Abstract
The role of intracellular Ca2+ (Ca2+i) on hematopoiesis was investigated in long term bone marrow cultures using cytokines and agonists of P2 receptors. Cytokines interleukin 3 and granulocyte/macrophage colony stimulator factor promoted a modest increase in Ca2+i concentration ([Ca2+]i) with activation of phospholipase Cgamma, MEK1/2, and Ca2+/calmodulin kinase II. Involvement of protein kinase C was restricted to stimulation with interleukin 3. In addition, these cytokines promoted proliferation (20 times) and an increase in the Gr-1(-)Mac-1+ population with participation of gap junctions (GJ). Nevertheless ATP, ADP, and UTP promoted a large increase in [Ca2+]i, moderate proliferation (6 times), a reduction in the primitive Gr-1(-)Mac-1(-)c-Kit+ population, and differentiation into macrophages without participation of GJ. It is likely that Ca2+i participates as a regulator of hematopoietic signaling: moderate increases in [Ca2+]i would be related to cytokine-dependent proliferation with participation of GJ, whereas high increases in [Ca2+]i would be related to macrophage differentiation without maintenance of the primitive population.
Highlights
Are sensitive to changes in [Ca2ϩ]i, translating these changes into cellular physiological effects
Hematopoietic Cytokines Promote Increase in [Ca2ϩ]i of Hematopoietic Cells—long term bone marrow cultures (LTBMC) reproduce myelopoiesis, the formation of granulocytic, monocyte/macrophage, and erythroid cells, erythrocyte formation depends on external erythropoietin [9]
LTBMC were incubated in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 0.5% fetal bovine serum (FBS) for 24 h before any stimulus to synchronize the cellular cycle; hematopoietic cells were sensitive to lower concentrations of FBS
Summary
Extraction of Bone Marrow and LTBMC—To establish the stroma layer, femur bones were excised from mice (C57Bl/6), and the medullary cavities were aseptically flushed with Iscove’s modified Dulbecco’s medium (IMDM; Invitrogen). LTBMC were stimulated with IL-3 (Sigma) or GM-CSF (Sigma) in 12-well plates in 0.5% FBS, IMDM (1 ml) to evaluate the proliferation and cell populations. Calcium Measurements in LTBMC—Bone marrow cells were seeded on cover glass slides (25 mm) in 6-well plates. Confocal Microscopy—Bone marrow cells were seeded on cover glass slides (13 mm) under conditions described above. These cells were cultured previously in 0.5% FBS (24 h) and stimulated with GM-CSF or IL-3 (5 min). Flow Cytometry Analysis—To determine the hematopoietic populations present in LTBMC, whole cells where collected after 3 days of stimulation with IL-3, GM-CSF, ATP (Sigma), ADP (Sigma), and UTP (Calbiochem). Statistical comparisons were performed by using Student’s t test or analysis of variance. p values Ͻ0.05 were considered statistically significant
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