Cytokine combinations differentially influence the SDF-1alpha-dependent migratory activity of cultivated murine hematopoietic stem and progenitor cells

Stromal cell-derived factor-1alpha (SDF-1alpha) is a strong migratory stimulant for hematopoietic stem and progenitor cells (HSPCs). The hematopoietic cytokines thrombopoietin (TPO), Flt3-ligand (FL), stem cell factor (SCF) and interleukin 11 (IL-11) are able to stimulate amplification of primitive murine hematopoietic stem cells (HSCs) in vitro. The effects of these cytokines on SDF-1alpha-induced migratory activity of murine Lin(-)c-kit+ HSPC were analyzed by cultivation of these cells in the presence of 12 combinations of FL, TPO, SCF and IL-11. Migratory activity was measured in a three-dimensional collagen matrix using time-lapse video microscopy. Each cytokine combination had a distinct effect on SDF-1alpha-stimulated migratory activity. For instance, FL- and SCF-cultivated cells showed a high migratory SDF-1alpha response, while cells cultivated with SCF, TPO and IL-11 did not react to SDF-1alpha stimulation with an elevated migration rate. Our data indicate that the differences in the migratory SDF-1alpha response are not related to different CXCR4 expression levels, but rather to the differential engagement of the CXCR4-dependent MAPK p42/44 and PI3K signal transduction pathways. This indicates that hematopoietic cytokines can have a significant impact on SDF-1alpha-stimulated migratory activity and the underlying intracellular signaling processes in cultivated HSPCs.     

Adenosine potentiates stimulatory effects on granulocyte-macrophage hematopoietic progenitor cells in vitro of IL-3 and SCF, but not those of G-CSF, GM-CSF and IL-11

The aim of the studies was to ascertain if adenosine is able to co-operate with selected hematopoietic growth factors and cytokines, namely with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), interleukin-3 (IL-3), and interleukin-11 (IL-11), in inducing the growth of colonies from hematopoietic progenitor cells for granulocytes and macrophages (GM-CFC) from normal bone marrow cells in vitro. Adenosine was found not to produce any colonies when present in the cultures as the only potential stimulator. All the tested cytokines and growth factors were observed to induce the growth of distinct numbers of GM-CFC colonies, with the exception of IL-11. When suboptimal concentrations of the evaluated cytokines and growth factors were tested in the cultures in which various concentrations of adenosine were concomitantly present, mutually potentiating effects were found in the case of IL-3 and SCF. These results confirm the role of adenosine in regulation of granulopoiesis and predict IL-3 and SCF as candidates for further in vivo studies of their combined administration with adenosine.     

The enzyme cytochemistry and composition of elasmobranch granulocytes

Peroxidase, alkaline phosphatase, acid phosphatase, β-glucuronidase, α-naphthyl acetate esterase (ANAE), α-naphthyl butyrate esterase, naphthol AS-D chloroacetate esterase, acetyl-L-tyrosine-α-naphthyl esterase (ATNE), tosyl-L-lysine-α-naphthyl esterase (TLNE) and periodic acid-Schiff (PAS) were studied in 17 species of elasmobranchs in which granulocytes had previously been identified at the ultrastructural level.
Eosinophils, eosinophilic and neutrophilic granulocytes contained variable acid phosphatase, esterases and PAS, but they were strongest in neutrophilic granulocytes; particularly ANAE. Esterases were released into surrounding plasma and therefore probably function as ectoenzymes. In eosinophils and some neutrophilic granulocytes there were indications of weak peroxidase, but this could not be conclusively demonstrated. Alkaline phosphatase was diffuse between granules in some eosinophils of Pavoraja , and (β-glucuronidase was diffuse in neutrophilic granulocytes of Etmopterus baxteri , otherwise granulocytes lacked these enzymes. Neutrophilic granulocytes stained moderately to strongly for ATNE and weakly and inconsistently for TLNE in Squalus acanthias and Dalatias licha . with a similar reaction in granular lymphocytoid and thrombocytoid cells of Galeorhinus ausiralis and Raja nasuta . The enzyme composition of these granulocytes is discussed.     

Modification of expression of stem cell factor by various cytokines.

The local production of stem cell factor (SCF) may be an important mechanism for regulating proliferation, differentiation, and migration of various cells bearing c-kit receptors, and might be susceptible to the cytokines that serve in inflammation and tissue repair. We have demonstrated that in three murine cell lines, Balb/3T3A31, MC3T3-E1, and C3H-2K, which constitutively produced SCF with different quantity, the SCF mRNA expression was greatly enhanced in response to basic fibroblast growth factor (bFGF) or transforming growth factor beta1 (TGF-beta1). The study was carried out by in situ hybridization utilizing nonradioactive oligonucleotide probes and quantitative image analysis. Leukemia inhibitory factor (LIF) or interleukin-4 (IL-4) moderately increased SCF mRNA in all cell lines, but IL-3 did not. The dot-blot enzyme-linked immunosorbent assay (ELISA) further confirmed that SCF protein production in these cell lines and bone marrow stromal cells was markedly enhanced by TGF-beta1, although TGF-beta1 suppressed the proliferation of all these cells. bFGF also enhanced the SCF production in these cell lines, but did not in bone marrow stromal cells, suggesting a difference in their susceptibility to the cytokine. Our results suggest that TGF-beta1 and bFGF potentially modulate the biological function of cells bearing c-kit receptors through the modulation of SCF production in fibroblasts.     

Cytokine-mediated protein kinase C activation is a signal for lineage determination in bipotential granulocyte macrophage colony-forming cells

Granulocyte macrophage colony-forming cells (GM-CFC) have the potential to develop into either macrophages and/or neutrophils. With a highly enriched population of these cells we have found that although GM-CFC are equally responsive to macrophage colony stimulating factor (M-CSF) and stem cell factor (SCF) in terms of DNA synthesis, M-CSF stimulated the development of colonies containing macrophages in soft gel assays, while SCF promoted neutrophilic colony formation. When SCF and M-CSF were combined, mainly macrophage development was stimulated both in soft agar colony-forming assays and liquid cultures. An analysis of some potential signaling mechanisms associated with cytokine-mediated developmental decisions in GM-CFC revealed that M-CSF, but not SCF, was able to chronically stimulate phosphatidylcholine breakdown and diacylglycerol production, indicating that protein kinase C (PKC) may be involved in the action of M-CSF. Furthermore, M-CSF, but not SCF, can increase the levels of PKC alpha (PKC alpha) expression and stimulate the translocation of PKC alpha to the nucleus. When the PKC inhibitor, calphostin C, was added to GM-CFC cultured in M-CSF then predominantly neutrophils were produced, conversely PKC activators added with SCF stimulated macrophage development. The data indicate a role for PKC in M-CSF-stimulated macrophage development from GM-CFC.     

Granulocyte interactions with GM-CSF and G-CSF secretion by endothelial cells and monocytes

We have, in previous studies, characterized the cytokine and cellular regulation of GM-CSF and G-CSF production by monocytes and endothelial cells. In this study, we investigated the regulatory role of granulocytes. The addition of granulocytes to endotoxin-stimulated monocytes dose-dependently decreased both GM-CSF and G-CSF concentrations, presumably by absorbing the cytokines. A similar dose-dependent decrease in GM-CSF concentration was found when granulocytes were added to IL-1-stimulated endothelial cells. In contrast, G-CSF secretion by endothelial cells responded to granulocytes in a biphasic fashion. At low granulocyte concentrations, endothelial cells responded with an increased G-CSF secretion, but at high concentrations of granulocytes G-CSF secretion was down modulated. Our results suggest that there exist two loops between granulocytes and endothelial cells for regulating G-CSF activity. Granulocytes can stimulate G-CSF secretion by activated endothelial cells but can also decrease the biological activity by absorbing the cytokine. These mechanisms might be involved in the regulation of the local and systemic levels of granulocytes.     

Cytokine-augmented culture of haematopoietic progenitor cells in a novel three-dimensional cell growth matrix

Studies aimed at the in vitro expansion of haematopoietic progenitor cells (HPCs) have suffered from the conflict of increasing cell numbers while maintaining long-term repopulating ability. We have developed a long-term bone marrow bioreactor culture system resembling the marrow-microenvironment that cultures HPCs in an inert, three-dimensional, porous biomatrix termed Cellfoam. Previous studies have shown that the short-term culture of CD34(+)cells in Cellfoam improved the maintenance and multipotency of haematopoietic stem cells compared to cells cultured on plastic dishes. In this study, we examined the effects of low concentrations of cytokines including stem cell factor (SCF), IL-3, and Flk-2/Flt-3 ligand, on the maintenance, preservation and multipotency of CD34(+) cells cultured for 3 or 6 weeks in Cellfoam. Analysis of cell yields using flow cytometry showed that in SCF and Flk-2/Flt-3 ligand-supplemented cultures as well as cytokine-free cultures, a higher number of CD45(+)34(+) and CD45(+)34(+)38(-) cells is observed in Cellfoam cultures as compared to plastic cultures. The function of cultured cells was evaluated in colony-forming assays. The data demonstrate that Cellfoam cultures supplemented with SCF and Flk-2/Flt-3 ligand resulted in a higher output of colony activity compared to plastic cultures. Analysis of CAFC (29 days) activity also demonstrated that primitive progenitors were maintained to a greater extent in Cellfoam cultures containing either no cytokines or low concentrations of early-acting cytokines. These data suggest that culture of HPCs in three-dimensional bioreactors such as Cellfoam for extended periods may benefit from the addition of low levels of early-acting cytokines, including SCF and Flk-2/Flt-3 ligand, resulting in high yields of cells that are enriched for multipotent haematopoietic progenitors. These findings demonstrate that a three-dimensional matrix promotes the survival of primitive HPCs in culture and may modulate the in vitro effects of cytokines.     

Effect of hematopoietic cytokines on renal function in cisplatin-induced ARF in mice

In this study, the effect of hematopoietic cytokines, i.e., granulocyte-colony stimulating factor (G-CSF), stem cell factor (SCF), and granulocyte-macrophage-colony stimulating factor (GM-CSF), on renal function was studied in cisplatin-induced acute renal failure in mice. Treatment with G-CSF significantly ameliorated both BUN and serum creatinine increase induced by cisplatin administration with concomitant alleviation in the degree of necrotic change, enhancement in DNA synthesis, and decrease in apoptosis of renal tubular cells. There was no significant change observed among these parameters following treatment with SCF or with GM-CSF. Serum hepatocyte growth factor level was significantly lower in mice treated with cisplatin and G-CSF compared with that in those treated with cisplatin only. In conclusion, G-CSF, but not SCF or GM-CSF, acts to accelerate regeneration and prevent apoptosis of renal tubular epithelial cells and leads to reduced renal injury in cisplatin-induced acute renal failure in mice.     

Expansion of megakaryocyte progenitors from cryopreserved leukocyte concentrates of human placental and umbilical cord blood in short-term liquid culture

Long-term severe thrombocytopenia following human placental and umbilical cord blood (CB) transplantation is a significant clinical problem. We studied the ex vivo expansion of megakaryocytic progenitor cells (CFU-Meg) from cryopreserved/thawed leukocyte concentrates (LC) of CB prepared by the Tokyo Cord Blood Bank protocol. The LC cells were cultured in serum-free culture medium supplemented with a combination of early-acting cytokines including thrombopoietin (TPO), flt3-ligand (FL), and stem cell factor (SCF). Combination of TPO plus FL, TPO plus SCF, and all of these cytokines together resulted in 8.9-, 7.7-, and 8.4-fold increases in CFU-Meg, respectively, by Day 5 of culture. Our results showed that this simple expansion strategy has the potential for expanding CFU-Meg from cryopreserved/thawed LC cells from CB.     

The cloning and expression of the cDNA for ovine stem cell factor (kit-ligand) and characterization of its in vitro haematopoietic activity.

The cDNA encoding the soluble form of ovine stem cell factor (SCF) has been cloned and expressed. The soluble protein is predicted to be 165/166 amino acids in length, one more than the human and murine SCFs with which it shares 87% and 81% identity respectively. Ovine SCF has 98.5%, 95% and 91% identity with cattle, pig and dog SCF, respectively. The recombinant ovine (rov) SCF protein has been expressed in Chinese hamster ovary (CHO) cells, purified, and its biological activity on ovine bone marrow cells compared with that of interleukin 3 (rovIL-3), granulocyte-macrophage colony-stimulating factor (rovGM-CSF), interleukin 5 (rovIL-5), human macrophage colony-stimulating factor (M-CSF) and human erythropoietin (epo). On its own rovSCF supported the development of small numbers of neutrophil, macrophage, eosinophil, granulocyte-macrophage, mixed cell phenotype, haemopoietic blast cell and basophilic granular cell colonies in a soft agar clonogenic assay. In combination with each of the above cytokines rovSCF supported an increase in the number and size of the lineage-specific colony types that were stimulated by the other cytokines on their own. In an assay for precursors of multipotential colony-forming cells (multi-CFC), rovSCF in combination with rovIL-3 (but neither cytokine alone) supported the development of these early haematopoietic progenitor cells.     

Effects of recombinant human stem cell factor (SCF) on the growth of human progenitor cells in vitro.

We have studied the effect of recombinant human Stem Cell Factor (SCF) on the growth of human peripheral blood, bone marrow, and cord blood progenitor cells in semisolid medium. While SCF alone had little colony-stimulating activity under fetal bovine serum (FBS)-deprived culture conditions, SCF synergized with erythropoietin (Epo), granulocyte/macrophage colony-stimulating factor (GM-CSF), and interleukin 3 (IL-3) to stimulate colony growth. Colony morphology was determined by the late-acting growth factor added along with SCF. Of all the combinations of growth factors, SCF plus IL-3 and Epo resulted in the largest number of mixed-cell colonies--a larger number than observed with IL-3 and Epo alone even in FBS-supplemented cultures. These results suggest that SCF is a growth factor that more specifically targets early progenitor cells (mixed-cell colony-forming cells) and has the capacity to synergize with a wide variety of other hematopoietic growth factors to cause the proliferation and differentiation of committed progenitor cells. Our studies indicate that SCF may be the earliest acting growth factor described to date.     

Application of proteoglycan extracted from the nasal cartilage of salmon heads for ex vivo expansion of hematopoietic progenitor cells derived from human umbilical cord blood

Highly purified proteoglycan (PG) extracted from the nasal cartilage of salmon heads was applied to the ex vivo expansion of hematopoietic progenitor cells prepared from human umbilical cord blood in serum-free cultures supplemented with the combination of early-acting cytokines, thrombopoietin (TPO), interleukin-3 (IL-3) and stem cell factor (SCF). PG showed no promoting effects on the cell proliferation rate; however, they promoted the generation of progenitor cells for granulocyte-macrophages, erythrocytes and/or megakaryocytes in culture with TPO alone or SCF plus TPO. However, no promoting effect was observed in a combination of IL-3 plus SCF, which showed the highest cell proliferation rate. PG failed to promote the generation of mixed colony-forming units (i.e. the relatively immature cells in hematopoiesis). These results suggest that PG acts on the relatively mature stem/progenitor cells, and may function as a regulatory factor in the differentiation pathway of hematopoiesis.     

Cytokine-pretreatment of CD34(+) cord blood stem cells in vitro reduces long-term cell engraftment in NOD/SCID mice

Stem cell homing, engraftment and organ regeneration are controlled by cytokines, chemokines and cell-cell interactions. In this paper, cytokine effects on homing- and engraftment-related characteristics of CD34(+) cord blood cells were examined. Untreated CD34(+) cells were mainly in the G(0)/G(1) cell cycle phase, expressed adhesion receptors on a low level, were positive for vimentin, and negative for the epithelial marker cytokeratin 8/18. Treatment with stem cell factor (SCF) stimulated cell proliferation, increased the number of cells in S and G(2)/M cell cycle phase as well as the expression of adhesion receptors. The expression of cytokeratin 8/18 was increased and that of vimentin remained unchanged. Hepatocyte growth factor (HGF) did not stimulate cell proliferation and expression of adhesion receptors, but increased expression of cytokeratin 8/18. In NOD/SCID mice, kinetics of stem cell distribution revealed a fast elimination of human cells from blood. An increase in the number of engrafted cells was observed in different mouse organs in a time-dependent manner, preferentially in bone marrow, spleen and liver. Pretreatment with SCF resulted in reduction of long-term engraftment in bone marrow. HGF pretreatment of cord blood cells showed no significant effects on long-term engraftment capacity in mouse organs compared to untreated cells. Our data provide in vivo evidence that pretreatment of CD34(+) cells with SCF reduces long-term cell engraftment in NOD/SCID mice.     

Characterization of cytokine interactions by flow cytometry and factorial analysis

BACKGROUND: Multiple cytokines are required for the growth and development of hematopoietic cells. The effect of many cytokines depends on the activity of other signaling pathways. These interactions are quantified using factorial experimental design and analysis. METHODS: Human umbilical cord blood (HUCB) CD34+ cells were cultured in fully defined media containing various combinations of recombinant cytokines as defined by resolution IV factorial (2(7-3)(IV)) or full factorial (2(4)) design experiments. The cytokines studied were stem cell factor (SCF), interleukin (IL)-3, megakaryocyte growth and development factor (MGDF), granulocyte-colony stimulating factor (G-CSF), Flt-3 ligand, IL-6, IL-11, and erythropoietin (EPO). In vitro cell divisions were tracked by staining CD34+ cells with 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester, followed by flow cytometric analysis at 4 days of culture. In separate experiments, lineage commitment and differentiation were determined at 7 days by immunophenotype. RESULTS: In addition to the main effects of single cytokines, cytokine interactions were identified. There was a negative interaction between IL-3 and MGDF that resulted in a less than additive effect of these factors on erythroid and megakaryocytic development. The effect of Flt-3 ligand and SCF factor on CD34+ cell production was also less than additive, although the response to both cytokines was greater than single cytokines. The only positive interaction that was identified was between EPO and SCF, which resulted in the synergistic production of erythroid cells. CONCLUSIONS: Factorial analysis provides a powerful methodology to study the integration of multiple signals at the cellular and molecular level.     

Phagocytosis and killing of Staphylococcus aureus by mouse granulocytes after interleukin-3 (IL-3) injection

Interleukin-3 is a multipotential hematopoietic growth factor, which like other colony stimulating factors (CSFs) is effective "in vitro" stimulation of the mature cells function. It was found that IL-3 synergistically with GM-CSF and G-CSF stimulated the proliferation of the granulocytes. Therefore the purpose of this investigation was the evaluation "in vivo" of the influence of IL-3 on the phagocytosis, bactericidal activity, and enzyme activities of granulocytes. IL-3 was injected into mice subcutaneously during 5 days in dose 1 microgram/kg/d. The examination of the percent of cells phagocytizing bacteria (Staphylococcus aureus), NBT test and bactericidal activity, were performed every day and evident increase of the tested parameters was found. Additionally the enzyme activities in primary granules were measured and showed on increase of acid phosphatase and peroxidase activity.     

Structural basis for stem cell factor-KIT signaling and activation of class III receptor tyrosine kinases

Stem cell factor (SCF) binds to and activates the KIT receptor, a class III receptor tyrosine kinase (RTK), to stimulate diverse processes including melanogenesis, gametogenesis and hematopoeisis. Dysregulation of KIT activation is associated with many cancers. We report a 2.5 A crystal structure of the functional core of SCF bound to the extracellular ligand-binding domains of KIT. The structure reveals a 'wrapping' SCF-recognition mode by KIT, in which KIT adopts a bent conformation to facilitate each of its first three immunoglobulin (Ig)-like domains to interact with SCF. Three surface epitopes on SCF, an extended loop, the B and C helices, and the N-terminal segment, contact distinct KIT domains, with two of the epitopes undergoing large conformational changes upon receptor binding. The SCF/KIT complex reveals a unique RTK dimerization assembly, and a novel recognition mode between four-helix bundle cytokines and Ig-family receptors. It serves as a framework for understanding the activation mechanisms of class III RTKs.     

Human intestinal mast cells are capable of producing different cytokine profiles: role of IgE receptor cross-linking and IL-4

Mast cells are recognized as a new type of immunoregulatory cells capable of producing different cytokines. So far, little is known about the cytokine profile of mature human mast cells isolated from intestinal tissue and cultured in the presence of stem cell factor (SCF). We observed that these cells express the proinflammatory cytokines TNF-alpha, IL-1 beta, IL-6, IL-8, IL-16, and IL-18 without further stimulation. Both IgE-dependent and IgE-independent agonists (e.g., Gram-negative bacteria) enhanced expression of TNF-alpha. Another set of cytokines consisting of IL-3, IL-5, IL-9, and IL-13 was expressed following activation by IgE receptor cross-linking. If mast cells were cultured in the presence of IL-4 and SCF, the production and release of IL-3, IL-5, and IL-13 was increased up to 4-fold compared with mast cells cultured with SCF alone. By contrast, IL-6 expression was completely blocked in response to culture with IL-4. In summary, our data show that mature human mast cells produce proinflammatory cytokines that may be up-regulated following triggering with IgE-independent agonists such as bacteria, whereas activation by IgE receptor cross-linking results in the expression of Th2-type cytokines. IL-4 enhances the expression of Th2-type cytokines but does not affect or even down-regulates proinflammatory cytokines.     

Modulation of the effects of osteoprotegerin (OPG) ligand in a human leukemic cell line by OPG and calcitonin

The discovery of osteoprotegerin (OPG), osteoprotegerin ligand (OPGL), and RANK has elucidated the mechanism by which osteoblasts and stromal cells regulate osteoclastic differentiation and function and mediate the effects exerted by other hormones and cytokines. We have investigated the effects of these novel cytokines on the preosteoclastic cell line FLG 29.1. We show that OPGL alone and in combination with macrophage colony-stimulating factor (CSF-1) dramatically reduced replication and increased tartrate-resistant acid phosphatase activity. However, although FLG29.1 cells appear to adhere to the bone surface, they are not able to form resorption lacunae. OPG and calcitonin completely abolished the differentiation induced by OPGL. RANK was detectable in FLG 29.1 and the number of positive cells was increased by OPGL/CSF-1 treatment while reduced by calcitonin. We propose that calcitonin could interact with the OPG/OPGL, and its effects on RANK may explain in part the action of this hormone in suppressing bone resorption.     

The non-osteogenic mouse pluripotent cell line, C3H10T1/2, is induced to differentiate into osteoblastic cells by recombinant human bone morphogenetic protein-2.

The possibility that the non-osteogenic mouse pluripotent cell line, C3H10T1/2 (10T1/2), could be induced to differentiate into osteogenic cells by various hormones and cytokines was examined in vitro. Of a number of agents tested, recombinant human bone morphogenetic protein-2 (rhBMP-2) and retinoic acid induced alkaline phosphatase (ALP) activity in 10T1/2 cells. rhBMP-2 also induced mRNA expression of ALP in the cells. Dexamethasone, 1 alpha, 25-dihydroxyvitamin D3, transforming growth factor-beta 1 and insulin-like growth factor-I did not stimulate ALP activity. Treatment with rhBMP-2 greatly induced cAMP production in response to parathyroid hormone in 10T1/2 cells. No ALP activity was induced in NIH3T3 fibroblasts treated with rhBMP-2 or retinoic acid. These results indicate that 10T1/2 cells have a potential to differentiate into osteogenic cells under the control of BMP-2.