Chloride channels in excised membrane patches from human platelets: effect of intracellular calcium

Interleukin-6 (IL-6) is one of the most important growth factors for myeloma cells. We examined the effect of recombinant IL-6 on the proliferation of five human myeloma cell lines, which were independently established AT Kawasaki Medical School. Only the KMS-11 cell line among these five lines showed growth enhancement induced by IL-6. Based on the results, a possible contribution of Ca(2+)-phospholipid-dependent protein kinase C (PKC) to the signal transduction in KMS-11 cells during growth enhancement was studied, since PKC may play an important role in malignant transformation or cell proliferation induced by some growth factors, such as IL-6. When exogenous IL-6 was added to KMS-11 culture, we observed (1) reduction of total PKC activity, and (2) translocation of PKC activity from its cytosol fraction to the membrane fraction. These findings may indicate that down regulation of PKC occurred during the myeloma cell proliferation induced by IL-6. However, IL-6 does not appear to be involved in cell proliferation and differentiation in the other cell lines studied.     

Developmental aspects of dendritic cells in vitro and in vivo

Our knowledge in immunology has been dramatically increased by several excellent investigations elucidating the role of the Fas (Apo-1/CD95) receptor/ligand (FasL) system in complex immunological processes such as the acquisition of self tolerance in T cells, progression of autoimmunity, clonal deletion of activated T cells, B-cell regulation and the establishment of "immune privileged" sites such as testis or retina. In addition to these regulatory immunological activities, Fas/FasL interaction was also shown to participate in active defense mechanisms of the host against infected or transformed cells thereby inducing apoptosis in target cells. However, the same mechanism seems also to be part of an escape strategy utilized by tumor cells in various neoplastic malignancies of both hematopoetic as also non-hematopoetic origin. We ourselves were able to demonstrate that neoplastic plasma cell lines, as well as native malignant myeloma cells constitutively express FasL mRNA and protein. The FasL molecule is functionally active and able to induce programmed cell death in Fas sensitive target T cells in vitro. These target T cells were protected from programmed cell death by preincubation of T cells with a Fas-blocking monoclonal antibody (mAb) or of myeloma cells with a FasL-neutralizing mAb. respectively. Furthermore, overexpression of the caspase inhibitor, cowpoxvirus protein CrmA, also protected target T cells from being killed by myeloma cells, identifying Fas/FasL mediated signaling as the effector pathway utilized by malignant plasma cells. Our observations strongly suggest the engagement of Fas/FasL interaction in the escape strategy of this malignancy. The molecular basis of this evasive mechanism differs in essential respects from those described in melanoma, lung cancer, hepatocellular carcinoma, or astrocytoma, since downregulation of Fas or instrinsic insensitivity towards Fas-mediated signaling were not prerequisites for the occurrence of this phenomenon in Fas-sensitive multiple myeloma cell lines. However, myeloma cell lines resisted cocultivation with FasL-expressing target T cells in vitro. The aim of this review is to discuss the role of Fas/FasL interaction in the establishment of malignant disease, in the light of our findings on myeloma cells and also by drawing upon similar observations of other investigators on different kinds of tumor cells and cell lines and further to consider its possible relevance in formulating novel approaches to cancer therapy.     

IFN-alpha is a survival factor for human myeloma cells and reduces dexamethasone-induced apoptosis

IFN-alpha is used as a maintenance therapy in patients with multiple myeloma, but its benefit is a matter of controversy. In vitro studies show that IFN-alpha can both stimulate and inhibit myeloma cell proliferation. We have tested the effect of IFN-alpha on the survival of myeloma cell lines and primary plasma cells. IFN-alpha significantly reduced the apoptosis induced by removal of IL-6 in four IL-6-dependent myeloma cell lines. It also reduced the level of apoptosis induced by dexamethasone in these cell lines as well as in purified primary myeloma cells from seven patients. IFN-alpha promoted the survival of myeloma cells, which, following removal of IL-6, were blocked in G1 and died. However, unlike IL-6, IFN-alpha-treated cells remained mainly blocked in the G1 phase of the cycle. While the effects of IL-6 are mediated through stimulation of its gp130 receptor subunit, the IFN-alpha-induced survival of myeloma cells was independent of gp130 transducer activation (as demonstrated using a neutralizing anti-gp130 Ab). However, the signal transduction cascades activated by these two cytokines share at least some common elements, since stimulation with either IFN-alpha or IL-6 resulted in STAT3 phosphorylation. These results indicate that IFN-alpha promotes the survival, but not the proliferation, of myeloma cells, preventing the apoptosis induced by removal of IL-6 or addition of dexamethasone. This survival factor activity may explain the conflicting reports on the effects of IFN-alpha on myeloma cell proliferation.     

Fas/APO-1 (CD95)-mediated apoptosis is activated by interferon-gamma and interferon- in interleukin-6 (IL-6)-dependent and IL-6-independent multiple myeloma cell lines

A poor response to Fas-induced apoptosis is evident in some multiple myeloma (MM) cell lines and primary cells. In this study, we have examined the possibility to increase the sensitivity to Fas-induced apoptosis by pretreatment of MM cells with interferon-gamma (IFN-gamma) or interferon-alpha (IFN-alpha). Both IFN-gamma and IFN-alpha markedly increased the Fas-induced apoptosis in all cell lines tested (U-266-1970, U-266-1984, and U-1958). In the U-266-1970 and U-1958 cell lines, pretreatment with either IFN-gamma or IFN-alpha also inhibited proliferation in a dose-dependent manner. In contrast, IFN-gamma activation of the Fas death pathway in the U-266-1984 cells was not accompanied by growth inhibition. Incubation with the IFNs increased the Fas antigen expression in one of three cell lines but did not alter the expression of Bcl-2 or Bax. The IFNs are important regulators of growth and survival in MM cells. Our results suggest that activation of Fas-mediated apoptosis is a novel mechanism by which the IFNs exert inhibitory effects on MM cells.     

Chemokine production and adhesion molecule expression by neural cells exposed to IL-1, TNF alpha and interferon gamma

We investigated the effect of TNF alpha, IL-1alpha and IFN gamma on two neuroblastoma (NB) cell lines (SK-N-SH and SK-N-MC). These lines responded differentially to IL-1alpha, TNF alpha and IFN gamma for MCP-1 and IL-8 production and expression of the ICAM-1 and VCAM-1 adhesion molecules. None of the cytokines induced MCP-1 or IL-8 on SK-N-MC cells. Both chemokines were produced in response to IL-1alpha by SK-N-SH cells, while TNF alpha induced mainly MCP-1 production. Addition of IFN gamma decreased IL-8, but not MCP-1 production. These responses correlated with monocyte and neutrophil chemotactic activity in NB culture supernatants. This activity was neutralized by antibodies to IL-8 and MCP-1. The expression of ICAM-1 on SK-N-MC was up-regulated by TNF alpha or IFN gamma, while IL-1alpha also upregulated ICAM-1 on SK-N-SH cells. VCAM-1 expression on SK-N-SH was induced by IL-1alpha and TNF alpha and IFN gamma synergized with TNF alpha in this respect on both NB cell lines. These results suggest that mechanisms for chemokine production and VCAM-1 and ICAM-1 upregulation by inflammatory cytokines differ and IFN gamma, in conjunction with TNF alpha, stimulate neural cell responses (high MCP-1 and VCAM-1 and decreased IL-8) favouring mononuclear cell recruitment.     

The dynamics of the dominant alpha-rhythm frequency in the perception and reproduction of time intervals

In contrast to cytotoxic agents inducing rapid cell death, biological agents such as hormones, vitamins (e.g., retinoids), cytokines, and antireceptor antibodies act slowly and may alter ratios between cell growth and programmed cell death (apoptosis). We showed previously that anti-interleukin 6 (IL-6) and antitransferrin (Tf) receptor antibodies inhibited in vitro growth and induced death of myeloma cells. Retinoids also inhibit in vitro growth of human cancer cells and decrease IL-6 receptor display and autosecretion by some myeloma cells. Retinoids may also antagonize in vitro growth-promoting effects of iron and transferrin. To develop a novel strategy for treating myeloma, we examined antiproliferative and cytotoxic effects of retinoids in combination with anti-Tf or anti-IL-6 receptor antibodies. Myeloma cell lines were cultured with retinoids with or without anti-growth factor receptor monoclonal antibodies. Both all-trans retinoic acid (ATRA) and 13-cis-retinoic acid showed variable, dose-dependent inhibition of myeloma cell line growth. ATRA also induced significant down-regulation of myeloma IL-6 receptors and inhibited IL-6 autosecretion by myeloma cells. Antiproliferative effects of ATRA were increased by coculture with anti-Tf but not anti-IL-6 receptor antibodies. Colony-forming assays showed that antiproliferative effects of anti-Tf receptor antibodies were largely reversible, but 1 microM ATRA was cytotoxic to myeloma cells. To assess apoptosis, a flow cytometry assay detecting DNA damage was used. Using previously studied cell line models, flow cytometry detected programmed cell death induced by transforming growth factor beta1 in leukemia cells and by anti-growth factor receptor antibody treatment of IL-6-dependent myeloma cells, treatments which caused only modest increases in the percentage of cells undergoing morphological apoptosis and increased internucleosomal DNA degradation. Flow cytometry analysis of ATRA and anti-Tf antibody-treated myeloma cells also showed evidence for apoptosis induced by ATRA, but not with anti-Tf receptor antibodies. These changes were apparent several days before detection of internucleosomal DNA degradation on agarose gels in 8226 cells but were not detected at any time in U266 cells, which underwent cell death but showed no DNA damage using flow cytometry or degradation on agarose gels. Retinoids merit further study as possible maintenance or chemoprevention therapies for clonal plasma cell disorders and for treating paraneoplastic disorders such as Castleman's disease. Flow cytometry rapidly detects apoptosis induced by biological agents and may be useful for in vitro screening of novel biological therapies.     

Regulation of B-CLL apoptosis through membrane receptors and Bcl-2 family proteins

The accumulation of monoclonal chronic lymphocytic leukemia B (B-CLL) cells may be due to excessive proliferation and longevity. Clinical progression may thus come from a constitutive but altered expression of a number of genes that results in extended B-CLL cells life span, increased proliferative capacity and diminished cell death. B-CLL cells express a number of surface markers that characterise the normal B-cells phenotype. However, B-CLL cells are CD5 positive and most of them also express CD6, surface receptors that are present in just a small subset of normal B-cells. When exploring CD6 function, we found out that cross-linking of CD6 protected B-CLL from anti-IgM-induced apoptosis. CD6 activation blocked anti-IgM- induced Bax(alpha) up-regulation and, by doing so, corrected an imbalance in the Bcl-2/Bax ratio that accompanies apoptosis. Here, we review all surface receptors and cytokines that have been described as participating in the induction or protection of B-CLL apoptosis together with data on chemosensitivity and gene modulation, data on the Fas receptor/Fas ligand system, and the implications of all the latter for B-CLL cell survival.     

Identification and regulation of testicular interferon-gamma (IFNgamma) receptor subunits: IFNgamma enhances interferon regulatory factor-1 and interleukin-1beta converting enzyme expression

Interferon-gamma (IFNgamma) transmits its signal through a specific cell surface receptor (IFNgammaR), which consists of a primary ligand binding alpha-chain (IFNgammaR alpha) and a signaling beta-chain (IFNgammaR beta). Recent studies identified the cytokines IFNgamma, interleukin-6 (IL-6), IL-1alpha, and tumor necrosis factor-alpha in testicular cells. Therefore, we: 1) examined the expression of IFNgammaR alpha and IFNgammaR beta subunits in freshly isolated and purified rat testicular cells; 2) examined the differential regulation of receptor components by cytokines using primary cultures of Sertoli cells; 3) identified the cell signaling pathway components of testicular IFNgammaR; and 4) characterized the functional role of testicular IFNgamma using primary Sertoli cells. We demonstrated the messenger RNAs for both chains of IFNgammaR in rat testicular cells using Northern hybridization analysis. Western blot analysis and immunocytochemistry showed that both specific IFNgammaR protein subunits were present in cultured primary Leydig and Sertoli cells prepared from the testes of immature rats. The expression of both IFNgammaR component messenger RNAs in cultured Sertoli cells was increased by its specific ligand (IFNgamma), as well as IL-1alpha and tumor necrosis factor-alpha, in both a time- and dose-dependent manner. IFNgamma-activation of the Janus (JAK) tyrosine kinases, JAK1 and JAK2 proteins, indicate that IFNgammaR, expressed in the Sertoli cell, is functional. Moreover, IFNgamma modulates the expression of interferon regulatory factor (IRF)-1 and IL-1beta converting enzyme genes in Sertoli cells. Thus, our data are suggestive of a role(s) for IFN-gamma in the regulation of distinct gene expression and cell-specific sensitivity to apoptosis in the testis.     

Induction of specific T cell tolerance by Fas ligand-expressing antigen-presenting cells

Autocrine interaction of Fas and Fas ligand leads to apoptosis of activated T cells, a process that is critical for the maintenance of peripheral T cell tolerance. Paracrine interactions of Fas ligand with T cells also may play an important role in the maintenance of tolerance, as Fas ligand can create immune-privileged sites and prevent graft rejection by inducing apoptosis in T cells. We surmised that APCs that express Fas ligand might directly induce apoptosis of T cells during presentation of Ag to the T cells, thus inducing Ag-specific, systemic T cell tolerance. Here, we show that profound, specific T cell unresponsiveness to alloantigen was induced by treatment of H-2k mice with H-2b APCs that expressed Fas ligand and that profound T cell unresponsiveness specific for the H-Y Ag was induced by treatment of H-2Db/H-Y TCR transgenic female mice with H-2Db/H-Y APCs that expressed Fas ligand. The induction of this systemic T cell tolerance required the expression of Fas ligand on the APCs as well as the expression of Fas on the T cells. The tolerance was restricted to the Ag presented by the APCs. The rapid and profound clonal deletion of the Ag-specific, peripheral T cells mediated by the Fas ligand-expressing APCs contributed to the induction of tolerance. These findings demonstrate that Ag-specific T cell tolerance can be induced by APCs that express Fas ligand and suggest a novel function for APCs in the induction of T cell apoptosis. Furthermore, they indicate a novel immunointervention strategy for treatment of graft rejection and autoantigen-specific autoimmune diseases.     

T cell apoptosis in human heart allografts: association with lack of co-stimulation?

It is unclear whether the intracardial immune reactivity after heart transplantation influences the peripheral immunological status (activation or nonresponsiveness) of the patient. Co-stimulation and activation-induced cell death (AICD) or apoptosis play an important role in determining the balance between lymphocyte reactivity and nonreactivity. Therefore, we studied the expression of co-stimulatory molecules and the process of apoptosis in biopsies of human heart allografts, using immunohistochemistry. Although a normal expression of co-stimulatory molecules on antigen-presenting cells was observed, the expression of their counter-structures on T cells was absent. This may be due to chronic T cell activation, which can lead to the induction of apoptosis via the Fas/Fas ligand pathway. In the infiltrates, a considerable percentage of the lymphocytes, but not the macrophages, were apoptotic. Apoptosis was confirmed by DNA fragmentation analysis. Increased numbers of Bax-expressing versus decreased numbers of Bcl2-expressing lymphocytes in comparison with normal lymphoid tissue confirmed a imbalance in favor of apoptosis. Apoptosis was biased towards CD4+ T cells (65.7% versus 26.6% in CD8+ T cells). Fas was expressed on most of the infiltrating cells. Fas ligand expression was also observed, not only on most of the T cells but also on all macrophages. Because macrophages were often detected in close contact with T cells, they may play a role in T cell regulation via the Fas/Fas ligand pathway. This study indicates that, during rejection, not only is tissue damage induced by infiltrating T cells, but also the infiltrating lymphocytes themselves are actively down-regulated (eg, AICD) by one another and by macrophages in the infiltrate. This regulatory process may affect the immunological status of the patient after heart transplantation.     

Expression of Apo-1/Fas (CD95), Bcl-2, Bax and Bcl-x in myeloma cell lines: relationship between responsiveness to anti-Fas mab and p53 functional status

The down-regulation of apoptosis may be an essential mechanism for tumour cell expansion in slowly proliferating tumours such as multiple myeloma. We studied eight myeloma cell lines for the presence of Bcl-2, which inhibits apoptosis, of Bax, which counteracts Bcl-2, of Bcl-x(L) and Bcl-x(S), which act in an anti- and pro-apoptotic fashion, respectively, and of Apo-1/Fas, which induces programmed cell death, when activated by the Apo-1/Fas ligand or the relevant monoclonal antibody (mab). All cell lines constitutively expressed homogenous amounts of Bcl-2, but displayed different amounts of Bax and Bcl-x proteins. The Apo-1/Fas antigen could be detected in seven out of eight myeloma lines, but expression levels varied considerably. The relative expression levels of Apo-1/Fas correlated with that of Bax, but not with that of Bcl-2 or Bcl-x subtypes. Furthermore, the effectiveness of the Apo-1/Fas mab was associated with the relative expression levels of the Apo-1/Fas and with that of the Bax antigen, but not with that of the Bcl-2 and Bcl-x antigens. We further showed that wild-type p53 function is not required for Apo-1/Fas-induced apoptosis, nor is it necessary for the expression of Bax or Apo-1/Fas antigens in myeloma. In conclusion, our results suggest a p53-independent co-regulation of Apo-1/Fas and Bax, as well as a role for Bax in Apo-1/Fas-induced apoptosis in myeloma.     

Tetrapeptide DEVD-aldehyde or YVAD-chloromethylketone inhibits Fas/Apo-1(CD95)-mediated apoptosis in renal-cell-cancer cells

The apoptotic machinery has been intensively investigated, and interleukin-1-beta-converting enzyme (ICE) and its homologs directly mediate apoptosis by means of their unique protease activity. Fas/Apo1 (CD95), a member of the TNF-receptor family, mediates apoptosis by binding to its ligand, which is mainly expressed on lymphocytes. Here, we investigated the expression and function of both molecules in renal-cell cancer (RCC). The expression of Fas was examined in 6 RCC cell lines by immunoblotting and all of them expressed Fas. ICE and CPP32/YAMA were also identified among the cell lines. We earlier examined ACHN cells expressing low levels of BCL-2, as well as KRC/Y cells with high levels of BCL-2. Here, we found that the anti-Fas monoclonal antibody, CH-11, induced apoptosis in a dose-dependent fashion more remarkably in ACHN cells. Pre-incubation with the tetrapeptide YVAD-chloromethyl-ketone or DEVD-aldehyde inhibited Fas-mediated apoptosis. These findings suggest that, in RCC, apoptosis is induced by lymphocytes bearing Fas-L, and that it is achieved through the proteolytic action of CPP32/YAMA and/or ICE, or another member of the ICE/ced-3 protease family.     

Potentiation of Fas-mediated apoptosis of murine granulosa cells by interferon-gamma, tumor necrosis factor-alpha, and cycloheximide

The Fas antigen is a transmembrane receptor belonging to the tumor necrosis factor-alpha (TNF) receptor family that, when activated by Fas ligand or agonistic antibodies, induces death by apoptosis. Although the presence of Fas antigen in ovarian tissues has been demonstrated, little is known about whether Fas antigen is functional in the ovary. This report shows that murine granulosa cells are initially resistant to antibody-induced Fas-mediated apoptosis, but will undergo apoptosis when cotreated with TNF and interferon-gamma (IFN) or cycloheximide (CX). Granulosa cells were obtained from follicles of 23-day-old mice 2 days after injection of PMSG. Twenty-four hours after plating, cells were pretreated with either 0 or 200 U/ml IFN, which has been shown to induce Fas antigen expression and is required for Fas-mediated killing in many cell types. At 48 h, cells were treated with 2 microg/ml control IgG, 2 microg/ml anti-Fas antigen antibody (Fas mAb), 10 ng/ml TNF, or Fas mAb and TNF. Cytotoxicity (percent killing) relative to control IgG was determined at 72 h by counting granulosa cells after trypsinization. In the absence of IFN, no cytotoxicity was observed. In the presence of IFN, neither TNF or Fas mAb alone was cytotoxic, but the combination of Fas mAb and TNF resulted in 25% killing (P < 0.05). Fas antigen messenger RNA (mRNA) was detectable in cultures not treated with cytokines and was increased 5-fold by TNF, 2-fold by IFN, and 17-fold by the combination of IFN and TNF. To test whether the presence of a labile inhibitor(s) of Fas-mediated killing in granulosa cells is the cause of resistance to Fas mAb, the protein synthesis inhibitor CX was used. Experiments were performed as described above, except that cells were treated with 0.5 microg/ml CX in conjunction with other treatments at 48 h. Fas mAb treatment in the presence of CX induced 25% cell death without IFN pretreatment and 38% with IFN (P < 0.05). TNF treatment in the presence of CX had no effect alone, but potentiated the effects of Fas mAb, resulting in 56% killing in the absence of IFN and 86% killing in the presence of IFN (P < 0.05). Cells stained positively for DNA fragmentation and annexin V binding, features characteristic of apoptosis. Because initial experiments showed that treatment with TNF alone increased Fas mRNA levels, the effect of pretreating cells for 24 h with TNF before treatment with Fas mAb was tested. Pretreatment with TNF or IFN alone did not promote Fas mAb-mediated killing, but combined pretreatment with TNF and IFN resulted in 25% killing in response to Fas mAb. Treatment of cells with the combination of IFN and TNF induced a 19-fold increase in Fas antigen mRNA levels. Corresponding increases in Fas antigen protein expression on the surface of cells in response to cytokine treatments were detected by immunocytochemistry. Human TNF did not duplicate the effects of mouse TNF in inducing Fas antigen mRNA expression and Fas mAb-induced killing. As human TNF interacts exclusively with the type I, but not the type II, TNF receptor in the mouse, potentiating effects of mouse TNF on the Fas pathway are probably mediated via the type II TNF receptor. The effects of cytokine treatments on levels of mRNA for FAP-1, an inhibitor of Fas-mediated apoptosis, were determined. FAP-1 mRNA was detectable in untreated granulosa cells, and levels were not altered by treatment with TNF and/or IFN. In summary, the Fas-mediated pathway of apoptosis is functional in mouse granulosa cells that are stimulated with IFN and TNF. These cytokines may function at least partially by increasing Fas antigen expression. Granulosa cells appear to have inhibitors of the Fas antigen pathway, as treatment with CX potentiates Fas-mediated death. TNF promotes Fas-mediated killing in the presence and absence of CX. Therefore, TNF is not likely to act simply by increasing Fas antigen expression or decreasing protein inhibitors of the Fas pathway, because TNF remains effec     

TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis in Fas ligand-resistant melanoma cells and mediates CD4 T cell killing of target cells

We have previously shown that melanoma cells were resistant to apoptosis induced by TNF family members Fas ligand (FasL), TNF-alpha, and CD40L. FasL also was not involved in CD4 T cell-mediated killing of melanoma cells. In the present study, we have tested melanoma cells for their susceptibility to apoptosis induced by human TNF-related apoptosis-inducing ligand (TRAIL) and the ability of a mAb against TRAIL to inhibit apoptosis and CD4 CTL-mediated killing of melanoma and Jurkat target cells. The results show that TRAIL-induced apoptosis in cells from 7 of 10 melanoma cell lines tested as well as in Jurkat T cells. Susceptibility to apoptosis was increased in some of the cell lines by treatment with cyclohexamide or actinomycin D. The melanoma cells were resistant to apoptosis induced by FasL, TNF-alpha, and CD40L. mAb M180 against TRAIL inhibited apoptosis induced by TRAIL. It was also found to inhibit CD4 CTL-mediated killing of Jurkat T cells as well as autologous and allogeneic melanoma cells. The degree of inhibition produced by the mAb varied between different clones of CTL and according to the susceptibility of the target cells to TRAIL-induced apoptosis. These results suggest that TRAIL is an important mediator of cell death induced by CTL and may have an important therapeutic role against human melanoma.     

Mechanisms of multiple myeloma cell growth

The mechanism of human multiple myeloma cell growth was studied utilizing eleven myeloma cell lines established in vitro or in vivo (Scid mouse). Four bone marrow derived cell lines grew dependently on IL-6 or bone marrow stromal cells. Seven extramedullary lesion derived cell lines grew spontaneously and additively proliferated in response to IL-6. All cell lines expressed the IL-6 receptor (IL-6R) and IL-6RmRNA, but none expressed IL-6mRNA. No IL-6 activity was detected in the myeloma cell culture supernatant. Both the anti-IL-6 antibody and anti-IL-6R antibody neutralized IL-6-induced proliferation, but did not inhibit spontaneous proliferation of extramedullary lesion derived cell lines. While establishing cell lines, it was found that the proliferating fraction was primarily included in a fraction which was non-adherent to stromal cells and composed of undifferentiated plasmablasts. Undifferentiated plasmablasts proliferated in response to IL-6, in contrast to the adherent, mature form of myeloma cells which did not proliferate in response to IL-6. Innoculation of myeloma cells into Scid mice induced subcutaneous tumor formation. These tumors were composed of undifferentiated plasmablasts, which also proliferated in response to IL-6. These results imply that the growth of bone marrow derived myeloma cell lines are dependent on the IL-6 paracrine mechanism and that the growth of extramedullary lesion derived cell lines primarily autonomous and additively dependent on the IL-6 paracrine mechanism.     

Fas (CD95)-transduced signal preferentially stimulates lupus peripheral T lymphocytes

Fas (CD95) is a cell surface receptor whose biological function in circulating peripheral T cells is not well understood. To address the question of abnormal T cell sensitivity to Fas stimulation in systemic lupus erythematosus (SLE), we studied Fas-transduced stimulation and apoptosis in peripheral blood T cells from patients with SLE and normal control. Immobilized anti-Fas monoclonal antibodies (mAb) (imCH-11; IgM type) significantly stimulated SLE T cell proliferation compared to T cells from normal donors and patients with rheumatoid arthritis (p < 0.003 and p < 0.005, respectively). The soluble form of CH-11 and other immobilized anti-Fas mAb (UB-2, ZB-4; IgG type) failed to stimulate lupus T cells while immobilized human Fas ligand did. Furthermore, imCH-11 induced IL-2 and IL-6 mRNA expression. However, imCH-11 activation failed to induce expression of the T cell activation surface molecules CD25 and CD69. Addition of exogenous ceramide, a second messenger for Fas-mediated apoptosis signaling, also induced T cell proliferation in SLE and normal controls. Moreover, fumonisin B1, a specific ceramide synthase inhibitor, and caspase inhibitors markedly suppressed imCH-11 induced T cell proliferation, suggesting that the ceramide pathway may be involved in Fas-transduced stimulation signals in SLE T cells. These results show that SLE T cells have an alteration in the Fas signal transduction pathway leading to cell proliferation. This defect may be important in Fas-mediated peripheral immune homeostasis.     

Human T cells require IL-2 but not G1/S transition to acquire susceptibility to Fas-mediated apoptosis

The interaction between Fas ligand and Fas, both expressed on activated T cells, is the major pathway in the regulation of activation-induced cell death. However, activated T cells that express membrane Fas are initially resistant to anti-Fas-induced apoptosis and become susceptible only after proliferation in vitro. Since IL-2 is known to regulate activation-induced cell death, we studied the effect of IL-2 on anti-Fas-mediated apoptosis. Interference with the IL-2 pathway was achieved by 1) inhibition of cytokine synthesis using cyclosporin A or FK506, 2) neutralization of IL-2 by anti-IL-2 Ab, 3) inhibition of binding to IL-2R by CD25 mAb, and 4) blocking of IL-2R signaling by rapamycin. We show that Fas expression is independent of the IL-2 pathway, whereas Fas-mediated apoptosis does not develop in the presence of inhibitors of IL-2 production or signaling. While the addition of rIL-2 reversed the inhibitory effect of cyclosporin A and FK506, the addition of rIL-4, rIL-7, or rIFN-gamma did not, although these cytokines induced progression into the S phase of the cell cycle. Aphidicolin-treated activated T cells that do not progress into the S phase were susceptible to Fas-mediated apoptosis. Therefore, Fas-mediated apoptosis is controlled by signals generated by IL-2 in agreement with the reported alteration of apoptosis in mice deficient in IL-2 or IL-2R.