Increased proliferation of bone marrow-derived fibroblasts in primitive hypertrophic osteoarthropathy with severe myelofibrosis

Pachydermoperiostosis or primary hypertrophic osteoarthropathy (HOA) is a rare congenital growth disorder of connective tissue. We report a case of severe myelofibrosis in a patient with HOA. When cultured in vitro, patient bone marrow-derived fibroblasts displayed a high proliferative potential with a shortened doubling time (24 hours v 36 to 48 hours for normal fibroblasts). The role of platelet-derived growth factor (PDGF), previously implicated in the pathogenesis of secondary acquired myelofibrosis, was studied. HOA fibroblasts expressed an increased number of PDGF-BB binding sites (300,000 sites/cell v 200,000 sites/cell for normal fibroblasts) without any modification of affinity. The increased expression of PDGF-R beta appeared to result from an accelerated rate of PDGF-R beta resynthesis with normal kinetics of endocytosis. As a consequence, a several-fold increase of PDGF-R beta tyrosine kinase activity was observed. No autocrine mechanism of growth was suspected as neither spontaneous PDGF-R beta autophosphorylation nor mitogenic activity in HOA fibroblast-conditioned medium was detected. Patient serum and platelet lysate were less potent than controls in inducing [3H]thymidine incorporation into HOA fibroblasts. This was inconsistent with a paracrine mechanism of growth. In vitro, human serum or PDGF-BB were not more mitogenic for HOA than normal fibroblasts. High levels of cyclin D1, a putative oncogene, were detected in serum-deprived HOA fibroblasts. Cyclin D1 overexpression could be implicated in the accelerated growth of these cells. Our results suggest that the mechanism of fibroblastic proliferation observed in this case of myelofibrosis might differ from those reported in other acquired myeloproliferative syndromes and could be associated with an intrinsic abnormality of HOA fibroblast growth.

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The effect of 5-fluorouracil on erythropoiesis

Blood ◽

10.1182/blood.v77.6.1164.1164 ◽

1991 ◽

Vol 77 (6) ◽

pp. 1164-1170 ◽

Cited By ~ 30

Author(s):

IN Rich

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Normal Values ◽

Biologically Active ◽

Stem Cell Population ◽

Enzyme Linked Immunosorbent Assay ◽

Proliferative Potential ◽

Colony Forming Unit ◽

Kinetics Of

Abstract The effects of a single dose (150 mg/kg) of 5-fluorouracil on mature erythroid and erythropoietic and multipotential in vitro precursor populations in the bone marrow and spleen and circulating biologically (erythroid colony forming unit [CFU-E] assay) and immunologically active (enzyme-linked immunosorbent assay) erythropoietin (Epo) are described. All mature erythroid (reticulocytes, erythrocytes) and in vitro erythropoietic precursors (CFU-E, erythroid burst-forming unit [BFU-E]) are severely reduced, if not eradicated. Transient repopulation of the pure BFU-E and CFU-E populations on days 6 and 7, respectively, produces a marked reticulocytosis after day 9. Circulating Epo increases to above normal values by day 2. However, whereas biologically active Epo remains constant at this level until day 9, immunologically active Epo continually increases; by day 12, however, both assays detect circulating Epo levels of about 400 mU/mL. In vitro multipotential stem cells (BFU-E mix) are reduced to 32% on day 1, 7.6% on day 2, and return to normal values between days 4 and 5. The survival and repopulation kinetics of the BFU-E mix imply a stem cell population more mature than the high proliferative potential colony-forming cells. However, the BFU-E mix may be responsible for erythropoiesis repopulating ability.

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Long-term generation and expansion of human primitive hematopoietic progenitor cells in vitro

Blood ◽

10.1182/blood.v81.3.661.661 ◽

1993 ◽

Vol 81 (3) ◽

pp. 661-669 ◽

Cited By ~ 92

Author(s):

EF Srour ◽

JE Brandt ◽

RA Briddell ◽

S Grigsby ◽

T Leemhuis ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Bone Marrow Cells ◽

Fold Increase ◽

Hematopoietic Progenitor Cells ◽

Proliferative Potential ◽

Hematopoietic Progenitor ◽

Hla Dr

Abstract Although sustained production of committed human hematopoietic progenitor cells in long-term bone marrow cultures (LTBMC) is well documented, evidence for the generation and expansion of human primitive hematopoietic progenitor cells (PHPC) in such cultures is lacking. For that purpose, we attempted to determine if the human high proliferative potential colony-forming cell (HPP-CFC), a primitive hematopoietic marrow progenitor cell, is capable of generation and expansion in vitro. To that effect, stromal cell-free LTBMC were initiated with CD34+ HLA-DR-CD15- rhodamine 123dull bone marrow cells and were maintained with repeated addition of c-kit ligand and a synthetic interleukin-3/granulocyte-macrophage colony-stimulating factor fusion protein. By day 21 of LTBMC, a greater than twofold increase in the number of assayable HPP-CFC was detected. Furthermore, the production of HPP-CFC in LTBMC continued for up to 4 weeks, resulting in a 5.5-fold increase in HPP-CFC numbers. Weekly phenotypic analyses of cells harvested from LTBMC showed that the number of CD34+ HLA-DR- cells increased from 10(4) on day 0 to 56 CD34+ HLA-DR- cells increased from 10(4) on day 0 to 56 x 10(4) by day 21. To examine further the nature of the in vitro HPP-CFC expansion, individual HPP- CFC colonies were serially cloned. Secondary cloning of individual, day 28 primary HPP-CFC indicated that 46% of these colonies formed an average of nine secondary colony-forming unit--granulocyte-macrophage (CFU-GM)--derived colonies, whereas 43% of primary HPP-CFC gave rise to between one and six secondary HPP-CFC colonies and 6 to 26 CFU-GM. These data show that CD34+ HLA-DR- CD15- rhodamine 123dull cells represent a fraction of human bone marrow highly enriched for HPP-CFC and that based on their regeneration and proliferative capacities, a hierarchy of HPP-CFC exists. Furthermore, these studies indicate that in the presence of appropriate cytokine stimulation, it is possible to expand the number of PHPC in vitro.

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The effect of 5-fluorouracil on erythropoiesis

Blood ◽

10.1182/blood.v77.6.1164.bloodjournal7761164 ◽

1991 ◽

Vol 77 (6) ◽

pp. 1164-1170

Author(s):

IN Rich

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Normal Values ◽

Biologically Active ◽

Stem Cell Population ◽

Enzyme Linked Immunosorbent Assay ◽

Proliferative Potential ◽

Colony Forming Unit ◽

Kinetics Of

The effects of a single dose (150 mg/kg) of 5-fluorouracil on mature erythroid and erythropoietic and multipotential in vitro precursor populations in the bone marrow and spleen and circulating biologically (erythroid colony forming unit [CFU-E] assay) and immunologically active (enzyme-linked immunosorbent assay) erythropoietin (Epo) are described. All mature erythroid (reticulocytes, erythrocytes) and in vitro erythropoietic precursors (CFU-E, erythroid burst-forming unit [BFU-E]) are severely reduced, if not eradicated. Transient repopulation of the pure BFU-E and CFU-E populations on days 6 and 7, respectively, produces a marked reticulocytosis after day 9. Circulating Epo increases to above normal values by day 2. However, whereas biologically active Epo remains constant at this level until day 9, immunologically active Epo continually increases; by day 12, however, both assays detect circulating Epo levels of about 400 mU/mL. In vitro multipotential stem cells (BFU-E mix) are reduced to 32% on day 1, 7.6% on day 2, and return to normal values between days 4 and 5. The survival and repopulation kinetics of the BFU-E mix imply a stem cell population more mature than the high proliferative potential colony-forming cells. However, the BFU-E mix may be responsible for erythropoiesis repopulating ability.

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The Potential Role of the Six1/Eya1 Pathway in the Establishment of Leukemia Stem Cells in MLL-ENL – Induced Leukemia

Blood ◽

10.1182/blood.v118.21.1362.1362 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1362-1362 ◽

Cited By ~ 1

Author(s):

Sylvia Takacova ◽

Pavla Luzna ◽

Viktor Stranecky ◽

Vladimir Divoky

Keyword(s):

Bone Marrow ◽

Cellular Transformation ◽

Leukemia Cells ◽

Leukemia Stem Cells ◽

Transcription Profile ◽

Self Renewal ◽

Kinetics Of

Abstract Abstract 1362 During the multistep pathogenesis of acute leukemia (AL), a pool of leukemia stem cells (LSCs) emerges that is capable of limitless self-renewal and ensuring disease maintenance. The molecular mechanism that controls the kinetics of cellular transformation and development of LSCs is largely unknown. Using our MLL-ENL-ERtm mouse model, we have previously shown (Takacova et al., Blood 2009, 114 (22): 947–947, ASH abstract) activation of the ATR/ATM-Chk1/Chk2-p53/p21 checkpoint leading to senescence at early stages of cellular transformation (myeloproliferation), thereby preventing AL development in vivo. Experimental ATM/ATR inhibition accelerated the transition to immature cell states, acquisition of LSC properties and AL development in these mice. The MLL-ENL-ERtm mouse model allows us to study the kinetics of MLL-ENL-ERtm LSC development. We raised the questions how the transformation process progresses from the pre-LSC to the LSC state, and how DNA damage response (DDR) - mediated senescence affects the transition in gene expression. Given that the threshold of DDR signaling events is rate-limiting, we determined the transcription profile of the pre- LSC–enriched cell states derived from bone marrow and spleen of the MLL-ENL-ERtm mice at the early disease stage, and we correlated this transcription profile with the level of DDR, proliferation rate and induction of senescence. Pair-wise comparisons revealed up-regulation of the Six1 transcription factor gene and its cofactor Eya1 in the MLL-ENL-ERtm pre-LSCs in association with aberrant proliferation in both tissues. The notable difference between the two tissues concerning the barrier induction was the higher threshold of DDR and senescence in the bone marrow due to cooperation with inflammatory cytokines that fine-tune the DDR level. Interestingly, the expression of Six1 and Eya1 genes was down-regulated in senescence exclusively in the bone marrow. Consistent with these in vivo data, we found Six1 expression decreased in response to inflammation/DDR-induced senescence in the MLL-ENL-ERtm bone marrow cells cultured in vitro and correlated with SA-beta-gal positivity and p16 up-regulation. Six1 mRNA level was decreased only transiently after ionizing radiation (4 Gy)-induced DDR in the same cell line. These data suggest that Six1 expression is down-regulated in response to high DDR and permanent cell-cycle arrest in the MLL-ENL-ERtm pre-LSCs. Furthermore, we identified the transcription profile of the LSC-enriched cell state after inhibition of DDR in caffeine-treated MLL-ENL-ERtm mice in vivo. Interestingly, the expression level of Six1 and Eya1 was significantly increased in the bone marrow and spleen of the MLL-ENL-ERtm AML mice compared to the early (preleukemia) stage. High expression of Six1 and Eya1 and higher cell number expressing these genes was further confirmed by immunohistochemical staining on tissue sections. The MLL-ENL-ERtm LSC-enriched spleen cells showed increased colony forming ability in vitro and leukemia-initiating potential in serial transplantation experiments compared to pre-LSCs. Moreover, we detected Six1 and Eya1 expression in the infiltrating leukemia cells in tissues of the caffeine-treated MLL-ENL-ERtm AML mice and in a subset of leukemia cells in transplanted mice. Based on these findings and correlations, we hypothesized that the Six1/Eya1 pathway might be involved in regulation of some of the aspects of LSC development as well as invasion and maintenance of leukemia in our MLL-ENL-ERtm mice. Notably, our data indicate that senescence represses a subset of the MLL-ENL-downstream transcription response and prevents full activation of self-renewal. Experiments leading to more detailed understanding of the role of the Six1/Eya1 pathway in the MLL-ENL-induced cellular transformation are ongoing. Disclosures: No relevant conflicts of interest to declare.

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Osteal Macrophages and Megakaryocytes Increase Adipogenesis

Proceedings of IMPRS ◽

10.18060/25739 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Nikhil Tewari ◽

Deepa Kanagasabapathy ◽

Rachel J. Blosser ◽

Edward F. Srour ◽

Angela Bruzzaniti ◽

...

Keyword(s):

Bone Marrow ◽

Cell Sorting ◽

Fetal Liver ◽

Fold Increase ◽

Wild Type ◽

Bone Marrow Adipose Tissue ◽

New Treatments

Bone marrow adipose tissue (MAT) increases with aging and contributes to low bone density and skeletal fractures. However, the cells and factors within the bone marrow (BM) that regulate adipogenesis remain poorly understood. In the current study, we examined the role of osteal macrophages (OMs) and megakaryocytes (MKs) on the regulation of adipogenesis. We cultured murine osteoblasts/osteoblast progenitors (OBs from hereon) derived from neonatal calvarial cells (CCs, a combination of OBs and OMs) or OBs isolated by fluorescence activated cell sorting (FACS) in the presence or absence of fetal liver derived murine MK. The cells underwent induced adipogenesis for 5-7 days by supplementation of media with insulin, indomethacin, and dexamethasone, and then the number of adipocytes was quantified. We found that co-culturing MKs and OMs with OBs results in up to a 7.8-fold and 11.7-fold increase in adipocytes, respectively. We also elucidated that thrombopoietin (TPO), the major growth factor for MKs, inhibits adipogenesis in both OBs and CCs by approximately 60%. Similarly, we found that CCs and OBs derived from mice deficient in the TPO receptor, Mpl, had approximately 30% more adipocytes than their wild-type (WT) counterparts. Finally, in vitro findings were corroborated in vivo through quantification of MKs and adipocytes in mice in which MK number was elevated or reduced. Mice with significantly higher numbers of BM-residing MKs also had significantly higher numbers of BM-residing adipocytes. Because there is typically an inverse relationship between adipogenesis and osteogenesis, understanding ways to inhibit adipogenesis could lead to an increase in OB number and bone formation, which in turn could lead to new treatments for bone loss diseases such as osteoporosis.

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Evidence of a Role for Monocytes in Dissemination and Brain Invasion by Cryptococcus neoformans

Infection and Immunity ◽

10.1128/iai.01065-08 ◽

2008 ◽

Vol 77 (1) ◽

pp. 120-127 ◽

Cited By ~ 224

Author(s):

Caroline Charlier ◽

Kirsten Nielsen ◽

Samira Daou ◽

Madly Brigitte ◽

Fabrice Chretien ◽

...

Keyword(s):

Bone Marrow ◽

Cryptococcus Neoformans ◽

Trojan Horse ◽

Brain Invasion ◽

Fungal Burden ◽

Trojan Horse Method ◽

Kinetics Of ◽

Intravenous Inoculation

ABSTRACT The pathogenesis of cryptococcosis, including the events leading to the production of meningoencephalitis, is still largely unknown. Evidence of a transcellular passage of Cryptococcus neoformans across the blood-brain barrier (BBB) and subsequent BBB disruption exists, but the paracellular passage of free yeasts and the role of monocytes in yeast dissemination and brain invasion (Trojan horse method) remain uncertain. We used our model of disseminated cryptococcosis, in which crossing of the BBB starts 6 h after intravenous inoculation, to study paracellular passage of the BBB. We prepared bone marrow-derived monocytes (BMDM) infected in vitro with C. neoformans (BMDM yeasts) and free yeasts and measured fungal loads in tissues. (i) Spleen and lung CFU were >2-fold higher in mice treated with BMDM yeasts than in those treated with free yeasts for 1 and 24 h (P < 0.05), while brain CFU were increased (3.9 times) only at 24 h (P < 0.05). (ii) By comparing the kinetics of brain invasion in naïve mice and in mice with preestablished cryptococcosis, we found that CFU were lower in the latter case, except at 6 h, when CFU from mice inoculated with BMDM yeasts were comparable to those measured in naïve mice and 2.5-fold higher than those in mice with preestablished cryptococcosis who were inoculated with free yeasts. (iii) Late phagocyte depletion obtained by clodronate injection reduced disease severity and lowered the fungal burden by 40% in all organs studied. These results provide evidence for Trojan horse crossing of the BBB by C. neoformans, together with mechanisms involving free yeasts, and overall for a role of phagocytes in fungal dissemination.

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Targeting Cyclin D1 in the Treatment of Multiple Myeloma: Preclinical Validation of a Novel Specific Small Molecule Cyclin D1 Inhibitor, P276-00.

Blood ◽

10.1182/blood.v108.11.3454.3454 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3454-3454

Author(s):

Noopur Raje ◽

Teru Hideshima ◽

Sonia Vallet ◽

Shweta Chhetri ◽

Constantine Mitsiades ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Bone Marrow ◽

Phase I ◽

Cyclin D1 ◽

Therapeutic Target ◽

Growth Arrest

Abstract Either an overexpression or dysregulation of cyclin D1, D2, or D3, has been reported in the majority of multiple myeloma (MM) tumors, suggesting a possible early unifying event in MM pathogenesis. This proposed critical role of cyclin D dysregulation in myeloma pathogenesis makes the cyclins, specifically cyclin D1, an attractive therapeutic target. We have evaluated a specific small molecule cyclin D1 inhibitor, P276-00 in MM. Its specificity has been confirmed in an in vitro kinase assay by potent inhibitory activity for Cdk4-D1 as compared to Cdk2-E. Additionally in vitro kinase assays against a broad range of other kinases have also confirmed specificity for D1 and B cyclins at nanomolar concentrations. P276-00 has been tested against a wide range of cancer cell types in both in vitro and tumor xenograft models. Based on these data, it is undergoing phase I clinical testing in North America. We have observed both time and dose dependent in vitro activity against a broad range of MM cells sensitive and resistant to conventional agents like dexamethasone, doxorubicin, and melphalan with IC50 ranging from 400–800nM. Spectral karyotyping confirmed t(11;14) (q13;q32) in KMS 12 MM cells which were sensitive to P276-00. Importantly, it has demonstrated activity in primary patient derived tumor cells. Cell cycle analysis confirmed that P276-00 induced either growth arrest or apoptosis in MM cells depending on the cell line. Apoptosis was in part caspase dependent suggested by partial reversal of cytotoxicity by Z-VAD Fmk. P276-00 inhibited Rb-1 phosphorylation as early as 6 hours in most of the MM cell lines tested associated with a decrease in cdk4 suggesting a regulatory role of P276-00 in cell cycle progression. These changes preceeded growth arrest and apoptosis of MM cells on cell cycle analysis. Ongoing studies are using SiRNA to Cyclin D1 to confirm this regulatory role of P276-00. As cyclin D1 dysregulation or overexpression can render MM cells more susceptible to proliferative stimuli such as IL-6, IGF-1, and the bone marrow microenvironment, we tested the effects of P276-00 in the presence of these cytokines and bone marrow stromal cells (BMSCs). Our data confirms that P276-00 was able to overcome these proliferative signals and induce apoptosis in MM cells. Next we evaluated in vivo efficacy of P276-00 in NOD-SCID mice bearing GFP+ MM xenografts. Animals were treated with either control PBS or P276-00 intraperitoneally at 25 mg/kg three times a week for 3 weeks. Our data confirms in vivo anti-tumor activity of P276-00 as suggested by a significant decrease in biluminesence of GFP+ MM cells (p<0.05) and a decrease in tumor volume. Immunohistochemistry on tumor tissue from P76.00 treated, and control animals validates our in vitro studies and will be presented. In vitro combination studies with bortezomib have been completed suggesting synergism. P276-00 and bortezomib combination is currently being tested in our in vivo model. These studies confirm cyclin D1 to be an important therapeutic target in MM and form the basis of a phase I/II study of P276-00 alone and in combination in the treatment of MM.

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

Blood ◽

10.1182/blood.v65.3.689.689 ◽

1985 ◽

Vol 65 (3) ◽

pp. 689-695 ◽

Cited By ~ 7

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.

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Long-term generation and expansion of human primitive hematopoietic progenitor cells in vitro

Blood ◽

10.1182/blood.v81.3.661.bloodjournal813661 ◽

1993 ◽

Vol 81 (3) ◽

pp. 661-669 ◽

Cited By ~ 1

Author(s):

EF Srour ◽

JE Brandt ◽

RA Briddell ◽

S Grigsby ◽

T Leemhuis ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Bone Marrow Cells ◽

Fold Increase ◽

Hematopoietic Progenitor Cells ◽

Proliferative Potential ◽

Hematopoietic Progenitor ◽

Hla Dr

Although sustained production of committed human hematopoietic progenitor cells in long-term bone marrow cultures (LTBMC) is well documented, evidence for the generation and expansion of human primitive hematopoietic progenitor cells (PHPC) in such cultures is lacking. For that purpose, we attempted to determine if the human high proliferative potential colony-forming cell (HPP-CFC), a primitive hematopoietic marrow progenitor cell, is capable of generation and expansion in vitro. To that effect, stromal cell-free LTBMC were initiated with CD34+ HLA-DR-CD15- rhodamine 123dull bone marrow cells and were maintained with repeated addition of c-kit ligand and a synthetic interleukin-3/granulocyte-macrophage colony-stimulating factor fusion protein. By day 21 of LTBMC, a greater than twofold increase in the number of assayable HPP-CFC was detected. Furthermore, the production of HPP-CFC in LTBMC continued for up to 4 weeks, resulting in a 5.5-fold increase in HPP-CFC numbers. Weekly phenotypic analyses of cells harvested from LTBMC showed that the number of CD34+ HLA-DR- cells increased from 10(4) on day 0 to 56 CD34+ HLA-DR- cells increased from 10(4) on day 0 to 56 x 10(4) by day 21. To examine further the nature of the in vitro HPP-CFC expansion, individual HPP- CFC colonies were serially cloned. Secondary cloning of individual, day 28 primary HPP-CFC indicated that 46% of these colonies formed an average of nine secondary colony-forming unit--granulocyte-macrophage (CFU-GM)--derived colonies, whereas 43% of primary HPP-CFC gave rise to between one and six secondary HPP-CFC colonies and 6 to 26 CFU-GM. These data show that CD34+ HLA-DR- CD15- rhodamine 123dull cells represent a fraction of human bone marrow highly enriched for HPP-CFC and that based on their regeneration and proliferative capacities, a hierarchy of HPP-CFC exists. Furthermore, these studies indicate that in the presence of appropriate cytokine stimulation, it is possible to expand the number of PHPC in vitro.

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

Blood ◽

10.1182/blood.v65.3.689.bloodjournal653689 ◽

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.

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