scholarly journals Growth autonomy and tumorigenicity of interleukin 6-dependent B cells transfected with interleukin 6 cDNA.

1990 ◽  
Vol 171 (2) ◽  
pp. 389-400 ◽  
Author(s):  
N Tohyama ◽  
H Karasuyama ◽  
T Tada
Keyword(s):  
B Cells ◽  
B Cell ◽  
Interleukin 6 ◽  
Expression Vector ◽  
Direct Function ◽  
Transfected Cells ◽  
Selective Growth Advantage ◽  
Malignant State ◽  
Self Stimulation ◽  
Growth Autonomy

We introduced an IL-6 cDNA expression vector into a murine B cell line, the growth of which definitely required the presence of exogenous IL-6. The transfected cells secreted substantial amounts of IL-6, to which they themselves responded by proliferating without further requirement of exogenous IL-6. The proliferation was a direct function of cell density and was inhibitable by antibodies to IL-6, indicating the autocrine nature of the growth. The IL-6 cDNA-transfected cells displayed greatly enhanced tumorigenicity when inoculated into syngeneic and nude mice. Our data suggest that an IL-6 autocrine self stimulation confers on B cells a selective growth advantage and results in the induction of progression of the malignant state of B cells.

scholarly journals FRI0111 TOCILIZUMAB MAY INDUCE SECONDARY HYPOGAMMAGLOBULINAEMIA. A RETROSPECTIVE CASE SERIES OF 42 PATIENTS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 636.2-636
Author(s):  
F. Vílchez-Oya ◽  
A. Pros ◽  
I. Carrión Barberà ◽  
J. A. Meraz Ostiz ◽  
T. C. Salman Monte ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
B Cells ◽  
B Cell ◽  
Interleukin 6 ◽  
Human Monoclonal Antibody ◽  
Case Series ◽  
Infection Risk ◽  
Immunoglobulin Levels ◽  
Igg Level

Background:Tocilizumab (TCZ) is a recombinant humanized, anti-human monoclonal antibody of the immunoglobulin G1ksubclass directed against soluble and membrane-bound interleukin 6 receptors (IL-6R) [1].Interleukin-6 (IL-6) has a pleiotropic effect on inflammation, immune response, and hematopoiesis. When it was first identified, it was named as B-cell-stimulating factor 2 (BSF-2) according to its ability to induce immunoglobulin production in Epstein-Barr virus-transformed B-cell lines or in Staphylococcus aureus Cowan 1-stimulated B cells [2-4].Nowadays, it is known that IL-6 controls the survival, population expansion and maturation of B cells and plasmablasts. In that way, the regulation of Blimp-1 by STAT3 is linked to antibody secretion and is associated with long-lived plasma cells that produce large amounts of immunoglobulin. Furthermore, the ability of IL-6 to promote humoral immunity has been linked to its effects on follicular helper T cells where they promote B cell proliferation and immunoglobulin class switching [5].Objectives:Hypogammaglobulinaemia is a known complication of some immunosuppressive drugs, not previously described in patients who received therapy with monoclonal antibody against the IL-6R. We aimed to analyzed the prevalence of hypogammaglobulinaemia in our series of patients treated with tocilizumab after a carefully diagnostic workup which ruled out other causes and analyzed whether is associated with a higher risk of infection.Methods:We conducted a retrospective review from 2010 to 2019 of forty-two patients affected with a rheumatic disease and treated with TCZ at our centre. In those patients in whom we had no record of immunoglobulin levels, we determined them in the blood analysis performed by usual clinical practice.Results:42 patients were identified, from whom 38 had rheumatoid arthritis. A 31% had immunoglobulin levels prior to starting treatment with TCZ but no one had hypogammaglobulinaemia. 2 patients were excluded due to their underlying disease could justify the IgG level abnormalities. During the treatment’s follow-up, we identified that a 30% of the patients (12/40) had hypogammaglobulinaemia. Of those patients in whom immunoglobulin levels had been determined prior to starting treatment with TCZ, a 36.3% of them (4/11) developed hypogammaglobulinaemia during the follow-up. From the series, we observed a statistical significance tendency (p=0.0057) for infection risk in those patients with hypogammaglobulinaemia in contrast to those with normal IgG level (41.5% vs 14.3%, respectively).Conclusion:Secondary hypogammaglobulinaemia may occurs in patients receiving anti-IL6 agents such as tocilizumab and this could be associated with an increasing infection risk. The prevalence is not precisely known, in part because measurement of IgG prior to or during the treatment has not been a standard of care. No medical data have been previously disclosed about this possible adverse effect of anti-interleukin-6 agents. Nevertheless, ideally randomized trials are needed to assess this initial hypothesis.References:[1]Sheppard M, Laskou F, Stapleton PP, Hadavi S, Dasgupta B. Tocilizumab (Actemra). Hum Vaccin Immunother. 2017;13(9):1972–1988.[2]Tanaka T, Kishimoto T. The biology and medical implications of interleukin-6. Cancer Immunol Res. 2014;2(4):288–294.[3]Tanaka T, Narazaki M, Kishimoto T. IL-6 in inflammation, immunity, and disease. Cold Spring Harb Perspect Biol. 2014;6(10):a016295. Published 2014 Sep 4.[4]Kishimoto T. Interleukin-6: discovery of a pleiotropic cytokine. Arthritis Res Ther. 2006;8 Suppl 2(Suppl 2):S2.[5]Hunter CA, Jones SA. IL-6 as a keystone cytokine in health and disease [published correction appears in Nat Immunol. 2017 Oct 18;18(11):1271]. Nat Immunol. 2015;16(5):448–457.Disclosure of Interests:Francisco Vílchez-Oya: None declared, Ana Pros: None declared, Irene Carrión Barberà Grant/research support from: I received a grant from the Spanish Rheumatology Foundation (FER) and laboratories KERN PHARMA for a brief stay abroad., Juan Antonio Meraz Ostiz: None declared, Tarek Carlos Salman Monte: None declared, Carolina Perez-Garcia: None declared

Dysregulation of Pax5 Activity Contributes to the Extinction of the B-Cell Phenotype in Reed-Sternberg Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 517-517 ◽  
Author(s):  
Graham P Collins ◽  
Jennifer C Paterson ◽  
Gillian E May ◽  
Rajeev Gupta ◽  
Teresa Marafioti ◽  
...  
Keyword(s):  
B Cells ◽  
Protein Expression ◽  
B Cell ◽  
Binding Site ◽  
Cell Lines ◽  
Target Genes ◽  
Epigenetic Modification ◽  
Methylation Status ◽  
Cell Phenotype ◽  
Transfected Cells

Abstract Hodgkin/Reed-Sternberg cells (HRS cells) are thought to be derived from post-germinal centre B-cells and yet have down-regulated the B-cell phenotype. The B-cell transcription factor Pax5 is important in the maintenance of B-cell identity and we demonstrate that it is down-regulated in HRS cells lines and in HRS cells of the majority of primary classical Hodgkin Lymphoma (cHL) cases. Specifically, 3/30 cases were negative for Pax5, 16/30 were weakly positive, 10/30 cases were moderately positive and 1/30 showed Pax5 staining of equivalent intensity to infiltrating, polyclonal B-cells. In order to functionally test the relevance of a reduced Pax5 expression level, the cHL cell lines L428 and L1236 were stably transfected with Pax5 using a lentiviral transfection system. Transfection of L1236 resulted in up-regulation of CD79a protein expression. However, CD79a was not upregulated in L428 and expression of the Pax5 target genes Cd19 and Blnk was unaffected by Pax5 transfection in both cell lines. Chromatin immunoprecipitation demonstrated that Pax5 failed to bind the high affinity binding site within the Cd19 promoter in the cHL lines despite high levels of Pax5 expression, appropriately localised to the nucleus. Pax5 could, however, bind synthetic oligonucleotide corresponding to this site (as shown by electrophoretic mobility shift assays) raising the possibility that epigenetic modification in vivo may be responsible for the failure to bind DNA. Bisulphite genome sequencing confirmed that in cHL cell lines, the region surrounding the Pax5 binding site in the Cd19 promoter was extensively methylated. Moreover, histone modification analysis also demonstrated an absence of markers of accessible, active chromatin (di- and trimethylated H3K4) and an enrichment of a marker indicating closed, repressive chromatin (trimethylated H3K27). Within the Cd79a promoter, previous studies have implicated the methylation status of a single cytosine residue within the binding site for a Pax5-Ets1 complex to be an important determinant of activation of the Cd79a gene. Interestingly, this residue was shown to be largely methylated in L428 cells but largely unmethyated in L1236 cells, providing a likely mechanism for the differential activation of this gene by transfected Pax5 protein. To investigate whether the observed epigenetic changes were responsible for preventing Pax5 binding and activity at the Cd19 and Cd79a promoters, Pax5 transfected cHL cell lines were cultured in the presence of the demethylating agent 5-aza-2-deoxycytidine. Up-regulation of Cd19 and Cd79a expression was significantly greater in Pax5 transfected cells than in control transfected cells. To conclude: our data suggests that dysregulation of Pax5 activity (at the levels of protein expression and epigenetic modification of the Pax5 binding sites) is important in mediating the extinction of the B-cell programme in HRS cells.

TRAF3 Over-Expression Contributes to the Enhanced Alternative NF-κB Activity in Hodgkin's Lymphoma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3665-3665
Author(s):  
Feng Guo ◽  
Peng Zhou ◽  
Liang Ma
Keyword(s):  
B Cells ◽  
B Cell ◽  
Expression Vector ◽  
Conflicts Of Interest ◽  
Negative Regulator ◽  
Activity Assay ◽  
Lymphoma Cells ◽  
Aberrant Expression ◽  
Wide Range ◽  
P100 Processing

Abstract Abstract 3665 Poster Board III-601 Introduction Hodgkin and Reed-Sternberg (H-RS) cells are originated from germinal center B cells. Constitutive nuclear factor κB (NF-κB) activation is one of the molecular characteristic futures of H-RS cells. TNFR-associated factors (TRAFs) participate in a wide range of biological processes, such as adaptive and innate immunity, stress response, and bone metabolism, which are mediated by the induction of cell survival, proliferation, and differentiation. Among those, TRAF3 are reported as a negative regulator of the alternative NF-κB signaling pathway in B cells. How TRAF3 functions in H-RS cells is currently unclear. Methods Electromobility shift assay (EMSA) was performed to examine the NF-κB activity in B cell-derived Hodgkin's cells (L428 and KM-H2). An ELISA-based NF-κB family transcription factor activity assay was performed to quantify NF-κB DNA-binding in nuclear extracts from L428 cells. p100 processing, the expression of other NF-κB family members in the cytoplasm, and TRAF3 expression were detected by Western blot analysis. The effects of TRAF3 in L428 cells were studied by transient expression of TRAF3 expression vector. Results In this study, we found that TRAF3 was minimally detected in B cell-derived Hodgkin's cell lines (L428 and KM-H2) either in mRNA or protein levels. Both the classical (p50-RelA) and the alternative (p52-RelB) NF-kB activity were consistently activated in L428 cells, measured by EMSA and TransAM NF-kB activity assay. The enhanced alternative NF-κB activity, accompanied by increased p100 processing and RelB accumulation in the cytoplasm were detected in L428 cells. Transient transfection of TRAF3-expression vector enforced the expression of TRAF3 and blocked the p100 processing in L428 cells. The alternative NF-kB activity was partially decreased whereas the classical NF-kB activity remained intact. In addition, the increased TRAF3 expression did not affect the anti-apoptotic effects in L428 cells. Conclusions Not only the classical NF-κB activity but also the alternative NF-κB activity characterized by p100 processing and p52-RelB nuclear localization is constitutively activated in B cell-derived lymphoma cells. Lack of TRAF3 expression might be one of the reasons for the aberrant expression of alternative NF-κB activity. TRAF3 is indeed an important molecule regulating the activation of the alternative NF-kB activity but not the classical NF-kB activity in H-RS cells. Disclosures: No relevant conflicts of interest to declare.

ZAP-70 Enhances Migration of Malignant B Cells towards Lymphoid Organs in a Burkitt Lymphoma Xenograft Model

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2844-2844
Author(s):  
Noelia Purroy ◽  
Eva Calpe ◽  
Pau Abrisqueta ◽  
Cecilia Carpio ◽  
Carles Palacio ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cells ◽  
Tumor Growth ◽  
B Cell ◽  
Cell Lines ◽  
Ectopic Expression ◽  
Xenograft Model ◽  
Lymphoid Organs ◽  
Transfected Cells

Abstract Abstract 2844 Introduction. ZAP-70 (ξ-associated protein) is a protein tyrosine kinase of the Syk/ZAP family that plays a crucial role in cellular activation in T and NK cells. High expression of ZAP-70 protein in malignant cells from Chronic Lymphocytic Leukemia (CLL) correlates with adverse clinical prognostic features, such as unmutated IgHV genes, short time to progression, and short survival. Moreover, ZAP-70 protein has been related to aggressive features of the CLL cells, such as enhanced B-cell receptor (BCR) signaling and higher migration capacity. To further investigate into the mechanisms by which ZAP-70 protein influences the clinical outcome of patients with CLL, we analyzed the functional consequences of ZAP-70 ectopic expression in malignant B-cells. For this, Ramos and Raji (Burkitt) B-cell lines were stably transfected with a ZAP-70 expressing vector (pEGFP-N2ZAP-70). Raji transfectant showed constitutively phosphorylated ZAP-70 protein, whilst Ramos cells required stimulation with 5 μg/ml F(ab') 2 anti-IgM to get ZAP-70 activated. ZAP-70 expression induced the upregulation of the chemokine receptor CCR7, thus giving the cells the ability to better respond and migrate towards CCL21 (own data, Blood 2011 pre-published). CCR7 ligands (chemokines CCL21 and CCL19) are mainly expressed in high endothelial venules and the T zones from secondary lymphoid organs. The aims of this study were firstly to evaluate in vivo the migratory/invasive capability of pEGFP-N2ZAP-70 transfected Raji and Ramos cell lines compared to pEGFP Raji and Ramos cell lines; and later, to compare the overall survival (OS) of mice injected with pEGFP-N2ZAP-70 transfected cells to those injected with only pEGFP transfected cells. Methods. For this, a total of 27 7- to 8-week old SCID (CB17Crl) mice were used. Mice were inoculated intravenously with 5×106 cells of each cell line (6 mice with Raji-GFP, 5 mice with Raji-GFP-ZAP-70, 5 mice with Ramos-GFP and 10 mice with Ramos-GFP-ZAP-70). Mice were observed for the onset of hind legs paralysis, dyspnea, or evidence of tumor growth, once symptoms appeared, mice were euthanized and lymphoid and non-lymphoid organs were obtained for further analysis of the presence of GFP-positive cells by flow cytometry and immunohistochemistry. Results. Twenty-six out of twenty-seven injected mice were included in the analysis. The excluded mouse was found dead before it could be euthanized to obtain the organs. In the Raji xenograft model, 11/11 (100%) of mice had hind legs paralysis as the first symptom to appear. The median survival was 19 days for GFP-ZAP-70 and 16 days for GFP injected mice. There were no statistically significant differences between survival of GFP-ZAP-70 and GFP injected mice (OS was 66.7% [95% CI 38.4–100] vs 33.3% [95% CI 0–71.1], p=0.784, at 19 and 16 days, respectively). In the Ramos xenograft model, 6/15 (40%) of mice showed hind legs paralysis as the first symptom to appear, as well as evidence of abdominal tumor growth in 6/15 (40%), whereas in 3/15 (20%) the established event was dyspnea. The median survival in Ramos xenograft model was 40 days for GFP-ZAP-70 and 38 days for GFP injected mice. Again there were no statistically significant differences between survival of GFP-ZAP-70 and GFP Ramos injected mice (OS was 50% [95% CI 18.4–81.6] vs 40% [95% CI 0–83.8], p=0.180, at 40 and 38 days, respectively). By flow cytometry analysis of GFP cells we found that in the Raji xenograft model there were statistically significant differences between the migration of GFP-ZAP-70 and GFP injected cells towards bone marrow (21.5% vs 5.17, p=0.011), spleen (0.08% vs 0.01%, p=0.006) and thymus (0.00% vs 0.02%, p=0.037). The highest percentages of GFP positive cells were found in bone marrow samples (mean, 9.85%), whereas in spleen and thymus the percentages of GFP positive cells were all below 0, 1%. There was no statistically significant difference between the cellular migration in the Ramos xenograft model in any of the organs analyzed. Conclusion. In conclusion, malignant B-lymphocytes with ectopic expression of activated ZAP-70 protein show enhanced ability to migrate towards and infiltrate lymphoid organs in a xenograft model, specially the bone marrow, although it does not translate into a worse survival of the animals. Further specific immunohistochemical assays to determine infiltrated areas by ZAP-70 expressing lymphocytes are in process. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus Latency Locus Compensates for Interleukin-6 in Initial B Cell Activation

2015 ◽  
Vol 90 (4) ◽  
pp. 2150-2154 ◽  
Author(s):  
Sang-Hoon Sin ◽  
Sun Ah Kang ◽  
Yongbaek Kim ◽  
Anthony Eason ◽  
Kelly Tan ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Interleukin 6 ◽  
Cell Activation ◽  
Marginal Zone ◽  
Kaposi Sarcoma ◽  
B Cell Activation ◽  
Marginal Zone B Cells ◽  
Survival Factor

Interleukin 6 (IL-6) is considered a proliferation and survival factor for B cells. To assess the role of IL-6 in Kaposi sarcoma-associated herpesvirus (KSHV) latency, KSHV latency locus-transgenic mice (referred to as latency mice) lacking IL-6 were evaluated. IL-6−/−latency mice had the same phenotypes as the latency mice, i.e., increased frequency of marginal zone B cells, hyperplasia, and hyperglobulinemia, indicating that the KSHV latency locus, which includes all viral microRNAs (miRNAs), can compensate for lack of IL-6 in premalignant B cell activation.

Interleukin-6 production by murine B cells and B cell lines

1991 ◽  
Vol 132 (2) ◽  
pp. 442-450 ◽  
Author(s):  
Monte V. Hobbs ◽  
Robert J. McEvilly ◽  
Rick J. Koch ◽  
Gustavo J. Cardenas ◽  
Daniel J. Noonan
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Interleukin 6

scholarly journals Identification of upstream signals regulating interleukin-6 gene expression during in vitro treatment of human B cells with pokeweed mitogen

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2243-2252 ◽  
Author(s):  
D Chauhan ◽  
S Kharbanda ◽  
H Uchiyama ◽  
M Urashima ◽  
R Fragoso ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Map Kinase ◽  
Interleukin 6 ◽  
Early Response ◽  
Immediate Early ◽  
Pokeweed Mitogen ◽  
Mrna Induction ◽  
Early Response Genes

Abstract The present studies have characterized the regulation of interleukin-6 (IL-6) gene expression during pokeweed mitogen (PWM)-driven human B- cell differentiation. PWM induced an early and transient increase in the expression of immediate-early response genes of the jun/fos leucine zipper family (c-jun, jun B, c-fos, and fos-B). The induction of c-jun mRNA by PWM was concentration dependent. Nuclear run-on assays showed that PWM treatment is associated with an increased rate of c-jun gene transcription. The induction of c-jun mRNA precedes the induction of IL- 6 gene expression and IL-6 secretion by the B cells. c-Jun antisense, but not sense, oligodeoxynucleotide (ODN) significantly decreases PWM- related B-cell (1) proliferation; (2) IL-6 mRNA induction; (3) IL-6 secretion; and (4) nuclear extract binding to AP-1 in electrophoretic mobility shift assay. In contrast, c-Fos anti-sense ODN did not effect either IL-6 mRNA induction or IL-6 secretion triggered in B cells by PWM. The results further show activation of c-Raf-1 kinase in PWM- treated B cells. Raf-1 acts upstream to mitogen-activated protein (MAP) kinase; therefore, studies were performed to assay for MAP kinase activation in these cells. The results show an increase in phosphorylation of myelin basic protein (MBP) and c-Jun “Y” peptide in PWM-treated B cells. Taken together, these findings suggest that PWM is able to initiate an intracytoplasmic signaling cascade in normal human splenic B cells, which, at least in part, involves serine/threonine protein kinases. These results show transient induction of immediate- early response genes in B cells and support a potential role for the c- jun gene product in regulation of IL-6 transcription and secretion.

scholarly journals Regulation of B-cell growth and immunoglobulin gene transcription by interleukin-6

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 452-459 ◽  
Author(s):  
JE Tanner ◽  
G Tosato
Keyword(s):  
B Cells ◽  
B Cell ◽  
Interleukin 6 ◽  
Cell Activation ◽  
Cell Number ◽  
Immunoglobulin Gene ◽  
Rna Transcription ◽  
Barr Virus ◽  
Dna And Rna ◽  
Cycle Dependent

Interleukin-6 (IL-6) stimulates growth and immunoglobulin (lg) secretion in Epstein-Barr virus (EBV)-infected B cells. In this study, we demonstrate that B-cell activation by IL-6 is associated with an initial induction of c-myc, a gene believed to act as a competence factor for increased RNA transcription and DNA replication, and by increases in DNA, RNA, and protein synthesis, as well as cell number. IL-6 increased the levels of lg mRNA per cell in comparison to a non- cycle-dependent cellular mRNA, tubulin. However, two other cell cycle- dependent cellular mRNAs, c-myc and actin, were also induced by IL-6 comparable to lg mRNAs. Increased levels of lg mRNA were not due to significant changes in RNA turnover, but appeared to reflect increased levels of RNA transcription. Together, these findings support the notion that IL-6 plays an important role as a stimulator of DNA and RNA synthesis in EBV-activated B cells.

scholarly journals Autocrine growth and tumorigenicity of interleukin 2-dependent helper T cells transfected with IL-2 gene.

1989 ◽  
Vol 169 (1) ◽  
pp. 13-25 ◽  
Author(s):  
H Karasuyama ◽  
N Tohyama ◽  
T Tada
Keyword(s):  
T Cells ◽  
Expression Vector ◽  
Interleukin 2 ◽  
Tumor Development ◽  
Latency Period ◽  
Helper T Cells ◽  
Autocrine Growth ◽  
Direct Function ◽  
Transfected Cells

We introduced a mouse IL-2 cDNA expression vector into an IL-2-dependent mouse helper T cell line HT-2. Transfected cells secreted substantial amounts of IL-2, to which they themselves responded by proliferating without further requirement for exogenous IL-2. The proliferation was a direct function of the cell density and was inhibitable by antibodies against IL-2 or IL-2-R, indicating the autocrine nature of the proliferation. Those producing higher amounts of IL-2 were found to be tumorigenic when inoculated into nude mice. The latency period of tumor development correlated inversely with the level of IL-2 secreted. Tumor cells proliferated in vitro in an IL-2 autocrine fashion indistinguishable from that of the inoculated cells. We thus provide evidence that the aberrant activation of the IL-2 autocrine circuit can lead T cells to malignant transformation.

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