Non-Hematopoietic Stromal Cells Sense Toll-Like Receptor 4 Agonists and Consequently Enhance Myelopoiesis.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2583-2583
Author(s):  
Steffen Boettcher ◽  
Patrick Ziegler ◽  
Michael A Schmid ◽  
Guido Garavaglia ◽  
Hitoshi Takizawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Myeloid Cell ◽  
Cell Production ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Human Bmscs ◽  
Emergency Myelopoiesis ◽  
Stimulation Of

Abstract Abstract 2583 Hematopoiesis is tightly regulated by growth factors acting on stem and progenitor cells (HSPCs) in the bone marrow. During systemic infections cytokines are elevated in serum, myelopoiesis is enhanced, and myeloid CFUs and granulocytes increase in circulation. However, the underlying mechanisms of this “emergency” myelopoietic response have not been defined. Sensing of conserved pathogen-associated products by specialized pattern-recognition receptors such as Toll-like receptors (TLRs) is crucial for rapid responses to infection. Based on the well-known regulatory function of the BM microenvironment, we hypothesized that bone marrow stromal cells (BMSCs) express TLRs and possess all functional properties required to sense microbes and drive emergency myelopoiesis. Human BMSCs expressed Tlr1, Tlr5, and Tlr6 at similar levels and Tlr3 and Tlr4 mRNA at about 2-log higher compared to dendrititc cells (DCs). Stimulation of BMSCs with the TLR4 agonist LPS led to de novo expression of G-csf and Gm-csf, and increased M-csf, Il-6, and Il-11 expression. In line with this, LPS induced production of G-CSF and GM-CSF protein and significantly enhanced the secretion of M-CSF, IL-6, and IL-11. Using LPS-stimulated BMSC culture supernatant in myeloid CFU assays led to a 2.5-fold higher myeloid CFU activity compared to un-stimulated BMSC supernatant. This effect was partly mimicked by adding G-, M-, and GM-CSF to the methylcellulose cultures. Importantly, direct LPS stimulation of CB CD34+ cells had no effect. Furthermore, co-culture of BMSCs and CB CD34+ cells together with LPS for 12 days led to approximate 2-fold higher recovery of immuno-phenotypically primitive CD34+ cells, and retained up to 8-fold more CD34+ cells in divisions 0–3 as compared to LPS-free co-cultures as measured by CFSE dilution. When subjected to cytokine-supplemented myeloid CFU assays or transplanted into newborn RAG2-/- γc-/- mice to evaluate lymphoid differentiation, recovered CD34+ cells from LPS-stimulated BMSC cultures gave rise to the full spectrum of myeloid colonies and T and B cells, respectively, thus proving maintenance of primitive hematopoietic progenitors. To elucidate the in vivo relevance of the findings and to clarify the contribution of stromal vs. hematopoietic cell expressed TLR4, we generated chimeras with TLR4-/- hematopoiesis in a wild-type (WT) background (hematopoietic-TLR4-/-) and WT hematopoiesis in a TLR4-/- background (non-hematopoietic-TLR4-/-). Chimeric, WT, and TLR4-/- mice were injected with LPS and hallmarks of myelopoietic responses such as G-CSF expression, myeloid cell mobilization from the BM, and increased myeloid cell production in the BM was evaluated. Significant G-csf mRNA induction could be observed in the BM of WT and hematopoietic-TLR4-/- mice. To a much lesser, non-significant extent, this effect could be observed also in non-hematopoietic-TLR4-/- mice, while no transcripts were detectable in TLR4-/- mice. Accordingly, serum G-CSF levels significantly increased 10-fold in WT and hematopoietic-TLR4-/- mice after LPS injection, but no increase was detectable in non-hematopoietic-TLR4-/- and TLR4-/- mice. LPS injection also resulted in a significant decrease in BM cellularity accompanied by an increase of spleen cell numbers only in WT and hematopoietic-TLR4-/- mice. Furthermore, Gr-1highCD11blow/+ mature myeloid cells were significantly reduced whereas Gr-1lowCD11blow/+ immature promyelocytes and myelocytes significantly increased (2.5-fold) in the BM of WT and hematopoietic-TLR4-/- mice. In contrast, similar changes in cellular composition could not be observed in TLR4-/- and non-hematopoietic-TLR4-/- mice, while a small, but still significant 1.25-fold increase in immature Gr-1lowCD11blow/+ cells was detectable in non-hematopoietic-TLR4-/- mice. Finally, inflammation-induced Sca-1 upregulation on HSPCs and increasing frequencies of GMPs were only observed in WT and hematopoietic-TLR4-/- mice. Collectively, our in vitro data demonstrate that human BMSCs are able to sense pathogens and stimulate emergency myelopoiesis but also prevent loss of HSPCs by enhancing their maintenance. Importantly, in vivo signaling via non-hematopoietic cell-expressed TLR4 is sufficient and is the main mechanism regulating both the release of mature myeloid cells from and the enhanced myeloid cell production in the bone marrow during systemic challenges. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Hepatocyte Growth Factor Is Constitutively Produced by Human Bone Marrow Stromal Cells and Indirectly Promotes Hematopoiesis

Blood ◽  
1997 ◽  
Vol 89 (5) ◽  
pp. 1560-1565 ◽  
Author(s):  
Kenji Takai ◽  
Junichi Hara ◽  
Kunio Matsumoto ◽  
Gaku Hosoi ◽  
Yuko Osugi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factor ◽  
Hepatocyte Growth Factor ◽  
Stromal Cells ◽  
Mononuclear Cells ◽  
Gm Csf ◽  
Tumor Growth Factor ◽  
Cd34 Cells ◽  
Factor Α ◽  
Hepatocyte Growth

Bone marrow (BM) stromal cells are required for normal hematopoiesis. A number of soluble factors secreted by these cells that mediate hematopoiesis have been characterized. However, the mechanism of hematopoiesis cannot be explained solely by these known factors, and the existence of other, still unknown stromal factors has been postulated. We showed that hepatocyte growth factor (HGF ) is one such cytokine produced by human BM stromal cells. BM stromal cells were shown to constitutively produce HGF and also to express the c-MET/HGF receptor. The production of HGF was enhanced by addition of heparin and phorbol ester. Dexamethasone and tumor growth factor-β (TGF-β) inhibited the production of HGF. Interleukin-1α (IL-1α) tumor necrosis factor-α (TNF-α), and N6,2′-o-dibutyryl-adenosine-3′:5′-cyclic monophosphate (dbc-AMP) showed no obvious influence on HGF production. Western blot analysis of HGF derived from BM stromal cells showed two bands at 85 and 28 kD corresponding to native and variant HGF, respectively. Addition of recombinant HGF significantly promoted the formation of burst-forming unit-erythroid (BFU-E) and colony-forming unit-granulocyte erythroid macrophage (CFU-GEM) by BM mononuclear cells in the presence of erythropoietin and granulocyte-macrophage colony-stimulating factor (GM-CSF ), but the formation of CFU-GM was not modified. However, HGF had no effects on colony formation by purified CD34+ cells. Within BM mononuclear cells, c-MET was expressed on a proportion of cells (CD34−, CD33+, CD13+, CD14+, and CD15+), but was not found on CD34+ cells. We conclude that HGF is constitutively produced by BM stromal cells and that it enhances hematopoiesis. In addition, expression of c-MET on the stromal cells suggests the presence of an autocrine mechanism, operating through HGF, among stromal cells.

Angptl-4 Is Upregulated Under Inflammatory Conditions In The BM Of Mice, Expands Myeloid Progenitors and Accelerates Reconstitution Of Platelets After Myelosuppressive Therapy

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3677-3677
Author(s):  
Anne Schumacher ◽  
Till Braunschweig ◽  
Bernd Denecke ◽  
Tim H. Brümmendorf ◽  
Patrick Ziegler
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Myeloid Cell ◽  
Relative Increase ◽  
T Test ◽  
Gm Csf ◽  
Emergency Situations ◽  
Inflammatory Conditions

Abstract The concerted action of hematopoiesis supporting cytokines such as G-CSF, GM-CSF or IL-6 regulates hematopoiesis during steady state and emergency situations. Respective knockout mice show defects both in production and function of myelopoietic effector cells. However, alternative pathways are likely to exist as mice with single or combined deficiencies for G-CSF, GM-CSF, and IL-6 or G-CSF and GM-CSF are still able to mount reactive neutrophilia responses during inflammatory conditions. In order to identify pathways for inflammation induced enhancement of hematopoiesis as well as to find new cytokines, which enhance myeloid cell regeneration, we analyzed the bone marrow (BM) of lipopolysaccharide (LPS) and vehicle injected wild type (WT) mice (single IP- injection) by gene expression microarray. Focusing on the identification of genes encoding for secreted or membrane proteins, we found 83 candidates to be up- and 14 to be downregulated after LPS treatment. Among known candiates, we found angiopoietin-like 4 (Angptl-4) as a predominantly upregulated gene in the BM of LPS-treated WT-mice. Upregulation was confirmed by RT-PCR as well as by Elisa in the BM of LPS treated mice and bone marrow stromal cells (BMSC) were identified as candidate producer cells. Functionally, we found recombinant Angptl-4 to stimulate the proliferation of myeloid colony-forming units (CFU) in vitro. In mice, repeated injections of Angptl-4 increased BM progenitor cell frequency and this was paralleled by a relative increase in phenotypically defined granulocyte-macrophage progenitors (GMPs). Furthermore, in vivo treatment with Angptl-4 resulted in elevated platelet counts both in untreated animals and after myelosuppressive therapy. After lethal irradiation and transplantation of syngeneic BM cells repetitive injections of recombinant Angptl-4 for 5 consecutive days resulted in an accelerated reconstitution of platelets starting at day 8 after transplantation. The 50% pre-treatment platelet count was reached on day 14 in Angptl-4-treated animals as compared to day 21 for transplanted controls receiving no Angptl-4 (n=8; p=0.03, student´s T test). In contrast, transplantation of BM cells from Angptl-4 pre-treated donor mice had no effect on the recovery of platelets in this setting. The frequency of CD41lowCD61+ immature megakaryocytes was significantly increased in the BM of Angptl-4 injected as compared to control mice (27% vs 19% of total megakaryocytes; p= 0.008, student´s T test). Furthermore, bone marrow cytology revealed local accumulation of megakaryocytes carrying dysplastic features in Angptl-4 injected mice. In summary, our data suggest that Angptl-4 plays a complementary role on hematopoiesis during emergency situations like sepsis. The use of Angptl-4 in the setting of autologous stem cell transplantation could represent a potential approach to accelerate the reconstitution of megakaryopoiesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Hepatocyte Growth Factor Is Constitutively Produced by Human Bone Marrow Stromal Cells and Indirectly Promotes Hematopoiesis

Blood ◽  
1997 ◽  
Vol 89 (5) ◽  
pp. 1560-1565 ◽  
Author(s):  
Kenji Takai ◽  
Junichi Hara ◽  
Kunio Matsumoto ◽  
Gaku Hosoi ◽  
Yuko Osugi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factor ◽  
Hepatocyte Growth Factor ◽  
Stromal Cells ◽  
Mononuclear Cells ◽  
Gm Csf ◽  
Tumor Growth Factor ◽  
Cd34 Cells ◽  
Factor Α ◽  
Hepatocyte Growth

Abstract Bone marrow (BM) stromal cells are required for normal hematopoiesis. A number of soluble factors secreted by these cells that mediate hematopoiesis have been characterized. However, the mechanism of hematopoiesis cannot be explained solely by these known factors, and the existence of other, still unknown stromal factors has been postulated. We showed that hepatocyte growth factor (HGF ) is one such cytokine produced by human BM stromal cells. BM stromal cells were shown to constitutively produce HGF and also to express the c-MET/HGF receptor. The production of HGF was enhanced by addition of heparin and phorbol ester. Dexamethasone and tumor growth factor-β (TGF-β) inhibited the production of HGF. Interleukin-1α (IL-1α) tumor necrosis factor-α (TNF-α), and N6,2′-o-dibutyryl-adenosine-3′:5′-cyclic monophosphate (dbc-AMP) showed no obvious influence on HGF production. Western blot analysis of HGF derived from BM stromal cells showed two bands at 85 and 28 kD corresponding to native and variant HGF, respectively. Addition of recombinant HGF significantly promoted the formation of burst-forming unit-erythroid (BFU-E) and colony-forming unit-granulocyte erythroid macrophage (CFU-GEM) by BM mononuclear cells in the presence of erythropoietin and granulocyte-macrophage colony-stimulating factor (GM-CSF ), but the formation of CFU-GM was not modified. However, HGF had no effects on colony formation by purified CD34+ cells. Within BM mononuclear cells, c-MET was expressed on a proportion of cells (CD34−, CD33+, CD13+, CD14+, and CD15+), but was not found on CD34+ cells. We conclude that HGF is constitutively produced by BM stromal cells and that it enhances hematopoiesis. In addition, expression of c-MET on the stromal cells suggests the presence of an autocrine mechanism, operating through HGF, among stromal cells.

scholarly journals Recombinant human interleukin-3 stimulation of hematopoiesis in humans: loss of responsiveness with differentiation in the neutrophilic myeloid series

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1797-1804
Author(s):  
AF Lopez ◽  
PG Dyson ◽  
LB To ◽  
MJ Elliott ◽  
SE Milton ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Bone Marrow Cells ◽  
Myeloid Cell ◽  
Neutrophil Function ◽  
Superoxide Anion Production ◽  
Interleukin 3 ◽  
Gm Csf ◽  
Mature Neutrophil ◽  
Stimulation Of

Recombinant human (rh) interleukin-3 (IL-3) stimulated the proliferation and differentiation of erythroid, granulocyte, macrophage, eosinophil (Eo), and mixed colonies as well as megakaryocytes from human bone marrow cells. rh IL-3 was a weaker stimulus than rh granulocyte-macrophage colony-stimulating factor (GM- CSF) for day 14 myeloid cell colonies. At day 7 of incubation, rh IL-3 stimulated a few G, M, and Eo clusters but no colonies. This loss of responsiveness of myeloid cells to rh IL-3 was accentuated with further differentiation of the cells. rh IL-3 stimulated very few or no clones after five-day incubation with enriched promyelocytes and myelocytes, whereas rh GM-CSF was an efficient stimulus. Responsiveness to rh IL-3 was completely lost in postmitotic mature neutrophils. Incubation of these cells with rh IL-3 did not result in enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) of tumor cells or superoxide anion production after stimulation with formyl-methyl-leucyl-phenylalanine (FMLP), although they could be stimulated by rh GM-CSF. In addition, preincubation of neutrophils with different concentrations of rh IL-3 failed to increase or decrease their response to rh GM-CSF. In contrast to neutrophils, mature Eos could be stimulated by rh IL-3 to kill antibody-coated tumor cells. These results show that cells of the neutrophilic myeloid series lose their responsiveness to h IL-3 as they differentiate and suggest that although h IL-3 may be an important therapeutic agent to use for hematopoietic regeneration in vivo, the lack of stimulation of mature neutrophil function makes it an unlikely sole candidate as adjunct therapy for treatment of infectious diseases.

JAK2-Mediated Extrinsic Survival of CML Stem Cells: Exploring the Potential Combination of BCR-ABL and JAK2 Inhibitors In Vivo

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 199-199
Author(s):  
Elie Traer ◽  
Ryan MacKenzie ◽  
Jennifer Snead ◽  
Brian J Druker ◽  
Michael W Deininger
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Median Survival ◽  
Conditioned Media ◽  
High Dose ◽  
Protective Effects ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Jak2 Inhibitors

Abstract Abstract 199 Background: The tyrosine kinase inhibitors (TKIs) imatinib, nilotinib and dasatinib are very effective for the treatment of chronic phase CML. However, the majority of these patients continue to have persistence of CML cells despite continued therapy, suggesting that TKIs fail to target leukemic stem cells (LSCs). There is increasing evidence that the bone marrow microenvironment provides a sanctuary to LSCs, thereby contributing to persistence. Results: We used the human stromal cell lines (HS-5, HS-23, HS-27a) to model the microenvironment. Conditioned media from HS-5, but not HS-23 or HS-27a, reduced apoptosis of CML cell lines treated with TKIs (K562, LAMA-84, KBM-5 and KYO-1), consistent with previous reports. Similarly, CML CD34+ cells were protected from 5 μ M imatinib in a 4 day co-culture with HS-5 cells, as assessed by CFU-GM colony survival (23% vs 9% when compared to untreated controls, N=6, p=0.018). We were also able to demonstrate protection from TKIs with transwells over HS-5 and with HS-5 conditioned media, which suggests that factors secreted by HS-5 cells protect CML cells from TKIs. Cytokine analysis of conditioned media revealed relatively higher concentrations of IL6, IL-8, MCP-1, MCP-3, G-CSF and GM-CSF from HS-5 as compared to HS-23 and HS-27a. Since IL-6, G-CSF and GM-CSF are known to signal via JAK2, we tested combinations of imatinib and JAK2 inhibitors (TG101209 or CYT387) using our in vitro assay. Combination treatment with imatinib and CYT387 or TG101209 abrogated the protective effects of HS-5 conditioned media in CML cell lines. Combination treatment of CML CD34+ in HS-5 co-culture assays also abrogated the protective effects of stroma on colony formation. However, we observed that both normal CD34+ and CML CD34+ colony formation was dramatically reduced by JAK2 inhibitors using our HS-5 co-culture system, particularly at higher doses. Thus, it was unclear if a potential therapeutic window existed in vivo. To test the potential of combination therapy in vivo, we infected marrow from Balb/c mice with a retrovirus that simultaneously expresses BCR-ABL and GFP, followed by transplantation into lethally irradiated syngeneic recipients. The mice were separated into five cohorts: vehicle control, TG101209 (200mg/kg/d), nilotinib (75mg/kg/d), nilotinib + low-dose TG101209 (50mg/kg/d) and nilotinib + high-dose TG101209 (200mg/kg/d). The vehicle-treated control group died rapidly of myeloproliferative disease (MPD) with a median survival of 15.5 days. The median survival of mice treated with TG101209 was slightly prolonged at 20.5 days (p=0.06); however, these mice also died of MPD with enlarged spleens/livers and lung hemorrhage. The survival curves of mice treated with nilotinib monotherapy and nilotinib + low-dose TG101209 were similar (median survival not reached at termination of experiment). Mice treated with nilotinib + high-dose TG101209 initially had minimal mortality, however on day 26 the mice began to die without signs of MPD (no definitive cause of death at autopsy) and thus the remaining cohort was sacrificed on day 27 for analysis. In stark contrast to the other cohorts, the spleens of these mice were very small, leukopenic, and largely devoid of normal follicles, with decreased spleen weight compared to mice treated with nilotinib monotherapy (0.025 vs. 0.072 gm, p<0.01). The bone marrow was also profoundly hypocellular, suggesting that myelosuppresion (anemia and leukopenia in particular) may have been a factor in mortality. However, despite toxicity this combination may retain a degree of selectivity for BCR-ABL cells since disease burden, as measured by the percentage of GFP-positive cells, was reduced compared to nilotinib monotherapy (spleen: 4% vs 11.7%, p=0.047; bone marrow: 8.7% vs 13.8%, p=0.22). Conclusions: (1) Factors secreted by human bone marrow stromal cells attenuate the effects of imatinib in CML cell lines and primary CML CD34+ cells in a JAK2-dependent fashion. (2) Simultaneous in vivo inhibition of BCR-ABL and JAK2 dramatically reduces BCR-ABL expressing cells, but at the cost of marrow toxicity. We speculate that this limitation may be overcome by intermittent rather than continuous JAK2 inhibition, a strategy that might avoid toxicity while reducing persistent BCR-ABL disease burden. Disclosures: Deininger: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Genzyme: Research Funding; Ariad: Consultancy.

scholarly journals Recombinant human interleukin-3 stimulation of hematopoiesis in humans: loss of responsiveness with differentiation in the neutrophilic myeloid series

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1797-1804 ◽  
Author(s):  
AF Lopez ◽  
PG Dyson ◽  
LB To ◽  
MJ Elliott ◽  
SE Milton ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Bone Marrow Cells ◽  
Myeloid Cell ◽  
Neutrophil Function ◽  
Superoxide Anion Production ◽  
Interleukin 3 ◽  
Gm Csf ◽  
Mature Neutrophil ◽  
Stimulation Of

Abstract Recombinant human (rh) interleukin-3 (IL-3) stimulated the proliferation and differentiation of erythroid, granulocyte, macrophage, eosinophil (Eo), and mixed colonies as well as megakaryocytes from human bone marrow cells. rh IL-3 was a weaker stimulus than rh granulocyte-macrophage colony-stimulating factor (GM- CSF) for day 14 myeloid cell colonies. At day 7 of incubation, rh IL-3 stimulated a few G, M, and Eo clusters but no colonies. This loss of responsiveness of myeloid cells to rh IL-3 was accentuated with further differentiation of the cells. rh IL-3 stimulated very few or no clones after five-day incubation with enriched promyelocytes and myelocytes, whereas rh GM-CSF was an efficient stimulus. Responsiveness to rh IL-3 was completely lost in postmitotic mature neutrophils. Incubation of these cells with rh IL-3 did not result in enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) of tumor cells or superoxide anion production after stimulation with formyl-methyl-leucyl-phenylalanine (FMLP), although they could be stimulated by rh GM-CSF. In addition, preincubation of neutrophils with different concentrations of rh IL-3 failed to increase or decrease their response to rh GM-CSF. In contrast to neutrophils, mature Eos could be stimulated by rh IL-3 to kill antibody-coated tumor cells. These results show that cells of the neutrophilic myeloid series lose their responsiveness to h IL-3 as they differentiate and suggest that although h IL-3 may be an important therapeutic agent to use for hematopoietic regeneration in vivo, the lack of stimulation of mature neutrophil function makes it an unlikely sole candidate as adjunct therapy for treatment of infectious diseases.

TLR Agonist Induced Cytokine Production in Human Multipotent Mesenchymal Stromal Cells: A Potential Mechanism How Hematopoiesis Is Enhanced during Generalized Inflammatory Conditions.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1411-1411
Author(s):  
Steffen Boettcher ◽  
Patrick Ziegler ◽  
Markus G. Manz
Keyword(s):  
Bone Marrow ◽  
Immune Responses ◽  
Cord Blood ◽  
Stromal Cells ◽  
Multipotent Mesenchymal Stromal Cells ◽  
Potential Mechanism ◽  
Gm Csf ◽  
Inflammatory Conditions ◽  
Cd34 Cells

Abstract Toll-like receptors (TLRs) function as receptors for different conserved pathogen associated products as well as certain host derived molecules. TLRs are expressed in several hematopoietic and non-hematopoietic cells. Their activation plays a key role in innate and adaptive immune responses to infectious agents, as well as in the development of pathologic conditions like tissue damage and cancer. Human multipotent mesenchymal stromal cells (MSCs) have been shown to differentiate into various mesenchymal tissues such as bone, cartilage, and fat, as well as marrow and lymphoid organ stroma cells. Human MSCs are able to maintain CD34+ cells to some extent in vitro. Furthermore, it has been demonstrated that upon intra bone marrow transplantation into adult immunodeficient mice MSC derived cells support human hematopoiesis in vivo. We hypothesized that MSCs express TLRs and are capable to respond to TLR agonists by changing their cytokine expression pattern in order to more efficiently support hematopoiesis according to respective needs in inflammatory conditions. MSCs from human bone marrow, cord blood, and umbilical cord whartons jelly were cultured by plastic adherence in IMDM 20% FCS, 1–8M dexamethasone (only during first 3 weeks), expanded for 2 passages, and subsequently analyzed. MSCs expressed gene-transcripts for IL-6, IL-7, IL-11, IL-15, SCF, TPO, FLT3L, M-CSF, GM-CSF, LIF, and SDF-1, while G-CSF was rarely detectable. Consistently, respective cytokines were measured in supernatants at the following, declining levels (pg/ml): IL-6 (10000–10E6) > SDF-1 > IL-11 > M-CSF > IL-7 > LIF > SCF, whereas GM-CSF was rarely detectable, G-CSF, FLT3-L, and TPO were not detectable by ELISA. MSCs were further analyzed for expression of TLRs by semiquantitative RT-PCR. TLR 1, 3, 4, 5, 6, and 9 expression, but not TLR 2, 7, 8, and 10 expression was detectable. Compared to human conventional (BDCA-1+, CD14−, CD19−) and plasmacytoid (BDCA-4+,CD14−,CD19−) blood dendritic cells, MSCs expressed TLR-3 and TLR-4 at levels up to 2 log higher than did conventional DCs, while TLR 9 expression was low. Upon in vitro stimulation with LPS (TLR-4 agonist), MSCs produced previously undetectable G-CSF and GM-CSF, and M-CSF levels increased about 4 fold compared to untreated MSCs, whereas stimulation with CpG motifs (TLR-9 agonists) did not lead to changes in cytokine release detected in supernatants. In co-culture experiments using MSCs as a feeder layer for cord blood CD34+ cells, MSCs supported hematopoiesis and the development of myeloid cells. Importantly, MSC preconditioning with LPS led to an 1,7 fold increase in total hematopoietic cell number, while preconditioning with CpG had no measurable effect. These results demonstrate that MSCs express certain TLRs, and are capable to respond to pathogen associated molecules with an increase in secretion of hematopoiesis-relevant cytokines, and thus reveal a potential mechanism how hematopoiesis is enhanced during generalized inflammatory conditions, supporting e.g. in case of gram-negative sepsis efficient innate immune responses.

The Neurexin I Alpha/Neurexophilin Axis as An Anti-Proliferative Factor In Human Cord Blood and Murine Bone Marrow Hematopoiesis.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2627-2627
Author(s):  
John Kinzfogl ◽  
Giao Hangoc ◽  
Hal E. Broxmeyer
Keyword(s):  
Cell Culture ◽  
Bone Marrow ◽  
High Concentration ◽  
Blocking Antibody ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Signaling Axis ◽  
Single Cell Culture

Abstract Abstract 2627 The membrane-bound receptors Neurexin I alpha (NRXN1α) and Dystroglycan (DAG1) serve as ligands for each other, and the Neurexophilins (NXPHs) are thought to function as natural antagonists for this interaction. This signaling axis centered on NRXN1α, DAG1 and the three highly homologous NXPHs, plays a well-defined role in neuronal development and synapse development. The failure of DAG1 knock-out mice to develop a mesoderm led us to hypothesize that this axis plays a role in hematopoiesis. Consistent with previous research, DAG1 is heavily (>95%) expressed in human (hu) umbilical cord blood (CB) CD34+ cells, including the primitive CD38- subpopulation. NRXN1α is expressed at similar levels in these populations as well. Hematopoietic cell populations are more precisely defined in the murine (mu) system, and adult mu bone marrow (BM) exhibits a similar trend to hu CB with NRXN1α expression being higher in the more primitive cells and decreasing as the cells differentiate. A high concentration of Neurexophilins is present in the hu CB plasma (900ng/ml), though it is absent in adult mu blood plasma and fetal bovine serum. Neither blocking DAG1 with a well-described blocking antibody, nor stimulation with recombinant NRXN1α has any observable effect on stimulation of hematopoietic progenitor cell (HPC) proliferation in vitro. This led us to investigate the other receptor present in the axis, NRXN1α. We first used alpha-latrotoxin, an excitatory toxin for NRXN1α. While no stimulatory or inhibitory proliferation effect was observed under normal growth conditions, alpha-latrotoxin did promote HPC survival under growth factor starved conditions as effectively as SDF1α/CXCL12 (a known HPC survival enhancing factor), suggesting a possible protective role for the DAG1-NRXN1α axis during stress. In contrast, we identified the Neurexophilins as anti-proliferative agents for immature hematopoietic cells. When injected in vivo, recombinant NXPH1 acts in a myelo- and lympho-suppressive manner. Absolute numbers of mu HPCs were suppressed in a dose- and time-dependent manner, with the maximal dosage decreasing HPC numbers by over 50%. The decrease in absolute colony number was mirrored by a decrease in the cycling population of HPCs in mice treated with NXPH1, as measured by the high specificity activated tritiated thymidine kill assay. Peripheral blood neutrophils, lymphocytes, monocytes and platelets, were also decreased in a time-dependant fashion after in vivo exposure to NXPH1. Intriguingly, the most primitive LTR-HSCs behave in the opposite manner, with a three-fold increase in cycling status induced via the Rapamycin sensitive mTOR pathway. During in vitro culture NXPH1 is only able to suppress colony forming ability of unseparated HPCs treated with both GM-CSF and SCF in combination in comparison to stimulation by other cytokines. This response is most likely due to increased NRXN1α expression in response to stimulation by GM-CSF and SCF. Given the high level of DAG1 expression on colony forming hematopoietic cells, we hypothesized that endogenous DAG1 may be competing with NXPH1 for NRXN1α binding. We thus exposed cells to a well studied DAG1 blocking antibody or plated CD34+ cells in single cell culture. The in vitro results were then able to recapitulate the results observed in vivo and a 50% decrease in colony forming ability is observed in combination with the following cytokines: GM-CSF, IL-3, GM-CSF+SCF, and IL-3+SCF as well as under maximally stimulatory conditions (IL-3, GM-CSF, SCF, and Epo for hu cells and SCF, PWMSCM, and Epo for mu cell). The ability of NXPH1 to inhibit proliferation of CD34+ cells in single cell culture suggests that NXPH1 acts directly on HPCs. The anti-proliferative function of the NXPHs are consistent with previous literature correlating NXPH expression in cancers with negative prognoses and these data may provide a mechanism for the immune system evasion of cancer. Additionally, the high concentration of NXPH in CB plasma may help explain the relatively long time to engraftment of transplanted CB cells. More broadly, these results demonstrate the presence and function of a tightly regulated signaling axis centered on NRXN1α in hematopoiesis. Disclosures: Broxmeyer: CordUse: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Sign in / Sign up

Export Citation Format

Share Document