp80 molecule with monoclonal antibodies.

Gp80 human IL-6R was studied using 7 murine mAb (M37, M91, M113, M139, M164, M182 and M195) obtained after fusion of splenocytes of Balb/c mice immunised with a mixture of recombinant IL-6 receptor (rIL-6R) and cells from 2 cell lines expressing IL-6R. These were U266, which is IL-6 independent and XG-1 which is IL-6-dependent. In ELISA the 7 mAb reacted against the rIL-6R and against the natural soluble form found in plasma (nIL-6R), which both lack transmembrane and cytoplasmic domains. However, M195 reacted less with the natural than with the recombinant soluble IL-6R. Using FACS analysis, the 7 mAb were shown to bind to U266 cells but not to the Namalva cell line which is deprived of IL-6R. This showed that they all recognised the membrane form of the IL-6R. Three of the anti-IL-6R mAb reacted with rIL-6R by Western blotting. Four different epitopes of the molecule were identified, either by cross-blocking experiments of mAb binding to IL6R in ELISA or by the biosensor Biacore technology. A group of 4 mAb (M37, M113, M139 and M164) and another mAb (M195) identified 2 different epitopes involved in IL-6 binding. These antibodies were able to inhibit the binding of IL-6 to IL-6R and the proliferation of the IL-6-dependent XG-1 cell line. M91 and M182 recognized 2 other epitopes that were not involved in IL-6 binding. As expected, M91 did not inhibit XG-1 proliferation; in contrast, M182 interfered with the proliferative response of the XG-1 cell line.(ABSTRACT TRUNCATED AT 250 WORDS)