Cxcl1 monomer-dimer equilibrium controls neutrophil extravasation.

The chemokine Cxcl1 plays a crucial role in recruiting neutrophils in response to infection. The early events in chemokine-mediated neutrophil extravasation involve a sequence of highly orchestrated steps including rolling, adhesion, arrest, and diapedesis. Cxcl1 function is determined by its properties of reversible monomer-dimer equilibrium and binding to Cxcr2 and glycosaminoglycans. Here, we characterized how these properties orchestrate extravasation using intravital microscopy of the cremaster. Compared to WT Cxcl1, which exists as both a monomer and a dimer, the trapped dimer caused faster rolling, less adhesion, and less extravasation. Whole-mount immunofluorescence of the cremaster and arrest assays confirmed these data. Moreover, the Cxcl1 dimer showed impaired LFA-1-mediated neutrophil arrest that could be attributed to impaired Cxcr2-mediated ERK signaling. We conclude that Cxcl1 monomer-dimer equilibrium and potent Cxcr2 activity of the monomer together coordinate the early events in neutrophil recruitment.

2.5D Traction Force Microscopy: Imaging three-dimensional cell forces at interfaces and biological applications.

The forces that cells, tissues, and organisms exert on the surface of a soft substrate can be measured using Traction Force Microscopy (TFM), an important and well-established technique in Mechanobiology. The usual TFM technique (two-dimensional, 2D TFM) treats only the in-plane component of the traction forces and omits the out-of-plane forces at the substrate interfaces (2.5D) that turn out to be important in many biological processes such as tissue migration and tumour invasion. Here, we review the imaging, material, and analytical tools to perform "2.5D TFM" and explain how they are different from 2D TFM. Challenges in 2.5D TFM arise primarily from the need to work with a lower imaging resolution in the z-direction, track fiducial markers in three-dimensions, and reliably and efficiently reconstruct mechanical stress from substrate deformation fields. We also discuss how 2.5D TFM can be used to image, map, and understand the complete force vectors in various important biological events of various length-scales happening at two-dimensional interfaces, including focal adhesions forces, cell diapedesis across tissue monolayers, the formation of three-dimensional tissue structures, and the locomotion of large multicellular organisms. We close with future perspectives including the use of new materials, imaging and machine learning techniques to continuously improve the 2.5D TFM in terms of imaging resolution, speed, and faithfulness of the force reconstruction procedure.

Recent advances in GPR35 pharmacology; 5-HIAA serotonin metabolite becomes a ligand.

GPR35, an orphan receptor, has been waiting for its ligand since its cloning in 1998. Many endogenous and exogenous molecules have been suggested to act as agonists of GPR35 including kynurenic acid, zaprinast, lysophosphatidic acid, and CXCL17. However, complex and controversial responses to ligands among species have become a huge hurdle in the development of therapeutics in addition to the orphan state. Recently, a serotonin metabolite, 5-hydroxyindoleacetic acid (5-HIAA), is reported to be a high potency ligand for GPR35 by investigating the increased expression of GPR35 in neutrophils. In addition, a transgenic knock-in mouse line is developed, in which GPR35 was replaced with a human ortholog, making it possible not only to overcome the different selectivity of agonists among species but also to conduct therapeutic experiments on human GPR35 in mouse models. In the present article, I review the recent advances and prospective therapeutic directions in GPR35 research. Especially, I'd like to draw attention of readers to the finding of 5-HIAA as a ligand of GPR35 and lead to apply the 5-HIAA and human GPR35 knock-in mice to their research fields in a variety of pathophysiological conditions.

Methods for Imaging Inflammation and Transendothelial Migration in Vivo and ex Vivo.

Inflammation is the body's response to injury and harmful stimuli and contributes to a range of infectious and noninfectious diseases. Inflammation occurs through a series of well-defined leukocyte-endothelial cell interactions, including rolling, activation, adhesion, transmigration, and subsequent migration through the extracellular matrix. Being able to visualize the stages of inflammation is important for a better understanding of its role in diseases processes. Detailed in this article are protocols for imaging immune cell infiltration and transendothelial migration in vascular tissue beds, including those in the mouse ear, cremaster muscle, brain, lung, and retina. Also described are protocols for inducing inflammation and quantifying leukocytes with FIJI imaging software. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Induction of croton oil dermatitis Alternate Protocol 1: Induction of croton oil dermatitis using genetically fluorescent mice Basic Protocol 2: Intravital microscopy of the mouse cremaster muscle Support Protocol: Making a silicone stage Basic Protocol 3: Wide-field microscopy of the mouse brain Basic Protocol 4: Imaging the lungs (ex vivo) Alternate Protocol 2: Inflating the lungs without tracheostomy Basic Protocol 5: Inducing, imaging, and quantifying infiltration of leukocytes in mouse retina.

Interleukin-6 trans-signaling induced laminin switch contributes to reduced trans-endothelial migration of granulocytic cells.

BACKGROUND AND AIMS: Laminins are essential components of the endothelial basement membrane, which predominantly contains LN421 and LN521 isoforms. Regulation of laminin expression under pathophysiological conditions is largely unknown. In this study, we aimed to investigate the role of IL-6 in regulating endothelial laminin profile and characterize the impact of altered laminin composition on the phenotype, inflammatory response, and function of endothelial cells (ECs). METHODS: HUVECs and HAECs were used for in vitro experiments. Trans-well migration experiments were performed using leukocytes isolated from peripheral blood of healthy donors. The BiKE cohort was used to assess expression of laminins in atherosclerotic plaques and healthy vessels. Gene and protein expression was analyzed using Microarray/qPCR and proximity extension assay, ELISA, immunostaining or immunoblotting techniques, respectively. RESULTS: Stimulation of ECs with IL-6+sIL-6R, but not IL-6 alone, reduces expression of laminin α4 (LAMA4) and increases laminin α5 (LAMA5) expression at the mRNA and protein levels. In addition, IL-6+sIL-6R stimulation of ECs differentially regulates the release of several proteins including CXCL8 and CXCL10, which collectively were predicted to inhibit granulocyte transmigration. Experimentally, we demonstrated that granulocyte migration is inhibited across ECs pre-treated with IL-6+sIL-6R. In addition, granulocyte migration across ECs cultured on LN521 was significantly lower compared to LN421. In human atherosclerotic plaques, expression of endothelial LAMA4 and LAMA5 is significantly lower compared to control vessels. Moreover, LAMA5-to-LAMA4 expression ratio was negatively correlated with granulocytic cell markers (CD177 and myeloperoxidase (MPO)) and positively correlated with T-lymphocyte marker CD3. CONCLUSIONS: We showed that expression of endothelial laminin alpha chains is regulated by IL-6 trans-signaling and contributes to inhibition of trans-endothelial migration of granulocytic cells. Further, expression of laminin alpha chains is altered in human atherosclerotic plaques and is related to intra-plaque abundance of leukocyte subpopulations.

Regulatory T cell therapy for multiple sclerosis: Breaching (blood-brain) barriers.

Multiple sclerosis (MS) is an autoimmune disorder causing demyelination and neurodegeneration in the central nervous system. MS is characterized by disturbed motor performance and cognitive impairment. Current MS treatments delay disease progression and reduce relapse rates with general immunomodulation, yet curative therapies are still lacking. Regulatory T cells (Tregs) are able to suppress autoreactive immune cells, which drive MS pathology. However, Tregs are functionally impaired in people with MS. Interestingly, Tregs were recently reported to also have regenerative capacity. Therefore, experts agree that Treg cell therapy has the potential to ameliorate the disease. However, to perform their local anti-inflammatory and regenerative functions in the brain, they must first migrate across the blood-brain barrier (BBB). This review summarizes the reported results concerning the migration of Tregs across the BBB and the influence of Tregs on migration of other immune subsets. Finally, their therapeutic potential is discussed in the context of MS.

Impact of Activation of EGFL7 within Microenvironment of High Grade Ovarian Serous Carcinoma on Infiltration of CD4+ and CD8+ Lymphocytes.

Background: It has been demonstrated that Egfl7 promotes tumor cell escape from immunity by downregulating the activation of tumor blood vessels. Aim: to analyze mRNA expression of EGFL7 within the tumor microenvironment of high-grade ovarian serous carcinoma and its association with a number of intraepithelial CD4+/CD8+ lymphocytes and ICAM-1 expression. Methods: qPCR analysis of EGFL7 mRNA in cancer cells and adjacent stromal endothelium microdissected from formalin-fixed paraffin-embedded tumors of 59 high-grade ovarian serous carcinoma patients, was performed. Infiltration of intraepithelial lymphocytes (CD4+/CD8+) and expression of ICAM-1 were evaluated by immunohistochemistry and compared between tumors with different statuses of EGFL7 expression. Results: EGFL7 was expressed in cancer cells (9/59, 15.25%), endothelium (8/59, 13.56%), or both cancer cells and adjacent endothelium (4/59, 6.78%). ICAM-1 was expressed on cancer cells (47/59, 79.66%), stromal endothelium (46/59, 77.97%), or both epithelium and endothelium (40 of 59, 67.8%). EGFL7-positivity of cancer cells and endothelium was associated with lower intraepithelial inflow of CD4+ (p = 0.022 and p = 0.029, respectively) and CD8+ lymphocytes (p = 0.004 and p = 0.031, respectively) but impact neither epithelial nor endothelial ICAM-1 expression (p = 0.098 and p = 0.119, respectively). The patients' median follow-up was 23.83 months (range 1.07-78.07). Lack of prognostic significance of EGFL7-status and ICAM-1 expression was notified. Conclusion: EGFL7 is activated in the cancer cells as frequently as in the endothelium of human high-grade ovarian serous carcinoma. Activation of EGFL7 in cancer cells and/or endothelial cells could negatively impact diapedesis regardless of localization.

The Actin-Binding Protein Cortactin Promotes Sepsis Severity by Supporting Excessive Neutrophil Infiltration into the Lung.

Sepsis is a systemic infection that can lead to multi-organ failure. It is characterised by an uncontrolled immune response with massive neutrophil influx into peripheral organs. Neutrophil extravasation into tissues depends on actin remodeling and actin-binding proteins such as cortactin, which is expressed ubiquitously, except for neutrophils. Endothelial cortactin is necessary for proper regulation of neutrophil transendothelial migration and recruitment to sites of infection. We therefore hypothesised that cortactin plays a crucial role in sepsis development by regulating neutrophil trafficking. Using a murine model of sepsis induced by cecal ligation and puncture (CLP), we showed that cortactin-deficient (KO) mice survive better due to reduced lung injury. Histopathological analysis of lungs from septic KO mice revealed absence of oedema, reduced vascular congestion and mucus deposition, and better-preserved alveoli compared to septic wild-type (WT) mice. Additionally, sepsis-induced cytokine storm, excessive neutrophil infiltration into the lung and oxidative stress were significantly reduced in KO mice. Neutrophil depletion 12 h after sepsis improved survival in WT mice by averting lung injury, similar to both neutrophil-depleted and non-depleted KO mice. Our findings highlight a critical role of cortactin for lung neutrophil infiltration and sepsis severity.

Superoxide Dismutase-3 Downregulates Laminin α5 Expression in Tumor Endothelial Cells via the Inhibition of Nuclear Factor Kappa B Signaling.

The balance between laminin isoforms containing the α5 or the α4 chain in the endothelial basement membrane determines the site of leukocyte diapedesis under inflammatory conditions. Extracellular superoxide dismutase (SOD3) induces laminin α4 expression in tumor blood vessels, which is associated with enhanced intratumor T cell infiltration in primary human cancers. We show now that SOD3 overexpression in neoplastic and endothelial cells (ECs) reduces laminin α5 in tumor blood vessels. SOD3 represses the laminin α5 gene (LAMA5), but LAMA5 expression is not changed in SOD1-overexpressing cells. Transcriptomic analyses revealed SOD3 overexpression to change the transcription of 1682 genes in ECs, with the canonical and non-canonical NF-κB pathways as the major SOD3 targets. Indeed, SOD3 reduced the transcription of well-known NF-κB target genes as well as NF-κB-driven promoter activity in ECs stimulated with tumor necrosis factor (TNF)-α, an NF-κB signaling inducer. SOD3 inhibited the phosphorylation and degradation of IκBα (nuclear factor of the kappa light polypeptide gene enhancer in B-cells inhibitor alpha), an NF-κB inhibitor. Finally, TNF-α was found to be a transcriptional activator of LAMA5 but not of LAMA4; LAMA5 induction was prevented by SOD3. In conclusion, SOD3 is a major regulator of laminin balance in the basement membrane of tumor ECs, with potential implications for immune cell infiltration into tumors.

HS1 deficiency protects against sepsis by attenuating neutrophil-inflicted lung damage.

Sepsis remains an important health problem worldwide due to inefficient treatments often resulting in multi-organ failure. Neutrophil recruitment is critical during sepsis. While neutrophils are required to combat invading bacteria, excessive neutrophil recruitment contributes to tissue damage due to their arsenal of molecular weapons that do not distinguish between host and pathogen. Thus, neutrophil recruitment needs to be fine-tuned to ensure bacterial killing, while avoiding neutrophil-inflicted tissue damage. We recently showed that the actin-binding protein HS1 promotes neutrophil extravasation; and hypothesized that HS1 is also a critical regulator of sepsis progression. We evaluated the role of HS1 in a model of lethal sepsis induced by cecal-ligation and puncture. We found that septic HS1-deficient mice had a better survival rate compared to WT mice due to absence of lung damage. Lungs of septic HS1-deficient mice showed less inflammation, fibrosis, and vascular congestion. Importantly, systemic CLP-induced neutrophil recruitment was attenuated in the lungs, the peritoneum and the cremaster in the absence of HS1. Lungs of HS1-deficient mice produced significantly more interleukin-10. Compared to WT neutrophils, those HS1-deficient neutrophils that reached the lungs had increased surface levels of Gr-1, ICAM-1, and L-selectin. Interestingly, HS1-deficient neutrophils had similar F-actin content and phagocytic activity, but they failed to polymerize actin and deform in response to CXCL-1 likely explaining the reduced systemic neutrophil recruitment in HS1-deficient mice. Our data show that HS1 deficiency protects against sepsis by attenuating neutrophil recruitment to amounts sufficient to combat bacterial infection, but insufficient to induce tissue damage.

The endothelial diapedesis synapse regulates transcellular migration of human T lymphocytes in a CX3CL1- and SNAP23-dependent manner.

Understanding how cytotoxic T lymphocytes (CTLs) efficiently leave the circulation to target cancer cells or contribute to inflammation is of high medical interest. Here, we demonstrate that human central memory CTLs cross the endothelium in a predominantly paracellular fashion, whereas effector and effector memory CTLs cross the endothelium preferably in a transcellular fashion. We find that effector CTLs show a round morphology upon adhesion and induce a synapse-like interaction with the endothelium where ICAM-1 is distributed at the periphery. Moreover, the interaction of ICAM-1:ß2integrin and endothelial-derived CX3CL1:CX3CR1 enables transcellular migration. Mechanistically, we find that ICAM-1 clustering recruits the SNARE-family protein SNAP23, as well as syntaxin-3 and -4, for the local release of endothelial-derived chemokines like CXCL1/8/10. In line, silencing of endothelial SNAP23 drives CTLs across the endothelium in a paracellular fashion. In conclusion, our data suggest that CTLs trigger local chemokine release from the endothelium through ICAM-1-driven signals driving transcellular migration.

ACKR1 favors transcellular over paracellular T-cell diapedesis across the blood-brain barrier in neuroinflammation in vitro.

The migration of CD4+ effector/memory T cells across the blood-brain barrier (BBB) is a critical step in MS or its animal model, EAE. T-cell diapedesis across the BBB can occur paracellular, via the complex BBB tight junctions or transcellular via a pore through the brain endothelial cell body. Making use of primary mouse brain microvascular endothelial cells (pMBMECs) as in vitro model of the BBB, we here directly compared the transcriptome profile of pMBMECs favoring transcellular or paracellular T-cell diapedesis by RNA sequencing (RNA-seq). We identified the atypical chemokine receptor 1 (Ackr1) as one of the main candidate genes upregulated in pMBMECs favoring transcellular T-cell diapedesis. We confirmed upregulation of ACKR1 protein in pMBMECs promoting transcellular T-cell diapedesis and in venular endothelial cells in the CNS during EAE. Lack of endothelial ACKR1 reduced transcellular T-cell diapedesis across pMBMECs under physiological flow in vitro. Combining our previous observation that endothelial ACKR1 contributes to EAE pathogenesis by shuttling chemokines across the BBB, the present data support that ACKR1 mediated chemokine shuttling enhances transcellular T-cell diapedesis across the BBB during autoimmune neuroinflammation.

Investigation of Evolutionary History and Origin of the Tre1 Family Suggests a Role in Regulating Hemocytes Cells Infiltration of the Blood-Brain Barrier.

Understanding the evolutionary relationship between immune cells and the blood-brain barrier (BBB) is important to devise therapeutic strategies. In vertebrates, immune cells follow either a paracellular or a transcellular pathway to infiltrate the BBB. In Drosophila, glial cells form the BBB that regulates the access of hemocytes to the brain. However, it is still not known which diapedesis route hemocytes cells follow. In vertebrates, paracellular migration is dependent on PECAM1, while transcellular migration is dependent on the expression of CAV1. Interestingly Drosophila genome lacks both genes. Tre1 family (Tre1, moody, and Dmel_CG4313) play a diverse role in regulating transepithelial migration in Drosophila. However, its evolutionary history and origin are not yet known. We performed phylogenetic analysis, together with HH search, positive selection, and ancestral reconstruction to investigate the Tre1 family. We found that Tre1 exists in Mollusca, Arthropoda, Ambulacraria, and Scalidophora. moody is shown to be a more ancient protein and it has existed since Cnidaria emergence and has a homolog (e.g., GPCR84) in mammals. The third family member (Dmel_CG4313) seems to only exist in insects. The origin of the family seems to be related to the rhodopsin-like family and in particular family α. We found that opsin is the nearest receptor to have a common ancestor with the Tre1 family that has diverged in sponges. We investigated the positive selection of the Tre1 family using PAML. Tre1 seems to have evolved under negative selection, whereas moody has evolved during positive selection. The sites that we found under positive selection are likely to play a role in the speciation of function in the case of moody. We have identified an SH3 motif, in Tre1 and, moody and Dmel_CG4313. SH3 is known to play a fundamental role in regulating actin movement in a Rho-dependent manner in PECAM1. Our results suggest that the Tre1 family could be playing an important role in paracellular diapedesis in Drosophila.

Autophagy modulates endothelial junctions to restrain neutrophil diapedesis during inflammation.

The migration of neutrophils from the blood circulation to sites of infection or injury is a key immune response and requires the breaching of endothelial cells (ECs) that line the inner aspect of blood vessels. Unregulated neutrophil transendothelial cell migration (TEM) is pathogenic, but the molecular basis of its physiological termination remains unknown. Here, we demonstrated that ECs of venules in inflamed tissues exhibited a robust autophagic response that was aligned temporally with the peak of neutrophil trafficking and was strictly localized to EC contacts. Genetic ablation of EC autophagy led to excessive neutrophil TEM and uncontrolled leukocyte migration in murine inflammatory models, while pharmacological induction of autophagy suppressed neutrophil infiltration into tissues. Mechanistically, autophagy regulated the remodeling of EC junctions and expression of key EC adhesion molecules, facilitating their intracellular trafficking and degradation. Collectively, we have identified autophagy as a modulator of EC leukocyte trafficking machinery aimed at terminating physiological inflammation.

Reduced Lamin A/C Does Not Facilitate Cancer Cell Transendothelial Migration but Compromises Lung Metastasis.

The mechanisms by which the nuclear lamina of tumor cells influences tumor growth and migration are highly disputed. Lamin A and its variant lamin C are key lamina proteins that control nucleus stiffness and chromatin conformation. Downregulation of lamin A/C in two prototypic metastatic lines, B16F10 melanoma and E0771 breast carcinoma, facilitated cell squeezing through rigid pores, and reduced heterochromatin content. Surprisingly, both lamin A/C knockdown cells grew poorly in 3D spheroids within soft agar, and lamin A/C deficient cells derived from spheroids transcribed lower levels of the growth regulator Yap1. Unexpectedly, the transendothelial migration of both cancer cells in vitro and in vivo, through lung capillaries, was not elevated by lamin A/C knockdown and their metastasis in lungs was even dramatically reduced. Our results are the first indication that reduced lamin A/C content in distinct types of highly metastatic cancer cells does not elevate their transendothelial migration (TEM) capacity and diapedesis through lung vessels but can compromise lung metastasis at a post extravasation level.

Age-related changes in the local milieu of inflamed tissues cause aberrant neutrophil trafficking and subsequent remote organ damage.

Aging is associated with dysregulated immune functions. Here, we investigated the impact of age on neutrophil diapedesis. Using confocal intravital microscopy, we found that in aged mice, neutrophils adhered to vascular endothelium in inflamed tissues but exhibited a high frequency of reverse transendothelial migration (rTEM). This retrograde breaching of the endothelium by neutrophils was governed by enhanced production of the chemokine CXCL1 from mast cells that localized at endothelial cell (EC) junctions. Increased EC expression of the atypical chemokine receptor 1 (ACKR1) supported this pro-inflammatory milieu in aged venules. Accumulation of CXCL1 caused desensitization of the chemokine receptor CXCR2 on neutrophils and loss of neutrophil directional motility within EC junctions. Fluorescent tracking revealed that in aged mice, neutrophils undergoing rTEM re-entered the circulation and disseminated to the lungs where they caused vascular leakage. Thus, neutrophils stemming from a local inflammatory site contribute to remote organ damage, with implication to the dysregulated systemic inflammation associated with aging.

Brain endothelial tricellular junctions as novel sites for T cell diapedesis across the blood-brain barrier.

The migration of activated T cells across the blood-brain barrier (BBB) is a critical step in central nervous system (CNS) immune surveillance and inflammation. Whereas T cell diapedesis across the intact BBB seems to occur preferentially through the BBB cellular junctions, impaired BBB integrity during neuroinflammation is accompanied by increased transcellular T cell diapedesis. The underlying mechanisms directing T cells to paracellular versus transcellular sites of diapedesis across the BBB remain to be explored. By combining in vitro live-cell imaging of T cell migration across primary mouse brain microvascular endothelial cells (pMBMECs) under physiological flow with serial block-face scanning electron microscopy (SBF-SEM), we have identified BBB tricellular junctions as novel sites for T cell diapedesis across the BBB. Downregulated expression of tricellular junctional proteins or protein-based targeting of their interactions in pMBMEC monolayers correlated with enhanced transcellular T cell diapedesis, and abluminal presence of chemokines increased T cell diapedesis through tricellular junctions. Our observations assign an entirely novel role to BBB tricellular junctions in regulating T cell entry into the CNS. This article has an associated First Person interview with the first author of the paper.

Once upon a time, inflammation.

Inflammation has accompanied humans since their first ancestors appeared on Earth. Aulus Cornelius Celsus (25 BC-50 AD), a Roman encyclopedist, offered a still valid statement about inflammation: "Notae vero inflammationis sunt quatuor: rubor et tumor cum calore and dolore", defining the four cardinal signs of inflammation as redness and swelling with heat and pain. While inflammation has long been considered as a morbid phenomenon, John Hunter (18th century) and Elie Metchnikoff (19th century) understood that it was a natural and beneficial event that aims to address a sterile or an infectious insult. Many other famous scientists and some forgotten ones have identified the different cellular and molecular players, and deciphered the different mechanisms of inflammation. This review pays tribute to some of the giants who made major contributions, from Hippocrates to the late 19th and first half of the 20th century. We particularly address the discoveries related to phagocytes, diapedesis, chemotactism, and fever. We also mention the findings of the various inflammatory mediators and the different approaches designed to treat inflammatory disorders.

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.

L-selectin regulates human neutrophil transendothelial migration.

The migration of circulating neutrophils towards damaged or infected tissue is absolutely critical to the inflammatory response. L-selectin is a cell adhesion molecule abundantly expressed on circulating neutrophils. For over two decades, neutrophil L-selectin has been assigned the exclusive role of supporting tethering and rolling - the initial stages of the multi-step adhesion cascade. Here, we provide direct evidence for L-selectin contributing to neutrophil transendothelial migration (TEM). We show that L-selectin co-clusters with PECAM-1 - a well-characterised cell adhesion molecule involved in regulating neutrophil TEM. This co-clustering behaviour occurs specifically during TEM, which serves to augment ectodomain shedding of L-selectin and expedite the time taken for TEM (TTT) to complete. Blocking PECAM-1 signalling (through mutation of its cytoplasmic tail), PECAM-1-dependent adhesion or L-selectin shedding, leads to a significant delay in the TTT. Finally, we show that co-clustering of L-selectin with PECAM-1 occurs specifically across TNF- but not IL-1ß-activated endothelial monolayers - implying unique adhesion interactomes forming in a cytokine-specific manner. To our knowledge, this is the first report to implicate a non-canonical role for L-selectin in regulating neutrophil TEM.