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Biomedical subjects

A Rolink

Publications and source records attributed to A Rolink.

At least 73 records · Page 4Linked to original sources
PubMed1993

Rearrangement and expression of kappa light chain genes can occur without mu heavy chain expression during differentiation of pre-B cells.

The kinetics of kappa light (kappa L) chain gene rearrangement and expression on mRNA and protein level has been studied with four stromal cell/IL-7 reactive, long-term in vitro proliferating pre-B cell lines and clones, two from fetal liver of normal mice and two from fetal liver of E microH-bcl-2 transgenic (bcl-2-tg) mice. These pre-B cell lines and clones are DJH-rearranged on both H chain alleles. Two of the clones harbor H chain rearrangements which do not allow the expression of VHDJH rearranged H chain genes as microH chain proteins. Upon removal of IL-7 from the pre-B cell cultures all four cell lines rearrange VH-DJH and VL-JL gene segments, loose the surface expression of c-kit, CD43, and surrogate light chain, as well as the capacity to be clonable on stromal cells in the presence of IL-7. Pre-B cells from normal mice die by apoptosis during differentiation, while those from bcl-2-tg mice do not. All four lines and clones express comparable levels of mRNA for microH and kappa L chains with the same time kinetics during 3 days of differentiation. However, only two of the four pre-B cell lines and clones express microH chain protein, whereas all four pre-B cell lines and clones express kappa L chain protein at comparable levels between 2 x 10(5) and 1.4 x 10(6) kappa L chain molecules per cell. These results suggest that microH chain expression is not mandatory for rearrangement and normal expression of kappa L chain genes when pre-B cells differentiate to B cells.

Animals↗
PubMed1992

A novel regulatory myosin light chain gene distinguishes pre-B cell subsets and is IL-7 inducible.

We describe a novel regulatory myosin light chain gene (termed precursor lymphocyte-specific regulatory light chain or PLRLC) that is expressed specifically in precursor B and T lymphocytes. PLRLC is the first example of a regulatory myosin light chain gene which displays specific expression in non-muscle cells. PLRLC is expressed in adult bone marrow derived normal and transformed pre-B cells; in the former, PLRLC expression levels are induced by the pre-B cell specific growth factor interleukin-7 (IL-7). PLRLC is not expressed in either transformed pre-B cells derived from fetal liver or in normal fetal liver pre-B clones grown in the presence of IL-7. Therefore this gene provides the first marker that clearly distinguishes these two pre-B subsets. Finally, several of the different PLRLC transcripts potentially encode regulatory myosin light chains with unique structural features. The unique distribution, regulation and structural features of the PLRLC gene products suggest an important role for PLRLC during lymphocyte development.

Amino Acid Sequence↗
PubMed1992

The expression of the mouse VpreB/lambda 5 locus in transformed cell lines and tumors of the B lineage differentiation pathway.

The expression of RNA transcripts from two pre B lymphocyte related genes, VpreB and lambda 5, has been studied in a series of transformed cell lines which appear frozen at different states of B lineage differentiation, from early progenitors to surface Ig positive B cells. In the HAFTL-1 cell line, which arose from fetal liver by transformation with a retrovirus containing the Hras oncogene, Northern analysis of poly A+ mRNA as well as in situ hybridization of RNA in single cells revealed that lambda 5 and VpreB are already expressed at the progenitor stage and increase in expression as the progenitors differentiate to precursor (preB) cells, or are turned off as the progenitors differentiate to myeloid cells. Continued rearrangements of Ig genes in pre B cell lines leading to Ig expression on the surface of NFS-5 pre B cells do not influence the continued expression of VpreB and lambda 5. Surface Ig-positive B lineage cell lines also express the pre B-related genes. Both Ly1+ as well as Ly1- pre B cells are VpreB- and lambda 5-positive. Lipopolysaccharide (LPS) stimulation of 70Z/3 pre B cells does not turn off lambda 5 expression. It therefore appears that, at least in transformed cell lines, the expression of VpreB and lambda 5 is not directly regulated by the expression of microH, kappa L, or lambda L chains, LPS reactivity, or the Ly1 surface antigen. Fusion of plasmacytoma cells with normal pre B cells to generate pre B hybridomas leads to down-regulation of VpreB/lambda 5 expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Amino acid sequence analysis of a mouse interleukin 5 receptor protein reveals homology with a mouse interleukin 3 receptor protein.

A polypeptide chain for the mouse interleukin 5 receptor (IL5R) was purified from detergent-lysed B13 cells, a mouse IL5-dependent pre-B cell line. Purification was by a single immunoaffinity chromatographic step using an anti-mouse IL5R monoclonal antibody, R52. Internal amino acid sequence was obtained from four trypsin-generated peptides. All peptides were found to be present in the published amino acid sequence of a mouse IL3R and the mouse IL3R-like protein deduced from the cDNA. This indicates that the mouse IL5R and the mouse IL3R have a homologous polypeptide in common and suggests that the specificity of these lymphokine receptors is mainly generated by association with another ligand-specific polypeptide chain.

Amino Acid Sequence↗
PubMed1991

The c-kit-encoded tyrosine kinase regulates the proliferation of early pre-B cells.

A monoclonal antibody (mAb; ACK2) recognizing the extracellular domains of the c-kit-encoded tyrosine kinase has been employed to demonstrate that c-kit is involved in B lymphocyte development. The c-kit-encoded tyrosine kinase is expressed on the surface of normal DHJH-rearranged murine pre-B cell clones which proliferate continuously at that stage in vitro on stromal cells and in the presence of recombinant interleukin 7. These pre-B cell clones, capable of differentiation to surface immunoglobulin-positive B cells in vitro and in vivo, are inhibited by the mAb in their proliferation while remaining capable of differentiation to surface immunoglobulin-positive B cells. Stimulation of mature B cells by mitogens is unimpaired by the mAb. This indicates that c-kit regulates early antigen-independent, but not late antigen-dependent, B cell development.

Animals↗
PubMed1991

The kappa/lambda ratio in surface immunoglobulin molecules on B lymphocytes differentiating from DHJH-rearranged murine pre-B cell clones in vitro.

The expression of kappa and lambda light chains in surface immunoglobulin (sIg) molecules on B lymphocytes differentiating from murine pre-B cell clones in vitro was analyzed. The four pre-B cell clones used represent a very early pre-B cell stage. They have their heavy chain loci DJ rearranged and their light chain loci in germ-line configuration. In order to grow in vitro, these clones require contact with stromal cells and the stimulatory activity of interleukin (IL) 7. Upon removal of IL 7 from the cultures, these clones differentiate within 3 days into sIg+ B cells. Between 7% and 12% of IgM+ B cells could be detected in these cultures. The majority (78%-92%) of the IgM+ B cells co-expressed kappa light chains. The percentage of lambda light chain expressing B cells was below detectable level. Upon lipopolysaccharide (LPS) stimulation, the percentages of IgM+ B cells increased dramatically (from 32%-64%). The majority (91%-97%) of the IgM+ B cells express kappa chains, but a very small percentage (3.1%-5.0%) express lambda. A similarly high kappa/lambda ratio was found in 418 hybridomas prepared from these LPS-stimulated B cells (388 kappa+ and 30 lambda+). Thus, the high kappa/lambda ratio characteristic of the mouse peripheral B cell repertoire is already evident in the antigen-independent transition from pre-B to B cells.

Animals↗
PubMed1991

Long-term proliferating early pre B cell lines and clones with the potential to develop to surface Ig-positive, mitogen reactive B cells in vitro and in vivo.

Cell lines and clones were established from PB76-positive mouse fetal liver at day 13 and 14 of gestation, which proliferated with division times of a day in serum-substituted cultures under the stimulatory influence of adherent stromal cells and the cytokine IL-7 for periods longer than half a year. These lines expressed varying levels of the B lymphocyte lineage related markers PB76, B220, BP-1, VpreB and lambda 5, but no surface Ig or MHC class II molecules. All clones expressed PB76, VpreB and lambda 5 in a high percentage of cells, while B220 and/or BP-1 expression was low or undetectable in some. A cell line, and several clones established from it, all had kappa and lambda light chain genes in germ-line configuration. Either one or both of their H-chain-gene containing chromosomes carried a DH to JH. These pre B cell lines and clones could be induced to VH to DH and VL to JL rearrangements. This resulted in the development of varying percentages of sIg-positive surface, MHC class II negative, LPS-reactive B cells within 2-3 days, in the absence of contacts with stromal cells and/or IL-7. When injected into SCID mice, the cultured pre B cells populated the spleen of these mice to 5% with surface Ig-, MHC class II-positive LPS-reactive cells for greater than 25 weeks. The long-term in vitro proliferative capacity of these DH-JH rearranged pre B cell clones makes them major candidates for committed stem cells of the B lineage.

Animals↗
PubMed1990

Expression of the murine interleukin-5 receptor on Xenopus laevis oocytes.

In this study we describe the use of Xenopus laevis oocytes for the detection of mRNA coding for a murine interleukin-5 (mI15) receptor. When injected with sucrose gradient fractionated polyA+ RNA derived from the murine 115-dependent pre B cell line B13, these oocytes could specifically bind 35S-methionine labeled mI15. A size of approximately 4000 nucleotides (25S) was estimated for the mRNA corresponding to the mIL5-binding activity. This binding was not blocked by a monoclonal antibody R52 specific for the MI15-receptor, suggesting that the oocytes express a different form of this receptor.

Animals↗
PubMed1989

One synchronous wave of B cell development in mouse fetal liver changes at day 16 of gestation from dependence to independence of a stromal cell environment.

Precursor cells of the B lineage can be enriched from mouse fetal liver by FACS with the aid of the pre-B cell-specific mAb G-5-2. The cells are concomitantly enriched for cells expressing the pre-B cell-specific gene lambda 5, and for cells developing to LPS-reactive mature B cells. The enriched purified precursors are not influenced by rIL-2 through -7, alone or in combination, to develop to mitogen-reactive, sIg+ cells. Marginal proliferation of the precursors is observed in response to IL-3 plus -4, and IL-6 plus -7, and this does not change in the presence of stromal cells. Development to mitogen-reactive, sIg+ cells is dependent on interactions with embryonic stromal cells from fetal liver. Two mAbs raised against the stromal cells inhibit this development. Two phases of precursor cell development can be distinguished in fetal liver. Between days 13 and 15 of gestation, it is dependent on stromal cell interactions, thereafter, from days 16 to 19, it is independent. A sudden increase in the number of mitogen-reactive, sIg+ B lineage cells occurs within 24 h between days 16 and 17. All these results indicate that B cell development occurs in one wave with synchronous steps of changes from a mitogen-insensitive, sIg-, stromal cell dependent to a mitogen-reactive, sIg+, stromal cell-independent B lineage line.

Animals↗
PubMed1989

The epigenetic influences of bone marrow and fetal liver stroma cells on the developmental potential of Ly-1+ pro-B lymphocyte clones.

The Ly-1+ Mac-1+ B-220+ CC11+ progenitor clones LyD9 and LyB9 were previously shown to give rise to Ly-1+IgM+ B lymphocytes either in vivo or in vitro by co-culture with nonlymphoid accessory cells from spleen and lipopolysaccharide. The clones did not generate T lymphocytes either in vivo or in vitro. We now find that both LyD9 and LyB9 progenitors are induced to differentiate in vitro by co-culture with the RP.0.10 bone marrow stroma clone or with heterogeneous marrow-adherent stroma cell populations obtained from adult mice into Ly-1-IgM+ B lymphocytes as well as myeloid GM1.2+ Mac-1+ cells. We could obtain evidence that a high proportion of LyD9 and LyB9 cells already switched off expression of Ly-1 and CC11 (interleukin 3 receptor) surface molecules 2 days after initiation of the cultures, and by days 8-10 of culture no detectable Ly-1+ cells and only about 20% CC11+ cells were observed. Ly-1 surface expression could not be re-induced on the progeny of LyD9 and LyB9 progenitors generated under the influence of marrow stroma cells. Remarkably, the LyD9 and LyB9 progenitors gave rise to both Ly-1+IgM+ and Ly-1-IgM+ B cells upon culture with heterogeneous stroma monolayers obtained from 18-day fetal liver. Finally, the differentiating property of the stroma cells for the LyD9 and LyB9 progenitors could not be replaced with soluble factors produced either spontaneously or after stimulation by the marrow stroma cells. Our results show the importance of epigenetic influences provided by a given microenvironment on the developmental potential of B cell progenitors. They provide direct evidence that the same pro-B lymphocyte can give rise to both Ly-1+ and Ly-1-IgM+ B cells depending on both the time of development and the tissue of origin of stroma cells with which the B cell progenitor interacts. Also, the results strongly suggest that cell contact between the stroma cell and the B cell progenitor is essential to induce rearrangement and expression of the Ig genes in pro-B lymphocytes.

Animals↗
PubMed1989

Cellular stages and molecular steps of murine B-cell development.

Development of B cells in fetal liver occurs in one synchronous wave and involves probably no more than four critical divisions. This leads us to suggest that the main pool of proliferating progenitors that replenish the peripheral B-cell pool are progenitors before Ig gene rearrangement, that the four Ig gene rearrangements (DH to JH, VH to DHJH, VK to JK, and V lambda to J lambda) might occur in four critical divisions, and that a stromal-cell-dependent phase of pre-B development in which all rearrangements are made is succeeded by a stromal-cell-independent phase of sIG+ pre-B-cell maturation to mature mitogen-reactive B cells. We speculate on the molecular nature of the tightly controlled steps of Ig rearrangements during pre-B-cell development that might involve the pre-B-cell specific genes Vpre-B and lambda 5 and the B-lineage-specific gene mb-1 in interactions with stromal cells.

Animals↗
PubMed1988

Recombinant interleukin 2 or 5, but not 3 or 4, induces maturation of resting mouse B lymphocytes and propagates proliferation of activated B cell blasts.

Plasmacytoma transformants of the X63-Ag8-653 cell line carrying an expression vector with either IL-2, -3, -4, or -5 cDNA were established that secrete the corresponding ILs at high rates. The four mouse ILs (mILs) were then tested as single ILs and in combinations for their effects on the maturation of resting and proliferation of activated normal mouse splenic B cells. mIL-3 and mIL-4 were inactive in all assays. mIL-2, as well as mIL-5, synergized with Ig-specific antibodies and B cell growth factor alpha (BCGF-alpha) to stimulate successive rounds of B cell division with LPS-activated B cells. This activity as BCGF-beta was effective at concentrations similar to those at which mIL-2 induced proliferation of the CTL-L T cell line, indicating a high-affinity interaction of both mIL-2 and mIL-5 with their corresponding receptors on activated B cells. mIL-5 and maybe IL-2 also induced maturation of resting B cells to Ig-secreting cells without proliferation. This B cell maturation factor (BMF) activity of mIL-5 was as effective as its BCGF-beta activity, while the BMF activity of mIL-2 was at least 10(2)-fold less effective. BMF activity of mIL-2, but not mIL-5, was blocked by anti-Il-2-R antibodies, indicating that mIL-2 and mIL-5 use separate receptors for B cell signaling. mIL-2, as well as mIL-5, furthermore, acted as filler activities when proliferation in the presence of Ig-specific antibodies and BCGF-alpha was measured with as little as 500 B cells. In the case of mIL-5, this was also true for maturation of that few cells. Limiting dilution analyses showed that approximately 1-2% of the resting B cells matured without division, while 30-100-fold fewer cells (0.03-0.06%) proliferated and matured in response to IL-5. A single IL, therefore, is capable of inducing maturation and of stimulating mitotic cell cycle progression of normal B cells.

Animals↗