Biomedical subjects

K Kaushansky

Publications and source records attributed to K Kaushansky.

138 records · Page 8Linked to original sources

PubMed1989

Mechanisms of tumor-induced neutrophilia: constitutive production of colony-stimulating factors and their synergistic actions.

Transplantation of a murine mammary carcinoma (CE maca) into mice induces marked granulocytosis and hypercalcemia secondary to excessive bone resorption. Such responses are not induced by another murine mammary carcinoma Bc66. In order to understand the mechanisms of these unique phenomena, we analyzed mRNA of tumor cells for expression of murine granulopoietic growth factors and studied interactions of tumor-derived factors using antiserum to a growth factor in vitro and in vivo. The Northern blot analysis of CE tumor clones revealed the expression of granulocyte colony stimulating factor (G-CSF) and macrophage colony stimulating factor (M-CSF), but no other CSF genes, while the Bc66 clone expressed only M-CSF. The G-CSF and M-CSF gene expression in CE tumor clones was accompanied by secretion of these proteins in culture. The granulocyte stimulating activity of CE tumor-derived G-CSF or recombinant human G-CSF was markedly enhanced by purified M-CSF in vitro. Significant but variable neutrophilia was observed in mice inoculated with CE tumor clones. Anti-M-CSF treatment of CE tumor-bearing mice significantly reduced neutrophilia, but did not affect hypercalcemia. These studies document that G-CSF and M-CSF are produced constitutively from the CE maca, and G-CSF is likely responsible for granulocytosis induced by this tumor. G-CSF and M-CSF function synergistically in granulocyte stimulation in vitro and this synergism may also play a role in marked granulocytosis of tumor-bearing animals, providing further evidence of the effect of CSFs in vivo.

Adenocarcinoma↗

PubMed1989

Interleukin-6 is not involved in the interleukin-1-induced production of colony-stimulating factors by human bone marrow stromal cells and fibroblasts.

Interleukin-6 (IL-6) is a multifunctional cytokine that plays a role in regulation of hematopoiesis. Because IL-6 is coinduced with colony-stimulating factors (CSFs) by various cell types in response to stimulation with IL-1, we investigated whether IL-6 is involved in the IL-1-induced production of CSF by human bone marrow (BM) cells in long-term culture or human fibroblasts. We showed that IL-6 does not induce CSF production by these cells. Neither addition of exogenous IL-6 nor neutralization of endogenous production of IL-6 by an anti-IL-6 monoclonal antibody (MoAb) diminished the IL-1-induced colony-stimulating activity (CSA), indicating that IL-6 did not act synergistically with IL-1. Finally, IL-6 did not influence the kinetics of IL-1-induced CSA production by human fibroblasts. We conclude that IL-6, either alone or in combination with IL-1, does not induce CSF production by human BM stromal cells or fibroblasts.

Bone Marrow↗

PubMed1988

Tumor necrosis factor-alpha and tumor necrosis factor-beta (lymphotoxin) stimulate the production of granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, and IL-1 in vivo.

A number of cell types have been shown to elaborate hematopoietic growth factors in response to inflammatory mediators in vitro. To determine if this response occurs in vivo, we have administered levels of TNF-alpha and TNF-beta (lymphotoxin) found during an inflammatory reaction to mice. Using Northern blot analysis to detect tissue levels of hematopoietic growth factor-specific transcripts, and specific biologic and immunologic assays to detect the presence of colony-stimulating factors in the serum, we have found that TNF-alpha and TNF-beta induce the transcription and production of granulocyte-macrophage-CSF, macrophage-CSF, and IL-1. These findings provide an in vivo mechanism for the hematopoietic response to inflammation.

Animals↗

PubMed1988

Interleukin 1 stimulates fibroblasts to synthesize granulocyte-macrophage and granulocyte colony-stimulating factors. Mechanism for the hematopoietic response to inflammation.

IL-1 is a family of polypeptides which play a critical role in the inflammatory response. Characteristics of this response include an enhanced release of bone marrow neutrophils, activation of circulating and tissue-phase phagocytes, and enhanced production of neutrophils and monocytes. We have sought to understand the hematopoietic response to acute and chronic inflammatory states on a cellular and molecular level. Colony-stimulating factors (CSFs) are glycoproteins involved in the production and activation of neutrophils and monocytes in vitro and in vivo. We have found that quiescent dermal fibroblasts constitutively release granulocyte-macrophage CSF (GM-CSF), granulocyte CSF (G-CSF), and macrophage CSF in culture, and that picomolar concentrations of the inflammatory mediator IL-1 stimulate by at least fivefold the transcription and release of GM-CSF and G-CSF. These findings establish the role of IL-1 in the hematopoietic response to inflammation through the stimulation of the production and release of GM-CSF and G-CSF.

Animals↗

PubMed1988

Human fibroblasts produce granulocyte-CSF, macrophage-CSF, and granulocyte-macrophage-CSF following stimulation by interleukin-1 and poly(rI).poly(rC).

Electrophoretically pure human interleukin-1 (IL-1) beta was found to stimulate human fibroblasts in a monolayer culture to elaborate colony-stimulating activity (CSA). Supernatant fluids from cultures induced with increasing concentrations of IL-1 were found to stimulate colony formation of myeloid (CFU-GM), erythroid (BFU-E), and multipotent (CFU-GEMM) progenitor cells in a dose-dependent fashion. The effect on mixed colony formation, however, was less than on CFU-GM and BFU-E growth. Similar to IL-1, the synthetical double-stranded RNA poly(rI).poly(rC) also stimulated release of CSA by fibroblasts. The kinetics of IL-1- and poly(rI).poly(rC)-induced CSA release were found to be different, in that poly(rI).poly(rC)-induced CSA production occurred more slowly. Anti-IL-1 antiserum was able to completely neutralize the IL-1-induced CSA release, but had no effect on poly(rI).poly(rC)-induced CSF production, suggesting that the latter effect was mediated by other mechanisms than IL-1 in supernatant. By the use of specific immunologic assays, G-CSF, M-CSF, and GM-CSF could be identified in media conditioned by fibroblasts treated with IL-1 or poly(rI).poly(rC). Poly(rI).poly(rC) appeared to be a better inducer for M-CSF than IL-1.

Colony-Forming Units Assay↗

PubMed1987

Role of carbohydrate in the function of human granulocyte-macrophage colony-stimulating factor.

cDNA clones for the human hematopoietic regulator granulocyte-macrophage colony-stimulating factor (hGM-CSF) were isolated from a lamba gt11 cDNA library prepared from RNA of COS cells transiently expressing the gene for hGM-CSF. As the RNA was a rich source of hGM-CSF mRNA, approximately 0.1% of the clones of this library contained hGM-CSF sequences. All of the clones analyzed were full length and were correctly processed. When subcloned into an expression vector and transfected into COS cells, the cDNA clones direct the synthesis of higher levels of the growth factor than the gene from which they were derived. The cDNA for native hGM-CSF was used to generate structural mutants which lack N-linked carbohydrate, O-linked carbohydrate, or both. Although the mutant proteins had differing specific activities, the nonglycosylated forms reproduce many, if not all, of the physiologic functions of authentic hGM-CSF. The role of carbohydrate in the secretion and function of hGM-CSF is discussed.

Animals↗

PubMed1987

Interleukin 1 stimulates human endothelial cells to produce granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor.

Endothelial cells are a potent source of hematopoietic growth factors when stimulated by soluble products of monocytes. Interleukin 1 (IL 1) is released by activated monocytes and is a mediator of the inflammatory response. We determined whether purified recombinant human IL 1 could stimulate cultured human umbilical vein endothelial cells to release hematopoietic growth factors. As little as 1 U/ml of IL 1 stimulated growth factor production by the endothelial cells, and increasing amounts of IL 1 enhanced growth factor production in a dose-dependent manner. Growth factor production increased within 2 to 4 hr and remained elevated for more than 48 hr. To investigate the molecular basis for these findings, oligonucleotide probes for granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), and multi-CSF were hybridized to poly(A)-containing RNA prepared from unstimulated and IL 1-stimulated endothelial cells. Significant levels of GM-CSF and G-CSF, but not M-CSF or multi-CSF, mRNA were detected in the IL 1-stimulated endothelial cells. Biological assays performed on the IL 1-stimulated endothelial cell-conditioned medium confirmed the presence of both GM- and G-CSF. These results demonstrate that human recombinant IL 1 can stimulate endothelial cells to release GM-CSF and G-CSF, and provide a mechanism by which IL 1 could modulate both granulocyte production and function during the course of an inflammatory response.

Biological Products↗

PubMed1987

The molecular biology of the colony-stimulating factors.

Our understanding of the biochemistry and molecular biology of the colony stimulating factors (CSF) and the regulation of their production has advanced rapidly with the application of recombinant DNA technology. This report reviews several aspects of the regulation of CSF production and the structural features relating to their function. The structural gene for hGM-CSF is present as a single copy, is 2.5 kilobase long region and is located on the long arm of chromosome 5. The genes for human multi-CSF and M-CSF are also on chromosome 5. The gene for hGM-CSF is organized into four exons. The intron-exon boundaries contain a single consensus splice donor and acceptor sequence. CSFs are elaborated by endothelial cells, fibroblasts, and T lymphocytes in response to inflammatory mediators. Many of these same cell types respond to inflammatory stimuli with production of additional immune mediators such as IL-1, IL-2, or the interferons. Insights are now emerging into important structure--function relationships of the hematopoietic growth factors and molecular events in the regulation of CSF gene expression.

Chromosomes, Human, Pair 5↗

PubMed1986

Genomic cloning, characterization, and multilineage growth-promoting activity of human granulocyte-macrophage colony-stimulating factor.

Through the use of long single-sequence oligonucleotide probes, the complete gene for human granulocyte-macrophage colony-stimulating factor (hGM-CSF) has been cloned from a human genomic library. The gene is 2.5 kilobases in length, contains three introns, and is present as a single copy in the human genome. When subcloned into the mammalian expression vector pD3, the gene directs the synthesis of authentic hGM-CSF. In addition to its stimulation of in vitro granulopoiesis and monopoiesis, recombinant hGM-CSF stimulates in vitro erythropoiesis and megakaryopoiesis.

Base Sequence↗

PubMed1986

Tumor necrosis factor type alpha stimulates human endothelial cells to produce granulocyte/macrophage colony-stimulating factor.

Tumor necrosis factor type alpha (TNF-alpha) is produced by monocytes and has been purified, sequenced, and cloned from the HL-60 cell line. Soluble products of monocytes stimulate endothelial cells to release multilineage hematopoietic colony-stimulating activity. To determine whether TNF-alpha could stimulate endothelial cells to produce these activities, we added recombinant human TNF-alpha to cultured human umbilical vein endothelial cells. Untreated endothelial cell conditioned medium and TNF-alpha-stimulated endothelial cell conditioned medium were tested for hematopoietic colony stimulating activity in colony-forming assays in methylcellulose. TNF-alpha stimulated growth factor production by endothelial cells. Fifth-passage human endothelial cells and multiply-passaged bovine aortic endothelial cells responded similarly to first-passage endothelial cells, indicating that the action of TNF-alpha on endothelial cells is direct and not due to contaminating lymphocytes or monocytes present in the first-passage cultures. To investigate the molecular basis for these findings, polyadenylylated RNA was prepared from the TNF-alpha-stimulated endothelial cells and probed for granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor mRNA. Granulocyte-macrophage colony-stimulating factor, but not granulocyte colony-stimulating factor, message was detected. This finding suggests that at least some of the hematopoietic colony-stimulating activity released by the TNF-alpha-stimulated endothelial cells is granulocyte-macrophage colony-stimulating factor. These results demonstrate that a purified monocyte product can stimulate endothelial cells to produce the multilineage growth factor granulocyte-macrophage colony-stimulating factor and extend the role of this immunoregulatory protein to the regulation of hematopoiesis in vitro.

Cells, Cultured↗

PubMedDate not supplied

The regulation of GM-CSF is dependent on a complex interplay of multiple nuclear proteins.

GM-CSF is an important mediator of hematopoiesis and its dysregulation may play a role in neoplastic and inflammatory conditions. Previous studies have demonstrated that GM-CSF production depends upon the accumulation of specific mRNA, which occurs by transcriptional and post-transcriptional mechanisms. In order to dissect the cis-acting sequences responsible for its regulation, we performed an extensive mutagenesis study spanning 54 nucleotides 5' of the GM-CSF coding region. Our analysis suggests that the previously-described functional elements of the GM-CSF promoter, kappa B and a repetitive CATTT/A motif, the former co-exists with an overlapping 9 nucleotide site which silences promoter activity, and the CATTT/A complex binds multiple polypeptides which differentially contribute to basal and inducible promoter activity. These two sites interact to provide tissue-appropriate and stimulus-specific promoter function. Using DNA-protein cross-linking and co-transfection studies, we demonstrate that the c-rel-related proteins p65 and p50 bind to the GM-CSF promoter and that p65 binding is primarily responsible for the enhancing effects at this site. In addition, we show that the GM-CSF kappa B decanucleotide is inadequate to provide full binding affinity; mutation of nucleotides flanking this site affect promoter function by altering NF-kappa B binding affinity. Together these results suggest that the transcriptional response of GM-CSF is dependent on a complex interplay of multiple DNA binding proteins.

Base Sequence↗

PubMedDate not supplied

Chronic destructive arthritis in familial Mediterranean fever: the predominance of hip involvement and its management.

Chronic destructive arthritis is a rare complication of Familial Mediterranean Fever (FMF). The hip joints are most commonly involved, but destructive changes may also occur in the knees, ankles, sacroiliac spine, shoulder, or temporomandibular joints. A 28-year-old man with bilateral advanced coxitis and FMF was successfully treated by total hip arthroplasty.

Adult↗