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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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SDF-1 level decreases in irradiated bone marrow (BM)Panel A: Conditioned media from irradiated BM cells enhance chemotactic activity of normal clonogeneic progenitors (black bars), but this effect was partially disrupted by blocking CXCR4 with AMD3100 (1μM, gray bars). Panel B: SDF-1 at both the mRNA level (real-time PCR, left panel) and protein level (ELISA, middle panel) decreases after 24 hours in lethally irradiated BM (1000 cGy). At the same time, we did not observe changes in the SDF-1 level in PB of irradiated mice (right panel). Panel C: Mass spectrometry analysis shows that ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) become upregulated in murine BM after conditioning for hematopoietic transplantation by lethal irradiation. The data shown in panels A–C represent the combined results from three independent experiments carried out in triplicate per group.
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Figure 2: SDF-1 level decreases in irradiated bone marrow (BM)Panel A: Conditioned media from irradiated BM cells enhance chemotactic activity of normal clonogeneic progenitors (black bars), but this effect was partially disrupted by blocking CXCR4 with AMD3100 (1μM, gray bars). Panel B: SDF-1 at both the mRNA level (real-time PCR, left panel) and protein level (ELISA, middle panel) decreases after 24 hours in lethally irradiated BM (1000 cGy). At the same time, we did not observe changes in the SDF-1 level in PB of irradiated mice (right panel). Panel C: Mass spectrometry analysis shows that ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) become upregulated in murine BM after conditioning for hematopoietic transplantation by lethal irradiation. The data shown in panels A–C represent the combined results from three independent experiments carried out in triplicate per group.

Mentions: It is widely accepted that irradiation induces expression of SDF-1 in BM 16, 17. Therefore, we became interested in the SDF-1-mediated chemotactic activity of conditioned media (CM) harvested from BM irradiated cells. Figure 2panel A shows that CM harvested from BM cells 24 hours after lethal irradiation chemoattracts CFU-GM. However, we also observed that exposure of BM cells to the CXCR4 antagonist AMD3100 before chemotaxis did not significantly affect chemotactic responsiveness of tested cells to these CM. This suggests that factors other than SDF-1 are involved in this process. In fact, Figure 2panel B shows that both SDF-1 mRNA and SDF-1 protein are downregulated in lethally irradiated BM. Of note, the SDF-1 level remained unchanged in PB of irradiated mice, as in non-irradiated control animals.


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)

SDF-1 level decreases in irradiated bone marrow (BM)Panel A: Conditioned media from irradiated BM cells enhance chemotactic activity of normal clonogeneic progenitors (black bars), but this effect was partially disrupted by blocking CXCR4 with AMD3100 (1μM, gray bars). Panel B: SDF-1 at both the mRNA level (real-time PCR, left panel) and protein level (ELISA, middle panel) decreases after 24 hours in lethally irradiated BM (1000 cGy). At the same time, we did not observe changes in the SDF-1 level in PB of irradiated mice (right panel). Panel C: Mass spectrometry analysis shows that ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) become upregulated in murine BM after conditioning for hematopoietic transplantation by lethal irradiation. The data shown in panels A–C represent the combined results from three independent experiments carried out in triplicate per group.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3197954&req=5

Figure 2: SDF-1 level decreases in irradiated bone marrow (BM)Panel A: Conditioned media from irradiated BM cells enhance chemotactic activity of normal clonogeneic progenitors (black bars), but this effect was partially disrupted by blocking CXCR4 with AMD3100 (1μM, gray bars). Panel B: SDF-1 at both the mRNA level (real-time PCR, left panel) and protein level (ELISA, middle panel) decreases after 24 hours in lethally irradiated BM (1000 cGy). At the same time, we did not observe changes in the SDF-1 level in PB of irradiated mice (right panel). Panel C: Mass spectrometry analysis shows that ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) become upregulated in murine BM after conditioning for hematopoietic transplantation by lethal irradiation. The data shown in panels A–C represent the combined results from three independent experiments carried out in triplicate per group.
Mentions: It is widely accepted that irradiation induces expression of SDF-1 in BM 16, 17. Therefore, we became interested in the SDF-1-mediated chemotactic activity of conditioned media (CM) harvested from BM irradiated cells. Figure 2panel A shows that CM harvested from BM cells 24 hours after lethal irradiation chemoattracts CFU-GM. However, we also observed that exposure of BM cells to the CXCR4 antagonist AMD3100 before chemotaxis did not significantly affect chemotactic responsiveness of tested cells to these CM. This suggests that factors other than SDF-1 are involved in this process. In fact, Figure 2panel B shows that both SDF-1 mRNA and SDF-1 protein are downregulated in lethally irradiated BM. Of note, the SDF-1 level remained unchanged in PB of irradiated mice, as in non-irradiated control animals.

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