Platelet-derived microparticles induce polymorphonuclear leukocyte-mediated damage of human pulmonary microvascular endothelial cells.

BACKGROUND: Platelets (PLTs) stored at 22°C accumulate microparticles and biologic response modifiers (BRMs) that induce inflammatory reactions in transfusion recipients. However, soluble BRMs are fully diluted in the recipient's blood circulation. The mechanisms by which BRMs exert their effects have not been elucidated. The objectives of this study were to determine the effect of PLT microparticles (PMPs) on polymorphonuclear leukocyte (PMN)-mediated human pulmonary microvascular endothelial cell (HMVEC) damage and determine the role of soluble CD40 ligand (sCD40L). STUDY DESIGN AND METHODS: PMPs were isolated from apheresis PLT concentrates. We used a two-insult in vitro model of HMVEC damage to investigate the effects of PMP and sCD40L and role of apocynin, an inhibitor of PMN respiratory burst. Their priming activities were measured using hydrogen peroxide production. The expression of intercellular cell adhesion molecule-1 (ICAM-1) and integrin αM (CD11b) were also determined. RESULTS: Lipopolysaccharide (LPS)-activated HMVEC damage and PMN respiratory burst depend on the presence of PMP and the concentration of sCD40L. PMP-induced PMN-mediated HMVEC damage was significantly reduced by apocynin-treated PMNs (p < 0.05). The surface expression of ICAM-1 on HMVEC was increased by LPS stimulation. The expression of CD11b on PMNs was increased by PMP priming. Blocking ICAM-1 with a monoclonal antibody (MoAb) CD54 significantly reduced HMVEC damage (p < 0.05). The treatment of endothelial cells but not PMN with a MoAb targeting CD40 failed to prevent the HMVEC damage caused by PMPs (p > 0.05). CONCLUSION: PMPs carry a concentrated CD40L signal, promote PMN-mediated HMVEC damage, and may affect the development of transfusion-related acute lung injury.

[The role of third-party tolerogenic dendritic cells in the prevention of acute graft-versus-host-disease following allogeneic bone marrow transplantation in mice].

OBJECTIVE: To explore the biological characteristic of third-party-derived tolerogenic DC(tDC) and the influence of third-party-derived tDC on acute graft-versus-host-disease (aGVHD) following allogeneic bone marrow transplantation (allo-BMT) in mice. METHODS: tDC from bone marrow cells of D1 mice was cultured with low doses of GM-CSF, IL-10 and TGF-ß1D1. The phenotype, expression of cytokines and function associated molecules were identified with FACS and RT-PCR. Mixed lymphocyte reaction was applied to analyze the influence of third-party-derived tDC on allo-CD4(+)T cells proliferation in vitro. Different doses of D1-tDC were adoptive transferred in the aGVHD model in allogeneic BMT which B6 mice as donors and D2 mice as recipients. Survival time, clinical GVHD score and the levels of Th1/2 cytokines in serum were monitored after allo-BMT using the aGVHD model as control. RESULTS: tDC expressed lower levels of MHC II and co-stimulatory molecules, such as CD80, CD86 and CD40, even when stimulated by LPS. The results by RT-PCR indicated that tDC expressed low levels of IL-12p40 and high levels of immunosuppressive molecules, such as IL-10, TGF-ß, Fas Ligand, indoleamine 2, 3-dioxygenase (IDO) and arginase. In the allogeneic MLR, third-party tDC suppressed allo-CD4(+)T cells proliferation, which was relative to the dose of tDC. In the B6→D2 mouse model, all aGVHD mice died within 18 days. Remarkably, if 10(4) third-party tDC were transferred, 60% mice survived at least 60 days. When the doses of tDC were reduced to 10(3) cells, only 20% of mice survived day 60, and when increased tDC to 10(5), all of the mice died within day 37 after allo-BMT. The cytokine levels in serum indicated that 10(4) tDC-treated mice secreted in vivo high level of IL-10 21d after BMT (P < 0.05), the levels of IL-10 in 10(3), 10(4) and 10(5) tDC-treated mice were (114.23 ± 7.78), (646.18 ± 212.02), (121.97 ± 10.47) ng/L, respectively. CONCLUSION: Third-party tDC could suppress allo-CD4(+)T cells proliferation in vitro and prevent aGVHD in allogeneic BMT mode, which may be mediated by modulating tolerogenic cytokines secretion, such as IL-10. And this effect was associated with the dose of tDC. Adoptive therapy by transfusing third-party tDC cultured with low doses of GM-CSF, IL-10 and TGF-ß1 could significantly prolong the survival of recipients and prevent aGVHD in allogeneic BMT.

[Effect of sphingosine 1-phosphate/sphingosine 1-phosphate receptor signal pathway on function of neutrophils].

The aim of this study was to examine the priming effect of sphingosine 1-phosphate (S1P) on fMLP-activated neutrophils, mainly to detect the neutrophil respiratory burst products, and to investigate the signaling pathway involved in S1P activity. Flow cytometry was used to evaluate the new isolated neutrophil; the superoxide anion output was detected indirectly by cytochrome C reduction in respiratory burst; the dihydro-rhodamine 123 was used to detect the intensity of respiratory burst; the signal transduction pathways of neutrophil respiratory burst were explored by Western blot. The results showed that after pretreated with S1P, the level of superoxide anion released by fMLP-activated neutrophils significantly increased; the Rhodamine 123 mean fluorescence intensity in S1P primed fMLP-activated neutrophils group was significantly higher than that in fMLP treatment group; PI3K and Akt proteins involved in the signal pathway of neutrophil respiratory burst. It is concluded that S1P is a new priming reagent, which primes respiratory burst of fMLP-activated neutrophils; this signal pathway may be that S1P interacts with its receptor, activates PI3K, then activates Akt-transmitting signals through NADPH oxidase, finally results in the respiratory burst.

[A preliminary study on the influence of human plasma exosomes-like vesicles on macrophage Wnt5A-Ca²+ pathway].

OBJECTIVE: To study the influence of human plasma exosomes-like vesicles on the regulatory function of macrophages. METHODS: The exosomes-like vesicles were purified from healthy donors plasma with a series of high-speed centrifugation and ultrafiltration. Macrophages were derived from cultured human blood monocytes. The molecular markers of macrophages were assayed by FACS. After cultured with exosomes-like vesicles, the changes of macrophages cytoplasma Ca(2+), and related genes and proteins were assayed by FACS, RT-PCR and Western Blot, respectively. RESULTS: After cultured with exosomes-like vesicles, mean fluorescent intensity (MFI) of macrophages cytoplasma Ca(2+) was increased. The vesicles enhanced macrophages to express cytokines genes, the expression of IL-1ß and TNF-α genes being increased by 0.85 and 1.69 times respectively at 2 h, and that of IL-6 gene 3.7 times compared with the control at 8 h. However, the vesicles inhibited the expression of macrophages IL-10 gene, had no influence on the Frizzled5 receptor expression and could induce CaMKII phosphorylation. CONCLUSIONS: Exosomes-like vesicles can up-regulat macrophages expression of inflammatory cytokines genes, and increase the secretion of inflammatory cytokines by activating the Wnt5A-Ca(2+) signaling pathway.