Expression of the IL-6 gene induces differentiation of a human monoclonal EBV-transformed B cell line.

To study the role of interleukin (IL)-6 as a growth and differentiation factor for Epstein-Barr virus (EBV)-transformed B lymphocytes, we transfected the cDNA coding for human IL-6 in a monoclonal IgG1-secreting EBV B cell line. Two independent clones were selected that constitutively secreted high amounts of IL-6. These clones showed enhanced levels of IL-6 and tumor necrosis factor alpha secretion when compared to non-IL-6 transfected controls. Moreover, they could efficiently be recovered from low cell density cultures in limiting dilutions when plated on a feeder layer of heterologous EBV B cells. IL-6-induced phenotypical changes comprised a significant rise in immunoglobulin secretion levels and enhanced membrane expression of CD25 (the beta chain of the IL-2 receptor) and of the B cell differentiation antigen CD40. IL-6-dependent down modulation of CD38 and of the adhesion structure VLA4 were also observed. Our data support the notion that IL-6 can serve as an growth and differentiation factor for EBV B cells.

Protein kinase C regulates secretion of lymphotoxin (LT/TNF beta) and TNF alpha by human EBV-transformed B cells.

Epstein-Barr virus (EBV)-transformed cell lines constitutively secrete lymphotoxin (LT/TNF beta) and not tumor necrosis factor-alpha (TNF alpha). To analyze the cellular processes that regulate LT and TNF alpha secretion by lymphoblastoid cell lines, we studied the role of two signal transduction pathways leading to either protein kinase C (PK-C) or PK-A activation. We demonstrate that PK-C activation, either after cross-linking of surface Ig or by direct activation with phorbolester, leads to increased production of both LT and TNF alpha, whereas no prominent role for PK-A was found. Interleukin (Il)-4 was found to synergize with PK-C activation in raising levels of secreted LT and TNF alpha. Increased levels of LT and TNF alpha did not correlate with augmented levels of immunoglobulin secreted by the cell lines nor with improved proliferation. These observations demonstrate that EBV B cells respond to B cell activation signals leading to PK-C activation with increased production of both LT and TNF alpha. It is, however, unlikely that these molecules serve as autostimulatory factors for EBV B cells, but in contrast might play a role in downregulation of biological functions in these cells.

Effects of IL-4, IL-5, and IL-6 on growth and immunoglobulin production of Epstein-Barr virus-infected human B cells.

In the present study we investigated whether interleukin-4 (IL-4), IL-5, and IL-6 could enhance the efficiency of Epstein-Barr virus (EBV) transformation for the generation of specific human monoclonal antibody (HuMAb)-producing B-cell lines directed against erythrocyte Rhesus(D) antigen. In newly EBV-infected B cells, IL-4 and IL-6 caused a comparable enhancement of proliferation and of total IgG and IgA production. IL-6 showed a much stronger effect than IL-4 on IgM production, whereas IL-4 was unique in inducing IgE production. No stimulatory effects of IL-5 on either growth or Ig production were observed. Although addition of IL-6 resulted during the early phase after EBV infection in high numbers of Ag-specific antibody-producing wells, this did not result in an increased number of stable HuMAb-secreting cell lines. When the effects of cytokines were tested on established polyclonal EBV B cells, in a high cell density culture system, only IL-6 was able to enhance Ig secretion, while no effect could be demonstrated on proliferation. These studies substantiate that IL-6 is an important regulator of proliferation and Ig production, and that it acts at distinct stages after EBV infection, but does not increase the final overall recovery of Ag-specific EBV B-cell lines.

Heterogeneity in both cytokine production and responsiveness of a panel of monoclonal human Epstein-Barr virus-transformed B-cell lines.

To optimize growth and Ig production of in vitro-cultured Epstein-Barr virus (EBV)-transformed B cells, a panel of six monoclonal EBV B-cell lines was analyzed for autocrine growth factor production and responsiveness to various cytokines. Three cell lines produced Il-I and four produced Il-6, although differences concerning the amount of lymphokines produced were observed. Interestingly a considerable tumor necrosis factor beta (lymphotoxin) activity was found in supernatants of all cell lines. One IgM-producing cell line that did not secrete either Il-1 or Il-6 was exceptional in its ability to respond to the addition of rIl-6 with a 5- to 10-times elevated IgM production. In contrast, cell lines in the panel capable of Il-6 production showed only a minimal elevation of Ig production on addition of exogenous Il-6. Ig production was slightly less in some cell lines when Il-6 was neutralized. Antibodies against lymphotoxin or Il-6 did not influence growth rate of the cell lines significantly, implying that neither Il-6 nor lymphotoxin had an autostimulatory effect on the analyzed cell lines. This study demonstrates a heterogeneity regarding the amount and type of lymphokines produced by long-term monoclonal EBV cell lines, which may account for the diverse responses exhibited by these cells towards exogenously added lymphokines.

Effect of IL-6 on proliferation and IG production of human EBV-transformed cell lines in serum free culture media.

To optimalize growth and Ig production of EBV transformed B cells for large scale tissue culture, we analyzed five stable monoclonal EBV-B cell lines for their responsiveness to interleukin (IL)-6 in standard medium with 5% FCS and in several serum-free media. As we previously demonstrated these cell lines were found to be heterogeneous with regard to their production of Il-1, Il-6 and lymphotoxin. We could not demonstrate an effect of Il-6 on proliferation on any of the cell lines in either standard medium with 5% FCS or in any of the serum-free culture media. In standard medium, cell lines capable of Il-6 production showed a slight elevation of Ig production in response to exogenous Il-6. Only one cell line, that did not secrete any of the lymphokines tested, responded strongly to Il-6 with a 12 times elevated IgM production. In contrast, in serum-free medium supplemented with Bovine Serum Albumin (BSA), Il-6 raised Ig secretion of all cell lines. This study shows that in some serum-free culture media EBV cell lines can be propagated as well as in standard medium with FCS. Responsiveness of these cells to cytokines is heterogeneous and is at least partially influenced by the culture medium used.

Structure and nucleotide sequence of the region encoding the mobilization proteins of plasmid CloDF13.

Mobilization of the non-conjugative plasmid CloDF13 requires both gene products of a conjugative plasmid and CloDF13-encoded information. About 30% of the CloDF13 genome is involved in plasmid transfer. The CloDF13 mobilization region comprises sequences acting in cis (bom, basis of mobilization) as well as protein-coding sequences (mob). Here we present the nucleotide sequence of the genes encoding the CloDF13 mobilization proteins. We confirmed the previous genetic data that the plasmid CloDF13 encodes two proteins involved in plasmid mobilization. The information for these proteins, designated B and C having Mrs of 57,890 and 15,870, respectively, is located within one operon directed by a promoter at 94% of the CloDF13 genome. The gene encoding the smaller protein is located distally within this operon. Transcription proceeds counter-clockwise and is terminated beyond gene C, although it can not be excluded that attenuation of the transcript occurs in the intergenic region. The role of the CloDF13 mobilization proteins in plasmid transfer will be discussed.