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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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Myeloablative conditioning for hematopoietic transplantation by lethal irradiation induces a proteolytic microenvironment in murine BMPanel A: Zymography revealed that the activity of MMP-9 is increased at 24 and 48 hours in conditioned media harvested from BM cells after lethal (1000 cGy) γ-irradiation (upper panel). The complement cascade becomes activated and MAC is deposited in the BM microenvironment of mice that were conditioned for transplantation by lethal γ-irradiation (lower panel). Because it is a peptide, SDF-1 was disrupted by proteolytic enzymes that are elevated in irradiated BM. Panel B: Chemotactic activity of SDF-1 against clonogenic progenitors decreases after exposure to MMP-2, MMP-9, cathepsin G (CG), or neutrophil elastase (NE). Disruption of SDF-1 was prevented by addition of protease-specific inhibitors. Panel C: SDF-1 was exposed to MMP-2 (100ng) or MMP-9 (100ng) with or without inhibitor (ARP101, 100ng) pre-incubation, and SDF-1 concentration was evaluated by ELISA. Panel D: The SDF-1 samples from Panel C were tested for chemotactic activity against BM-derived CFU-GM. The data shown in panels B–D represent the combined results from three independent experiments carried out in triplicate per group. * p<0.0001.
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Figure 1: Myeloablative conditioning for hematopoietic transplantation by lethal irradiation induces a proteolytic microenvironment in murine BMPanel A: Zymography revealed that the activity of MMP-9 is increased at 24 and 48 hours in conditioned media harvested from BM cells after lethal (1000 cGy) γ-irradiation (upper panel). The complement cascade becomes activated and MAC is deposited in the BM microenvironment of mice that were conditioned for transplantation by lethal γ-irradiation (lower panel). Because it is a peptide, SDF-1 was disrupted by proteolytic enzymes that are elevated in irradiated BM. Panel B: Chemotactic activity of SDF-1 against clonogenic progenitors decreases after exposure to MMP-2, MMP-9, cathepsin G (CG), or neutrophil elastase (NE). Disruption of SDF-1 was prevented by addition of protease-specific inhibitors. Panel C: SDF-1 was exposed to MMP-2 (100ng) or MMP-9 (100ng) with or without inhibitor (ARP101, 100ng) pre-incubation, and SDF-1 concentration was evaluated by ELISA. Panel D: The SDF-1 samples from Panel C were tested for chemotactic activity against BM-derived CFU-GM. The data shown in panels B–D represent the combined results from three independent experiments carried out in triplicate per group. * p<0.0001.

Mentions: Our zymography data (Figure 1panel A, top) show that at 24 and 48 hours after irradiation, the concentration of MMP-9 is upregulated in conditioned media harvested from BM cells. This was paralleled by activation of CC and deposition of C5b-C9 (MAC) in the BM microenvironment, as demonstrated by immunohistochemical staining (Figure 1panel A, bottom).


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)

Myeloablative conditioning for hematopoietic transplantation by lethal irradiation induces a proteolytic microenvironment in murine BMPanel A: Zymography revealed that the activity of MMP-9 is increased at 24 and 48 hours in conditioned media harvested from BM cells after lethal (1000 cGy) γ-irradiation (upper panel). The complement cascade becomes activated and MAC is deposited in the BM microenvironment of mice that were conditioned for transplantation by lethal γ-irradiation (lower panel). Because it is a peptide, SDF-1 was disrupted by proteolytic enzymes that are elevated in irradiated BM. Panel B: Chemotactic activity of SDF-1 against clonogenic progenitors decreases after exposure to MMP-2, MMP-9, cathepsin G (CG), or neutrophil elastase (NE). Disruption of SDF-1 was prevented by addition of protease-specific inhibitors. Panel C: SDF-1 was exposed to MMP-2 (100ng) or MMP-9 (100ng) with or without inhibitor (ARP101, 100ng) pre-incubation, and SDF-1 concentration was evaluated by ELISA. Panel D: The SDF-1 samples from Panel C were tested for chemotactic activity against BM-derived CFU-GM. The data shown in panels B–D represent the combined results from three independent experiments carried out in triplicate per group. * p<0.0001.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Myeloablative conditioning for hematopoietic transplantation by lethal irradiation induces a proteolytic microenvironment in murine BMPanel A: Zymography revealed that the activity of MMP-9 is increased at 24 and 48 hours in conditioned media harvested from BM cells after lethal (1000 cGy) γ-irradiation (upper panel). The complement cascade becomes activated and MAC is deposited in the BM microenvironment of mice that were conditioned for transplantation by lethal γ-irradiation (lower panel). Because it is a peptide, SDF-1 was disrupted by proteolytic enzymes that are elevated in irradiated BM. Panel B: Chemotactic activity of SDF-1 against clonogenic progenitors decreases after exposure to MMP-2, MMP-9, cathepsin G (CG), or neutrophil elastase (NE). Disruption of SDF-1 was prevented by addition of protease-specific inhibitors. Panel C: SDF-1 was exposed to MMP-2 (100ng) or MMP-9 (100ng) with or without inhibitor (ARP101, 100ng) pre-incubation, and SDF-1 concentration was evaluated by ELISA. Panel D: The SDF-1 samples from Panel C were tested for chemotactic activity against BM-derived CFU-GM. The data shown in panels B–D represent the combined results from three independent experiments carried out in triplicate per group. * p<0.0001.
Mentions: Our zymography data (Figure 1panel A, top) show that at 24 and 48 hours after irradiation, the concentration of MMP-9 is upregulated in conditioned media harvested from BM cells. This was paralleled by activation of CC and deposition of C5b-C9 (MAC) in the BM microenvironment, as demonstrated by immunohistochemical staining (Figure 1panel A, bottom).

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