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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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C5-deficient mice show an engraftment defect in transplanted HSPCsPanel A: Wild type (C5+/+) and C5-deficient (C5−/−) animals, which do not generate MAC, were lethally irradiated and transplanted with wild type bone marrow mononuclear cells. We followed the subsequent recovery of hematopoietic parameters in these animals. C5−/− mice have recovery of peripheral blood leukocytes (upper panel) and platelets (lower panel) delayed by 3–5 days. The data shown represent the combined results from two independent experiments performed with 10 mice/group. * p<0.001. Panel B: By day 12, there is a decrease in the number of CFU-GM in BM of C5−/− mice transplanted with wt BM cells. Panel C: The number of CFU-S colonies formed in the spleens of wt mice were counted after transplantation of wt BMMNC cells, which were pre-stimulated or unstimulated before transplantation by sC5b-9 (10μg/ml), and injected intravenously into lethally irradiated wild type animals. Twelve days later, spleen was isolated and the number of CFU-S evaluated. Panel D: The number of CFU-GM progenitors in BM of wt transplanted mice with SC5b-9-primed or unprimed BM cells. The data shown in panels C and D represent the combined results from three independent experiments carried out in triplicate per group (n=9). Panel E: Wild type mice were lethally irradiated and transplanted with wild type BMMNC after SC5b-9 treatment (primed) or not (unprimed control). We followed the subsequent recovery of hematopoietic parameters in these animals. Mice that were transplanted with SC5b-9-primed BMMNCs have a much faster recovery rate for peripheral blood leukocytes (left panel) and platelets (right panel). * p<0.05. Panel F: Soluble MAC, like SDF-1, activates MAPKp44/42 and Akt in normal murine Sca-1+ cells. “Mix” indicates SDF-1 (0.05μg/ml) plus SC5b-9 (1μg/ml). Experiments were repeated independently three times with similar results. A representative western blot is shown.
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Figure 5: C5-deficient mice show an engraftment defect in transplanted HSPCsPanel A: Wild type (C5+/+) and C5-deficient (C5−/−) animals, which do not generate MAC, were lethally irradiated and transplanted with wild type bone marrow mononuclear cells. We followed the subsequent recovery of hematopoietic parameters in these animals. C5−/− mice have recovery of peripheral blood leukocytes (upper panel) and platelets (lower panel) delayed by 3–5 days. The data shown represent the combined results from two independent experiments performed with 10 mice/group. * p<0.001. Panel B: By day 12, there is a decrease in the number of CFU-GM in BM of C5−/− mice transplanted with wt BM cells. Panel C: The number of CFU-S colonies formed in the spleens of wt mice were counted after transplantation of wt BMMNC cells, which were pre-stimulated or unstimulated before transplantation by sC5b-9 (10μg/ml), and injected intravenously into lethally irradiated wild type animals. Twelve days later, spleen was isolated and the number of CFU-S evaluated. Panel D: The number of CFU-GM progenitors in BM of wt transplanted mice with SC5b-9-primed or unprimed BM cells. The data shown in panels C and D represent the combined results from three independent experiments carried out in triplicate per group (n=9). Panel E: Wild type mice were lethally irradiated and transplanted with wild type BMMNC after SC5b-9 treatment (primed) or not (unprimed control). We followed the subsequent recovery of hematopoietic parameters in these animals. Mice that were transplanted with SC5b-9-primed BMMNCs have a much faster recovery rate for peripheral blood leukocytes (left panel) and platelets (right panel). * p<0.05. Panel F: Soluble MAC, like SDF-1, activates MAPKp44/42 and Akt in normal murine Sca-1+ cells. “Mix” indicates SDF-1 (0.05μg/ml) plus SC5b-9 (1μg/ml). Experiments were repeated independently three times with similar results. A representative western blot is shown.

Mentions: Because our data demonstrated that CC is activated and MAC is deposited in lethally irradiated BM (Figure 1panel A, right), we asked whether MAC plays a role in homing of HSPCs. To address this question, we transplanted lethally irradiated C5-deficient mice that do not generate MAC, as well as wild type (wt) mice, with wt BM cells. Figure 5panel A shows that the recovery of leucocytes and platelets was significantly delayed in C5-deficient mice, which do not generate and deposit MAC. In further support of this finding, the bones of mice at day 12 after transplantation exhibited a much lower number of transplant-derived CFU-GM (Figure 5panel B).


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)

C5-deficient mice show an engraftment defect in transplanted HSPCsPanel A: Wild type (C5+/+) and C5-deficient (C5−/−) animals, which do not generate MAC, were lethally irradiated and transplanted with wild type bone marrow mononuclear cells. We followed the subsequent recovery of hematopoietic parameters in these animals. C5−/− mice have recovery of peripheral blood leukocytes (upper panel) and platelets (lower panel) delayed by 3–5 days. The data shown represent the combined results from two independent experiments performed with 10 mice/group. * p<0.001. Panel B: By day 12, there is a decrease in the number of CFU-GM in BM of C5−/− mice transplanted with wt BM cells. Panel C: The number of CFU-S colonies formed in the spleens of wt mice were counted after transplantation of wt BMMNC cells, which were pre-stimulated or unstimulated before transplantation by sC5b-9 (10μg/ml), and injected intravenously into lethally irradiated wild type animals. Twelve days later, spleen was isolated and the number of CFU-S evaluated. Panel D: The number of CFU-GM progenitors in BM of wt transplanted mice with SC5b-9-primed or unprimed BM cells. The data shown in panels C and D represent the combined results from three independent experiments carried out in triplicate per group (n=9). Panel E: Wild type mice were lethally irradiated and transplanted with wild type BMMNC after SC5b-9 treatment (primed) or not (unprimed control). We followed the subsequent recovery of hematopoietic parameters in these animals. Mice that were transplanted with SC5b-9-primed BMMNCs have a much faster recovery rate for peripheral blood leukocytes (left panel) and platelets (right panel). * p<0.05. Panel F: Soluble MAC, like SDF-1, activates MAPKp44/42 and Akt in normal murine Sca-1+ cells. “Mix” indicates SDF-1 (0.05μg/ml) plus SC5b-9 (1μg/ml). Experiments were repeated independently three times with similar results. A representative western blot is shown.
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Related In: Results  -  Collection

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Figure 5: C5-deficient mice show an engraftment defect in transplanted HSPCsPanel A: Wild type (C5+/+) and C5-deficient (C5−/−) animals, which do not generate MAC, were lethally irradiated and transplanted with wild type bone marrow mononuclear cells. We followed the subsequent recovery of hematopoietic parameters in these animals. C5−/− mice have recovery of peripheral blood leukocytes (upper panel) and platelets (lower panel) delayed by 3–5 days. The data shown represent the combined results from two independent experiments performed with 10 mice/group. * p<0.001. Panel B: By day 12, there is a decrease in the number of CFU-GM in BM of C5−/− mice transplanted with wt BM cells. Panel C: The number of CFU-S colonies formed in the spleens of wt mice were counted after transplantation of wt BMMNC cells, which were pre-stimulated or unstimulated before transplantation by sC5b-9 (10μg/ml), and injected intravenously into lethally irradiated wild type animals. Twelve days later, spleen was isolated and the number of CFU-S evaluated. Panel D: The number of CFU-GM progenitors in BM of wt transplanted mice with SC5b-9-primed or unprimed BM cells. The data shown in panels C and D represent the combined results from three independent experiments carried out in triplicate per group (n=9). Panel E: Wild type mice were lethally irradiated and transplanted with wild type BMMNC after SC5b-9 treatment (primed) or not (unprimed control). We followed the subsequent recovery of hematopoietic parameters in these animals. Mice that were transplanted with SC5b-9-primed BMMNCs have a much faster recovery rate for peripheral blood leukocytes (left panel) and platelets (right panel). * p<0.05. Panel F: Soluble MAC, like SDF-1, activates MAPKp44/42 and Akt in normal murine Sca-1+ cells. “Mix” indicates SDF-1 (0.05μg/ml) plus SC5b-9 (1μg/ml). Experiments were repeated independently three times with similar results. A representative western blot is shown.
Mentions: Because our data demonstrated that CC is activated and MAC is deposited in lethally irradiated BM (Figure 1panel A, right), we asked whether MAC plays a role in homing of HSPCs. To address this question, we transplanted lethally irradiated C5-deficient mice that do not generate MAC, as well as wild type (wt) mice, with wt BM cells. Figure 5panel A shows that the recovery of leucocytes and platelets was significantly delayed in C5-deficient mice, which do not generate and deposit MAC. In further support of this finding, the bones of mice at day 12 after transplantation exhibited a much lower number of transplant-derived CFU-GM (Figure 5panel B).

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