Transcellular Diapedesis Research Articles

Conditions that promote transcellular neutrophil migration in vivo

Circulating leukocytes enter tissue either through endothelial junctions (paracellular) or via a pore through the body of endothelial cells (transcellular). We have previously shown that genetically replacing VE-cadherin with a VE-cadherin-α-catenin (VEC-αC) fusion construct—which binds constitutively to actin—obstructs junctions, and blocks leukocyte extravasation in lung, skin and postcapillary venules of cremaster muscle. However, neutrophil recruitment into the inflamed peritoneal cavity was unimpaired. Investigating reasons for this, here, we visualized neutrophil diapedesis by 3D intravital video microscopy in the cremaster muscle and omentum, the major site of neutrophil recruitment into the peritoneal cavity. We found that 80% of neutrophil-extravasation occurred through HEVs in the omentum, which was unimpaired by VEC-αC. In addition, in larger venules (60–85 µm) of both tissues, less than 15% of neutrophils extravasated transcellularly in WT mice. However, in VEC-α-C mice, transcellular diapedesis increased severalfold in the omentum, but not in the cremaster. In line with this, omental venules expressed higher levels of ICAM-1 and atypical chemokine receptor 1. Furthermore, only in the omentum, VEC-αC expression caused reduced elongation of venular endothelium in flow-direction, suggesting different biomechanical properties. Collectively, VEC-αC does not inhibit paracellular transmigration in all types of venules and can modulate the diapedesis route.

Paracellular and Transcellular Leukocytes Diapedesis Are Divergent but Interconnected Evolutionary Events.

Infiltration of the endothelial layer of the blood-brain barrier by leukocytes plays a critical role in health and disease. When passing through the endothelial layer during the diapedesis process lymphocytes can either follow a paracellular route or a transcellular one. There is a debate whether these two processes constitute one mechanism, or they form two evolutionary distinct migration pathways. We used artificial intelligence, phylogenetic analysis, HH search, ancestor sequence reconstruction to investigate further this intriguing question. We found that the two systems share several ancient components, such as RhoA protein that plays a critical role in controlling actin movement in both mechanisms. However, some of the key components differ between these two transmigration processes. CAV1 genes emerged during Trichoplax adhaerens, and it was only reported in transcellular process. Paracellular process is dependent on PECAM1. PECAM1 emerged from FASL5 during Zebrafish divergence. Lastly, both systems employ late divergent genes such as ICAM1 and VECAM1. Taken together, our results suggest that these two systems constitute two different mechanical sensing mechanisms of immune cell infiltrations of the brain, yet these two systems are connected. We postulate that the mechanical properties of the cellular polarity is the main driving force determining the migration pathway. Our analysis indicates that both systems coevolved with immune cells, evolving to a higher level of complexity in association with the evolution of the immune system.

Immunological response to bacterial infection in a pelagic tunicate: Inflammation in the salp Thalia democratica

Thaliaceans are pelagic tunicates that play a key role in trophic chains of the oceans. In the field of tunicate immunity, a notable gap is the lack of data on their inflammatory response. The common salp, Thalia democratica, possesses scant immunocytes, represented by a phagocytic line (hyaline amebocytes) and a mast cell-like line (granular cells). We aimed to provide the first investigation of defense reactions upon exposure to a large amount of bacteria (Bacillus clausii). We detected (i) bacterial phagocytosis by hyaline amebocytes, (ii) degradation of phagocytizing hyaline amebocytes in the tunic after transcellular diapedesis from the hemocoel, and (iii) release of heparin, histamine, and TNF-α by granular cells. Cell degranulation and phagocytosis occurred in epidermal cells lining the hemocoel, and an excess of mucus was observed in the post-branchial gut, causing a functional inhibition of cilia and microvilli. These findings indicate multi-step events comparable to an inflammation involving responses at both tissue and organismal levels.

Metabolic Remodeling of Neutrophils at Inflammatory Site Drives Invadosome Formation Favoring Transcellular Migration

Neutrophils traffic in and out of underlying vascular bed during hematopoiesis and immunosurveillance. However, during inflammatory conditions such as ischemia reperfusion injury or atherosclerosis, excessive neutrophil infiltration into tissue drives disease pathogenesis. Yet, the relationship between neutrophil transmigration and inflammation is ill-defined. Neutrophil extravasation can occur either between two endothelial cells (paracellular) or directly through an endothelial cell body (transcellular). During transcellular migration, neutrophils interact with underlying endothelial cells (EC) via invadosomal structures, which forms a ‘pore’ into endothelial cell membrane, thus facilitating neutrophil migration through EC body. We have recently reported that deficiency in Rap1b, a member of Ras superfamily of GTPase, enhanced neutrophil transcellular migration, invadosomal structures and metalloproteinase (MMP) release (Kumar et al, JEM, 2014), in a manner dependent on high Akt activity. Further, Rap1-deficiency increased neutrophil recruitment to inflamed lungs and enhanced susceptibility to endotoxin shock, suggesting mode of neutrophil migration may influence inflammatory outcome. Here, to further understand which factors drive neutrophil transcellular migration, we analyzed protein content of Rap1b-/- invadosomal structures during transcellular diapedesis. For this, neutrophils were stimulated in transwell filters of 1µM pore size, with FMLP placed in the lower chamber, allowing only invadosomal protrusions into the pores. After removing the cell body from top of the filter, mass spectrometric analysis was performed on the invadosomal fraction. About 680 proteins were identified in protrusions isolated from WT or Rap1b-/- neutrophils. As expected, majority of them were cytoskeleton and adhesion proteins. Interestingly, Rap1b-/- invadosomal structures contained more enzymes of glycolytic pathways, including HK1, Lactate dehydrogenase A (LDHA) and phosphoglycerate kinase1 (PGK1). Immunofluorescent staining and western blotting confirmed this observation. Importantly, glycolytic enzymes were present at the tip of the protrusions in colocalization with F-actin suggesting site specific glycolytic activity, raising the hypothesis that metabolic remodeling may influence the route of neutrophil migration. LDHA converts pyruvate to lactate and subsequent milieu acidification, which can then cause MMP activation. Consistently, Rap1b-/- neutrophils exhibited increased uptake of glucose analogue (2-NBDG) and concurrent intracellular acidification, as detected by pH sensitive dye. To investigate the importance of LDHA activity during transcellular migration, Rap1b-/- neutrophils were treated with a specific pharmacological inhibitor of LDHA, namely FX11. In vitro, FX11 treatment significantly decreased transcellular migration of Rap1b-/- neutrophils. It also reduced invadosome formation of Rap1b-/- neutrophils within transwell pores, as well as neutrophil acidity and MMP activity. Furthermore, during neutrophil-endothelial cell interactions in vitro, Rap1b-/- neutrophils caused F-actin depolymerization in EC, likely facilitating transcellular passage; this was inhibited by FX11. To examine its effect in vivo, under same inflammatory microenvironment, Rap1b-/- and WT neutrophils were tagged with cell tracker dyes and transferred to recipient mice, treated with FX11 or DMSO control. Ear microvasculature was stimulated with FMLP and labeled with PECAM antibody to visualize EC junctions. Rap1b-/- neutrophils migrated out of vessels at higher frequency than WT cells, which was abrogated by FX11 treatment. Moreover, treatment with FX11 reduced the number of Rap1b-/- neutrophils located away from EC junction (transcellular route), in vivo. These results suggest enhanced local glycolytic metabolism and LDHA activity could act as critical regulators of transcellular migration. Increase in extracellular acidification mediated by LDHA activity, could affect endothelial permeability and alter neutrophil migratory behavior affecting outcome of inflammation. Since milieu acidification plays a major role in ischemic damage to the heart, these findings may be clinically important for our understanding of hyperinflammatory disorders. DisclosuresNo relevant conflicts of interest to declare.

Leukocyte transcellular diapedesis: Rap1b is in control

The neutrophil transmigration across the blood endothelial cell barrier represents the prerequisite step of innate inflammation. It is well known that neutrophils cross the endothelial barrier by transmigrating at the endothelial cell junction (‘paracellular’). However, in vivo and in vitro evidence have clearly demonstrated occurrence of an alternate mode of migration directly through the endothelial cell body (‘transcellular’). Despite our knowledge on mechanisms of transendothelial migration, it remains unclear which factors determine distinct modes of migration. We recently found that the Ras-like Rap1b GTPase limits neutrophil transcellular migration. Rap1b restrains transcellular migration by suppressing Akt-driven invasive protrusions while leaving the paracellular route unaffected. Furthermore, Rap1b limits neutrophil tissue infiltration in mice and prevents hyper susceptibility to endotoxin shock. These findings uncover a novel role for Rap1b in neutrophil migration and inflammation. Importantly, they offer emerging evidences that paracellular and transcellular migration of neutrophils are regulated by separate mechanisms. Here, we discuss the mechanisms of neutrophil transmigration and their clinical importance for vascular integrity and innate inflammation.

Cell surface levels of endothelial ICAM-1 influence the transcellular or paracellular T-cell diapedesis across the blood-brain barrier.

The extravasation of CD4(+) effector/memory T cells (TEM cells) across the blood-brain barrier (BBB) is a crucial step in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) or multiple sclerosis (MS). Endothelial ICAM-1 and ICAM-2 are essential for CD4(+) TEM cell crawling on the BBB prior to diapedesis. Here, we investigated the influence of cell surface levels of endothelial ICAM-1 in determining the cellular route of CD4(+) TEM -cell diapedesis across cytokine treated primary mouse BBB endothelial cells under physiological flow. Inflammatory conditions, inducing high levels of endothelial ICAM-1, promoted rapid initiation of transcellular diapedesis of CD4(+) T cells across the BBB, while intermediate levels of endothelial ICAM-1 favored paracellular CD4(+) T-cell diapedesis. Importantly, the route of T-cell diapedesis across the BBB was independent of loss of BBB barrier properties. Unexpectedly, a low number of CD4(+) TEM cells was found to cross the inflamed BBB in the absence of endothelial ICAM-1 and ICAM-2 via an obviously alternatively regulated transcellular pathway. In vivo, this translated to the development of ameliorated EAE in ICAM-1(null) //ICAM-2(-/-) C57BL/6J mice. Taken together, our study demonstrates that cell surface levels of endothelial ICAM-1 rather than the inflammatory stimulus or BBB integrity influence the pathway of T-cell diapedesis across the BBB.

The physiological roles of ICAM-1 and ICAM-2 in neutrophil migration into tissues.

Neutrophil extravasation from the blood into tissues is initiated by tethering and rolling of neutrophils on endothelial cells, followed by neutrophil integrin activation and shear resistant arrest, crawling, diapedesis and breaching the endothelial basement membrane harbouring pericytes. Endothelial intercellular cell adhesion molecule (ICAM)-1 and ICAM-2, in conjunction with ICAM-1 on pericytes, critically contribute to each step. In addition, epithelial ICAM-1 is involved in neutrophil migration to peri-epithelial sites. The most recent findings on the role of ICAM-1 and ICAM-2 for neutrophil migration into tissues will be reviewed here. Signalling via endothelial ICAM-1 and ICAM-2 contributes to stiffness of the endothelial cells at sites of chronic inflammation and junctional maturation, respectively. Endothelial ICAM-2 contributes to neutrophil crawling and initiation of paracellular diapedesis, which then proceeds independent of ICAM-2. Substantial transcellular neutrophil diapedesis across the blood-brain barrier is strictly dependent on endothelial ICAM-1 and ICAM-2. Endothelial ICAM-1 or ICAM-2 is involved in neutrophil-mediated plasma leakage. ICAM-1 on pericytes assists the final step of neutrophil extravasation. Epithelial ICAM-1 rather indirectly promotes neutrophil migration to peri-epithelial sites. ICAM-1 and ICAM-2 are involved in each step of neutrophil extravasation, and have redundant but also distinct functions. Analysis of the role of endothelial ICAM-1 requires simultaneous consideration of ICAM-2.

The small GTPase Rap1b negatively regulates neutrophil chemotaxis and transcellular diapedesis by inhibiting Akt activation

Neutrophils are the first line of cellular defense in response to infections and inflammatory injuries. However, neutrophil activation and accumulation into tissues trigger tissue damage due to release of a plethora of toxic oxidants and proteases, a cause of acute lung injury (ALI). Despite its clinical importance, the molecular regulation of neutrophil migration is poorly understood. The small GTPase Rap1b is generally viewed as a positive regulator of immune cell functions by controlling bidirectional integrin signaling. However, we found that Rap1b-deficient mice exhibited enhanced neutrophil recruitment to inflamed lungs and enhanced susceptibility to endotoxin shock. Unexpectedly, Rap1b deficiency promoted the transcellular route of diapedesis through endothelial cell. Increased transcellular migration of Rap1b-deficient neutrophils in vitro was selectively mediated by enhanced PI3K-Akt activation and invadopodia-like protrusions. Akt inhibition in vivo suppressed excessive Rap1b-deficient neutrophil migration and associated endotoxin shock. The inhibitory action of Rap1b on PI3K signaling may be mediated by activation of phosphatase SHP-1. Thus, this study reveals an unexpected role for Rap1b as a key suppressor of neutrophil migration and lung inflammation.

The small GTPase Rap1b negatively regulates neutrophil chemotaxis and transcellular diapedesis by inhibiting Akt activation.

Neutrophils are the first line of cellular defense in response to infections and inflammatory injuries. However, neutrophil activation and accumulation into tissues trigger tissue damage due to release of a plethora of toxic oxidants and proteases, a cause of acute lung injury (ALI). Despite its clinical importance, the molecular regulation of neutrophil migration is poorly understood. The small GTPase Rap1b is generally viewed as a positive regulator of immune cell functions by controlling bidirectional integrin signaling. However, we found that Rap1b-deficient mice exhibited enhanced neutrophil recruitment to inflamed lungs and enhanced susceptibility to endotoxin shock. Unexpectedly, Rap1b deficiency promoted the transcellular route of diapedesis through endothelial cell. Increased transcellular migration of Rap1b-deficient neutrophils in vitro was selectively mediated by enhanced PI3K-Akt activation and invadopodia-like protrusions. Akt inhibition in vivo suppressed excessive Rap1b-deficient neutrophil migration and associated endotoxin shock. The inhibitory action of Rap1b on PI3K signaling may be mediated by activation of phosphatase SHP-1. Thus, this study reveals an unexpected role for Rap1b as a key suppressor of neutrophil migration and lung inflammation.

Intracellular delivery of desulfated heparin with bile acid conjugation alleviates T cell-mediated inflammatory arthritis via inhibition of RhoA-dependent transcellular diapedesis

Heparin has a potential regulatory role in inflammatory diseases. However, the anticoagulant activity and poor oral bioavailability of heparin limit its use as an anti-inflammatory agent. Conjugation of bis-deoxycholic acid to 6-O-desulfated low molecular weight heparin (6DSHbD) was efficiently internalized by activated endothelial cells via a 2-step model, in which heparin attaches to adhesion molecules that facilitate accessibility of the bile acid conjugate to membrane transporters. The critical role of P-selectin during endothelial cell uptake of 6DSHbD by arthritic tissue was confirmed in p-selectin−/− arthritic mice. Intracellular 6DSHbD inhibited transcellular diapedesis of T cells through activated endothelial cells and impaired both the formation of ICAM-1-rich docking structures at the T cell contact surface and subsequent cytoskeletal rearrangement. Furthermore, 6DSHbD blocked activation of RhoA-GTPase and phosphorylation of ezrin/radixin/moesin induced by ICAM-1 cross-linking on activated endothelial cells, thereby impairing lymphocyte transcellular transmigration. After oral administration 6DSHbD was preferentially delivered to inflamed joint tissue, particularly in and around post-capillary venular endothelium and inhibited effector T cell homing to arthritic joints. Aggravation of collagen-induced arthritis conferred by the transfer of effector T cells was suppressed by oral 6DSHbD. Thus, intracellular heparin exerts anti-inflammatory effects through the inhibition of RhoA-dependent transendothelial recruitment of T cells and may have applications in the treatment of chronic inflammatory arthritis.

β2 Integrin–Mediated Crawling on Endothelial ICAM-1 and ICAM-2 Is a Prerequisite for Transcellular Neutrophil Diapedesis across the Inflamed Blood–Brain Barrier

In acute neuroinflammatory states such as meningitis, neutrophils cross the blood-brain barrier (BBB) and contribute to pathological alterations of cerebral function. The mechanisms that govern neutrophil migration across the BBB are ill defined. Using live-cell imaging, we show that LPS-stimulated BBB endothelium supports neutrophil arrest, crawling, and diapedesis under physiological flow in vitro. Investigating the interactions of neutrophils from wild-type, CD11a(-/-), CD11b(-/-), and CD18(null) mice with wild-type, junctional adhesion molecule-A(-/-), ICAM-1(null), ICAM-2(-/-), or ICAM-1(null)/ICAM-2(-/-) primary mouse brain microvascular endothelial cells, we demonstrate that neutrophil arrest, polarization, and crawling required G-protein-coupled receptor-dependent activation of β2 integrins and binding to endothelial ICAM-1. LFA-1 was the prevailing ligand for endothelial ICAM-1 in mediating neutrophil shear resistant arrest, whereas Mac-1 was dominant over LFA-1 in mediating neutrophil polarization on the BBB in vitro. Neutrophil crawling was mediated by endothelial ICAM-1 and ICAM-2 and neutrophil LFA-1 and Mac-1. In the absence of crawling, few neutrophils maintained adhesive interactions with the BBB endothelium by remaining either stationary on endothelial junctions or displaying transient adhesive interactions characterized by a fast displacement on the endothelium along the direction of flow. Diapedesis of stationary neutrophils was unchanged by the lack of endothelial ICAM-1 and ICAM-2 and occurred exclusively via the paracellular pathway. Crawling neutrophils, although preferentially crossing the BBB through the endothelial junctions, could additionally breach the BBB via the transcellular route. Thus, β2 integrin-mediated neutrophil crawling on endothelial ICAM-1 and ICAM-2 is a prerequisite for transcellular neutrophil diapedesis across the inflamed BBB.

The Small Gtpase Rap1b Negatively Regulates Neutrophil Migration During Inflammation By Limiting Trans-Cellular Diapedesis

Neutrophils are the first line of cellular defense against infecting microorganisms by moving rapidly toward sites of infection. Impaired neutrophil recruitment and functions can cause life-threatening infections, while excessive neutrophil tissue infiltration contributes to inflammatory disorders and tissue injury. A number of key positive regulators of neutrophil tissue infiltration have been identified. However, the mechanisms that protect from unwanted inflammation by negative regulation of neutrophil recruitment and functions are still unrecognized.Rap1b is an evolutionary conserved protein of the Ras-like GTPase superfamily. The mammalian genome encodes two Rap1 genes, Rap1a and Rap1b, which are highly homologous. Rap1 is historically known to control functional activation of integrins to positively regulate a number of cellular processes, including cell adhesion, cell polarity, cell migration, platelet aggregation. Rap1b is the main Rap1 isoform expressed in neutrophils; yet, its functions in neutrophils are poorly understood.Here, we found that, quite unexpectedly, Rap1b is a key suppressor of neutrophil migration, and inflammation. Rap1b loss enhanced neutrophil emigration into lungs, associated with increased susceptibility to endotoxin shock. To further understand the role of Rap1b in neutrophil migration, we used a 3-D migration assay in which neutrophils are plated onto activated endothelial cells. This assay enables examination of critical steps of the extravasation cascade, ie neutrophil adhesion onto the endothelium, lateral crawling to the nearest endothelial cell junction and permissive sites for transmigration. This assay confirmed increased transendothelial migration of Rap1b-/- neutrophils compared to WT cells. However, Rap1b-/- neutrophils were unable to reach endothelial junction and shifted their mode of transmigration to a trans-cellular (through endothelial cells) diapedesis instead of the canonical paracellular route (between two endothelial cells). Indeed, using immunostaining with VE-Cadherin, ICAM-1 and CD11b to identify endothelial junctions and neutrophils, respectively, we found only 5-10% WT neutrophils used the transcellular route of diapedesis. Up to 30-35% Rap1b–/– neutrophils transmigrated via the transcellular route. Transcellular diapedesis requires the formation of invadopodia-like actin protrusions, an extracellular matrix-degrading structure enabling penetration into tissue. Transmission electron microscopy indicated increased invadopodia-like structures in Rap1b–/– neutrophils that penetrated deeper into endothelial surfaces than WT cells. Likewise, Rap1b–/– neutrophils had increased ability to degrade gelatin matrix in vitro. Mechanistically, the Rap1b-null phenotype was mediated by enhanced Pi3K-Akt activation. Rap1b-/- neutrophils manifested increased phosphorylation of Akt, in response to chemokine and integrin stimuli. Treatment of a small molecule Akt inhibitor, MK-2206 reversed elevated transmigration, matrix degradation and rescued crawling of Rap1b–/– neutrophils to endothelial junctions. Importantly, Akt inhibition in vivo suppressed excessive Rap1b–/– neutrophil migration into lungs and associated endotoxin shock. The inhibitory action of Rap1b on PI3K signaling may be mediated by the phosphatase SHP-1, as its activation and localization was Rap1b-dependent. These findings uncover a novel mechanism of neutrophil migration, and reveal an unexpected role for Rap1b as a key suppressor of neutrophilic lung inflammation. This work has far reaching importance for inflammatory processes. It may represent new avenues for the treatment of pathological inflammation conditions. Disclosures:No relevant conflicts of interest to declare.

Dendritic Cell Podosome Dynamics Does Not Depend on the F-actin Regulator SWAP-70

In addition to classical adhesion structures like filopodia or focal adhesions, dendritic cells similar to macrophages and osteoclasts assemble highly dynamic F-actin structures called podosomes. They are involved in cellular processes such as extracellular matrix degradation, bone resorption by osteoclasts, and trans-cellular diapedesis of lymphocytes. Besides adhesion and migration, podosomes enable dendritic cells to degrade connective tissue by matrix metalloproteinases. SWAP-70 interacts with RhoGTPases and F-actin and regulates migration of dendritic cells. SWAP-70 deficient osteoclasts are impaired in F-actin-ring formation and bone resorption. In the present study, we demonstrate that SWAP-70 is not required for podosome formation and F-actin turnover in dendritic cells. Furthermore, we found that toll-like receptor 4 ligand induced podosome disassembly and podosome-mediated matrix degradation is not affected by SWAP-70 in dendritic cells. Thus, podosome formation and function in dendritic cells is independent of SWAP-70.

Mesenchymal Stem Cells Transmigrate Between and Directly Through Tumor Necrosis Factor‐α‐Activated Endothelial Cells Via Both Leukocyte‐Like and Novel Mechanisms

Systemically administered adult mesenchymal stem cells (MSCs), which are being explored in clinical trials to treat inflammatory disease, exhibit the critical ability to extravasate at sites of inflammation. We aimed to characterize the basic cellular processes mediating this extravasation and compare them to those involved in leukocyte transmigration. Using high-resolution confocal and dynamic microscopy, we show that, like leukocytes, human bone marrow-derived MSC preferentially adhere to and migrate across tumor necrosis factor-α-activated endothelium in a vascular cell adhesion molecule-1 (VCAM-1) and G-protein-coupled receptor signaling-dependent manner. As several studies have suggested, we observed that a fraction of MSC was integrated into endothelium. In addition, we observed two modes of transmigration not previously observed for MSC: Paracellular (between endothelial cells) and transcellular (directly through individual endothelial cells) diapedesis through discrete gaps and pores in the endothelial monolayer, in association with VCAM-1-enriched "transmigratory cups". Contrasting leukocytes, MSC transmigration was not preceded by significant lateral migration and occurred on the time scale of hours rather than minutes. Interestingly, rather than lamellipodia and invadosomes, MSC exhibited nonapoptotic membrane blebbing activity that was similar to activities previously described for metastatic tumor and embryonic germ cells. Our studies suggest that low avidity binding between endothelium and MSC may grant a permissive environment for MSC blebbing. MSC blebbing was associated with early stages of transmigration, in which blebs could exert forces on underlying endothelial cells indicating potential functioning in breaching the endothelium. Collectively, our data suggest that MSC transmigrate actively into inflamed tissues via both leukocyte-like and novel mechanisms.

Mechanisms for transcellular diapedesis: probing and pathfinding by `invadosome-like protrusions'

Immune-system functions require that blood leukocytes continuously traffic throughout the body and repeatedly cross endothelial barriers (i.e. diapedese) as they enter (intravasate) and exit (extravasate) the circulation. The very earliest studies to characterize diapedesis directly in vivo suggested the coexistence of two distinct migratory pathways of leukocytes: between (paracellular pathway) and directly through (transcellular pathway) individual endothelial cells. In vivo studies over the past 50 years have demonstrated significant use of the transcellular diapedesis pathway in bone marrow, thymus, secondary lymphoid organs, various lymphatic structures and peripheral tissues during inflammation and across the blood-brain barrier and blood-retinal barrier during inflammatory pathology. Recently, the first in vitro reports of transcellular diapedesis have emerged. Together, these in vitro and in vivo observations suggest a model of migratory pathfinding in which dynamic 'invadosome-like protrusions' formed by leukocytes have a central role in both identifying and exploiting endothelial locations that are permissive for transcellular diapedesis. Such 'probing' activity might have additional roles in this and other settings.

Settings and mechanisms for trans-cellular diapedesis

Immune system functions require blood leukocytes to continuously traffic throughout the body and repeatedly cross endothelial barriers (i.e., diapedese) as they enter (intravasation) and exit (extravasation) the circulation. The earliest studies to directly characterize diapedesis in vivo suggested co-existence of two distinct migratory pathways: between (para-cellular) and through (trans-cellular) individual endothelial cells. However, in the absence of conclusive in vitro observations, the latter pathway remained poorly accepted. The recent emergence of unambiguous in vitro reports of trans-cellular diapedesis has begun to illuminate mechanisms for this pathway and has renewed interest in its physiological roles. A thorough reevaluation of the existing literature reveals a large number of studies documenting significant use of the trans-cellular pathway in diverse in vivo settings. These include constitutive trafficking in bone marrow and lymphoid organs as well as upregulated extravasation in peripheral tissues during inflammation. Here we collectively summarize these in vivo observations alongside the emerging in vitro data in order to provide a framework for understanding the settings, mechanisms and roles for the trans-cellular route of diapedesis.

Mechanisms by which E-Selectin Regulates Diapedesis of Colon Cancer Cells under Flow Conditions

Diapedesis, the passage of circulating tumor cells across the endothelium, is a critical determinant in most cases of metastasis. Using a laminar flow chamber and a tissue-engineered blood vessel, we found that E-selectin is required not only for the initial adhesion and rolling of circulating HT-29 colon cancer cells on the endothelium but also for their subsequent diapedesis. These processes require both the intracellular and extracellular domains of E-selectin. We also identified three distinct mechanisms by which circulating cancer cells interact with E-selectin to initiate their diapedesis: formation of a mosaic between cancer cells and endothelial cells, paracellular diapedesis at the junction of three endothelial cells, and transcellular diapedesis. We also obtained evidence indicating that E-selectin-dependent paracellular extravasation is independent of intercellular adhesion molecule and vascular cell adhesion molecule and that it requires the activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase downstream of E-selectin. This is supported by the observation that the adenoviral-mediated expression of the E-selectin mutant Y603F is associated with both an inhibition of ERK and paracellular extravasation. Our study is the first to clearly establish, under dynamic and shear stress conditions, how E-selectin regulates diapedesis of circulating cancer cells. These results provide new insights in understanding the metastatic process.

Nano-surgery at the leukocyte–endothelial docking site

The endothelium has an important role in controlling the extravasation of leukocytes from blood to tissues. Endothelial permeability for leukocytes is influenced by transmembrane proteins that control inter-endothelial adhesion, as well as steps of the leukocyte transmigration process. In a cascade consisting of leukocyte rolling, adhesion, firm adhesion, and diapedesis, a new step was recently introduced, the formation of a docking structure or “transmigratory cup.” Both terms describe a structure formed by endothelial pseudopods embracing the leukocyte. It has been found associated with both para- and transcellular diapedesis. The aim of this study was to characterize the leukocyte–endothelial contact area in terms of morphology and cell mechanics to investigate how the endothelial cytoskeleton reorganizes to engulf the leukocyte. We used atomic force microscopy (AFM) to selectively remove the leukocyte and then analyze the underlying cell at this specific spot. Firmly attached leukocytes could be removed by AFM nanomanipulation. In few cases, this exposed 8–12 μm wide and 1 μm deep footprints, representing the cup-like docking structure. Some of them were located near endothelial cell junctions. The interaction area did not exhibit significant alterations neither morphologically nor mechanically as compared to the surrounding cell surface. In conclusion, the endothelial invagination is formed without a net depolymerization of f-actin, as endothelial softening at the site of adhesion does not seem to be involved. Moreover, there were no cases of phagocytotic engulfment, but instead the formation of a transmigratory channel could be observed.

Transcellular Diapedesis Is Initiated by Invasive Podosomes

Diapedesis is critical for immune system function and inflammatory responses. This occurs by migration of blood leukocytes either directly through individual microvascular endothelial cells (the "transcellular" route) or between them (the "paracellular" route). Mechanisms for transcellular pore formation in endothelium remain unknown. Here we demonstrate that lymphocytes used podosomes and extended "invasive podosomes" to palpate the surface of, and ultimately form transcellular pores through, the endothelium. In lymphocytes, these structures were dependent on Src kinase and the actin regulatory protein WASP; inhibition of podosome formation selectively blocked the transcellular route of diapedesis. In endothelium, membrane fusion events dependent on the SNARE-containing membrane fusion complex and intracellular calcium were required for efficient transcellular pore formation in response to podosomes. These findings provide insights into basic mechanisms for leukocyte trafficking and the functions of podosomes.

Leukocyte Podosomes Sense Their Way through the Endothelium

Whether leukocytes can transmigrate through endothelial cells, rather than between them, is controversial. In this issue, Carman et al. (2007) demonstrate that lymphocytes can extend podosomes to palpate endothelial cells searching for areas permissive for transcellular diapedesis.