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Biomedical subjects

K Dorshkind

Publications and source records attributed to K Dorshkind.

At least 37 records · Page 2Linked to original sources
PubMed1996

Defective B cell development in Snell dwarf (dw/dw) mice can be corrected by thyroxine treatment.

Snell dwarf (dw/dw) mice are deficient in anterior pituitary hormones due to a mutation in the gene encoding the Pit-1 transcription factor. Bone marrow B cell development is also suppressed in the mice, providing circumstantial evidence that one or more anterior pituitary-derived products, or factors induced by them, are required for normal B lymphopoiesis. However, concluding that this is the case is dependent on showing that hormonal treatment of dwarf mice reverses their B cell defects. dw/dw mice were treated with growth hormone (GH), insulin-like growth factor-I (IGF-I), or thyroxine in an attempt to restore bone marrow B lymphopoiesis. GH and IGF-I increased the number of B lineage cells in the bone marrow and spleen but did not restore the frequency of bone marrow pre-B cells to normal. However, bone marrow cellularity in thyroxine-treated dw/dw mice was comparable to that in control animals, and both the frequency and absolute number of B lineage cells had increased to normal or even above normal. Taken together, these data indicate that endocrine factors, especially those regulated by the hypothalamic-pituitary-thyroid axis, are potent B lymphopoietic factors.

Animals↗
PubMed1996

Long-term culture of triple-negative thymocytes.

The role of the thymic microenvironment in regulating events that occur subsequent to the acquisition of CD4, CD8, and the TCR, such as positive and negative selection, has been studied extensively. However, comparatively less is known about how thymic stromal cells regulate growth and differentiation within the CD4-CD8-TCR- triple negative (TN) cell compartment. Long-term culture systems have played a pivotal role in the understanding of microenvironmental regulation of myelopoiesis and B lymphopoiesis, but establishment of comparable cultures for T lineage cells has proved challenging. This report describes a culture system in which a thymic stromal cell line preferentially supports the long-term growth of TN thymocytes. In addition to demonstrating that TN cells have a considerable proliferative potential, the results provide insights into the effects of IL-7 on the growth and/or survival of TN cells and its role in regulating TCR gene rearrangements. The ability of the cultured TN cells to mature into CD4- and/or CD8-expressing thymocytes, some of which express TCR-alpha beta, suggests that the system will be valuable for the identification of microenvironmental stimuli that regulate the maturation of TN cells.

Animals↗
PubMed1996

Thymic stromal cells can support B cell differentiation from intrathymic precursors.

T lineage cells are the predominant intrathymic population, but low numbers of thymic B cells are also present in the thymus. Recent studies describing the contribution of thymic B cells to the shaping of the T cell repertoire have raised the question as to whether they are produced intrathymically and the potential role of the thymic stroma in this process. During the course of studies aimed at testing the B cell developmental potential of CD4-CD8-CD3/TCR- triple-negative thymocytes present in a newly developed, stromal cell-dependent, long term culture system, the ability of selected thymic stromal cells to support distinct stages of B cell development was revealed. Some thymic stromal cell lines maintained cells with B cell developmental potential but could not support their differentiation, while other selected lines efficiently supported the development of surface IgM-expressing cells from triple-negative precursors. Thymic surface IgM+ B cells were generated most efficiently from thymocytes harvested from relatively young mice. Taken together, the results of this study demonstrate that the thymic microenvironment exhibits a functional heterogeneity in its ability to support different stages of B cell development.

Aging↗
PubMed1994

In vivo administration of insulin-like growth factor-I stimulates primary B lymphopoiesis and enhances lymphocyte recovery after bone marrow transplantation.

Recombinant human insulin-like growth factor-I (IGF-I) (100 micrograms/day) was infused by osmotic minipumps into mice to evaluate its effects on bone marrow B lymphopoiesis. IGF-I treatment resulted in a significant increase in the total number of bone marrow B lineage cells by 2 wk post-treatment in both normal, adult mice and animals that had been lethally irradiated and reconstituted with syngeneic bone marrow. Effects were still apparent at wk 3, even though pumps delivered IGF-I for only 14 days. In addition, the results confirm previous observations that IGF-I treatment results in an increase in numbers of splenic B cells and suggest that this is in part the result of stimulation of B cell proliferation in that organ.

Animals↗
PubMed1994

Differential expression of bone marrow stromal cell-surface antigens on myeloid and lymphoid cells.

Two monoclonal antibodies (MAb) that recognize cell-surface determinants present on bone marrow stromal cells have been generated. The 5B3 antibody recognizes a major histocompatibility class I-like molecule. This antigen is coexpressed on all bone marrow B-lineage cells and myeloid progenitors responsive to recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF). The 9D3 antibody recognizes a protein similar in size to those encoded by the Ly-6 locus. A comparable bone marrow staining pattern and inhibition of 9D3 binding to bone marrow cells by anti-Ly-6C antibodies strongly suggests that the 9D3 antigen is Ly-6C. These data provide a further phenotypic characterization of bone marrow stroma and indicate that these cells express cell-surface determinants also present on myeloid and lymphoid cells.

Animals↗
PubMed1994

Pan/E2A expression precedes immunoglobulin heavy-chain expression during B lymphopoiesis in nontransformed cells, and Pan/E2A proteins are not detected in myeloid cells.

A newly developed rat long-term bone marrow culture system was used to study the role of Pan/E2A basic helix-loop-helix transcription factors during B-cell development. In this system, B-lymphocyte progenitors actively differentiate into mature B cells. Monoclonal (Yae) and polyclonal (anti-Pan) antibodies were employed to characterize the expression of Pan proteins by Western blot assay during hematopoiesis and to examine the components of immunoglobulin heavy-chain gene enhancer element-binding species by electrophoretic mobility shift assay. During B-cell development, the appearance of Pan/E2A proteins preceded the expression of immunoglobulin heavy-chain protein. A Pan-containing immunoglobulin heavy-chain enhancer element (mu E5)-binding species (BCF1), composed of immunoreactive Pan-1/E47 but not Pan-2/E12, was observed concomitantly with the detection of Pan/E2A proteins. In addition to BCF1, other mu E5-binding species were detected which were not recognized by the Yae antibody. Two of these species were present in primary B-lymphocyte and myeloid cultures and were recognized by an anti-upstream stimulatory factor antiserum. Although Pan/E2A proteins have been proposed to be ubiquitous, Pan/E2A proteins were not detected in primary myeloid cultures composed mainly of granulocytes and macrophages or in the macrophage cell line J774. The absence of Pan/E2A proteins in differentiated myeloid cells correlated with low steady-state levels of Pan/E2A RNA. However, Pan/E2A proteins were present in a promyeloid cell line, 32DCL3, suggesting that extinction of Pan/E2A expression may play a role in myelopoiesis.

Adenovirus E2 Proteins↗
PubMed1993

Kinetics of E2A basic helix-loop-helix-protein expression during myelopoiesis and primary B cell differentiation.

The DNA-binding factors encoded by the E2A gene, which include the basic helix-loop-helix proteins E12 and E47, are thought to regulate Ig gene expression. In the rat these factors are products of the Pan locus and are referred to as Pan-2 and Pan-1, respectively. To determine when during B cell differentiation Pan (E2A) DNA-binding proteins are first expressed, cells from a newly developed rat long term lymphoid bone marrow culture system were analyzed molecularly and phenotypically using a newly defined anti-Pan mAb. The data indicate that Pan-expressing cells appear in B cell progenitors before the appearance of the CD45R B lineage-associated Ag or microH chain protein. The results also indicate that Pan proteins are present in cells that had undergone D-JH rearrangements. Finally, although Pan (E2A) proteins are present in myeloid precursors, expression is extinguished as differentiation into mature myeloid cells occurs.

Animals↗
PubMed1993

Antisense inhibition of the p65 subunit of NF-kappa B blocks tumorigenicity and causes tumor regression.

The NF-kappa B transcription factor, composed of two proteins, p50 and p65, is a pleiotropic activator that participates in the induction of a wide variety of cellular genes. Various cell adhesion molecules have NF-kappa B binding sites and may play an important role in inflammatory response, tumorigenicity, and metastasis. In an earlier study, we demonstrated that adhesion of diverse transformed cells was blocked by antisense inhibition of the p65 subunit of NF-kappa B. Since cell-substratum interactions play an important role in tumorigenicity, we reasoned that antisense p65 could inhibit tumorigenicity. In diverse transformed cell lines, phosphorothioate antisense oligonucleotides to p65 inhibited in vitro growth, reduced soft-agar colony formation, and eliminated the ability of cells to adhere to an extracellular matrix. Stable transfectants of a fibrosarcoma cell line expressing dexamethasone-inducible antisense RNA to p65 showed inhibition of in vitro growth and in vivo tumor development. In response to inducible expression of antisense RNA, a pronounced tumor regression was seen in nude mice. The administration of antisense but not sense p65 oligonucleotides caused a pronounced inhibition of tumorigenicity in nude mice injected with diverse tumor-derived cell lines. Inhibitors of NF-kappa B function may thus be useful in the treatment of cancer.

Animals↗
PubMed1993

In vivo and in vitro suppression of primary B lymphocytopoiesis by tumor-derived and recombinant granulocyte colony-stimulating factor.

Transplantation of a granulocytosis-inducing murine CE mammary carcinoma into mice suppresses primary B lymphopoiesis in the marrow. The mechanisms of this tumor-induced B-cell suppression were investigated using Whitlock-Witte-type lymphoid cultures. When seeded with normal marrow progenitors, stromal cells of tumor-bearing mice supported the production of B220+ cells as well as did either stomal cells derived from control mice or the stromal cell line S17. Cultured over normal stroma, marrow cells of tumor-bearing mice depleted of adherent cells and B220+ cells generated B220+ cells as effectively as a similar cell population from control mice. However, interleukin-7-responsive progenitors, were completely depleted from the marrow of tumor-bearing mice. When conditioned medium (CM) of cloned CE tumor cells known to produce granulocyte colony-stimulating factor (G-CSF) and macrophage-CSF, or recombinant murine G-CSF was added to the cultures established with S17 cells, B220+ cell production was significantly diminished. Antiserum to murine G-CSF blocked these effects. These in vitro observations were corroborated by the elimination of marrow B220+ cells in mice injected with G-CSF. These in vitro and in vivo studies suggest that G-CSF plays an inhibitory role in primary B lymphopoiesis by blocking stromal cell-mediated differentiation of early B-cell progenitors into phenotypically recognizable B220+ pre-B cells.

Animals↗
PubMed1993

Connexin-43-type gap junctions mediate communication between bone marrow stromal cells.

Several morphologic studies have suggested that gap junctions exist between bone marrow stromal cells. This possibility was examined by analysis of stromal cells present in the adherent layer of primary long-term lymphoid bone marrow cultures and in additional studies using a stromal cell line. Results showing that the fluorescent dye lucifer yellow, when microinjected into a single stromal cell, transferred between most other contacting stroma and that stromal cells were electronically coupled provided support that cell-cell communication occurs between these microenvironmental elements. Additional studies showed that transcripts for connexin (Cx) 43, but not for Cx26 or Cx32, were present in a stromal cell line. To examine the potential for regulated cell-cell communication between the stroma, cells were treated with interleukin-1 (IL-1), a cytokine known to affect stromal cell function, and the effects on dye transfer were examined. IL-1 treatment resulted in a reversible decrease in the ability of dye to transfer between stromal cells in contact. Taken together, these studies show that gap junctions exist between stromal cells and that their permeability can be regulated. However, gap junction-mediated cell-cell communication could not be shown between the stroma and developing lymphoid cells.

Animals↗
PubMed1992

Status of kappa L chain gene rearrangements and c-kit and IL-7 receptor expression in stromal cell-dependent pre-B cells.

Studies from several laboratories have provided evidence that distinct stromal cell-derived signals are involved in the maturation of pre-B cells into surface Ig expressing B lymphocytes. In order to define the stage of development at which these stimuli act, various polymerase chain reaction strategies were used to characterize the status of kappa L chain gene rearrangements in nontransformed, stromal cell dependent pre-B cells. These cells were obtained from lymphoid colonies whose growth was potentiated by factors from a stromal cell line. kappa L chain genes in cells from many of these colonies were rearranged, and analysis of the Jk genes used indicated a bias toward the most 3' loci. However, the use of a reverse transcriptase PCR strategy failed to detect mature kappa transcripts, indicating that stromal cell mediators exist that allow pre-B cells to progress to the stage at which L chain genes are rearranged but not expressed. Reverse transcriptase PCR further revealed that no transcripts for c-kit (the receptor for kit-ligand) and the IL-7R could be detected in these cells. This suggests that these receptors are no longer expressed by the time cells have undergone kappa rearrangements and minimize a role for stromal cell-derived kit-ligand and IL-7 in mediating the pre-B to B cell transition.

Animals↗
PubMed1992

Insulin-like growth factor-I regulates pro-B cell differentiation.

Progression of B-lymphocyte development in the bone marrow of postnatal mammals is marked by progressive rearrangement and expression of immunoglobulin (Ig) heavy- and light-chain genes. Following productive VHDJH gene rearrangement in the Ig heavy-chain gene complex, mu-heavy chain is the first Ig gene product expressed in cells committed to the B-lymphoid differentiation pathway. Interleukin (IL)-7 has been shown to stimulate proliferation of pre-B cells following c mu expression and this proliferative stimulus is potentiated by kit ligand (KL). However, it appears that neither of these cytokines contributes to differentiation of pro-B cells or initiation of expression of Ig gene products. We previously demonstrated that differentiation of pro-B cells and expression of mu-heavy chain is stimulated by either bone marrow stromal cell line S17 or cell-free supernatants from that line. This biological activity was attributed to molecules with an apparent M(r) of less than 10 Kd and approximately 40 to 60 Kd. We now report that this biological activity resides with stromal cell-derived insulin-like growth factor-I (IGF-I). Recombinant IGF-I stimulated the expression of cytoplasmic mu-heavy chain in short-term bone marrow cultures and this stimulus was abrogated in the presence of anti-IGF-I antibody. We also demonstrate that either anti-IGF-I antibody or pretreatment of S17 cells with antisense oligonucleotide for IGF-I abrogated the pro-B cell differentiation activity of S17 stromal cell supernatants. Although IGF-I did not directly stimulate proliferation of B-lineage cells, like KL, it potentiated the proliferative stimulus provided by IL-7. Taken together, these data strongly suggest that IGF-I produced by bone marrow stromal cells in the hematopoietic microenvironment plays a key role in regulating primary B lymphopoiesis.

Animals↗
PubMed1992

Differential roles of stromal cells, interleukin-7, and kit-ligand in the regulation of B lymphopoiesis.

Newly formed B lymphocytes are a population of rapidly renewed cells in the bone marrow of mammals and their steady state production presumably depends on a cascade of regulatory cells and cytokines. Although considerable information has been forthcoming about the role of interleukin-7 (IL-7) in potentiating pre-B-cell proliferation, few studies have addressed the possibility that multiple cytokines are involved in the progression of early events in cellular differentiation and proliferation in this hematopoietic lineage. Our laboratory previously described pre-B-cell differentiation mediated by the bone marrow stromal cell line S17. In this study, we further delineate the role of stromal cells in differentiation and proliferation of pre-B cells. These experiments show that the stromal cell line S17 potentiates the proliferative effect of IL-7 on B-lineage cells and that this S17-derived potentiator can be replaced with recombinant kit-ligand (KL). Our results further show that pre-B-cell formation from B220-, Ig- progenitor cells and expression of mu heavy chain of immunoglobulin is uniquely dependent on the presence of S17 stromal cells and cannot be reproduced with IL-7, KL, or costimulation with both IL-7 and KL. These data contribute to a rapidly evolving model of stromal cell regulation of B-cell production in the marrow and suggest unique roles for IL-7, KL, and as yet uncharacterized stromal cell-derived lymphokines in this process.

Animals↗
PubMed1992

The stromal cell line S17 supports the growth of lipopolysaccharide-stimulated CBA/N spleen cell colonies in vitro.

The expression of an X-linked defect in the CBA/N strain of mice has been found to result in a number of immune abnormalities. These include low responsiveness to antigens and a greatly reduced ability to respond to many of the common B cell mitogens. An in vitro manifestation of this condition is the virtual inability of CBA/N B cells to form colonies in lipopolysaccharide (LPS)-containing semisolid agar cultures. In this report we show evidence that the colony-forming ability of CBA/N spleen cells can be effectively restored by the bone marrow stromal-derived cell line S17. Spleen cells from 4-5-week-old homozygous CBA/N female mice were grown in double-layer agar cultures containing S17 feeder layers. Control cultures contained the fibroblast-like cell line 95.17 or were treated with medium alone. It was found that at an input cell concentration of 10(4) cells per plate, CBA/N colony formation was increased from a frequency of approximately 1 in 5,000 to 1 in 50 total splenic cells. Studies with purified surface immunoglobulin-positive cells indicate the direct involvement of S17 in this process. The CBA/N colonies formed were dependent on the presence of a mitogen (LPS) and secreted detectable amounts of IgM. Major implications of these findings and the application of this assay system to study the CBA/N defect have been discussed.

Animals↗
PubMed1992

Regulation of B cell differentiation by bone marrow stromal cells.

B lymphocyte development occurs in the intersinusoidal spaces of bone marrow in association with a sessile population of stromal cells. Development of long-term bone marrow culture systems that support B lymphopoiesis has allowed the isolation of stromal cells that support that process and permitted analysis of their role in controlling the growth and differentiation of B lineage cells in culture. In addition to direct interactions with developing lymphocytes, stromal cells secrete a variety of cytokines that affect lymphocyte growth and differentiation, and many of these molecules have been expressed in recombinant form. These relatively recent advances have made it possible to formulate a working model of the cells and molecules involved in regulating primary B cell production.

Animals↗
PubMed1992

Regulatory cells and cytokines involved in primary B lymphocyte production.

Based upon the above data, it is now possible to formulate a working model that defines the stages of B cell development on which stromal cells and their products act. During the initial stages of this process, pro-B cells which do not express Ig heavy or light chain protein or other non-Ig B lineage associated molecules develop into B220 and c mu expressing pre-B cells in response to a low (< 10 kD) molecular weight stromal cell derived factor. No defined interleukin or colony stimulating factor, including molecules such as KL and IL-7, can replace stromal cell conditioned medium in mediating this developmental step. There appears to be little cell proliferation associated with the differentiation of pro-B cells into pre-B cells. However, our data indicate that as precursors develop into B220 expressing B cell progenitors, they become sensitive to the proliferation stimulating effects of IL-7 and KL. These results are in accord with findings that progenitor cells that have undergone DJH rearrangements are particularly sensitive to KL and IL-7(18,19). The analysis of pre-B cells present in individual lymphoid colonies indicates that once cells have rearranged and expressed their Ig heavy chain genes, they are no longer sensitive to KL and IL-7. These observations are based on the fact that receptors for these cytokines are not expressed in stromal cell dependent pre-B cells and are consistent with kinetic studies showing that the maturation of pre-B cells into surface Ig expressing B lymphocytes is not dependent upon cell proliferation21.(ABSTRACT TRUNCATED AT 250 WORDS)

B-Lymphocyte Subsets↗