Abstract
BackgroundIntravascular leukocyte recruitment in most vertebrate tissues is restricted to postcapillary and collecting venules, whereas capillaries and arterioles usually support little or no leukocyte adhesion. This segmental restriction is thought to be mediated by endothelial, rather than hemodynamic, differences. The underlying mechanisms are largely unknown, in part because effective tools to distinguish, isolate, and analyze venular endothelial cells (V-ECs) and non-venular endothelial cells (NV-ECs) have been unavailable. We hypothesized that the atypical chemokine receptor DARC (Duffy Antigen Receptor for Chemokines, a.k.a. ACKR1 or CD234) may distinguish V-ECs versus NV-ECs in mice.MethodsWe generated a rat-anti-mouse monoclonal antibody (MAb) that specifically recognizes the erythroid and endothelial forms of native, surface-expressed DARC. Using this reagent, we characterized DARC expression and distribution in the microvasculature of murine tissues. ResultsDARC was exquisitely restricted to post-capillary and small collecting venules and completely absent from arteries, arterioles, capillaries, veins, and most lymphatics in every tissue analyzed. Accordingly, intravital microscopy showed that adhesive leukocyte-endothelial interactions were restricted to DARC+ venules. DARC was detectable over the entire circumference of V-ECs, but was more concentrated at cell-cell junctions. Analysis of single-cell suspensions suggested that the frequency of V-ECs among the total microvascular EC pool varies considerably between different tissues.ConclusionsImmunostaining of endothelial DARC allows the identification and isolation of intact V-ECs from multiple murine tissues. This strategy may be useful to dissect the mechanisms underlying segmental microvascular specialization in healthy and diseased tissues and to characterize the role of EC subsets in tissue-homeostasis, immune surveillance, infection, inflammation, and malignancies.
Highlights
Intravascular leukocyte recruitment in most vertebrate tissues is restricted to postcapillary and collecting venules, whereas capillaries and arterioles usually support little or no leukocyte adhesion
The peripheral circulatory system is segmentally organized into sparse arteries and arterioles that feed a dense capillary network drained by postcapillary venules (PCVs) that merge into collecting venules (CVs) and veins
Validation of anti-mouse DARC mAb specificity After immunization of a rat with DARC-eGFP transfected PC-12 cells, hybridoma lines were generated and supernatants screened by FACS
Summary
Intravascular leukocyte recruitment in most vertebrate tissues is restricted to postcapillary and collecting venules, whereas capillaries and arterioles usually support little or no leukocyte adhesion. Studies employing intravital microscopy (IVM) have demonstrated that the initial attachment and arrest of free flowing leukocytes to the microvascular wall in most tissues (except in lung, liver, and spleen) is a feature of PCVs and CVs, whereas capillaries and arterioles usually do not support leukocyte trafficking [5]. This specialization of venular endothelium is found in vertebrates ranging from agnathans to mammals [1, 6] and in both adults and fetuses [7]. The underlying differentiation program that enables venular endothelial cells (V-ECs) to express leukocyte traffic molecules, but prohibits non-venular endothelial cells (NV-ECs) in capillaries and arterioles from doing so is unknown
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