Human umbilical vein endothelial cells (HUVEC)2 were cultured in the HUVEC-specific culture medium. To purify the exosomes, the culture supernatant was collected and processed following published procedures (14). Briefly, the supernatant was subjected to three successive centrifugation steps at 12,000 × g for 1 h, 35,000 × g for 1 h, and 70,000 × g for 3 h at 4 °C. The supernatant was filtered sequentially through 10.0-, 0.45-, and 0.22-μm filters. The supernatant was then ultracentrifuged at 100,000 × g for 1 h at 4 °C. The pellet of exosomes was used for further experiments. The purified exosomes were lysed with lysing buffer; the proteins were analyzed by Western blotting to assess the levels of αvβ6 and the endosome marker LAMP1.

Flow Cytometry

For analysis by flow cytometry, brefildin A (10 μg/ml) was added to the culture medium for the last 3 h of culture. Cells were fixed with 2% paraformaldehyde (in case of the intracellular staining, 0.1% Triton X-100 was added to the fixatives) for 1 h. After washing, the cells were blocked by 5% skim milk. The cells were incubated with fluorescence-labeled antibodies as indicated in figures at concentrations of 0.5–1 μg/ml for 1 h at room temperature. The cells were analyzed by a flow cytometer (FACSCanto II; BD Bioscience). The gating was performed when necessary. Briefly, cells were stained with the indicated fluorescence-labeled antibodies. One positively stained cell population was gated first; by double clicking the gated cell population, a new window popped out for further analysis, and so on.

Test the Immune Suppressor Function of CX3CR1+ B Cells

CX3CR1+ B cells and CD4+ CD25− T cells (labeled with carboxyfluorescein diacetate, succinimidyl ester) were obtained from healthy subjects. The cells were cultured at a ratio of 1:5 (B cell:T cell) for 3 days (the treatment is denoted in Fig. 5). The cells were collected and analyzed by the carboxyfluorescein diacetate, succinimidyl ester dilution assay.

Methodology/Principal Findings

We demonstrate unambiguously that highly purified human B cells from tonsil and peripheral blood expressed CX3CR1 at mRNA and protein levels as assessed by quantitative PCR, flow cytometry and competition binding assays. In particular, naïve, germinal centre and memory B cells expressed CX3CR1 but only germinal centre B cells were attracted by soluble CX3CL1 in a transwell assay. CX3CL1 signalling in germinal centre B cells involved PI3K, Erk1/2, p38, and Src phosphorylation, as assessed by Western blot experiments. CX3CR1+ germinal centre B cells were devoid of centroblasts and enriched for centrocytes that migrated to soluble CX3CL1. ELISA assay showed that soluble CX3CL1 was secreted constitutively by follicular dendritic cells and T follicular helper cells, two cell populations homing in the germinal centre light zone as centrocytes. At variance with that observed in humans, soluble CX3CL1 did not attract spleen B cells from wild type mice. OVA immunized CX3CR1−/− or CX3CL1−/− mice showed significantly decreased specific IgG production compared to wild type mice.

Abstract

Chemokine receptors play an important role in leukocyte migration, both in physiological and pathological conditions, and the interest in new methodologies for their detection is increasing. In this review, we focused on chemokine receptors detection through flow cytometric approaches, including the use of specific antibodies and fluorescent chemokines, and on approaches aimed at the analysis of their functions, from intracellular trafficking to signaling activities. © 2014 International Society for Advancement of Cytometry

Abstract

Chemokines and their receptors play an essential role within the immune system by dictating cellular migration. In vivo, receptor–ligand interactions rarely occur in isolation as cellular recruitment and migration are complex and highly coordinated processes often involving networks of multiple chemokines and multiple receptors. Simultaneous detection of multiple chemokine receptors on the single cell level is necessary to allow immunophenotyping studies that will help understand the intricacies of these networks. Chemokine receptors undergo a basal level of ongoing internalization, intracellular trafficking, and recycling back to the cell surface, even in the absence of the ligand. In the presence of ligand, receptor–ligand interactions enhance receptor internalization, reducing the cell surface receptor concentration, making precise determination of intrinsic levels challenging. Using multicolor flow cytometry, we sought to evaluate and optimize the simultaneous detection of cell surface expression levels of CCR2, CX3CR1, and CCR5 in primary human monocytes using a single antibody panel. We observed that staining for CCR2 alone or for CX3CR1 alone showed greater expression levels than when the cells were stained with the full panel of antibodies. Fluorescent-minus-one (FMO) controls revealed that ligation of the CCR5 monoclonal antibody to the receptor interfered with detection of CX3CR1 and CCR2. Sequential addition of antibodies during the staining procedure was sufficient to restore the detection levels, suggesting close proximity and possible functional interactions between CCR2/CCR5 and CX3CR1/CCR5 in monocytes. This study highlights the importance of optimizing staining procedures and using proper controls when simultaneously evaluating expression levels of multiple chemokine receptors by flow cytometry. Concurrent assessment of multiple receptors will provide insight and greater understanding of the complex interactions involved in cellular migration. © 2013 International Society for Advancement of Cytometry