Fig. 4.

Interaction of leukocytes with, and entry across, the BBB. The neurovascular unit is comprised of a single layer of ECs, contractile pericytes, and smooth muscle cells embedded in the basement membrane, as well as astrocytes and neurons. Neurons and astrocytes send signals to the mural contractile cells to regulate CBF. Perivascular $\mathrm{M}\mathrm{\Phi }$ reside in the perivascular space that is lined with the end-feet of astrocytes forming the glia limitans. Microglia are present in the brain parenchyma but can make direct contact with pericytes via their numerous processes. Infectious agents and tissue damage are detected by these tissue resident immune cells via pattern recognition receptors such as toll-like receptors, which in turn induce transcription and translation of inflammatory cytokines to activate ECs, promoting leukocyte entry and increasing permeability to small molecules. Leukocyte entry occurs across post-capillary vascular segments adjoining ascending venules, where ECs initially upregulate P and E selectin to capture leukocytes in the blood via their respective carbohydrate ligands (e.g., P selectin/PSGL1 interactions). Rolling reduces the speed of leukocytes, allowing prolonged contact time with the endothelium. Subsequent firm adhesive interactions occur via immobilized chemokine ligation to GPCRs on leukocytes, which switches leukocyte integrins into a high affinity state (e.g., ICAM-1/LFA-1 interactions). In the periphery, the majority of leukocyte migration occurs in a paracellular manner, through the junctions of adjacent ECs.⁸³ However, in the CNS during exacerbated inflammation T cells can migrate through the body of the EC: a transcellular route⁸⁴ which occurs due to a lack of T cell crawling induced by high ICAM-1 expression.⁸⁵ Additionally, ACKR1, a non-signaling atypical chemokine receptor, mediates the shuttling of chemokines across the BBB, which could also contribute to the mediation of transcellular migration.⁸⁶ It is thought the transcellular route is the preferred mode by which neutrophils move through the brain EC layer.⁸⁷ Approximately 50% of paracellular movement of leukocytes across the vessel wall occurs at tricellular junctions of ECs of the CNS (where three ECs meet).⁸⁸ After crossing the endothelial layer into the perivascular space, leukocytes must then pass the glia limitans layer⁸⁹ to enter the brain parenchyma. Figure created using BioRender.⁵⁴