scholarly journals Monocyte-derived recruiting activity: kinetics of production and effects of endotoxin

Blood ◽  
1985 ◽  
Vol 65 (3) ◽  
pp. 689-695
Author(s):  
E McCall ◽  
GC Jr Bagby
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Bone Marrow Cells ◽  
Umbilical Vein ◽  
Calf Serum ◽  
Fold Increase ◽  
Umbilical Vein Endothelial Cells ◽  
Kinetics Of ◽  
Marrow Cells

Cultured monocytes release a factor, monocyte-derived recruiting activity (MRA), which stimulates fibroblasts, endothelial cells, and T lymphocytes to produce colony-stimulating activity (CSA). We studied the kinetics of MRA production using a technique in which MRA levels were measured in a two stage bioassay. We used umbilical vein endothelial cells as the MRA-responsive (CSA-producing) cells, and normal colony-forming unit granulocyte-macrophage (CFU-GM)-enriched bone marrow cells (T lymphocyte- and monocyte-depleted, low density bone marrow cells) as the CSA-responsive cells. MRA stimulated a 30- fold increase in CSA production by endothelial cells. MRA production was detected in supernatants from as few as 10(3) monocytes per milliliter, required the presence of fetal calf serum, and was inhibited by cycloheximide (10 to 100 micrograms/mL) and puromycin (10 to 50 micrograms/mL). Production was detectable after 24 hours of monocyte incubation, was maintained for three days, and fell to undetectable levels by seven days. With the addition of bacterial endotoxin (lipopolysaccharide [LPS]) (50 micrograms per 10(6) cells), MRA was detectable after only three hours of incubation, and levels peaked at 24 hours. Further, maximum MRA levels in the supernatants of LPS-stimulated monocytes were up to ten times greater than peak levels in the supernatants of unstimulated monocytes. Endotoxin augmented monocyte production of MRA to a greater extent than it did CSA production, indicating that the stimulation of CSA production by endotoxin may be at least partly indirect. The responsiveness of MRA production to endotoxin in vitro is consistent with the notion that MRA may be a biologically relevant regulator of CSA production by cells of the hematopoietic microenvironment.

scholarly journals Monocyte-derived recruiting activity: kinetics of production and effects of endotoxin

Blood ◽  
1985 ◽  
Vol 65 (3) ◽  
pp. 689-695 ◽  
Author(s):  
E McCall ◽  
GC Jr Bagby
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Bone Marrow Cells ◽  
Umbilical Vein ◽  
Calf Serum ◽  
Fold Increase ◽  
Umbilical Vein Endothelial Cells ◽  
Kinetics Of ◽  
Marrow Cells

Abstract Cultured monocytes release a factor, monocyte-derived recruiting activity (MRA), which stimulates fibroblasts, endothelial cells, and T lymphocytes to produce colony-stimulating activity (CSA). We studied the kinetics of MRA production using a technique in which MRA levels were measured in a two stage bioassay. We used umbilical vein endothelial cells as the MRA-responsive (CSA-producing) cells, and normal colony-forming unit granulocyte-macrophage (CFU-GM)-enriched bone marrow cells (T lymphocyte- and monocyte-depleted, low density bone marrow cells) as the CSA-responsive cells. MRA stimulated a 30- fold increase in CSA production by endothelial cells. MRA production was detected in supernatants from as few as 10(3) monocytes per milliliter, required the presence of fetal calf serum, and was inhibited by cycloheximide (10 to 100 micrograms/mL) and puromycin (10 to 50 micrograms/mL). Production was detectable after 24 hours of monocyte incubation, was maintained for three days, and fell to undetectable levels by seven days. With the addition of bacterial endotoxin (lipopolysaccharide [LPS]) (50 micrograms per 10(6) cells), MRA was detectable after only three hours of incubation, and levels peaked at 24 hours. Further, maximum MRA levels in the supernatants of LPS-stimulated monocytes were up to ten times greater than peak levels in the supernatants of unstimulated monocytes. Endotoxin augmented monocyte production of MRA to a greater extent than it did CSA production, indicating that the stimulation of CSA production by endotoxin may be at least partly indirect. The responsiveness of MRA production to endotoxin in vitro is consistent with the notion that MRA may be a biologically relevant regulator of CSA production by cells of the hematopoietic microenvironment.

Thrombin Induces Thromboplastin Synthesis in Cultured Vascular Endothelial Cells

1985 ◽  
Vol 54 (02) ◽  
pp. 373-376 ◽  
Author(s):  
K S Galdal ◽  
T Lyberg ◽  
S A Evensen ◽  
E Nilsen ◽  
H Prydz
Keyword(s):  
Endothelial Cells ◽  
De Novo ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Calf Serum ◽  
Phosphodiesterase Inhibitors ◽  
Fold Increase ◽  
Homocysteine Thiolactone ◽  
Umbilical Vein Endothelial Cells

SummaryCultured human umbilical vein endothelial cells responded to thrombin (10−2 – 10 NIH u/ml) with a 2-5 fold increase in thromboplastin activity. The maximum response was reached after 4 hr in serum-free medium. The effect of thrombin was fully inhibited by the presence of 50% (v/v) fetal calf serum or more in the medium, by preincubation of thrombin with hirudin or by treatment of thrombin with N-bromosuccinimide or phenylmethylsulfonyl fluoride. The thrombin-induced thromboplastin activity was inhibited by incubation of the cells with cycloheximide (2 μg/ml) or actinomycin D (2 μg/ml) showing that the response depended on de novo protein and RNA synthesis. It was also suppressed by exposure of the cells to two different phosphodiesterase inhibitors, 3-butyl-l-methyl-xanthine (5 · 10−4 M) and rac-4 (3-butoxy-4-methoxybenzyl)-2-imidazole (5 · 10−4 M), to the transmethylation inhibitors 3-deazaadenosine (10−5 M) and 1-homocysteine thiolactone (2 · 10−5 M) in combination and to the intracellular calcium antagonist 8-(N,N-diethylamino)-octyl 3,4,5,-tri-methoxybenzoate hydrochloride (8 · 10−5 M). Our results suggest that small amounts of thrombin can induce thromboplastin synthesis in endothelial cells in vitro and that this synthesis probably is regulated by the intracellular level of cAMP, by cytoplasmic Ca2+ and possibly also by transmethylation reactions.

The Hemoglobin-Haptoglobin Receptor (CD163) Is Expressed by a Subpopulation of Erythroid and Granulo-Macrophagic Progenitors and Can Be Stimulated in Vitro and in Vivo by Physiological and Pharmacological Ligands to Stimulate Hematopoiesis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1221-1221
Author(s):  
Kathryn Matthews ◽  
Nicole Worsham ◽  
Neeta Rugg ◽  
Jose A. Cancelas ◽  
David Bell
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Fold Increase ◽  
Hematopoietic Progenitor ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Marrow Cells

Abstract Abstract 1221 The receptor for the hemoglobin (Hb)-haptoglobin (Hp) complex, CD163, is expressed on the surface of a subpopulation of hematopoietic stem/progenitor cells (HPCs) (Matthews et al, 2006). The purpose of the studies presented here were two-fold – to demonstrate that the CD34+CD163+ double positive population could be isolated from normal adult bone marrow cells and these cells were functional as HPCs and, in addition, that these cells could be stimulated in vivo by ligands to CD163 to affect hematopoiesis. To investigate the clonogenic potential of CD34+/CD163+ HPCs, bone marrow CD34+ cells were examined for CD163 co-expression, sorted by fluorescence activated cell sorting (FACS) and plated into colony-forming assays (CFAs). 4.2% ± 1.4% (n=4) of CD34+ cells were found to co-express CD163 and this population consisted of two distinct sub-populations, CD34++ (hi)CD163+ and CD34+(lo)CD163+, each of which represented approximately half of the total CD34+CD163+ population. All three sorted populations (CD34+(all)CD163−, CD34++(hi) CD163+, CD34+(lo)CD163+) were plated into CFAs (n=4) and were assessed for erythroid and myeloid colony formation. The clonogenic efficiency of CD34++(hi)CD163+ had a 2.5-fold increase in the number CFU-E and CFU-GM when compared to both CD34+ (total) CD163− and CD34+(lo) CD163+ cells. In contrast, CD34+(hi an low)CD163+cells produced fewer BFU-E. To determine how the expression of CD163 expression on progenitor cells may play a role in hematopoiesis, we investigated the effects of the natural ligand to CD163 (Hb/Hp) as well as an agonistic antibody to CD163 (TBI 304) on HPCs in vivo. NOD-scid IL2R gammanull (NSG) mice (HuMurine Technologies) were engrafted with human CD34+cells and animals with < 30% human CD45+ cells in the peripheral blood were administered either 2 mg Hb/mouse, or 100 or 500 μg/mouse TBI 304 every 4 days. At study termination (day 14), bone marrow cells (BMC) were examined by flow cytometry and enriched for CD34+ cells for enumeration in CFAs. Hb administration resulted in an increase of human CD34+cells ranging from 4% to 7% of BMC and a corresponding 57% increase in colony-forming cells (CFC) when compared to control (PBS-administered) animals. In contrast, TBI 304 produced a dose dependent decrease in CD34+ and CFC, possibly reflecting a depletion of CD34+/CD163+ cells from overstimulation due to the longer circulating antibody. To investigate this, human CD34+ cell engrafted animals were given a single dose of 10 or 100 μg/mouse of TBI 304 and bone marrow cells were examined on day 7. TBI 304 provided a 3.5-fold increase in human CD34+ cells as well as a 1.8 to 6.7-fold increase in bone marrow erythroid lineage engraftment (huGlyA+, huCD36+ and huCD71+) and a 2-fold increase in erythroid and myeloid colony-forming cells. No overall toxicities were observed with the administration of TBI 304 or Hb. We have demonstrated that CD163 is expressed on a population of CD34+ hematopoietic progenitor cells, these cells have increased hematopoietic progenitor activity in vitro and that administration of physiological or pharmacological agonists of the CD163 receptor can measurably stimulate hematopoiesis in vivo. Disclosures: Matthews: Therapure Biopharma: Employment. Bell:Therapure Biopharma: Employment.

Elucidating the contribution of the elemental composition of fetal calf serum to antigenic expression of primary human umbilical-vein endothelial cells in vitro

2011 ◽  
Vol 31 (3) ◽  
pp. 199-210 ◽  
Author(s):  
Nicholas Bryan ◽  
Kirstie D. Andrews ◽  
Michael J. Loughran ◽  
Nicholas P. Rhodes ◽  
John A. Hunt
Keyword(s):  
Cell Culture ◽  
Endothelial Cells ◽  
Elemental Composition ◽  
Fetal Calf Serum ◽  
Umbilical Vein ◽  
Calf Serum ◽  
Human Umbilical Vein ◽  
Antigenic Expression ◽  
Umbilical Vein Endothelial Cells

One of the major obstacles to obtaining human cells of a defined and reproducible standard suitable for use as medical therapies is the necessity for FCS (fetal calf serum) media augmentation in routine cell culture applications. FCS has become the supplement of choice for cell culture research, as it contains an array of proteins, growth factors and essential ions necessary for cellular viability and proliferation in vitro. It is, however, a potential route for the introduction of zoonotic pathogens and makes defining the cell culture milieu impossible in terms of reproducibility, as the precise composition of each batch of serum not only changes but is in fact extremely variable. The present study determined the magnitude of donor variations in terms of elemental composition of FCS and the effect these variations had on the expression of a group of proteins associated with the antigenicity of primary human umbilical-vein endothelial cells, using a combination of ICPMS (inductively coupled plasma MS) and flow cytometry. Statistically significant differences were demonstrated for a set of trace elements in FCS, with correlations made to variations in antigenic expression during culture. The findings question in detail the suitability of FCS for the in vitro supplementation of cultures of primary human cells due to the lack of reproducibility and modulations in protein expression when cultured in conjunction with sera from xenogeneic donors.

Mad2 (Mitotic Arrest Deficiency 2) Is Essential for the Synergistic Proliferation Induced by Stem Cell Factor Combined with GM-CSF in Hematopoietic Progenitor Cells, Effects Reflecting the Association of c-kit with Mad2.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 71-71
Author(s):  
Shigeki Ito ◽  
Charlie Mantel ◽  
Myung-Kwan Han ◽  
Seiji Fukuda ◽  
Yoji Ishida ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Cytokine Signaling ◽  
Mouse Bone Marrow ◽  
Wild Type ◽  
Gm Csf ◽  
Kinetics Of ◽  
Marrow Cells

Abstract Mitotic spindle checkpoint protein, Mad2, is required for proper functioning of the mitotic checkpoint which ensures correct chromosome segregation during cell division. Homozygous Mad2 gene deletion is embryonic-lethal. Mad2 interacts with mitosis-associated molecules such as Mad1 and anaphase promoting complex/cyclosome to ensure proper cell cycle progression. Recently, Mad2 was shown to physically associate with the common beta chain of the GM-CSF receptor which raises the possibility that Mad2 may also be involved in cytokine signaling and regulation of mitosis in hematopoietic progenitor cells. To investigate this, we studied hematopoiesis and cytokine signaling in Mad2-haploinsufficient (+/−) mutant mice (M2MT). Colony formation by granulocyte macrophage progenitor cells (CFU-GM) from bone marrow of wild type (WT) mice is synergistically stimulated in vitro by the combination of stem cell factor (SCF) and GM-CSF. We found that bone marrow CFU-GM from M2MT mice are deficient in the synergistic proliferative/colony formation response in vitro to stimulation with the combination of GM-CSF plus SCF. In contrast, there was no difference in stimulation of CFU-GM formation in response to the individual cytokines, GM-CSF or SCF alone, nor a difference in response to pokeweed mitogen mouse spleen cell conditioned medium between M2MT and WT mice. Because there was no difference in the frequency of c-kit+Sca-1+Lin- (KSL) cells nor a difference in the intensity of c-kit surface expression on KSL cells from wild type and M2MT mice, we considered whether the suppression of the SCF/GM-CSF synergy response was due to a difference in intracellular growth-factor receptor signaling pathways. We found that the kinetics of Erk1/2 phosphorylation signaling differ in M2MT Lin- cells compared to WT Lin- cells and that the duration of Erk1/2 phosphorylation in M2MT cells was at least one half of that in WT Lin- cells. On the other hand, we found no difference in the kinetics of Akt phosphorylation between WT and M2MT Lin- cells suggesting a specificity of involvement of the MAP-kinase pathways. To understand how Mad2 plays a role in SCF/GM-CSF synergy, we tested the physical interaction between Mad2 and c-kit in primary Lin- mouse bone marrow cells. Primary Lin- bone marrow cells from WT mice were expanded in liquid culture with SCF and thrombopoietin for 5 days. We found that Mad2 physically associated with c-kit as indicated by co-immunoprecipitation. These results suggest that Mad2 is required for the SCF/GM-CSF proliferative-synergy response in primary Lin- mouse bone marrow cells and that Mad2 is involved in growth-factor signaling pathways, such as the MAP-kinase cascade, in addition to spindle checkpoint function in primary hematopoietic cells. These effects are likely mediated through Mad2 interaction with c-kit and the beta chain of the GM-CSF receptor.

scholarly journals A gene transfer strategy for making bone marrow cells resistant to trimetrexate

Blood ◽  
1996 ◽  
Vol 87 (6) ◽  
pp. 2579-2587 ◽  
Author(s):  
HT Spencer ◽  
SE Sleep ◽  
JE Rehg ◽  
RL Blakley ◽  
BP Sorrentino
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Fold Increase ◽  
Retroviral Vectors ◽  
Hematopoietic Progenitor ◽  
High Level ◽  
Variant Enzyme ◽  
Marrow Cells

Trimetrexate (TMTX) is an anticancer drug with potential advantages over the more commonly used antifolate, methotrexate (MTX); however, its use has been limited by severe myelosuppression. Retroviral vectors containing mutant dihydrofolate reductase (DHFR) genes have been used to protect bone marrow cells from MTX, suggesting a similar approach could be used for TMTX. We first screened six variants of human DHFR to determine which allowed maximal TMTX resistance in fibroblasts. A variant enzyme containing a Leu-to-Tyr mutation in the 22nd codon (L22Y) was best, allowing a 100-fold increase in resistance over controls. Murine hematopoietic progenitor cells transduced with an L22Y- containing retroviral vector also showed high-level TMTX resistance in vitro. Mice reconstituted with L22Y-transduced bone marrow cells were challenged with a 5-day course of TMTX to determine whether hematopoiesis could be protected in vivo. Transfer of the L22Y vector resulted in consistent protection from TMTX-induced neutropenia and reticulocytopenia at levels that correlated with the proviral copy number in circulating leukocytes. We conclude that the L22Y vector is highly effective in protecting hematopoiesis from TMTX toxicity and may provide a means for increasing the therapeutic utility of TMTX in certain cancers.

scholarly journals Interferon gamma induces synthesis of complement alternative pathway proteins by human endothelial cells in culture.

1988 ◽  
Vol 168 (5) ◽  
pp. 1917-1922 ◽  
Author(s):  
J Ripoche ◽  
J A Mitchell ◽  
A Erdei ◽  
C Madin ◽  
B Moffatt ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Alternative Pathway ◽  
Fold Increase ◽  
Factor H ◽  
Complement Alternative Pathway ◽  
Ifn Gamma ◽  
Umbilical Vein Endothelial Cells ◽  
High Level

Human umbilical vein endothelial cells grown in vitro under standard conditions contain a high level of mRNA specific for the complement regulatory factors H and I. An additional 1.8-kb mRNA encoding a truncated form of factor H is also present. IFN-gamma stimulation of the cells causes a 6-7 fold increase in both factor H mRNA species, and a greater than 10-fold increase in factor I mRNA. IL-1 and LPS slightly suppressed factor H mRNA, while TNF had no effect. mRNA for factor B is also detectable in IFN-gamma-stimulated cells, but messengers for C1q, C4bp, and CR3 beta chain were not found. Secretion of factor H protein was also stimulated by IFN-gamma. The presence of mRNA for factors H, B, and I, together with C3 secretion, demonstrated by others, suggests that endothelial cells can assemble the complete alternative complement pathway. Endothelial cell complement may be involved in leukocyte-endothelium interactions mediated by leukocyte C3 receptors.

Endothelial-borne platelet-activating factor and interleukin-8 rapidly immobilize rolling neutrophils

1997 ◽  
Vol 272 (1) ◽  
pp. H114-H122 ◽  
Author(s):  
G. E. Rainger ◽  
A. C. Fisher ◽  
G. B. Nash
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Contact Point ◽  
Platelet Activating Factor ◽  
Adhesive Contact ◽  
Interleukin 8 ◽  
Median Response ◽  
Umbilical Vein Endothelial Cells ◽  
Kinetics Of

The kinetics of the response of integrins to activating signal(s) must be rapid to ensure that rolling neutrophils are localized at the sites of inflammation. From video records, we analyzed the adhesion of individual neutrophils in a flow-based in vitro model of endothelial hypoxia and reoxygenation. There were numerous rolling interactions between flowing neutrophils and P-selectin on human umbilical vein endothelial cells after hypoxia, but 90% lasted for < 1 s, with approximately 30% converted to stationary attachment via beta 2-integrin(s). Interleukin-8 (IL-8) and platelet-activating factor (PAF) were responsible for neutrophil activation in this model [G. E Rainger, A. Fisher, C. Shearman, and G. B. Nash. Am. J. Physiol. 269 (Heart Circ. Physiol. 38): H1398-H1406, 1995]. In the presence of a PAF-receptor antagonist, IL-8 acting alone induced conversion of rolling to stationary adhesion in as little as 80 ms after the initial attachment of a neutrophil, with a median response time of 240 ms. In the presence of a monoclonal antibody that neutralized IL-8 activity, PAF acting alone required a minimum duration of rolling of 560 ms to promote stationary adhesion, with a significantly longer median duration of 720 ms. In a reconstituted model, treatment of endothelial cells with hydrogen peroxide induced short-lived rolling of neutrophils supported by P-selectin. Exogenously added IL-8 and/or PAF bound to the endothelial surface and successfully induced the immobilization of neutrophils. Rapid and distinct kinetics of the conversion to stationary adhesion were observed again for IL-8 or PAF. Thus although endothelial-presented signals differed in their rate of action, neutrophils could be localized within one or two endothelial cell diameters of their initial adhesive contact point.

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