Pericytes form an integral component of all blood vessels but, due to a lack of well accepted protocols for their isolation and culture, and availability of unequivocal pericyte markers, very little attention has been paid to investigations of leukocyte interactions with these mural cells. To address this fundamental issue, we have investigated the profile, dynamics, and mechanisms of leukocyte–pericyte interactions in inflamed tissues in vivo using an IVM platform with high spatiotemporal resolution. The findings report on previously unidentified modes by which pericytes facilitate leukocyte transmigration through venular walls, highlighting the need for further exploration of the role of pericytes under both physiological and pathological inflammatory conditions.
To enable direct analysis of neutrophil–pericyte interactions, we applied confocal IVM to observations of events within the microcirculation of inflamed cremaster muscles of αSMA-RFPcherry
transgenic mice crossed with Lys-EGFP-ki
mice. The resultant mouse colony expressed red pericytes and green leukocytes, and thus provided a uniquely powerful tool for analysis and investigations of leukocyte–pericyte interactions. The imaging platform was optimized for tracking neutrophils (as opposed to monocytes) and also incorporated a highly effective mode of in vivo immunolabeling of EC junctions (Woodfin et al., 2011
). This approach allowed investigations of neutrophil behavior and dynamics at different steps of the transmigration response, initiating with TEM and finishing with breaching of the pericyte sheath. The application of this technique to the analysis of TNF-stimulated tissues in 3D revealed that neutrophils could breach the endothelium rapidly, largely via a paracellular route (average duration, ~4 min), in line with our previous findings with multiple other inflammatory reactions (Woodfin et al., 2011
). After TEM, neutrophils exhibited a significant crawling response within the pericyte layer lasting ~30 min and covering distances of ~54 µm before crossing the pericyte sheath and entering the adjacent interstitial tissue. This abluminal crawling occurred in a highly meandering manner and crawling neutrophils clearly used pericyte processes as tracks. Indeed, neutrophil crawling was rarely seen in regions deficient of pericytes, i.e., within gaps between adjacent pericytes, suggesting a role for these mural cells in regulation of neutrophil motility within venular walls.
In investigating the adhesive mechanisms that support neutrophil–pericyte interactions, a role for ICAM-1 (on pericytes) and the integrins Mac-1 and LFA-1 (on neutrophils) was identified. Specifically, local application of blocking mAbs reduced the speed, track length, and displacement of neutrophil abluminal crawling in TNF-stimulated tissues. These effects were directly in line with enhanced duration of neutrophils within the venular wall and/or reduced neutrophil transmigration into the surrounding tissue, collectively suggesting a key role for neutrophil–pericyte interactions in regulation of neutrophil transmigration. Furthermore, TNF-stimulated tissues exhibited enhanced levels of ICAM-1 and the chemokine KC on both ECs and pericytes as compared with unstimulated tissues. These results indicate that, as found with ECs (Phillipson et al., 2006
), neutrophil crawling on pericytes is driven by pericyte-expressed chemokines and ICAM-1. ICAM-1 has also previously been reported on TNF-stimulated human brain pericytes (Verbeek et al., 1995
) and, more recently, on both unstimulated and cytokine-stimulated human placental pericytes (Maier and Pober, 2011
), although its in vivo functional role had not previously been explored. As the inhibitory effect of the anti–ICAM-1 mAb on abluminal neutrophil crawling was partial, other pericyte-associated adhesion molecules may also contribute to this phenomenon. In this context, cultured human pericytes have been reported to express VCAM-1, but not ICAM-2 or E-selectin (Maier and Pober, 2011
). Although pericyte-expressed VCAM-1 appears to support T cell adhesion in vitro (Verbeek et al., 1995
), at present nothing is known about the role of pericyte VCAM-1 in vivo. Finally, although our findings provide evidence for the ability of pericyte ICAM-1 to support neutrophil abluminal crawling, we cannot rule out the possibility that ICAM-1 may also be abluminally expressed on ECs, and as such may have contributed to the ICAM-1–dependent neutrophil abluminal crawling reported here.
We observed that neutrophils following other neutrophils along pericyte processes exhibited markedly reduced meandering motility within venular walls, indicating that the slow and wandering first line neutrophils provided some directional cues and or paved the way for the followers. The resultant effect of this phenomenon was that clear hot spots were noted where multiple neutrophils were seen to exit venular walls through the same pericyte gap. Although preferred sites of neutrophil migration through the endothelium and the venular basement membrane have previously been reported (Burns et al., 1997
; Wang et al., 2006
; Voisin et al., 2009
; Sumagin and Sarelius, 2010
), the present findings are the first to report this phenomenon with respect to breaching the pericyte layer in real time. Potential mechanisms through which leading neutrophils may facilitate migration of the followers within the venular wall include release of chemoattractants, such as chemokines and lipid mediators, from the leading cells, or paving the way through the venular basement membrane. The latter may occur in a protease-dependent manner, a process that, in addition to remodeling the basement membrane, may also lead to generation of basement membrane–derived chemotactic molecules (Steadman et al., 1993
; Wang et al., 2005
; Pham, 2006
; Mydel et al., 2008
Crawling neutrophils eventually exited the venular wall through gaps between adjacent pericytes and, in contrast to a previous study (Feng et al., 1998
), no transcellular pericyte breaching was observed in our models. We have previously reported that gaps between adjacent pericytes are colocalized with regions within the venular basement membrane, where lower levels of certain basement membrane constituents, such as collagen type IV, laminin-8, and laminin-10, exist. These sites, termed low expression regions (LERs), are preferentially used by transmigrating neutrophils and monocytes (Wang et al., 2006
; Voisin et al., 2009
). Interestingly, LERs are rich in perlecan (Voisin et al., 2010
), a basement membrane constituent that is antiadhesive for neutrophils in vitro (Sixt et al., 2001
), a phenomenon that may contribute to lack of neutrophil crawling on gaps between adjacent pericytes. In the present study, we show that only ~9% of the gaps present within the pericyte sheath are used by transmigrating neutrophils. Furthermore, transmigrating leukocytes appeared to preferentially use enlarged pericyte gaps in that ~70% of all the migratory events quantified occurred through gaps of 8–50 µm2
in size. These accounted for ~50% of gaps in inflamed tissues and ~30% of gaps in unstimulated tissues. A key question raised by these findings is what are the determinants that govern the preferential use of certain gaps during neutrophil transmigration? As it has previously been shown that ICAM-1 enrichment is associated with preferential leukocyte TEM through tricellular EC junctions (Sumagin and Sarelius, 2010
), we sought to analyze the distribution of KC and ICAM-1 on pericytes in relation to different size gaps. In this context, our results indicated that, on average, the pericyte cell body expressed higher levels of KC and ICAM-1 than pericyte regions that acted as borders to gaps between adjacent cells. These findings are in line with our observations that pericytes support effective neutrophil crawling in a chemokine- and ICAM-1–dependent manner, with almost no crawling being seen on pericyte-deficient regions. Interestingly however, a small number of gaps (~10%) were bordered by pericyte regions expressing higher levels of KC and ICAM-1 than the average cell body levels. Furthermore, >70% of these “KC/ICAM-1 high” borders were associated with pericyte gaps of 8–50 µm2
size, a gap size range preferentially used by transmigrating neutrophils. These associations suggest that expressions of KC and/or ICAM-1 near pericyte gaps may guide neutrophils to exit sites within the pericyte sheath. Although this presents a tantalizing possibility, at present we are unable to confirm this hypothesis in real-time neutrophil-tracking studies because of the lack of availability of a nonblocking anti–ICAM-1 mAb that can be used for in vivo labeling of pericyte ICAM-1.
Pericytes are contractile cells and, as such, several studies have shown the ability of pericytes to exhibit shape change after stimulation with vasoactive mediators in vitro, such as histamine (Murphy and Wagner, 1994
; Speyer et al., 1999
). As pericyte shape change appeared to facilitate neutrophil migration through the pericyte sheath, this response was investigated in more detail. Cremaster muscles or ear skin stimulated with TNF and IL-1β showed significantly enlarged gaps between adjacent pericytes, increasing by ~80% after a 2–4-h stimulation. Full time course analysis of pericyte shape change and neutrophil transmigration indicated that in response to TNF, the former preceded the response of neutrophil transmigration, suggesting that pericyte shape change occurs independently of neutrophils. This is in contrast to inflammation-induced remodeling of BM permissive regions (LERs), a response that we have previously shown to be strictly neutrophil dependent (Voisin et al., 2009
; Wang et al., 2006
). The ability of pericytes to exhibit shape change in a neutrophil-independent manner was conclusively demonstrated in studies where TNF and IL-1β were found to induce enlarged pericyte gaps in both control and neutrophil-depleted animals. To investigate the mechanism of this response, the potential expression of receptors for TNF and IL-1β on pericytes was explored. Immunofluorescence staining of unstimulated cremaster muscles revealed significant levels of TNFRI, TNFRII, and ILRI on pericytes comparable to levels detected on ECs. Parallel experiments conducted with the pericyte-like cell line C3H/10T1/2 (Reznikoff et al., 1973
) showed the ability of these cells to exhibit change in morphology in response to TNF and IL-1β and to express TNFRI, TNFRII, and ILRI. Collectively, the present results demonstrate that pericytes express receptors for TNF and IL-1β and that activation of these receptors can lead to increased expression of ICAM-1 and KC that support neutrophil abluminal crawling after TEM to preferred pericyte gaps. However, our results show that pericyte shape change as induced by TNF and IL-1β in vivo can contribute to the generation of enlarged gaps between adjacent pericytes that are preferentially used by transmigrating neutrophils. Details of the signaling pathways that regulate pericyte shape change in vivo are at present unclear, but both TNF and IL-1β are known to activate small GTPases that play a key role in actin cytoskeleton rearrangement (Puls et al., 1999
), and both cytokines have previously been reported to induce morphological changes in rat lung pericytes in vitro (Kerkar et al., 2006
). In addition, the disassembly of αSMA stress fibers in isolated bovine retina pericytes causing reduced cell size in vitro is reportedly mediated via the small GTPase RhoA (Kolyada et al., 2003
; Kutcher et al., 2007
; Kutcher and Herman, 2009
), but the potential role of such pathways in vivo has yet to be clarified.
In summary, through development of an imaging platform that allows direct quantitative analysis of neutrophil–pericyte interactions, we have unraveled several modes through which pericytes can facilitate neutrophil transmigration through venular walls. Hence, in addition to their accepted role in regulation of vascular tone, integrity, and barrier function (Shepro and Morel, 1993
; Hirschi and D’Amore, 1996
; Edelman et al., 2006
), the present results demonstrate a role for pericytes in inflammation, indicating the need for further analysis of the role of these mural cells in vascular functions.