Mast cells migrate from blood to brain

Abstract

It is well established that mast cells (MCs) occur within the CNS of many species. Furthermore, their numbers can increase rapidly in adults in response to altered physiological conditions. In this study we found that early postpartum rats had significantly more mast cells in the thalamus than virgin controls. Evidence from semithin sections from these females suggested that mast cells were transiting across the medium-sized blood vessels. We hypothesized that the increases in mast cell number were caused by their migration into the neural parenchyma. To this end, we purified rat peritoneal mast cells, labeled them with the vital dyes PKH26 or CellTracker Green, and injected them into host animals. One hour after injection, dye-filled cells, containing either histamine or serotonin (mediators stored in mast cells), were located close to thalamic blood vessels. Injected cells represented approximately 2-20% of the total mast cell population in this brain region. Scanning confocal microscopy confirmed that the biogenic amine and the vital dye occurred in the same cell. To determine whether the donor mast cells were within the blood-brain barrier, we studied the localization of dye-marked donor cells and either Factor VIII, a component of endothelial basal laminae, or glial fibrillary acidic protein, the intermediate filament found in astrocytes. Serial section reconstructions of confocal images demonstrated that the mast cells were deep to the basal lamina, in nests of glial processes. This is the first demonstration that mast cells can rapidly penetrate brain blood vessels, and this may account for the rapid increases in mast cell populations after physiological manipulations.

Fig. 1.

Sections (1 μm) through two mast cells from a non-injected, postpartum female. A, Low magnification photomicrograph showing the location of an elongated, granulated MC (arrowhead) and a thin-walled blood vessel (bv). The single arrow indicates the nucleus of the endothelial cell, the double arrowsindicate the nucleus of the pericyte, and the single arrowhead indicates the region where the MC is in close contact with the basal lamina of the blood vessel. A neuron (N) is shown adjacent to the MC. Magnification, 850×. B, A higher magnification of the same MC as in A. The elongate shape of the MC is associated with migration. The two arrowheads indicate the boundaries of basal lamina of the blood vessel. The trailing end of the MC is just external to the basal lamina. Magnification, 1900×.C, Another example of an MC with one end (arrowhead) adjacent to a blood vessel (bv) and the opposite pole stretching into the neuropil (double arrowheads). The single arrowindicates the position of a portion of the MC nucleus, whereas thedouble arrows indicate a glancing section through the nucleus of a neuron that is outside of the plane of focus.N is a neuron near the MC process. M, Myelinated axon bundles. Magnification, 1900×.

Fig. 2.

Single 1 μm optical section through a CTG-positive mast cell (A) that contains serotonin (B). The overlay (C) shows that the two fluorophores are in the same cell. Small cellular processes are evident in this cell (A, C, arrowhead), and serotonergic granules or clusters of granules can be seen inB and C (arrows). Scale bar, 5 μm.

Fig. 5.

A depth projection of an MC marked by CTG (A, B) and astrocytes marked immunocytochemically by glial fibrillary acidic protein (A′, B′). These are two levels through an MC that are compilations of the middle (A, A′) and lower third of the cell (B, B′), each micrograph representing ∼3 μm of depth. The lumen of the blood vessel associated with the glial ensheathment (G, thick processes) is below this optical plane. The box in A contains the MC; the same box in A′ is devoid of thick glial process, indicating that the MC lies above the ensheathment and is in the neuropil. In B, the CTG signal has almost disappeared, indicating that there is very little of the MC cytoplasm remaining (box). At this level (B′), one can see the basal lamina of the blood vessel and the glial ensheathment (G). The arrow lies on the basal lamina and indicates the trajectory of the blood vessel. Scale bar, 5 μm.