Red pulp macrophages in the human spleen are a distinct cell population with a unique expression of Fc-γ receptors.

Tissue-resident macrophages in the spleen play a major role in the clearance of immunoglobulin G (IgG)-opsonized blood cells, as occurs in immune thrombocytopenia (ITP) and autoimmune hemolytic anemia (AIHA). Blood cells are phagocytosed via the Fc-γ receptors (FcγRs), but little is known about the FcγR expression on splenic red pulp macrophages in humans, with only a few previous studies that showed conflicting results. We developed a novel method to specifically isolate red pulp macrophages from 82 human spleens. Surface expression of various receptors and phagocytic capacity was analyzed by flow cytometry and immunofluorescence of tissue sections. Red pulp macrophages were distinct from splenic monocytes and blood monocyte-derived macrophages on various surface markers. Human red pulp macrophages predominantly expressed the low-affinity receptors FcγRIIa and FcγRIIIa. In contrast to blood monocyte-derived macrophages, red pulp macrophages did not express the inhibitory FcγRIIb. Red pulp macrophages expressed very low levels of the high-affinity receptor FcγRI. Messenger RNA transcript analysis confirmed this expression pattern. Unexpectedly and despite these differences in FcγR expression, phagocytosis of IgG-opsonized blood cells by red pulp macrophages was dependent on the same FcγRs as phagocytosis by blood monocyte-derived macrophages, especially in regarding the response to IV immunoglobulin. Concluding, we show the distinct nature of splenic red pulp macrophages in human subjects. Knowledge on the FcγR expression and usage of these cells is important for understanding and improving treatment strategies for autoimmune diseases such as ITP and AIHA.

The composition of the mesenchymal stromal cell compartment in human bone marrow changes during development and aging.

Life-long hematopoiesis depends on the support of mesenchymal stromal cells within the bone marrow. Therefore, changes in the hematopoietic compartment that occur during development and aging probably correlate with variation in the composition of the stromal cell microenvironment. Mesenchymal stromal cells are a heterogeneous cell population and various subtypes may have different functions. In accordance with others, we show that CD271 and CD146 define distinct colony-forming-unit-fibroblast containing mesenchymal stromal cell subpopulations. In addition, analysis of 86 bone marrow samples revealed that the distribution of CD271(bright)CD146(-) and CD271(bright)CD146(+) subsets correlates with donor age. The main subset in adults was CD271(bright)CD146(-), whereas the CD271(bright)CD146(+) population was dominant in pediatric and fetal bone marrow. A third subpopulation of CD271(-)CD146(+) cells contained colony-forming-unit-fibroblasts in fetal samples only. These changes in composition of the mesenchymal stromal cell compartment during development and aging suggest a dynamic system, in which these subpopulations may have different functions.

Inside-out regulation of ICAM-1 dynamics in TNF-alpha-activated endothelium.

BACKGROUND: During transendothelial migration, leukocytes use adhesion molecules, such as ICAM-1, to adhere to the endothelium. ICAM-1 is a dynamic molecule that is localized in the apical membrane of the endothelium and clusters upon binding to leukocytes. However, not much is known about the regulation of ICAM-1 clustering and whether membrane dynamics are linked to the ability of ICAM-1 to cluster and bind leukocyte integrins. Therefore, we studied the dynamics of endothelial ICAM-1 under non-clustered and clustered conditions. PRINCIPAL FINDINGS: Detailed scanning electron and fluorescent microscopy showed that the apical surface of endothelial cells constitutively forms small filopodia-like protrusions that are positive for ICAM-1 and freely move within the lateral plane of the membrane. Clustering of ICAM-1, using anti-ICAM-1 antibody-coated beads, efficiently and rapidly recruits ICAM-1. Using fluorescence recovery after photo-bleaching (FRAP), we found that clustering increased the immobile fraction of ICAM-1, compared to non-clustered ICAM-1. This shift required the intracellular portion of ICAM-1. Moreover, biochemical assays showed that ICAM-1 clustering recruited beta-actin and filamin. Cytochalasin B, which interferes with actin polymerization, delayed the clustering of ICAM-1. In addition, we could show that cytochalasin B decreased the immobile fraction of clustered ICAM-1-GFP, but had no effect on non-clustered ICAM-1. Also, the motor protein myosin-II is recruited to ICAM-1 adhesion sites and its inhibition increased the immobile fraction of both non-clustered and clustered ICAM-1. Finally, blocking Rac1 activation, the formation of lipid rafts, myosin-II activity or actin polymerization, but not Src, reduced the adhesive function of ICAM-1, tested under physiological flow conditions. CONCLUSIONS: Together, these findings indicate that ICAM-1 clustering is regulated in an inside-out fashion through the actin cytoskeleton. Overall, these data indicate that signaling events within the endothelium are required for efficient ICAM-1-mediated leukocyte adhesion.

ICAM-1 clustering on endothelial cells recruits VCAM-1.

In the initial stages of transendothelial migration, leukocytes use the endothelial integrin ligands ICAM-1 and VCAM-1 for strong adhesion. Upon adhesion of the leukocyte to endothelial ICAM-1, ICAM-1 is clustered and recruited to the adhered leukocyte, promoting strong adhesion. In this study, we provide evidence for the colocalization of VCAM-1 at sites of ICAM-1 clustering. Anti-ICAM-1 antibody-coated beads were used to selectively cluster and recruit ICAM-1 on primary human endothelial cells. In time, co-localization of ICAM-1 and VCAM-1 around the adherent beads was observed. Biochemical pull-down assays showed that ICAM-1 clustering induced its association to VCAM-1, suggesting a physical link between these two adhesion molecules. The association was partly dependent on lipid rafts as well as on F-actin and promoted adhesion. These data show that VCAM-1 can be recruited, in an integrin-independent fashion, to clustered ICAM-1 which may serve to promote ICAM-1-mediated leukocyte adhesion.