Monocytes participate critically in atherosclerosis. There are 2 major subsets expressing different chemokine receptor patterns: CCR2 + CX3CR1 + Ly-6C hi and CCR2 -CX3CR1 ++ Ly-6C lo monocytes. Both C-C motif chemokine receptor 2 (CCR2) and C-X 3 -C motif chemokine receptor 1 (CX3CR1) are linked to progression of atherosclerotic plaques. Here, we analyzed mouse monocyte subsets in apoE-deficient mice and traced their differentiation and chemokine receptor usage as they accumulated within atherosclerotic plaques. Blood monocyte counts were elevated in apoE -/-mice and skewed toward an increased frequency of CCR2 + Ly-6C hi monocytes in apoE -/-mice fed a high-fat diet. CCR2 + Ly-6C hi monocytes efficiently accumulated in plaques, whereas CCR2 -Ly-6C lo monocytes entered less frequently but were more prone to developing into plaque cells expressing the dendritic cell-associated marker CD11c, indicating that phagocyte heterogeneity in plaques is linked to distinct types of entering monocytes. CCR2 -monocytes did not rely on CX3CR1 to enter plaques. Instead, they were partially dependent upon CCR5, which they selectively upregulated in apoE -/-mice. By comparison, CCR2 + Ly-6C hi monocytes unexpectedly required CX3CR1 in addition to CCR2 and CCR5 to accumulate within plaques. In many other inflammatory settings, these monocytes utilize CCR2, but not CX3CR1, for trafficking. Thus, antagonizing CX3CR1 may be effective therapeutically in ameliorating CCR2 + monocyte recruitment to plaques without impairing their CCR2-dependent responses to inflammation overall.

Some monocytes normally take up residence in tissues as sessile macrophages, but others differentiate into migratory cells resembling dendritic cells that emigrate to lymph nodes. In an in vitro model of a vessel wall, lipid mediators lysophosphatidic acid and platelet-activating factor, whose signals are implicated in promoting atherosclerosis, blocked conversion of monocytes into migratory cells and favored their retention in the subendothelium. In vivo studies revealed trafficking of monocyte-derived cells from atherosclerotic plaques during lesion regression, but little emigration was detected from progressive plaques. Thus, progression of atherosclerotic plaques may result not only from robust monocyte recruitment into arterial walls but also from reduced emigration of these cells from lesions.

The recognition events that mediate adaptive cellular immunity and regulate antibody responses depend on intercellular contacts between T cells and antigen presenting cells (APC)1. T cell signaling is initiated at these contacts when surface-expressed antigen receptors (TCR) recognize peptide fragments (antigens) of pathogens bound to Major Histocompatibility Complex molecules (pMHC) on APCs. This, along with engagement of adhesion receptors, leads to the formation of a specialized junction between T cells and APCs, known as the immunological synapse (IS)3, which mediates efficient delivery of effector molecules and intercellular signals across the synaptic cleft2. T cell recognition of pMHC and the adhesion ligand Intercellular Adhesion Molecule-1 (ICAM-1) on supported planar bilayers recapitulates the domain organization of the immunological synapse (IS)4–5, which is characterized by central accumulation of TCR5, adjacent to a secretory domain3, both surrounded by an adhesive ring4–5. Although accumulation of TCR at the IS center correlates with T cell function4, this domain is itself largely devoid of TCR signaling activity5–6, and is characterized by an unexplained immobilization of TCR-pMHC complexes relative to the highly dynamic IS periphery4–5. Here we show that centrally accumulated TCR is located on the surface of extracellular microvesicles that bud at the IS center. Tumor susceptibility gene 101 (TSG101)6 sorts TCR for inclusion in microvesicles, while vacuolar protein sorting 4 (VPS4) 7–8 mediates scission of microvesicles from the T cell plasma membrane. The HIV polyprotein GAG co-opts this process for budding of virus-like particles. B cells bearing cognate pMHC receive TCR from T cells and initiate intracellular signals in response to isolated synaptic microvesicles. We conclude that the immunological synapse orchestrates TCR sorting and release in extracellular microvesicles. These microvesicles deliver transcellular signals across antigen-dependent synapses by engaging cognate pMHC on APC.

Studying the influence of chemokine receptors (CCRs) on monocyte fate may reveal information about which subpopulations of monocytes convert to dendritic cells (DCs) and the migration pathways that they use. First, we examined whether prominent CCRs on different monocyte subsets, CCR2 or CX3CR1, mediated migration events upstream of the accumulation of monocyte-derived DCs in lymph nodes (LNs). Monocytes were labeled and traced by uptake of latex microspheres in skin. Unexpectedly, neither CCR2 nor CX3CR1 were required. However, absence of CCR2 led to an increased labeling of the minor Gr-1int monocyte population, and the number of latex+ DCs that emigrated to LNs was correspondingly increased. Characterization of Gr-1int monocytes revealed that they selectively expressed CCR7 and CCR8 mRNA in blood. CCR7 and CCR8 pathways were used by monocyte-derived DCs during mobilization from skin to LNs. The role of CCR8 in emigration from tissues also applied to human monocyte-derived cells in a model of transendothelial trafficking. Collectively, the data suggest that Gr-1int monocytes may be most disposed to become a lymphatic-migrating DCs. When these monocyte-derived DCs exit skin to emigrate to LNs, they use not only CCR7 but also CCR8, which was not previously recognized to participate in migration to LNs.

We investigated the fate of latex (LX) particles that were introduced into mice intranasally. Macrophages acquired the vast majority of particles and outnumbered LX particle-bearing airway dendritic cells (DCs) by at least two orders of magnitude. Yet alveolar macrophages were refractory to migration to the draining lymph node (DLN), and all transport to the DLN could be ascribed to the few LX+ airway DCs. Upon macrophage depletion, markedly greater numbers of DCs were recruited into the alveolar space. Consequently, the number of DCs that carried particles to the DLN was boosted by 20-fold. Thus, a so far overlooked aspect of macrophage-mediated suppression of airway DC function stems from the modulation of DC recruitment into the airway. This increase in DC recruitment permitted the development of a robust assay to quantify the subsequent migration of DCs to the DLN. Therefore, we determined whether lung DCs use the same molecules that skin DCs use during migration to DLNs. Like skin DCs, lung DCs used CCR7 ligands and CCR8 for emigration to DLN, but the leukotriene C4 transporter multidrug resistance-related protein 1 did not mediate lung DC migration as it does in skin, indicating that pathways governing DC migration from different tissues partially differ in molecular regulation.

Immunity 24, 203-215; February 2006) The text related to Figure 2F in the Results section of our paper conveys an incorrect calculation that affects the interpretation of the experiments in Figure 2F, although not the overall conclusion we drew. The text as stated indicates that very few DCs entered efferent lymph to arrive at a lymph node (LN) further downstream. However, this extremely low magnitude (0.01%) is incorrect because it was mistakenly derived from the total fraction of LN cells, not the fraction of migrating, labeled DCs. The correct value, as is evident from the figure itself, is that 12% of traceable DCs traversed a primary draining lymph node (popliteal) to enter the downstream iliac lymph node (397 6 84 labeled DCs in the popliteal LN versus 49 6 21 in the iliac LN). This value was not affected by immunization that induced inflammation in the popliteal LN, as originally stated. Although these data indicate that DCs enter efferent lymph to a reasonable extent, the magnitude is far too low to account for the difference in the number of DCs found in LNs that primarily drain nonimmunized and immunized LNs (such as in Figure 1C).

Dendritic cell (DC) migration from the periphery to lymph nodes is regulated by the pattern of genes expressed by DCs themselves and by signals within the surrounding peripheral environment. Here, we report that DC mobilization can also be regulated by signals initiated within the downstream lymph nodes, particularly when lymph nodes enlarge as a consequence of immunization. Lymph node B lymphocytes orchestrate expansion of the lymphatic network within the immunized lymph node. This expanded network in turn supports increased DC migration from the periphery. These results reveal unique relationships between B cells, lymphatic vessels, and migratory DCs. Knowledge that DC migration from the periphery is augmented by B cell-dependent signals reveals new potential strategies to increase DC migration during vaccination.

High LDL and/or low HDL are risk factors for atherosclerosis and are also a common clinical feature in systemic lupus erythematosus, rheumatoid arthritis, and psoriasis. Here, we show that changes in lipid profiles that reflect atherosclerotic disease led to activation of skin murine dendritic cells (DCs) locally, promoted dermal inflammation, and induced lymph node hypertrophy. Paradoxically, DC migration to lymph nodes was impaired, suppressing immunologic priming. Impaired migration resulted from inhibitory signals generated by platelet-activating factor (PAF) or oxidized LDL that acts as a PAF mimetic. Normal DC migration and priming was restored by HDL or HDL-associated PAF acetylhydrolase (PAFAH), which mediates inactivation of PAF and oxidized LDL. Thus, atherosclerotic changes can sequester activated DCs in the periphery where they may aggravate local inflammation even as they poorly carry out functions that require their migration to lymph nodes. In this context, HDL and PAFAH maintain a normally functional DC compartment.