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Biomedical subjects

F Melchers

Publications and source records attributed to F Melchers.

At least 91 records · Page 5Linked to original sources
PubMed1994

The structure of an alternate form of complement C3 that displays costimulatory growth factor activity for B lymphocytes.

In this study, the structure of a novel 1.9-kb transcript coding for complement component 3 (C3) is described. This alternate C3 is identical to the 3' end of the C3 message beginning at position 3300 of the C3 cDNA. Its transcription appears to be driven by an alternate promoter located within intron 8 of the C3 gene. This alternate C3 message contains an open reading frame that may encode a 536-amino acid-long protein identical to the 3' part of the C3 alpha chain. The resulting protein contains the complement receptor CR2 binding site. The suggested 5' end of coding region of the alternate C3 includes information for a potential hydrophobic leader peptide that would allow secretion of the protein. In vitro assays with macrophage-depleted mouse splenic B cells indicate that an activity is secreted from cell lines transfected with the alternate C3 cDNA. Together with Sepharose-bound immunoglobulin M-specific monoclonal antibodies and interleukin 2, it costimulates the proliferation of B cells. Implications for possible in vivo functions are discussed.

Alternative Splicing↗
PubMed1994

Influence of immunoglobulin heavy- and light-chain expression on B-cell differentiation.

To study the influence of immunoglobulin heavy-chain (HC) and light-chain (LC) expression in promoting B-cell differentiation, we have introduced functional immunoglobulin HC and/or LC transgenes into the recombinase activating gene-2-deficient background (RAG-2-/-). RAG-2-/- mice do not undergo endogenous V(D)J rearrangement events and, therefore, are blocked in B- and T-cell development at the early pro-B- and pro-T-cell stages. Introduction of immunoglobulin HC transgenes into the RAG-2-/- background promotes the development of a B-lineage cell population that phenotypically has the characteristics of pre-B cells. We have shown further that this population has altered growth characteristics as measured by interleukin-7 responsiveness in culture. Bone marrow cells from immunoglobulin HC transgenic RAG-2-/- mice have up-regulated expression of germ-line kappa LC gene transcripts and down-regulated expression of lambda 5 surrogate LCs (SLCs). Although mu HC/SLC complexes are detectable intracellularly in HC/RAG-2-/- pre-B-cell populations, HC expression is not readily detectable on the surface of these cells. lambda LC RAG-2-/- mice had a bone marrow B-lineage cell phenotype indistinguishable from that of RAG-2-/- littermates, indicating that LC expression by itself has no influence on pro-B cell differentiation. Strikingly, simultaneous introduction of mu HC and lambda LC transgenes into RAG-2-/- mice led to the generation of a substantial population of "monoclonal" peripheral B-cells that were functional with regard to immunoglobulin secretion, indicating that T cells or diverse immunoglobulin repertoires are not necessary for peripheral B-cell development.

Animals↗
PubMed1994

The expression of Vpre-B/lambda 5 surrogate light chain in early bone marrow precursor B cells of normal and B cell-deficient mutant mice.

Precursor B (pre-B) cells in bone marrow of normal and B cell-deficient mutant mice were analyzed for the expression of Vpre-B/lambda 5 surrogate light chain (SL). The surface expression of SL is confined to the early stages (pro-B and pre-B-I) of pre-B cell development and becomes undetectable once mu heavy chain (microH) is produced. The cell-cycle analysis revealed that cytoplasmic microH+ large cells (large pre-B-II), approximately 30% of which coexpressed SL in the cytoplasm, were most actively cycling, whereas cytoplasmic microH+ small cells (small pre-B-II) were SL- and not in cycle. The analysis of pre-B cells in B cell-deficient mice suggests that the large pre-B-II stage is a critical step for the selection and amplification of cells carrying functionally rearranged microH genes.

Animals↗
PubMed1994

Influence of surrogate L chain on DHJH-reading frame 2 suppression in mouse precursor B cells.

DHJH rearrangements start in progenitor and precursor B cells and occur in three reading frames (rf). A strong bias for rf I has been noticed in murine and chicken antibodies, while the representation of rf II has been found suppressed both in peripheral as well as in precursor B cells. H chain gene loci DHJH rearranged in rf II are potentially capable of expressing a truncated DHJHC mu protein on the cell surface. Mice incapable of expressing this protein on the surface have previously been shown to have all reading frames represented in near equal frequency, suggesting that membrane-bound DHJHC mu protein is involved in the suppression of rf II. In this paper we show that suppression of rf II is not yet established in c-kit+ CD43+ IL-7/stromal cell-reactive pre-B I cells of fetal liver at day 15 of gestation, but becomes established when such precursor cell populations are expanded in vitro on stromal cells in the presence of IL-7. H chain gene loci using the DQ52 segment for rearrangements (which contains a stop codon in rf II, thus being unable to make DHJHC mu protein) do not show rf II suppression under these conditions. The same type of fetal liver-derived pre B-I cells from lambda 5 deficient mice also do not show rf II suppression after in vitro expansion. Bone marrow-derived pre B-I cells from normal mice assayed ex vivo and expanded in vivo show rf II suppression, while the corresponding pre-B I cells from lambda 5T mice do not. Collectively these experiments suggest that surrogate L chain is involved in rf II suppression. This may happen by inhibition of proliferation of pre-B cells expressing a complex of DHJHC mu protein and surrogate L chain.

Animals↗
PubMed1994

Pre-B cell-specific lambda 5 gene expression due to suppression in non pre-B cells.

The lambda 5 gene is expressed specifically in pre-B cells; during B lymphocyte differentiation the expression of the lambda 5 gene is turned on in pre-B cells and turned off again at the mature B cell stage. No other cell type has yet been found to express the lambda 5 gene. The pre-B cell-specific expression of the lambda 5 gene is regulated at the level of transcription. We asked whether the region 5' of the lambda 5 gene (5' lambda 5) could explain the stage- and tissue-specific expression. The lambda 5 promoter lacks a TATA box and transcription is initiated at multiple sites. In the presence of a heterologous enhancer, 5' lambda 5 confers pre-B cell-specific expression on the reporter gene chloramphenicol acetyl-transferase. Deletion analysis of 5' lambda 5 defines two separate regions, referred to as A lambda 5 and B lambda 5. Region B lambda 5 suppresses the expression in non pre-B cells since deletion of B lambda 5 allows A lambda 5 to promote transcription of the reporter gene in in all cell types tested. In addition, B lambda 5 acts as an enhancer on the heterologous kappa light chain promoter in pre-B cells but not in B cells. Thus region A lambda 5 functions as a basal promoter in all cell types tested. Region B lambda 5, in concert with a heterologous enhancer, acts as a suppressing region in non-pre-B cells and therefore confers pre-B cell specificity on the expression of the lambda 5 gene.

Animals↗
PubMed1994

IL-2 receptor alpha chain (CD25, TAC) expression defines a crucial stage in pre-B cell development.

The analysis of the expression of the alpha chain of the IL-2 receptor (CD25, TAC) on the surface of B lineage cells in mouse bone marrow reveals that it is a useful marker to distinguish pre-B-I from pre-B-II cells. CD25 is not expressed on CD45R(B220)+ c-kit+ CD43+ TdT+ lambda 5+ c mu- sIg-IgH chain locus DJH-rearranged pre-B-I cells of mouse bone marrow. It is expressed on large cycling CD45R(B220)+ c-kit- CD43+ TdT- lambda 5+ c mu+ sIg- and on small resting CD45R(B220)+ c-kit- CD43- TdT- lambda 5- c mu- sIg- IgH chain locus VHDJH-rearranged pre-B-II cells. Therefore, the transition from pre-B-I to large pre-B-II cells is marked by the downregulation of c-kit and terminal deoxynucleotidyl transferase (TdT), and by the upregulation of CD25. SCID, RAG-2T, microMT and lambda 5T mutant mice do have normal, if not elevated numbers of pre-B-I cells but lack all CD25+ pre-B-II cells in their bone marrow. The expression of a transgenic H chain under control of the microH chain enhancer in RAG-2T bone marrow B lineage precursors allows the development of large and small CD25+ pre-B-II cells. The results suggest that the differentiation of pre-B-I to pre-B-II cells in mouse bone marrow requires the expression of microH chains and surrogate L chains in membranes, probably on the surface of precursor B cells.

Animals↗
PubMed1994

Roles of IgH and L chains and of surrogate H and L chains in the development of cells of the B lymphocyte lineage.

Proteins expressed from productively rearranged H and L chain gene loci have been implied in the regulation of Ig gene rearrangements during B lymphopoiesis. However, recent findings suggest that early B cell development can occur without expression of surrogate L chain, without deposition of microH chains into membranes, without productive H chain gene rearrangements, and even without any rearrangements of Ig gene loci. In bone marrow, 2-5% of all B220-, sIgM-, c-kit+ cells are pro B cells that undergo differentiation from B220- via B220+, c-kit+, CD43+, clonable long-term proliferating pre B-I cells to B220+, c-kit-, CD43-, IL-2 receptor+ pre B-II cells and immature B cells, only to die by apoptosis in situ within less than 4 days. A membrane-bound complex of surrogate H chain (gp130/gp35-65) and surrogate L chain expressed on pro B and pre B-I cells has apparently no influence on this early development. Pre B-I cells carrying DHJH-rearrangements in reading frame (rf) II are counter-selected, probably because they can express an Ig-like complex of truncated DHJHC mu-protein and surrogate L chain, while pre B-I cells DHJH-rearranged in rf I or III are not suppressed. Immature sIg+ B cells, also from bcl-2 transgenic mice, can continue to rearrange L chain gene loci. Thus, mere membrane deposition of Ig, even with concomitant expression of bcl-2, terminates neither expression of RAG-1 and 2, nor secondary L chain gene rearrangements, nor does it allow the development of mature B cells. Membrane-bound expression of an Ig-like complex of microH chains and surrogate L chains appears to be needed to generate the 50-70 million pre B-II cells in bone marrow. However, the membrane-bound expression of Ig is mandatory for negative and positive selection of immature B cells. Autoantigens delete or anergize self-reactive B cells. We speculate that all mature, resting, primary antigen-reactive B cells in the periphery have been selected from immature sIg+ B cells by unknown antigens and have, thereby, changed their lifestyle from rapid death by apoptosis to longevity.

Animals↗
PubMed1993

Immature surface Ig+ B cells can continue to rearrange kappa and lambda L chain gene loci.

Pro and pre B cells possess the long-term capacity to proliferate in vitro on stromal cells and interleukin 7 (IL-7) and can differentiate to surface immunoglobulin (sIg+) cells upon removal of IL-7 from the cultures. A key event in this differentiation is the extensive cell loss due to apoptosis. Because the proto-oncogene bcl-2 can promote cell survival, we established pre-B cell lines from E mu-bcl-2 transgenic mice. These pre-B cells have the same properties as those derived from non-bcl-2 transgenic mice except that they do not die by apoptosis. This allowed us to study the fate of newly formed B cells in vitro for a longer period of time. Here we show that early during the differentiation of pre-B cells, upregulation of RAG-1 and RAG-2 expression go hand in hand with rearrangements of the Ig gene loci. Moreover, the newly formed sIg+ B cells continue to express RAG-1 and RAG-2 and continue to rearrange L chain gene loci, even in the absence of proliferation, in an orderly fashion, so that kappa L+ sIg+ cells can become lambda L+ sIg+ or sIg- cells, whereas lambda L+ sIg+ cells can become sIg-, but not kappa L+ sIg+ cells. Thus, deposition of a complete Ig molecule on the surface of a B cell does not automatically stop the Ig-rearrangement machinery.

Animals↗
PubMed1993

A complex of glycoproteins is associated with VpreB/lambda 5 surrogate light chain on the surface of mu heavy chain-negative early precursor B cell lines.

Monoclonal antibodies (mAbs) have been made specific for the pre-B cell-specific proteins VpreB and lambda 5 which together form the surrogate light (L) chain. mAbs specific for VpreB protein identified the 16-kD molecule associated on precursor B cell lines with lambda 5 protein as the product of the VpreB gene. Surrogate L chain was detectable even in the absence of mu heavy (H) chain on the surface of early precursor cell lines such as pro-B cell lines where all immunoglobulin (Ig) loci are in the germline configuration, as well as early pre-B cell lines where Ig H chain loci are DHJH rearranged in reading frame I or III, which does not allow the expression of a DHJHC mu protein. A complex of glycoproteins (200, 130, 105, and 65-35 kD) was identified as coprecipitated with the Vpreb/lamba 5 surrogate L chain in mu H chain-negative precursor B cell lines. The 130-kD protein was most strongly labeled with iodine and most consistently detected in noncovalent association with surrogate L chain. This protein turned out to be a N-linked glycoprotein with a 100-kD protein core and isoelectric point 5.8, indicating that it is distinct from CD43 and the BP-1/6C3 antigen. The surface deposition of the surrogate L chain in association with the newly identified glycoproteins suggests that the surrogate L chain may function as a receptor even before the association with mu H chain in early precursor B cells.

Animals↗
PubMed1993

Changes in frequencies of clonable pre B cells during life in different lymphoid organs of mice.

Progenitor and precursor B lymphocytes with the capacity of long-term proliferation on stromal cells in the presence of interleukin-7 (IL-7) can be cloned ex vivo from fetal liver, neonatal blood, and spleen, and from adult bone marrow (BM) in frequencies that are similar in different strains of mice and that change with age. A wave of clonable cells appears before birth and disappears after birth in liver. Up to 2 weeks after birth, high frequencies of clonable cells are present in spleen but become undetectable at 6 to 8 weeks of age. In BM, high frequencies (1 in 50) of clonable cells are present early after birth, and then decrease continuously to 10- to 20-fold lower levels at 6 to 8 months of age. The earliest clonable cells have at least part of their IgH genes in germline configuration. Clones of pro/pre B cells apparently continue to rearrange DH to JH segments on both chromosomes. Rearrangements without insertion of N-sequences at the DHJH joints are found in fetal liver, while DHJH joints in pre B cells of spleen and BM throughout life have N-regions inserted. At least half of all primary pre B-cell clones develop mitogen-reactive B cells after differentiation to sIg+ B cells. Clonable pro and pre B cells are enriched in B220- c-kit(low) as well as in B220+ c-kit+ and B220+ CD43+ cell populations of BM. The frequencies of clonable cells in the B220- c-kit(low) BM cell population decrease 10- to 20-fold during 8 months of life, while those in the B220+ c-kit+ population remain constant, although their absolute numbers drop 5- to 10-fold during that time. All long-term proliferating clones express the surrogate L chain VpreB/lambda 5 as well as c-kit and CD43 on all cells. The number of total clonable pro and pre B cells is at best 10% of the number of cells required to produce the estimated daily output of 5 x 10(7) B-lineage cells in a mouse. This suggests that the production of a relatively constant number of B cells during adulthood may be effected by precursors, which are not clonable on stromal cells and IL-7 with long-term proliferative capacity. On the other hand, BM transplantation experiments indicate that a mouse retains B220- progenitors throughout life, from which pre B and B cells can be generated in old mice in frequencies characteristic of young mice.

Aging↗
PubMed1993

Interferon-gamma arrests proliferation and causes apoptosis in stromal cell/interleukin-7-dependent normal murine pre-B cell lines and clones in vitro, but does not induce differentiation to surface immunoglobulin-positive B cells.

Normal pre-B cells from fetal liver or bone marrow of the mouse proliferate for long periods of time in tissue culture on stromal cells in the presence of interleukin-7 (IL-7). Their IgH loci are partly in germ-line, partly in DHJH-rearranged configuration, while their light chain loci are in germ-line configuration. They express the pre-B cell-specific genes VpreB and lambda 5. Proliferation of these pre-B cells is inhibited by interferon (IFN)-gamma, with half-maximal inhibition at concentrations between 0.1 and 1 unit/ml. Normal pre-B cells exposed to IFN-gamma die by apoptosis, as is evidenced by the disintegration of pre-B cell DNA into oligonucleosomal multimers of 180-200 bp. While the proliferation of pre-B cells from E mu-bcl-2 transgenic (tg) mice is inhibited by IFN-gamma, these cells do not die by apoptosis. IFN-gamma does not induce differentiation to more mature B lineage cells. In the absence of IL-7 normal pre-B cells differentiate to VHDHJH/VLJL-rearranged, surface immunoglobulin-positive B cells expressing the alpha chain of the IL-2 receptor. They also down-regulate the expression of VpreB and lambda 5, and lose the capacity to proliferate on stromal cells in the presence of IL-7. In contrast, both normal and E mu-bcl-2 tg pre-B cells exposed to IFN-gamma in the presence of stromal cells and IL-7 fail to differentiate, i.e. do not express surface immunoglobulin, retain expression of VpreB and lambda 5, do not express the alpha chain of the IL-2 receptor, and retain the capacity to proliferate on stromal cells in the presence of IL-7, once IFN-gamma is removed. The potential usefulness of a treatment of acute lymphocytic leukemia of the B cell lineage (pre B-ALL) with IFN-gamma is discussed.

Animals↗
PubMed1993

B cell development in mice with a defective lambda 5 gene.

The surrogate light chain encoded by the two pre-B cell-specific genes VpreB and lambda 5 plays a critical role in B cell development of the mouse. It has been shown that targeted disruption of the lambda 5 gene results in a depletion of B220+ CD43- IgM-pre-B cells in bone marrow, and in a delayed appearance both of CD5+ as well as CD5- surface immunoglobulin (sIg)+ B cells in the periphery. In this report we show that DHJH-rearranged B220- and B220+, CD43+, c-kit+, sIgM- pro- and pre-B-I cells with long-term capacity to proliferate in vitro on stromal cells in the presence of interleukin-7 are present in normal numbers in the bone marrow of lambda 5 T/lambda 5 T mice at various ages. They express normal levels of VpreB mRNA but, in contrast to normal pre-B-I cells, do not express surrogate light chain on their surface. Pre-B-I cells from fetal liver and bone marrow of lambda 5 T/lambda 5 T mice differentiate with normal kinetics and in normal numbers to sIg+, mitogen-reactive B cells. These results suggest that the delayed generation of sIg+ B cells in the peripheral, mature compartments of CD5+ and CD5- cells could be accounted for by the daily production of approximately 5 x 10(5) sIg+ B cells from the pre-B-I cell pool in the absence of a normal pool of pre-B-II cells.

Animals↗
PubMed1993

The surrogate light chain in B-cell development.

The proteins encoded by the VpreB and lambda 5 genes associate with each other to form a light (L) chain-like structure, the surrogate L chain. It can form Ig-like complexes with three partners-the classical heavy (H) chain, the DHJHC mu-protein, or the newly discovered p55 chain; these are expressed on the surface of pre-B cells at different stages of development. Here, Fritz Melchers and colleagues review the structures of the VpreB and lambda 5 genes in mouse and their relatives in humans, describe their pattern of expression, and speculate on their possible evolution and functions.

Amino Acid Sequence↗
PubMed1993

Generation and regeneration of cells of the B-lymphocyte lineage.

B lymphopoiesis in the mouse and in man can be divided into two phases, one in which the compartments of the B lineage are filled with cells, and a second in which these compartments are maintained by regeneration, turnover and selection. Once the immune system has been built it contains around 5 x 10(8) and 10(12) cells of the B-lymphocyte lineage in the mouse and in man, respectively, of which nearly 10 per cent are precursors that are active in regeneration, whereas over 90 per cent are resting, mature B cells.

Animals↗