Biomedical subjects

K Kaushansky

Publications and source records attributed to K Kaushansky.

At least 19 recordsLinked to original sources

PubMed1992

Role of carbohydrate modification in the production and secretion of human granulocyte macrophage colony-stimulating factor in genetically engineered and normal mesenchymal cells.

Colony-stimulating factors (CSFs) are a group of acidic glycoproteins which stimulate the proliferation and differentiation of hematopoietic progenitor cells in vitro and stimulate hemopoiesis in vivo. Human GM-CSF contains two N-linked carbohydrate side chains of the complex acidic type and several sites of O-linked carbohydrate clustered on serine and threonine residues near the N-terminus of the molecule. Previous studies have failed to detect a significant functional role for the carbohydrate modification characteristic of human GM-CSF. Using permanent cell lines and transient expression systems which produce moderate to high levels of native or carbohydrate-deficient forms of the growth factor, the role of carbohydrate modification in the biosynthesis and secretion of GM-CSF was studied. Unlike a number of other secreted glycoproteins, the transient time and secretory efficiency of several carbohydrate-deficient mutants of GM-CSF are indistinguishable from those of the native growth factor in BHK, 293, COS, and ldlD cells. Furthermore, normal human endothelial cells and fibroblasts, which normally produce the growth factor, can synthesize and secrete GM-CSF that lacks all forms of carbohydrate modification. These studies help to point out the range of roles played by carbohydrate modification in the biosynthesis, assembly, and secretion of glycoprotein hormones.

Animals↗

PubMed1992

Structure-function relationships of the hematopoietic growth factors.

The hematopoietic growth factors are a family of glycoproteins involved in the production of blood cells from their bone marrow precursors and in the activation of mature blood cells. Much has been learned about the structural features of these molecules responsible for their characteristic biological activities. Most studies have been based upon mutagenesis strategies of intact polypeptides and on epitope mapping of informative monoclonal antibodies to the growth factors. A more limited amount of physical data is available. This review will summarize these findings, highlight the growing body of evidence suggesting that many of these proteins share common evolutionary origins and structural elements, and hopefully point to the directions being taken for further investigations of these scientifically informative and clinically useful group of proteins.

Amino Acid Sequence↗

PubMed1992

Structure-function relationships of interleukin-3. An analysis based on the function and binding characteristics of a series of interspecies chimera of gibbon and murine interleukin-3.

IL-3 is a glycoprotein cytokine involved in the hematopoietic response to infectious, immunologic, and inflammatory stimuli. In addition, clinical administration of recombinant IL-3 augments recovery in states of natural and treatment-related marrow failure. IL-3 acts by binding to high affinity cell surface receptors present on hematopoietic cells. To determine the site(s) at which IL-3 binds to it receptor, we analyzed a series of interspecies chimera of the growth factor for species-specific receptor binding and biological activity. The results suggest that IL-3 binds to its receptor and triggers a proliferative stimulus through two noncontiguous helical domains located near the amino terminus and the carboxy terminus of the molecule. To corroborate these findings, we have also mapped the binding epitopes of 10 mAb of human or murine IL-3, and have defined four distinct epitopes. Two of these epitopes comprise the amino-terminal receptor binding domain. A third epitope corresponds to the carboxy-terminal receptor interactive domain, and the fourth epitope, apparently not involved in the interaction of IL-3 and its receptor, lies between these sites. And on the basis of sandwich immunoassays using pairs of these mAbs, the two receptor interactive regions appear to reside in close juxtaposition in the tertiary structure of the molecule. These results provide a correlation of the structure-function relationships of IL-3 that should prove useful in evaluating the details of IL-3-IL-3 receptor interaction and in the rational design of clinically useful derivatives of this growth factor.

Adult↗

PubMed1991

Antisense oligonucleotides from the stage-specific myeloid zinc finger gene MZF-1 inhibit granulopoiesis in vitro.

Zinc finger proteins are transcriptional regulators of other genes, often controlling developmental cascades of gene expression. A recently cloned zinc finger gene, MZF-1, was found to be preferentially expressed in myeloid cells. Using complementary radiolabeled MZF-1 RNA hybridized to human bone marrow smears in situ, it was discovered that the expression of MZF-1 is essentially limited to the myelocyte and metamyelocyte stages of granulopoiesis. Antisense but not sense oligonucleotides from MZF-1 significantly inhibited granulocyte colony-stimulating factor-driven granulocyte colony formation in vitro.

Adult↗

PubMed1991

In vivo production of interleukin-5, granulocyte-macrophage colony-stimulating factor, macrophages colony-stimulating factor, and interleukin-6 during intravenous administration of high-dose interleukin-2 in cancer patients.

Recombinant human interleukin-2 (IL-2), administered to cancer patients by continuous intravenous (IV) infusion (3 x 10(6) U/m2/d), was found to induce the in vivo production of colony-stimulating factors (CSF). Plasma obtained from patients during IL-2 treatment stimulated in vitro colony formation of normal human bone marrow cells, depleted of mononuclear phagocytes and T lymphocytes. This colony-stimulating activity (CSA) was identified as IL-5, granulocyte-macrophage CSF (GM-CSF), and macrophage CSF (M-CSF), by the ability of specific antibodies against these factors to neutralize their effects. The presence of IL-2-induced GM-CSF and M-CSF was also demonstrated by specific radioimmunoassays. During IL-2 treatment, plasma also contained detectable levels of IL-6, which was measured in a bioassay. Using a cDNA-polymerase chain reaction (PCR) with specific primer sets for the various CSF, we showed that IL-2 treatment induced the expression of mRNA for M-CSF, GM-CSF, IL-3, and IL-5, but not for granulocyte CSF (G-CSF) in peripheral blood mononuclear cells, suggesting differential expression of CSF in vivo in response to IL-2. Furthermore, no negative regulators of hematopoiesis, such as interferon gamma (IFN-gamma) or tumor necrosis factor-alpha (TNF-alpha), were found in plasma. These data illustrate that in vivo administration of high-dose IL-2 may result in a stimulatory effect on hematopoiesis. The induction of detectable levels of IL-5 and GM-CSF in the circulation may explain the eosinophilia and neutrophilia observed in these patients.

Carcinoma, Renal Cell↗

PubMed1991

Hem-1, a potential membrane protein, with expression restricted to blood cells.

Overlapping cDNAs 3.8 kb in length containing a long open reading frame were obtained that hybridized exclusively to transcripts from hematopoietic cells. Sequence analysis found eight potential membrane domains and two possible cAMP/cGMP phosphorylation sites. This sequence exhibited no homologies with the EMBL/Genbank nucleic acid, SwissProt or GenPept amino acid data bases. The gene is located at 12q13.1, a region of occasional translocations in hematopoietic neoplasia and a rare folic acid fragile site, Fra 12A.

Animals↗

PubMed1991

A retinoic acid-responsive human zinc finger gene, MZF-1, preferentially expressed in myeloid cells.

Zinc finger genes encode metal-binding proteins that can act as transcriptional regulators of other genes. In an effort to identify activators of the genetic cascade in hemopoietic differentiation, we used degenerate synthetic oligonucleotides to the conserved zinc finger histidine-cysteine link to probe a human myeloid lambda gt11 cDNA library. One of the cDNA clones obtained hybridized preferentially to mRNA from myeloid cells. This cDNA was used to isolate clones encompassing the coding region for this gene. Sequence analysis found 13 zinc finger regions and a glycine-proline-rich region between the fourth and fifth zinc finger domain. The gene was localized to chromosome 19q13.2-4, a chromosome that has a large cluster of zinc finger genes. The gene was preferentially expressed in myeloid leukemia cell lines with the highest mRNA levels noted in HL-60 cells induced to differentiate with retinoic acid. Thus, this new zinc finger gene (designated MZF-1) may be one regulator of transcriptional events during hemopoietic development.

Amino Acid Sequence↗

PubMed1991

Cytokines in chronic inflammatory arthritis. VI. Analysis of the synovial cells involved in granulocyte-macrophage colony-stimulating factor production and gene expression in rheumatoid arthritis and its regulation by IL-1 and tumor necrosis factor-alpha.

Granulocyte-macrophage CSF (GM-CSF) is a potent stimulator of macrophages and neutrophils and is produced by rheumatoid arthritis (RA) synovium. We now report studies that identify some of the synovial cells and cytokines responsible for local GM-CSF production and gene expression in RA. GM-CSF was assayed by ELISA in supernatants from cultured RA fibroblast-like synoviocytes stimulated with various cytokines (IL-1 beta, TNF-alpha, macrophage-CSF, IFN-gamma, IL-6, and TGF-beta). Immunoreactive GM-CSF was detected in IL-1 beta and TNF-alpha-stimulated cultures, but not in cells cultured in medium or stimulated with any of the other cytokines. IL-1 and TNF-alpha had a synergistic effect on GM-CSF production. GM-CSF gene expression by fibroblast-like synoviocytes was analyzed by ribonuclease protection assay, Northern blot analysis, and in situ hybridization. Both IL-1 beta and TNF-alpha induced GM-CSF mRNA accumulation, with a maximum effect after 4 h of stimulation. We then studied GM-CSF production by macrophage-like synoviocytes (MLS) isolated from fresh synovial specimens by flow microfluorimetry. Fresh MLS spontaneously secreted the cytokine and exogenous IL-1 beta or TNF-alpha had no effect. After 1 wk in culture, additional stimulation with IL-1 beta or TNF-alpha was required for GM-CSF production. Finally, in situ hybridization performed on freshly isolated subpopulations of synovial cells, identified GM-CSF RNA transcripts in MLS.

Arthritis, Rheumatoid↗

PubMed1991

Identification of functionally distinct domains of human granulocyte-macrophage colony-stimulating factor using monoclonal antibodies.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein that is required for the survival, growth, and differentiation of hematopoietic progenitor cells. Although the primary structure of GM-CSF is known from cDNA cloning, the relationship between structure and function of GM-CSF is not fully understood. Fifteen different monoclonal antibodies (MoAbs) to human GM-CSF were generated to map immunologically distinct areas of the molecule. Each of the MoAbs was biotinylated and shown by enzyme-linked immunosorbent assay to bind to recombinant GM-CSF that had been affixed to a solid phase. Each of the 15 unconjugated MoAbs was then used to compete with each biotinylated MoAb for binding to GM-CSF. These cross-blocking studies identified eight distinct epitopes of native GM-CSF. Seven of these epitopes were also present in denatured GM-CSF by Western blotting, and four of the epitopes were at least partially conserved on GM-CSF that was reduced in beta-mercaptoethanol. MoAbs to four of eight epitopes neutralized both recombinant (glycosylated and nonglycosylated) and natural human GM-CSF in a GM colony-forming unit (CFU-GM) assay and blocked GM-CSF-induced activation of neutrophils. For most of the antibodies there was a good correlation between neutralizing activity and the capacity to block binding of 125I-GM-CSF to neutrophils or blasts. Non-neutralizing antibodies to one epitope partially blocked binding of 125I-GM-CSF to neutrophils. None of the MoAbs neutralized interleukin-3, G-CSF, or M-CSF. The locations of seven of the epitopes could be partially mapped with regard to the amino acid structure by determining reactivity to GM-CSF synthetic peptides or to human-mouse chimeric GM-CSFs. The neutralizing antibodies were found to map to amino acids 40-77, 78-94, or 110-127. Thus, these MoAbs are useful to identify functional domains of GM-CSF and in identifying regions that are likely to be involved in receptor interaction.

Amino Acid Sequence↗

PubMed1991

Hematopoietic colony-stimulating factors.

In summary, hematopoietic growth factors have been discovered, biochemically characterized, cloned, produced by recombinant DNA technology, and put into clinical use in a period of 25 years. We are approaching a greater understanding of the cellular anatomy and molecular mechanisms that regulate production of the CSFs, the ways in which the CSFs interact with their cell surface receptors and trigger their biological effects, the nature of these receptors themselves and their mechanisms of signal transduction, and the effects of the CSFs in vitro and in vivo on hematopoietic progenitor cells and mature leukocytes. However, many questions remain. What is the mechanism that couples growth-factor binding to the triggering of cellular proliferation? How do multi-CSF and GM-CSF cross-compete at the level of the cell-surface receptor, and yet show no primary amino acid sequence homology? What are the mechanisms that regulate the tissue expression profile of multi-CSF compared to the genetically similar growth factor GM-CSF? And, what are the optimal dosages, schedules of administration, and combinations of CSFs optimal for each of several conditions of marrow failure? These are but a few of the questions that continue to occupy much current research interest.

Agranulocytosis↗

PubMed1991

A neutralizing monoclonal antibody binds to an epitope near the amino terminus of murine granulocyte-macrophage colony-stimulating factor.

A rat anti-murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) monoclonal antibody, A2, that neutralizes bioactivity in vitro was isolated. The binding epitope recognized by this antibody was identified using human-murine hybrid GM-CSF proteins. A2 was unable to immunoprecipitate a hybrid (hm7) protein containing the human GM-CSF sequence for the first 11 amino terminal amino acids, and the mGM-CSF sequence for amino acids 12-124. In contrast, A2 did recognize a hybrid which substitutes human GM-CSF amino acids 23-36 in the murine sequence. These data suggest that this neutralizing antibody recognizes an epitope at the amino terminus of mGM-CSF. Because hm7 did maintain in vitro bioactivity, it is probable that the epitope recognized by the neutralizing antibody is not itself part of the receptor-binding domain of mGM-CSF; rather, it is likely that neutralization occurs as a result of antibody binding near the receptor-binding site, with steric inhibition of mGM-CSF binding to its receptor. Interestingly, monoclonal antibody A2 does not recognize mGM-CSF glycosylation species corresponding to predicted maximal O-glycosylation variants. The presence of O-glycosylation sites within the antibody-binding epitope was confirmed using site-directed mutagenesis. Potential O-glycosylation sites in native mGM-CSF were removed by introducing conservative amino acid substitutions, and expected molecular weight reductions were obtained. These findings are consistent with previous reports that suggest the importance of the integrity of residues near the amino terminus to GM-CSF bioactivity.

Amino Acid Sequence↗

PubMed1990

Interleukin-1 alpha also induces granulocyte-macrophage colony-stimulating factor in immature normal bone marrow cells.

The cytokine interleukin-1 (IL-1) plays a role in the regulation of normal as well as leukemic hematopoiesis. In acute myeloid leukemia (AML), IL-1 induces autocrine granulocyte/macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF) production, and these factors may then synergistically induce proliferation in AML blast cells. In this report, we show that IL-1 stimulates DNA synthesis of highly enriched normal bone marrow blast cells (CD34 positive, adherent cell depleted, CD3/CD14/CD15 negative). The stimulative effect of IL-1 can be blocked with neutralizing anti-TNF alpha and anti-GM-CSF antibodies and, most efficiently, by the combination of anti-TNF alpha and anti-GM-CSF, but not with anti-G-CSF antibody, suggesting that IL-1-induced proliferation was initiated through TNF and GM-CSF release. Concentrations of TNF and GM-CSF increased in the culture medium of normal bone marrow blast cells after IL-1 induction. Of the IL-1-induced cells, 12% were positive for GM-CSF mRNA by in situ hybridization, as opposed to 6% of non-induced cells. Thus, in addition to its effect on leukemic blast cells, IL-1 also acts on normal marrow blast cells. We propose a scheme where IL-1 stimulation of normal bone marrow blast cells leads to the induction of TNF alpha and GM-CSF, which in association stimulate DNA synthesis efficiently according to a paracrine or autocrine mechanism within the marrow blast cell compartment.

Adult↗

PubMed1990

Muramyl dipeptide induces production of hemopoietic growth factors in vivo by a mechanism independent of tumor necrosis factor.

Monocyte products including TNF and IL-1 can stimulate hemopoietic growth factor production in vitro and in vivo. Endotoxin-resistant C3H/HeJ mice have a cellular defect in the regulation of TNF production and provide a model in which to examine the relative importance of TNF and IL-1. To assess the relative role of TNF and IL-1, we injected the synthetic bacterial cell wall derivative muramyl dipeptide (MDP) or LPS into C3H/HeJ mice. Both MDP and LPS stimulated hemopoietic growth factor production in a dose-dependent manner. Northern blot analysis of splenic and lung mRNA revealed that MDP treatment induced granulocyte-macrophage CSF and macrophage-CSF transcripts, whereas LPS treatment induced macrophage-CSF transcripts. Messenger RNA for granulocyte-CSF, IL-3, IL-4, and IL-5 was not detected in control or treated mice. MDP treatment induced IL-1 mRNA but not TNF mRNA, and TNF bioactivity was not detected in the serum of MDP-treated mice. In contrast, LPS treatment induced TNF production. These results identify the hemopoietic growth factors induced by MDP and LPS in vivo, and suggest that MDP stimulates growth factor production by a mechanism independent of TNF.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed1990

Two neutralizing monoclonal antibodies against human granulocyte-macrophage colony-stimulating factor recognize the receptor binding domain of the molecule.

Using a series of mutant and chimeric human-mouse granulocyte-macrophage-CSF molecules the binding epitopes of two neutralizing mAb antibodies to human GM-CSF have been mapped. Both intact antibody and Fab fragments neutralize the biologic activity of human GM-CSF. The epitope of one of the antibodies contains residues widely separated in the primary structure of the growth factor that suggests that these two regions are adjacent in the tertiary structure of the molecule. In addition, evidence is presented that both mAb neutralize the activity of this cytokine by blocking the receptor binding domain of human GM-CSF.

Animals↗

PubMed1990

Differential expression of M-CSF, G-CSF, and GM-CSF by human monocytes.

The colony-stimulating factors (CSF) belong to a group of proteins which regulate blood cell production. Human monocytes allowed to adhere express high levels of M-CSF transcripts and secreted protein at 24 h in the presence but not in the absence of indomethacin (Indo), an inhibitor of prostaglandin E (PGE) production. When induced with lipopolysaccharide (LPS), adherent monocytes express M-CSF, G-CSF, and GM-CSF transcripts and secrete these proteins and TNF. M-CSF and GM-CSF messages increase in LPS-induced monocytes by the addition of Indo, while G-CSF mRNA appears to decrease. Exogenous addition of PGE-2 to LPS-induced monocytes down-modulates the expression of M-CSF and GM-CSF transcripts. G-CSF message is elevated, suggesting an alternate pathway to G-CSF regulation. PGE-2 inhibits the secretion of CSFs and TNF. In contrast, LPS-induced monocytes held 24 h in nonadherent culture express G- and GM-CSF but not M-CSF. Monocytes that are adhered for 24 h and then treated with LPS for an additional 24 h express only M-CSF message and secrete M-CSF and TNF. PGE-2 added with LPS during the 24-48 h induction blocks M-CSF and TNF production, but appears to enhance M-CSF message expression, in contrast to its effect on 0 h inductions. These results suggest that adherence alone induces M-CSF gene expression, but low levels of PGE or other arachidonic acid metabolites limit this expression. Other events in 1 d-cultured monocytes block the ability to induce G-CSF and GM-CSF expression with LPS, and block the suppressive effect of PGE-2 on M-CSF expression at the RNA level.

Cells, Cultured↗

PubMed1990

Molecular modeling of human granulocyte-macrophage colony-stimulating factor.

The colony-stimulating factors (CSFs) are a group of acidic glycoproteins which are required for the proliferation of hematopoietic progenitor cells and for their differentiation into mature blood cells. Receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF) are present on a wide spectrum of cells including erythroid, mixed erythroid-non-erythroid, mixed myeloid and megakaryocytic progenitors, and on mature neutrophils, eosinophils and monocytes. A number of studies are now available which provide insights into the structure-function relationships of human GM-CSF. In an attempt to further understand the interaction between GM-CSF and its cell surface receptor, we have constructed models of the tertiary structure of human GM-CSF using the known disulfide bonding pattern, predictions of the secondary structure of the growth factor and a model based on conformational homologies among cytokines (Parry et al., J Mol Recognition 1988;1:107-110). When compared to a number of functional mapping studies, structural features of the model are consistent with the experimental data, and the model, in turn, leads to the generation of a number of testable hypotheses. The implications of these features in terms of receptor-ligand interaction are discussed.

Algorithms↗

PubMed1990

Transcriptional regulation of interleukin 3 gene expression in T lymphocytes.

Interleukin 3 (IL-3 or multi-colony-stimulating factor) plays an important role in the hematopoietic response to inflammatory stimuli through its action on both immature and mature blood cells. Like other lymphokines, IL-3 is produced in response to activation of the T-cell receptor and protein kinase C pathways. By using nuclear run-on assays of quiescent and stimulated T-cell lines, we demonstrate that IL-3 gene expression is controlled, at least in part, at the level of transcription. Functional reporter gene analysis was used to delineate two regions of the IL-3 5' flanking sequence responsible for transcriptional stimulation. DNA binding proteins that potentially mediate these responses were then recognized by mobility-shift and DNase footprinting assays. One region responsible for transcriptional enhancement was localized to the sequence GATGAATAAT, the cognate site of a transcription factor, here termed NF-IL3-A. A second region of functional activity and protein binding was localized to a single transcription factor AP-1 site. In addition three functionally inhibitory regions were identified. These results, along with the further characterization of NF-IL3-A, will contribute to the understanding of IL-3 gene regulation in stimulated T cells.

Base Sequence↗

PubMed1990

Mechanisms of cytomegalovirus-mediated myelosuppression: perturbation of stromal cell function versus direct infection of myeloid cells.

Infection with cytomegalovirus (CMV) continues to be one of the most common complications following allogeneic bone marrow transplantation. To study the role of CMV in the suppression of hemopoiesis that frequently accompanies infection, we investigated the effect of CMV on the growth of isolated committed myeloid progenitors and on hemopoiesis in long-term bone marrow cultures. Laboratory strain AD169 had no effect on the growth and development of progenitor cells. In contrast, 40% of clinical isolates of CMV inhibited colony formation by up to 100%. In long-term bone marrow cultures all CMV isolates resulted in myelosuppression, which in the majority of cases was associated with the infection of stromal elements. Analysis of RNA from stromal cells infected with AD169 and one clinical isolate demonstrated a specific deficiency of granulocyte colony-stimulating factor transcripts. For a small proportion of the clinical isolates tested in long-term bone marrow cultures, suppression of hemopoiesis was correlated with infection of developing granulocytes. These studies suggest that CMV can impair hemopoiesis either through infection of stromal cells and consequent perturbation of growth factor production or by direct infection of myeloid cells.

Antigens, Viral↗