The lysosomal transfer of LDL/cholesterol from macrophages into vascular smooth muscle cells induces their phenotypic alteration.

AIMS: Macrophages (MPs) and vascular smooth muscle cells (VSMCs) closely interact within the growing atherosclerotic plaque. An in vitro co-culture model was established to study how MPs modulate VSMC behaviour. METHODS AND RESULTS: MPs were exposed to fluorescence-labelled-acetylated LDL (FL-acLDL) prior to co-culture with VSMCs. Fluorescence microscopy visualized first transport of FL-acLDL within 6 h after co-culture implementation. When MPs had been fed with FL-acLDL in complex with fluorescence-labelled cholesterol (FL-Chol), these complexes were also transferred during co-culture and resulted in cholesterol positive lipid droplet formation in VSMCs. When infected with a virus coding for a fusion protein of Rab5a and fluorescent protein reporter (FP) to mark early endosomes, no co-localization between Rab5a-FP and the transported FL-acLDL within VSMCs was detected implying a mechanism independent of phagocytosis. Next, expression of lysosome-associated membrane glycoprotein 1 (LAMP1)-FP, marking all lysosomes in VSMCs, revealed that the FL-acLDL was located in non-acidic lysosomes. MPs infected with virus encoding for LAMP1-FP prior to co-culture demonstrated that intact fluorescence-marked lysosomes were transported into the VSMC, instead. Xenogenic cell composition (rat VSMC, human MP) and subsequent quantitative RT-PCR with rat-specific primers rendered induction of genes typical for MPs and down-regulation of the cholesterol sensitive HMG-CoA reductase. CONCLUSION: Our results demonstrate that acLDL/cholesterol-loaded lysosomes are transported from MPs into VSMCs in vitro. Lysosomal transfer results in a phenotypic alteration of the VSMC towards a foam cell-like cell. This way VSMCs may lose their plaque stabilizing properties and rather contribute to plaque destabilization and rupture.

Alemtuzumab depletes dendritic cells more effectively in blood than in skin: a pilot study in patients with chronic lymphocytic leukemia.

The antibody alemtuzumab (anti-CD52) is effective in preventing acute graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (aHSCT). As well as depleting donor T cells, alemtuzumab may also work by targeting host dendritic cells (DC). To determine whether this second mechanism of action is significant, we investigated the effects of intravenous alemtuzumab by comparing skin and blood DC numbers of patients with chronic lymphocytic leukemia, before and after a 4-week course of alemtuzumab treatment. Although skin DC express CD52, the epitope is only weakly detectable and their numbers were not consistently reduced by alemtuzumab. In contrast, circulating blood DC, with stronger CD52 expression, were invariably diminished by alemtuzumab. Because DC depletion in the transplant recipient remains a promising approach for GvHD prophylaxis and therapy, more potent techniques, such as an antibody of different specificity, may be required for effective DC eradication in GvHD target organs.

Fast appearance of donor dendritic cells in human skin: dynamics of skin and blood dendritic cells after allogeneic hematopoietic cell transplantation.

BACKGROUND: Both number and origin (donor vs. host) of dendritic cells (DC) are associated with acute graft-versus-host disease (aGvHD), relapse and graft failure after human allogeneic hematopoietic cell transplantation (aHCT). METHODS: We prospectively and simultaneously investigated skin and blood DC subtypes, their donor/recipient origin, and the correlation of DC reconstitution kinetics with treatment, clinical outcome, and incidence of aGvHD in patients undergoing aHCT. RESULTS: A significant reduction of skin and a marked decrease of blood DC were observed in patients compared to healthy volunteers. A dominant donor chimerism of migratory Langerhans cells (LC) and dermal-dendritic-cells (DDC) was detected even early after transplantation, and developed independently from chemotherapy regimen, graft manipulation or time point after transplantation. Before start of the therapy patients showed significantly decreased numbers of peripheral blood CD123+ preDC2, whereas CD11c+ preDC1 numbers appeared to be diminished, but were statistically indistinguishable from controls. Host derived pB preDC were virtually absent following aHCT. After a further reduction in cell number around day 56 both preDC subtypes reconstituted and stabilized to pretransplant numbers by day 112. Occurrence of aGvHD and its treatment diminished numbers of both preDC subtypes. Furthermore conditioning therapy with Alemtuzumab apparently affected reconstitution of both preDC subsets negatively. CONCLUSION: Given that induction of GvHD in humans is as host DC dependent as in mouse models, investigation of DC chimerism and number at different sites and especially in GvHD target organs might provide important insights into the pathogenesis of the main obstacle of aHCT.