Neutrophils emigrate from venules by a transendothelial cell pathway in response to FMLP

Abstract

Circulating leukocytes are thought to extravasate from venules through open interendothelial junctions. To test this paradigm, we injected N-formyl-methionyl-leucyl-phenylalanine (FMLP) intradermally in guinea pigs, harvesting tissue at 5-60 min. At FMLP-injected sites, venular endothelium developed increased surface wrinkling and variation in thickness. Marginating neutrophils formed contacts with endothelial cells and with other neutrophils, sometimes forming chains of linked leukocytes. Adherent neutrophils projected cytoplasmic processes into the underlying endothelium, especially at points of endothelial thinning. To determine the pathway by which neutrophils transmigrated endothelium, we prepared 27 sets of serial electron microscopic sections. Eleven of these encompassed in their entirety openings through which individual neutrophils traversed venular endothelium; in 10 of the 11 sets, neutrophils followed an entirely transendothelial cell course unrelated to interendothelial junctions, findings that were confirmed by computer-assisted three-dimensional reconstructions. Having crossed endothelium, neutrophils often paused before crossing the basal lamina and underlying pericytes that they also commonly traversed by a transcellular pathway. Thus, in response to FMLP, neutrophils emigrated from cutaneous venules by a transcellular route through both endothelial cells and pericytes. It remains to be determined whether these results can be extended to other inflammatory cells or stimuli or to other vascular beds.

Figure 1

Large venule in guinea pig skin harvested 60 min after intradermal injection of 10⁻⁵ FMLP. Many neutrophils and a single eosinophil (eos) are captured at various stages of attachment to and extravasation across vascular endothelium and underlying pericytes (p). Two neutrophils (single joined arrow), one in the lumen and another partway across the endothelium, are tethered together. Another neutrophil (long arrow) has projected a cytoplasmic process into an underlying EC. Other neutrophils (arrowheads) and the eosinophil have crossed the EC barrier, but remain superficial to pericytes, forming dome-like structures that bulge into the vascular lumen. Still another neutrophil (open arrow) that has already crossed the endothelium has extended a process into the basal lamina and indents an underlying pericyte. Other neutrophils (some indicated by n) have crossed both the EC and pericyte barriers and have entered the surrounding connective tissues. L, lumen. Bar, 10 μm.

Figure 2

Progression of neutrophil migration across venular endothelium at 60 min after intradermal injection of 10⁻⁵ M FMLP. (A) Three neutrophils in the vascular lumen (L) are tethered together at point contacts; two of these also attach to the endothelium (e) at disparate sites. Three additional neutrophils (n) have already crossed the endothelial barrier. (B) An adherent neutrophil (n) projects two separate pseudopods into an EC (e). As confirmed in deeper serial sections, the smaller of these (arrowhead) projects into the EC at a point adjacent to the nucleus, whereas the larger (dominant) pseudopod forms a blunt projection into a thinned region of EC cytoplasm nearby. The EC shows extensive wrinkling of its abluminal surface, except over the nucleus, which is rounded with pinched folds and bulges into the lumen. (C) A chain of three interconnected neutrophils. One of these (elongate cell) has traversed both endothelium (e), basal lamina, and pericyte (p) layers and extends into the underlying connective tissue while its trailing edge remains within the vascular lumen. The second neutrophil has formed attachments to endothelium at two discontinuous sites and the third remains in the vascular lumen with no attachments to endothelium. (D) Higher magnification of a portion of Fig. 1, illustrating two neutrophils and an eosinophil (eos) that have crossed the endothelium but have not penetrated the pericyte (p) layer; each is covered over with a thin overlay of flattened, relatively smooth endothelium that, together with the leukocytes, form dome-like structures that bulge into the vascular lumen. L, lumen. Bar, 3 μm.

Figure 3

Transmigration of a neutrophil (n) across a thinned portion of venular endothelium (e) at a skin site injected 60 min earlier with 10⁻⁵ M FMLP. 12 of a series of 45 consecutive serial sections (sections 10–19 and 21–22) are illustrated and together encompass in its entirety the transendothelial pore (F–J) through which the neutrophil is migrating. Maximum pore diameter was 0.75 μm. L, lumen. Bar, 1 μm.

Figure 6

Neutrophil (n) that has already crossed endothelium (e) is at an early stage of transmigration through a subendothelial pericyte (p) 60 min after intradermal injection of 10⁻⁵ M FMLP. 8 of a series of 90 consecutive serial sections (sections 60–66 and 68) are illustrated. B–G illustrate the transpericyte pore through which the neutrophil has advanced a cytoplasmic projection. Maximum pore diameter was 0.83 μm. Bar, 1 μm.

Figure 7

Neutrophil (n) migrating through a subendothelial pericyte (p) 60 min after intradermal injection of 10⁻⁵ M FMLP. Four (sections 12–14 and 16) of a series of 46 consecutive serial sections illustrate a transcytoplasmic pore in the pericyte through which the neutrophil has extended a blunt cytoplasmic projection. The projecting neutrophil pseudopod is filled with a feltwork of microfilaments. Note also the prominent, horizontally arrayed microfilaments in the immediately adjacent pericyte cytoplasm (C, arrowheads). Bar, 0.1 μm.

Figure 8

Schematic diagram summarizing different aspects of neutrophil diapedesis in response to FMLP. In A, neutrophil n1 extends a cytoplasmic process that forms contact with a thinned portion of EC e1 as in Fig. 2, A and B; in B–D n1 has progressed through e1, the underlying basal lamina (bl) and a pericyte (p1). This pattern of near-simultaneous trans-migration through endothelium as well as other components of the vascular wall is illustrated in Fig. 2 C, but was less common than that exhibited by neutrophils n2 and n3 that, after crossing the EC barrier, paused for a time before progressing farther. In A, n2 initially makes contact with the endothelial surface with two processes, as in Fig. 2 B; one of these becomes dominant and leads the way as the neutrophil begins to traverse EC e2 in B, again as in Fig. 2 B. Having crossed the endothelial cell barrier, n2 does not immediately progress through the basal lamina; instead it remains covered over for a time (C and D) by a thin overlying rim of flattened EC cytoplasm (as in Fig. 1, arrowheads) and Fig. 2 D) before extending a pseudopod through the basal lamina and underlying pericyte in D. As it progresses through e2 (C and D), it is embraced by cytoplasmic processes that extend from abluminal portions of both e2 and an adjacent EC, e1 (as in Fig. 5). Transmigration of n3 is similar to that of n1 and n2, except that n3 persists for a longer time in the subendothelial space superficial to the basal lamina. n3 in A corresponds to the neutrophil marked by a long arrow in Fig. 1; n3 in C and D models neutrophils illustrated in Fig. 1, arrowheads and Fig. 2 A and D. Extravasation of all three neutrophils is transendothelial and inter-EC junctions remain closed.

Figure 5

Computer-generated three-dimensional reconstruction of the transmigrating neutrophil illustrated in Fig. 4. The panels portray successive rotations toward the viewer around a horizontal axis at angles of 60°, 120°, 240°, and 300° as indicated. 0° (not shown) would represent a vascular cross section at right angles to the direction of blood flow and 90° (also not shown) would represent a view looking directly down on the luminal surface. Emigrating neutrophil (n), purple, upper and lower left; in the other panels, the neutrophil was subtracted electronically to visualize the pore that passes cleanly through the cytoplasm of EC e1 (orange-brown) distinctly apart from the junction of e1 with e2 (yellow). Cytoplasmic arms of both e1 and e2 embrace the neutrophil luminally and, to a lesser extent, abluminally. L, lumen.

Figure 4

Transmigration of a neutrophil (n) across a thinned portion of venular endothelium at 60 min. after local intradermal injection of 10⁻⁵ M FMLP. 12 sections (numbers 13, 14, 16, 19, 21, 24, 25, 27, 29, and 30–32) of a series of 74 consecutive serial sections are illustrated. Portions of the neutrophil's nucleus (E–G) are included within the pore. The pore passes through a single EC (e1), but a junction of e1 with a second EC, e2, is indicated (arrow); this junction is intact and maintained a distance of >1 μm from the pore margin at all levels of sectioning. L, lumen. Bar, 1 μm.