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Conditioning for hematopoietic transplantation activates the complement cascade and induces a proteolytic environment in bone marrow: a novel role for bioactive lipids and soluble C5b-C9 as homing factors.

Kim CH, Wu W, Wysoczynski M, Abdel-Latif A, Sunkara M, Morris A, Kucia M, Ratajczak J, Ratajczak MZ - Leukemia (2011)

Bottom Line: As a result, BM is enriched for proteolytic enzymes and the soluble form of the terminal product of CC activation, the membrane attack complex C5b-C9 (MAC).Next, we observed that C5-deficient mice that do not generate MAC show impaired engraftment of HSPCs.We conclude that an increase in BM levels of proteolytic enzyme-resistant S1P and C1P and activation of CC, which leads to the generation of MAC, has an important and previously underappreciated role in the homing of transplanted HSPCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Stem Cell Institute at the James Graham Brown Cancer Center, University of Louisville, Louisville, KY 40202, USA.

ABSTRACT
We have observed that conditioning for hematopoietic transplantation by lethal irradiation induces a proteolytic microenvironment in the bone marrow (BM) that activates the complement cascade (CC). As a result, BM is enriched for proteolytic enzymes and the soluble form of the terminal product of CC activation, the membrane attack complex C5b-C9 (MAC). At the same time, proteolytic enzymes induced in irradiated BM impair the chemotactic activity of α-chemokine stromal-derived factor-1 (SDF-1). As SDF-1 is considered a crucial BM chemoattractant for transplanted hematopoietic stem/progenitor cells (HSPCs), we sought to determine whether other factors that are resistant to proteolytic enzymes have a role in this process, focusing on proteolysis-resistant bioactive lipids. We found that the concentrations of sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) increase in the BM after conditioning for transplantation and that both S1P and, as we show here for the first time, C1P are potent chemoattractants for HSPCs. Next, we observed that C5-deficient mice that do not generate MAC show impaired engraftment of HSPCs. In support of a role for MAC in homing and engraftment, we found that soluble MAC enhances in a CR3 (CD11b/CD18)-dependent manner the adhesion of HSPCs to BM stromal cells and increases the secretion of SDF-1 by BM stroma. We conclude that an increase in BM levels of proteolytic enzyme-resistant S1P and C1P and activation of CC, which leads to the generation of MAC, has an important and previously underappreciated role in the homing of transplanted HSPCs.

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MAC affects several steps crucial for stem cell homingPanel A: Normal murine BM-derived stromal cells were incubated with soluble MAC (sC5b-9, 10μg/ml for 3 hrs) and subsequently conditioned media were harvested from these cells and assayed for their chemotactic activity against normal murine hematopoietic progenitors (black bars). Importantly, this chemotactic effect was totally inhibited after blockage of the CXCR4 receptor by AMD3100 treatment (gray bars). Panel B: Soluble MAC (sC5b-9), like SDF-1, strongly increases adhesiveness of murine hematopoietic progenitor cells (Sca-1+) to murine bone marrow-derived stroma. In inhibition studies, Sca-1+ cells were treated with MAPK inhibitor (U0126, 10μM) or Akt inhibitor (LY294002, 20μM) for 1 h before adhesion assay. The data shown represent the combined results from three independent experiments carried out in triplicate per group.
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Figure 6: MAC affects several steps crucial for stem cell homingPanel A: Normal murine BM-derived stromal cells were incubated with soluble MAC (sC5b-9, 10μg/ml for 3 hrs) and subsequently conditioned media were harvested from these cells and assayed for their chemotactic activity against normal murine hematopoietic progenitors (black bars). Importantly, this chemotactic effect was totally inhibited after blockage of the CXCR4 receptor by AMD3100 treatment (gray bars). Panel B: Soluble MAC (sC5b-9), like SDF-1, strongly increases adhesiveness of murine hematopoietic progenitor cells (Sca-1+) to murine bone marrow-derived stroma. In inhibition studies, Sca-1+ cells were treated with MAPK inhibitor (U0126, 10μM) or Akt inhibitor (LY294002, 20μM) for 1 h before adhesion assay. The data shown represent the combined results from three independent experiments carried out in triplicate per group.

Mentions: Finally, we became interested in sMAC-mediated molecular mechanisms that may enhance engraftment of HSPCs. While sMAC did not affect expression of CXCR4 or VLA-4 in murine cells (Supplementary Figure 6), chemotactic responsiveness of BM-derived CFU-GM to supernatants harvested from BM stromal cells (stimulated or unstimulated by sMAC in the presence or absence of AMD3100, Figure 6panel A), suggest that MAC may enhance SDF-1 secretion by murine BM stroma. Furthermore, sMAC like SDF-1, enhanced adhesion of HSPCs to bone marrow stroma in an AKT- and MAPKp42/44-dependent manner (Figure 6panel B). However, in contrast to SDF-1, S1P, and C1P (Figure 4panel C), this pro-adhesive effect of sMAC was not dependent on increasing interactions between VLA-4 and VCAM1 (data not shown).


Conditioning for hematopoietic transplantation activates the complement cascade and induces a proteolytic environment in bone marrow: a novel role for bioactive lipids and soluble C5b-C9 as homing factors.

Kim CH, Wu W, Wysoczynski M, Abdel-Latif A, Sunkara M, Morris A, Kucia M, Ratajczak J, Ratajczak MZ - Leukemia (2011)

MAC affects several steps crucial for stem cell homingPanel A: Normal murine BM-derived stromal cells were incubated with soluble MAC (sC5b-9, 10μg/ml for 3 hrs) and subsequently conditioned media were harvested from these cells and assayed for their chemotactic activity against normal murine hematopoietic progenitors (black bars). Importantly, this chemotactic effect was totally inhibited after blockage of the CXCR4 receptor by AMD3100 treatment (gray bars). Panel B: Soluble MAC (sC5b-9), like SDF-1, strongly increases adhesiveness of murine hematopoietic progenitor cells (Sca-1+) to murine bone marrow-derived stroma. In inhibition studies, Sca-1+ cells were treated with MAPK inhibitor (U0126, 10μM) or Akt inhibitor (LY294002, 20μM) for 1 h before adhesion assay. The data shown represent the combined results from three independent experiments carried out in triplicate per group.
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Related In: Results  -  Collection

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Figure 6: MAC affects several steps crucial for stem cell homingPanel A: Normal murine BM-derived stromal cells were incubated with soluble MAC (sC5b-9, 10μg/ml for 3 hrs) and subsequently conditioned media were harvested from these cells and assayed for their chemotactic activity against normal murine hematopoietic progenitors (black bars). Importantly, this chemotactic effect was totally inhibited after blockage of the CXCR4 receptor by AMD3100 treatment (gray bars). Panel B: Soluble MAC (sC5b-9), like SDF-1, strongly increases adhesiveness of murine hematopoietic progenitor cells (Sca-1+) to murine bone marrow-derived stroma. In inhibition studies, Sca-1+ cells were treated with MAPK inhibitor (U0126, 10μM) or Akt inhibitor (LY294002, 20μM) for 1 h before adhesion assay. The data shown represent the combined results from three independent experiments carried out in triplicate per group.
Mentions: Finally, we became interested in sMAC-mediated molecular mechanisms that may enhance engraftment of HSPCs. While sMAC did not affect expression of CXCR4 or VLA-4 in murine cells (Supplementary Figure 6), chemotactic responsiveness of BM-derived CFU-GM to supernatants harvested from BM stromal cells (stimulated or unstimulated by sMAC in the presence or absence of AMD3100, Figure 6panel A), suggest that MAC may enhance SDF-1 secretion by murine BM stroma. Furthermore, sMAC like SDF-1, enhanced adhesion of HSPCs to bone marrow stroma in an AKT- and MAPKp42/44-dependent manner (Figure 6panel B). However, in contrast to SDF-1, S1P, and C1P (Figure 4panel C), this pro-adhesive effect of sMAC was not dependent on increasing interactions between VLA-4 and VCAM1 (data not shown).

Bottom Line: As a result, BM is enriched for proteolytic enzymes and the soluble form of the terminal product of CC activation, the membrane attack complex C5b-C9 (MAC).Next, we observed that C5-deficient mice that do not generate MAC show impaired engraftment of HSPCs.We conclude that an increase in BM levels of proteolytic enzyme-resistant S1P and C1P and activation of CC, which leads to the generation of MAC, has an important and previously underappreciated role in the homing of transplanted HSPCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Stem Cell Institute at the James Graham Brown Cancer Center, University of Louisville, Louisville, KY 40202, USA.

ABSTRACT
We have observed that conditioning for hematopoietic transplantation by lethal irradiation induces a proteolytic microenvironment in the bone marrow (BM) that activates the complement cascade (CC). As a result, BM is enriched for proteolytic enzymes and the soluble form of the terminal product of CC activation, the membrane attack complex C5b-C9 (MAC). At the same time, proteolytic enzymes induced in irradiated BM impair the chemotactic activity of α-chemokine stromal-derived factor-1 (SDF-1). As SDF-1 is considered a crucial BM chemoattractant for transplanted hematopoietic stem/progenitor cells (HSPCs), we sought to determine whether other factors that are resistant to proteolytic enzymes have a role in this process, focusing on proteolysis-resistant bioactive lipids. We found that the concentrations of sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) increase in the BM after conditioning for transplantation and that both S1P and, as we show here for the first time, C1P are potent chemoattractants for HSPCs. Next, we observed that C5-deficient mice that do not generate MAC show impaired engraftment of HSPCs. In support of a role for MAC in homing and engraftment, we found that soluble MAC enhances in a CR3 (CD11b/CD18)-dependent manner the adhesion of HSPCs to BM stromal cells and increases the secretion of SDF-1 by BM stroma. We conclude that an increase in BM levels of proteolytic enzyme-resistant S1P and C1P and activation of CC, which leads to the generation of MAC, has an important and previously underappreciated role in the homing of transplanted HSPCs.

Show MeSH
Related in: MedlinePlus