Role In Transendothelial Migration Research Articles

Elucidating the Biomechanics of Leukocyte Transendothelial Migration by Quantitative Imaging.

Leukocyte transendothelial migration is crucial for innate immunity and inflammation. Upon tissue damage or infection, leukocytes exit blood vessels by adhering to and probing vascular endothelial cells (VECs), breaching endothelial cell-cell junctions, and transmigrating across the endothelium. Transendothelial migration is a critical rate-limiting step in this process. Thus, leukocytes must quickly identify the most efficient route through VEC monolayers to facilitate a prompt innate immune response. Biomechanics play a decisive role in transendothelial migration, which involves intimate physical contact and force transmission between the leukocytes and the VECs. While quantifying these forces is still challenging, recent advances in imaging, microfabrication, and computation now make it possible to study how cellular forces regulate VEC monolayer integrity, enable efficient pathfinding, and drive leukocyte transmigration. Here we review these recent advances, paying particular attention to leukocyte adhesion to the VEC monolayer, leukocyte probing of endothelial barrier gaps, and transmigration itself. To offer a practical perspective, we will discuss the current views on how biomechanics govern these processes and the force microscopy technologies that have enabled their quantitative analysis, thus contributing to an improved understanding of leukocyte migration in inflammatory diseases.

Calcium Signaling Regulates Leukocyte Transendothelial Migration through the Action of Endothelial Cell IQGAP1, Calmodulin, and CaMKIIδ

When properly contained, inflammation leads to the recruitment and activation of circulating leukocytes to restore tissue and organism homeostasis. Although this process is critical for successful wound healing and the elimination of pathogens and infections, misdirected inflammation can exacerbate pathology and cause substantial morbidity and mortality. Inflammation a fundamental process that underlies the pathology of virtually all diseases including arthritis, atherosclerosis, and multiple sclerosis. As such, the regulation of inflammation represents a key target for potential therapeutic intervention.The transendothelial migration (TEM) of leukocytes out of the bloodstream and into tissue is the rate‐limiting and putative committed step of the inflammatory process. We recently identified the scaffolding protein IQ‐motif containing GTPase Activating Protein 1 (IQGAP1) in a proteomic screen for factors with a potential role in TEM. Knockdown of endothelial IQGAP1 substantially reduced leukocyte TEM. Using IQGAP1 domain truncation constructs we found that the IQ domain is required for its function in TEM. In other systems, the IQ domain binds calmodulin (CaM), a known calcium binding protein. Our group has also shown that a transient calcium influx, mediated by TRPC6, is required to support TEM. We hypothesized that IQGAP1 integrates the calcium signaling from TRPC6 through calmodulin to mediate TEM.We first confirmed that calcium signaling occurs during TEM using live animal intravital microscopy and a GCaMP3 reporter mouse. In this mouse, the fluorescence of a modified GFP construct increases with calcium concentration. Indeed, during leukocyte TEM we observed a local increase in endothelial calcium signal surrounding the transmigrating leukocyte. In vitro we then confirmed that IQGAP1 interacts with CaM via its IQ motif domain. We suspected that CaM propagated the calcium signal through calmodulin kinase II (CaMKII), an important CaM‐regulated kinase. Here we show that CaM interacts with CaMKIIδ in a calcium‐dependent manner. Both the inhibition of CaM with trifluoperazine and disruption of the CaM‐CaMKII interaction with the inhibitor KN‐93 caused a marked reduction in TEM. Similarly, the expression of both a dominant negative CaMKIIδ and CaMKIIN, a peptide inhibitor of CaMKII, substantially reduced leukocyte TEM in vitro.To further validate these findings, we developed two separate mouse models: one with an endothelial‐specific deletion of CaMKIIδ, the other with an endothelial‐specific expression of the CaMKIIN inhibitory peptide used in vitro. Both animal models showed significantly reduced TEM in the croton oil dermatitis model of inflammation confirming that endothelial CaMKIIδ is required for efficient leukocyte TEM in vivo. Overall, these findings uncover a novel connection between IQGAP1, CaM, and CaMKIIδ in coordinating the calcium signal that facilitates leukocyte TEM.Support or Funding InformationThis work was supported by NIH R01 HL046849 and R37 HL064774 to W.A.M.; F30HL134202, T32GM8152, and an Alpha Omega Alpha Carolyn L. Kuckein Student Research Fellowship to P.J.D.; and the Intramural Research Program of the NIH to D.B.S.

Salivary Del-1, IL-17, and LFA-1 levels in periodontal health and disease.

Developmental endothelial locus-1 (Del-1), lymphocyte function-associated antigen-1 (LFA-1), and interleukin 17 (IL-17) play critical roles in transendothelial migration of neutrophils in periodontal diseases. The aim of this study was to evaluate salivary Del-1, IL-17, and LFA-1 protein levels in patients with gingivitis (G), chronic periodontitis (CP), and generalized aggressive periodontitis (GAP). A total of 180 systemically healthy, non-smoking patients (45 periodontally healthy (H) and 45 G, 50 CP, and 40 GAP) individuals (between March 2014 and February 2016) were included in this study according to Armitage's (1999) classification. Clinical periodontal parameters, including clinical attachment level, probing depth, plaque index, and gingival index, were recorded. Del-1, IL-17, and LFA-1 protein expression levels were measured in unstimulated saliva samples collected from patients by using enzyme-linked immunosorbent assays. Kruskal-Wallis and Mann-Whitney U tests were used for multiple comparisons and post hoc statistical analyses, respectively. ROC curve analysis was used to evaluate the sensitivity and specificity of Del-1, IL-17, and LFA-1 in distinguishing periodontal disease from health and gingivitis. It was found a high level of IL-17 and a low level of Del-1 in the CP and GAP, as compared to the G and H groups (P<.001). Nevertheless, we found LFA-1 levels were higher in the GAP than in the CP or G groups (P=.00). Consistently, LFA-1 levels were lower in the H and G groups than in the CP and GAP groups (P=.00). The combination of three biomarkers was found as the best predictor yielded exhibited the highest AUC [0.893, 0.845-0.94 (%95 CI) P<.001] in discriminating periodontal disease from health and gingivitis. Salivary Del-1, LFA-1, and IL-17 levels might be useful markers for determining the clinical health and disease status of patients with periodontitis. However, further studies that evaluate the level of salivary Del-1, LFA-1, and IL-17 before and after periodontal therapy are required to understand the exact roles of these cytokines during the periodontal healing period.

Endothelial Calmodulin and CaMKII Play a Role in Leukocyte Transmigration

Inflammation results in the dilation of blood vessels and recruitment of circulating leukocytes into damaged tissues for repair. A transient increase in endothelial cytosolic free calcium (↑[Ca2+]i) is required for leukocyte transendothelial migration (TEM). We have also found that IQ‐domain GTPase‐activating protein 1 (IQGAP1) is essential for TEM. Specifically, the calmodulin‐binding isoleucine‐glutamine (IQ) motif domain is critical for its function during TEM. Calmodulin (CaM) is an intracellular calcium‐binding protein, however, its role in TEM is undescribed. Therefore, we hypothesized that it is involved in the endothelial calcium signaling cascade essential for supporting leukocyte transmigration.In endothelial cells IQGAP1 knockdown reduced TEM substantially. Re‐expressing IQGAP1 truncation mutants containing both the actin‐binding and IQ‐motif domains rescued TEM but truncation mutants lacking the IQ motif domain but did not support TEM.Human endothelial cells treated with the calmodulin inhibitor trifluoperazine (TFP) demonstrated a marked reduction in leukocyte TEM. Only 13% of leukocytes transmigrated through endothelial monolayers pretreated with 100 mM TFP compared to 84% TEM in control, untreated monolayers (p&lt;0.05). We also looked at the role of calmodulin kinase II (CaMKII). Treating endothelial monolayers with 10 mM KN‐93, a CaMKII inhibitor, substantially reduced leukocyte TEM. Here 22% of leukocytes transmigrated which was significantly reduced compared to control TEM rates (p&lt;0.05). Calmodulin pulldowns also showed that without the IQ‐motif domain IQGAP1 cannot substantially interact with calmodulin further suggesting a link between endothelial calcium, IQGAP1 and CaM.Current experiments focus on inhibiting CaM and CaMKII in endothelial cells using genetically encoded selective inhibitory peptides and inducible endothelial‐specific CaMKII‐deficient mice. Identifying the mechanism by which endothelial Ca2+ influx mediates TEM will provide novel insight into the signaling and also provide potential targets for anti‐inflammatory therapy.Support or Funding InformationR01 HL046849, R01 HL064774, F30 HL134202This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of an intercellular cell adhesion molecule-1 homologue from grass carp: Evidence for its involvement in the immune cell adhesion in teleost

Intercellular cell adhesion molecule-1 (ICAM-1) is a single-chain transmembrane glycoprotein which plays key roles in transendothelial migration of leukocytes and interaction between antigen presenting cells and T cells. In teleost, information of cell adhesion-related molecules is still lacking. In this study, we identified a gene from grass carp sharing similar exon and intron organization with human ICAM-1. Cloning and in silico analysis of its homologues in zebrafish and other two cyprinid fishes, respectively demonstrated the existence of the gene in these fishes. Moreover, the molecular features of these genes in fishes were conserved compared with human ICAM-1. In grass carp, the transcripts of this gene were detected with high levels in heart and liver and its mRNA expression in headkidney leukocytes was induced by Il-1β. Overexpression of this molecule in COS-7 cells could increase the adhesion of the cells with grass carp peripheral blood lymphocytes (PBLs), and the adhesion was further enhanced by lipopolysaccharide stimulation on PBLs. Further studies revealed that the mRNA levels of lymphocyte function-associated antigen-1, a ligand for ICAM-1, were much higher in the PBLs adhering to the COS-7 cells with overexpressing this molecule than in the PBLs alone. These results collectively showed that the newly cloned cDNA encodes grass carp intercellular cell adhesion molecule-1 (Icam-1) and it can mediate the adhesion of PBLs. This provides functional evidence for the existence of Icam-1 in teleost and will facilitate investigation on the transendothelial migration of leukocytes in fish species.

Nuclear Magnetic Resonance Solution Structure of the Recombinant Fragment Containing Three Fibrin-Binding Cysteine-Rich Domains of the Very Low Density Lipoprotein Receptor.

Our previous studies revealed that interaction of fibrin with the very low density lipoprotein (VLDL) receptor plays a prominent role in transendothelial migration of leukocytes and thereby inflammation. The major goal of our subsequent studies is to establish the structural basis for this interaction. As the first step toward this goal, we localized the fibrin-binding sites within cysteine-rich (CR) domains 2-4 of the VLDL receptor. In this study, we have made a next step toward this goal by establishing the nuclear magnetic resonance solution structure of the recombinant VLDLR(2-4) fragment containing all three fibrin-binding CR domains of this receptor. The structure revealed that all three CR domains have a similar general fold. Each domain contains a calcium-binding loop, and the loop in the CR3 domain has a unique conformation relative to the other two. Domains CR2 and CR3 interact with each other, while CR4 is flexible relative to the other two domains. In addition, analysis of the electrostatic potential surface of VLDLR(2-4) revealed extended negatively charged regions in each of its CR domains. The presence of these regions suggests that they may interact with three positively charged clusters of the fibrin βN domain whose involvement in interaction with the VLDL receptor was demonstrated earlier. Altogether, these findings provide a solid background for our next step toward establishing the structural basis for fibrin-VLDL receptor interaction.

CD99 facilitates rearrangement of actin cytoskeleton during T cell activation.

Abstract CD99 is an O-glycosylated transmembrane protein expressed on most hematopoietic cells and has been known to play roles in transendothelial migration of leukocytes. Several reports showed that blocking CD99 on T cells rendered the cells unable to infiltrate into inflamed tissue sites. To understand how CD99 affects T cell migration, we examined the dynamics of antigen-specific T cells in the presence or absence of CD99 during allo-response. When we adoptively transferred WT or CD99-deficient T cell into B6 mice followed by intraperitoneal immunization of splenocytes from allogeneic mice, CD99-deficient antigen-specific T cells were rarely detected in the peritoneum, the antigen-existing target site, relative to WT T cells on day 3 post-immunization, consistent with previous studies. Unexpectedly, however, CD99-deficient antigen-specific T cells were detected in the peritoneum with comparable numbers to WT antigen-specific T cells at later time point, day 7. This delayed migration in CD99-deficient T cells was caused by slower activation than CD99-sufficient T cells in the spleen. We also found that actin dynamics and microcluster movement were impaired in CD99-deficient T cells during synapse formation, and these defects were due to insufficient Rac1 activation in the absence of CD99. Thus, these results demonstrate that CD99 is an important mediator of actin cytoskeleton rearrangement for adequate T cell activation and migration.

Exploring the Role of Calmodulin and Calcium Signaling in Leukocyte Transmigration

Inflammation results in the dilation of local blood vessels, the activation of endothelium by cytokines and the recruitment of circulating leukocytes into areas of tissue damage. A transient increase in endothelial cytosolic free calcium (Ca2+) is required for leukocyte transendothelial migration (TEM) and transient receptor potential canonical 6 (TRPC6) has been found to mediate this required Ca2+ influx. Furthermore, we have also found that IQ‐domain GTPase‐activating protein 1 (IQGAP1) is essential for TEM. Specifically, the actin‐binding domain and the calmodulin‐binding isoleucine‐glutamine (IQ) domain of IQGAP1 are critical for its function during TEM. In endothelial cells re‐expression of IQGAP1 truncation mutants lacking the N‐terminal actin‐binding domain did not localize to endothelial borders and did not support TEM. Re‐expression of truncated IQGAP1 constructs containing the actin‐binding domain and the IQ domain localized to cell borders and supported TEM while constructs containing the actin‐binding domain but lacking the IQ domain localized to the cell borders but did not restore TEM.Calmodulin (CaM) is a small, highly conserved intracellular calcium‐binding protein, which has been shown to interact with IQGAP1 through the IQ domain. It has also been previously found to regulate TRPC channel activation; however, not much is known regarding its role in TEM. Therefore, we hypothesized that it may be involved in the calcium signaling cascade within endothelial cells essential for supporting leukocyte transmigration.Human endothelial cells treated with calmodulin inhibitor trifluoperazine (TFP) demonstrated a marked reduction in leukocyte TEM. Endothelial monolayers pretreated with 100 mM TFP for 1 hour drastically reduced leukocyte TEM. Only about 13% of leukocytes were able to transmigrate compared to 84% TEM in control, untreated monolayers (p=0.0025).After identifying the potential role of calmodulin, we generated CaM shRNA to deplete endogenous CaM within endothelial cells, wildtype Flag‐tagged CaM re‐expression constructs and CaM calcium‐binding mutants that we are currently in the process of testing. Identifying the specific mechanism by which endothelial Ca2+ influx through TRPC6 mediates the events of TEM will provide novel insight into the signaling involved in TEM and also provide potential therapeutic targets for anti‐inflammatory therapy.Support or Funding InformationF30HL134202, R01HL046849, R01HL064774This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 5814: Engineered microfluidic 3D human microvasculature identifies Talin-1-dependent adhesion and FAK activation as the key promoter of cancer cell trans-endothelial migration

Abstract In extravasation cancer cells and vascular niche are involved in a tight cross-talk which has been defined as the rate-regulating event for metastases establishment [1]. Recent, animal studies support the hypothesis that metastatic deficiency lies in focal adhesion complex alterations, however, it still needs to be elucidated which are the specific regulators of each event composing extravasation. Focal adhesion proteins Talin-1 (TLN-1) and Focal Adhesion Kinase (FAK) are up-regulated in breast cancer. Both targets due to their structural and functional role, dramatically influence cancer mechanotransduction leading to endothelial junction disruption, critical in extravasation process [2]. Here, we generated ad hoc engineered models for each extravasation step allowing single cell behavior analyses through high resolution real time imaging in a reliable and quantitative way in a physiological environment. Through this novel approach we analyzed the effect of TLN-1 and FAK and their genetic and chemical inhibition in breast cancer extravasation. The 3D-microfluidic vasculature displayed maturation markers and physiological permeability (1.5±0.76*10-6cm/s) and allowed cancer cell injection through the hollow vessels. Western blot confirmed TLN-1 and FAK knock down (KD) in MDA-MB231. Both targets significantly affected morphology and proliferation (p&amp;lt;0.001). We demonstrated the involvement of both targets in adhesion to the endothelium (p&amp;lt;0.001). Trans endothelial migration (TEM) was decreased in TLN-1 KD (p&amp;lt;0.05) and in FAK KD (P&amp;lt;0.01). Both TLN-1 and FAK KD cells were trapped into the vessels and were not able to extend pseudopodia. Cancer migration tracking in 3D matrix was statistically lower in both KD (p&amp;lt;0.001). The calculated ratios of adhesion/TEM and invasion/TEM were lower in TLN-1 compared to FAK KD, demonstrating that TLN-1 plays a major role in adhesion to endothelium and early invasion while FAK was identified as the main driver in TEM. Additive (TLN-1 and FAK) KD did not show significant difference respect to the target that mostly affected adhesion and invasion demonstrating TLN-1 role in these events. Additive KD was statistically lower compared to TLN-1 KD (P&amp;lt;0.05), supporting FAK crucial role in TEM. Inhibition of TLN-1 and FAK phosphorylation revealed that actin polymerization and pseudopodia formation (required for extravasation) were dependent by their structural role and were independent by their phosphorylation. However, the inhibition of FAK activity showed FAK phosphorylation as the key driver of TEM mechanism (p&amp;lt;0.01). Concluding, our results supported by in vivo data (p&amp;lt;0.05) showed that TLN-1 and FAK inhibition may represent novel strategies challenging mechanisms leading to metastatic establishment. 1. Cell,2011.147(2):p.275-92; 2. Proc Natl Acad Sci USA,2015.112(1):p.214-9 Citation Format: Mara Gilardi, Simone Bersini, Rosa Maria Moresco, Marco Vanoni, Roger D. Kamm, Matteo Moretti. Engineered microfluidic 3D human microvasculature identifies Talin-1-dependent adhesion and FAK activation as the key promoter of cancer cell trans-endothelial migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5814. doi:10.1158/1538-7445.AM2017-5814

Identifying Key Domains in Endothelial IQGAP1 Critical for Leukocyte Transmigration

Inflammation results in the dilation of local blood vessels, the activation of endothelium by cytokines and the recruitment of circulating leukocytes into areas of tissue damage. The majority of leukocytes undergo transendothelial migration (TEM) by using a unique endothelial cell membrane system referred to as the lateral border recycling compartment (LBRC). IQ‐domain GTPase‐activating protein 1 (IQGAP1) was identified as an LBRC‐associated protein and subsequently found to be essential for TEM. IQGAP1 is a scaffolding protein comprised of six major interaction domains. It has been reported to interact productively with over 135 cellular proteins. In endothelial cells IQGAP1 partially localizes to cell borders and knocking down its expression prevents TEM. However, it is not known which of the many proteins it is known to interact with carries out its role in TEM. Since these proteins bind to specific IQGAP1 domains, as a first step toward understanding the mechanism by which IQGAP1 regulates TEM, we explored which of its six protein interactions domains were critical for its function. We knocked down IQGAP1 in endothelial cells and re‐expressed truncated forms of IQGAP1 with specific regions deleted to identify the regions (and thereby the potential binding partners) that are critical for TEM. We show that the actin‐binding domain and the calmodulin‐binding isoleucine‐glutamine (IQ) domain of IQGAP1 are critical for its function during TEM.Human endothelial cells in which &gt;80% of endogenous IQGAP1 was depleted by shRNA did not support TEM; re‐expression of wild‐type IQGAP1 restored TEM to control levels. Re‐expression of IQGAP1 with a GFP‐tagged construct after knockdown also showed IQGAP1 enrichment around transmigrating leukocytes. Re‐expression of IQGAP1 truncation mutants lacking the N‐terminal actin‐binding domain did not localize to endothelial borders and did not support TEM. Re‐expression of truncated IQGAP1 constructs containing the actin‐binding domain and the IQ domain localized to cell borders and supported TEM. Constructs containing the actin‐binding domain but lacking the IQ domain localized to the cell borders but did not restore TEM.After identifying the IQ domain as having a critical role in TEM, we generated specific point mutations in this domain within the full‐length construct. In particular, the isoleucine (I)‐glutamine (Q) motif after which the IQ domain is named has been identified in the literature as having an essential role in IQGAP1 function and we are currently testing whether they are also important in TEM. Using these constructs in future studies will help identify the role of the IQ domain in TEM and its interacting partner(s). Identifying the binding partners of IQGAP1 that are critical for inflammation will provide clues to its mechanism of action and potential therapeutic targets for anti‐inflammatory therapy.Support or Funding InformationFunded by R01 HL046849, R01 HL064774, Alpha Omega Alpha Carolyn Kuckein Student Research Fellowship

Poliovirus Receptor-Related 2: A Cholesterol-Responsive Gene Affecting Atherosclerosis Development by Modulating Leukocyte Migration.

Recently, poliovirus receptor-related 2 (Pvrl2) emerged as a top gene in a global gene expression study aiming to detect plasma cholesterol-responsive genes causally related to atherosclerosis regression in hypercholesterolemic mice. PVRL2 is an adherens junction protein implied to play a role in transendothelial migration of leukocytes, a key feature in atherosclerosis development. In this study, we investigated the effect of Pvrl2 deficiency on atherosclerosis development and transendothelial migration of leukocytes activity. APPROACH AND RESULTS: Pvrl2-deficient mice bred onto an atherosclerosis-prone background (Pvrl2-/-Ldlr-/-Apob100/100) had less atherosclerotic lesions and more stable plaques compared with littermate controls (Pvrl2+/+Ldlr-/-Apob100/100). Pvrl2-/-Ldlr-/-Apob100/100 mice also showed a 49% decrease in transendothelial migration of leukocytes activity observed using the in vivo air pouch model. In accordance, augmented arterial wall expression of Pvrl2 during atherosclerosis progression coincided with an increased gene expression of migrating leukocytes into the vessel wall. Both in human and mice, gene and protein expression of PVRL2 was predominantly observed in the vascular endothelium according to the immunohistochemical and gene expression data. In addition, the cholesterol responsiveness of PVRL2 was also observed in humans. PVRL2 is a plasma cholesterol-responsive gene acting at endothelial sites of vascular inflammation that could potentially be a new therapeutic target for atherosclerosis prevention through its suggested transendothelial migration of leukocytes modulating activity.

Borrelia burgdorferi Keeps Moving and Carries on: A Review of Borrelial Dissemination and Invasion.

Borrelia burgdorferi is the etiological agent of Lyme disease, a multisystemic, multistage, inflammatory infection resulting in patients experiencing cardiac, neurological, and arthritic complications when not treated with antibiotics shortly after exposure. The spirochetal bacterium transmits through the Ixodes vector colonizing the dermis of a mammalian host prior to hematogenous dissemination and invasion of distal tissues all the while combating the immune response as it traverses through its pathogenic lifecycle. The innate immune response controls the borrelial burden in the dermis, but is unable to clear the infection and thereby prevent progression of disease. Dissemination in the mammalian host requires temporal regulation of virulence determinants to allow for vascular interactions, invasion, and colonization of distal tissues. Virulence determinants and/or adhesins are highly heterogenetic among environmental B. burgdorferi strains with particular genotypes being associated with the ability to disseminate to specific tissues and the severity of disease, but fail to generate cross-protective immunity between borrelial strains. The unique motility of B. burgdorferi rendered by the endoflagella serves a vital function for dissemination and protection from immune recognition. Progress has been made toward understanding the chemotactic regulation coordinating the activity of the two polar localized flagellar motors and their role in borrelial virulence, but this regulation is not yet fully understood. Distinct states of motility allow for dynamic interactions between several B. burgdorferi adhesins and host targets that play roles in transendothelial migration. Transmigration across endothelial and blood–brain barriers allows for the invasion of tissues and elicits localized immune responses. The invasive nature of B. burgdorferi is lacking in proactive mechanisms to modulate disease, such as secretion systems and toxins, but recent work has shown degradation of host extracellular matrices by B. burgdorferi contributes to the invasive capabilities of the pathogen. Additionally, B. burgdorferi may use invasion of eukaryotic cells for immune evasion and protection against environmental stresses. This review provides an overview of B. burgdorferi mechanisms for dissemination and invasion in the mammalian host, which are essential for pathogenesis and the development of persistent infection.

Selective Inhibition of α4β1 Integrin (VLA-4) Mitigates GvHD

Allogeneic hematopoietic cell transplantation (allo-HCT) is considered a curative treatment for many malignant and non-malignant hematological conditions. However, treatment related mortality (TRM) limits the use of such treatment modality. Acute graft versus host disease (aGvHD) is responsible for more than 50% of TRM. Alloantigen presentation to donor T cells results in alloactivation of donor T cells and GvHD. Alloreactive donor T cells are also responsible for the beneficial graft versus leukemia (GVL) effect. Furthermore, alloactivation is also crucial for the development of immune tolerance. Thus, direct inhibition of alloreactive T cells might negatively impact GVL and the development of immune-regulatory cells. Modulation of alloreactive T cell trafficking and migration especially to the GI tract has been of great interest. α4 integrin plays a major role in transendothelial migration of immune cells to secondary lymphoid tissues. Thus, we hypothesize that α4 deficient donor T cells will fail to effectively traffic to GVHD target organs. To test this hypothesis, we generated Tie-2 cre+ α4fl/fl mice (B6, H-2b, CD45.2+) from which T cells were obtained. After in vitro activation of murine pan T cells using mixed lymphocyte reaction assay, we found α4 and β1 integrin are more highly expressed than β7 and αE integrin on WT T cell. Using (B6 to Balb/c) fully MHC mismatched mouse allo-HCT model we found that only donor derived alloreactive T cells (Ly5.1; derived from donor splenocytes) overexpressed α4 integrin but not bone marrow derived T cells (Ly5.2) at day +17 and +30 after transplant. As previously described (Alahmari B et al. 2015 ASH), we found that α4(-/-) T cells transplanted into allogeneic transplant recipients (B6 to Balb/c) resulted in significantly reduced acute GVHD and increased survival compared to recipients infused with WT T cells (Fig. A). We hypothesized that defective T cell transendothelial migration may contribute to less GvHD observed with α4(-/-) T cells. We performed bioluminescence imaging (BLI) to track CBRluc-transduced murine pan T cells (2 × 106 cells) after allo-HCT in vivo. We found a significant difference in the percentage of BLI signal intensity between α4 deficient and control T cells in spleen and gut at day 14 and 21 post allo-HCT. Using luciferase expressing mouse A20 leukemia cells (BALB) we found α4(-/-) T cells were able to clear leukemia cells as effectively as WT T cells. Of interest in vitro activation of T cells using anti-CD3/CD28 antibody-coated beads resulted in significantly increased expression of CTLA-4 expression on α4(-/-) compared to WT T cells. In spite of this, in vivo blockade of CTLA-4 after allo-HCT did not accelerate the onset or increase the severity of GVHD in mice receiving α4(-/-) T cells compared to recipients receiving WT T cells, suggesting that the protective effect of α4(-/-) T cell is independent of T cell immunoregulatory function. α4 integrin can form a heterodimer with either β1 to form α4β1 integrin (VLA-4) or β7 to form α4β7 (LPAM-1). Previous results of others (Petrovic A et al. 2004 Blood; Waldman E et al 2006 Blood) implicated β7 integrin in aGvHD, especially GI GvHD. To define which α4 heterodimer (α4β1 or α4β7/αEβ7) is the more potent in GVHD induction, we performed (B6 to Balb/c) allo-HCT and treated mice with either BIO5192 (a highly selective VLA-4 inhibitor) from day +2 to + 28, or with LPAM-1 monoclonal antibody (DATK32) 3 times per week for one month. Although both interventions resulted in reduced GVHD and increased survival, treatment with BIO5192 appeared to be more effective as an approach to reduce GvHD (Fig. B and C).In conclusion, α4 integrins is involved in GVHD pathology likely through facilitating the migration of alloreactive T cells to GVHD target organs (especially the GI tract). In addition, we report for the first time that selective inhibition of α4β1 integrin (VLA-4) is more effective than that of α4β7 (LPAM-1) in prevention of GVHD in a fully MHC mismatched allo-HCT model. Blockade of α4 integrins (natalizumab, vedolizumab) especially α4β1 should be considered a valid approach to treat or prevent GvHD in humans and can be exploited to treat other inflammatory settings such as solid organ transplantation, chronic inflammatory diseases and autoimmune diseases. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract PR11: Increased matrix stiffness induces CCN1 mediated upregulation of N-Cadherin in endothelial cells: Implications for cancer cell metastasis

Abstract The formation of a stiff lump in soft tissue is one of the most common symptoms that people associate with cancer. This is a sign of abnormal tissue stiffness, which derives from changes in the extracellular matrix (ECM) driven by cancer and stromal cells. This physical property is known to be a consequence and promoter of tumor development. While it is well established that high stiffness promotes invasive behavior of cancer cells, the role of abnormal stiffness on endothelial cells (ECs) is still largely unknown. Here we use unbiased mass spectrometry (MS)-based proteomics to ascertain how increased tissue stiffness affects ECs, and the impact of these alterations in cancer. To do this we used an in vitro system of polyacrylamide gels representing ECM stiffness of normal and a tumor tissue. Using high resolution MS combined with stable isotope labelling of amino acids in cell culture (SILAC), we quantified the global proteome of ECs cultured at each ECM stiffness and unveiled that many cell adhesion proteins were upregulated at tumor stiffness, including N-Cadherin and the matricellular protein CCN1. By silencing and overexpressing CCN1 in ECs, we demonstrated that stiffness-induced CCN1 induces a β-catenin mediated increase in N-Cadherin expression. N-Cadherin is known to play a key role in trans-endothelial migration of cancer cells through the blood vessels, and we show that CCN1 induces cancer cell adhesion to ECs via N-Cadherin in vitro. This indicates that CCN1 can increase cancer cell metastasis by aiding the exit of cancer cells from the primary tumor and entry at distant sites. We confirmed this hypothesis in vivo, by using C57Bl/6 Ccn1loxP/loxP mice and syngeneic B16 melanoma model. Ccn1 was acutely knocked out in the vasculature by injecting the Tat-Cre fusion protein intravenously, and this showed that knocking out Ccn1 decreased the number of circulating tumor cells and subsequent metastases in the lungs. Thus, CCN1 loss in the vasculature can decrease cancer cell intravasation. Our work determines an unprecedented mechanism through which CCN1 in the stromal cells promotes invasion of cancer cells. This complements the findings that CCN1 can induce invasion in the cancer cells, and provides evidence that targeting CCN1 therapeutically in the clinic may decrease the spread of cancer from the perspective of the endothelial cells. This abstract is also presented as Poster C34. Citation Format: Steven Reid, Jens Serneels, Lisa J. Neilson, Anne-Theres Henze, Juan R. Hernández-Fernaud, Ralf H. Adams, Karen Blyth, Massimiliano Mazzone, Sara Zanivan. Increased matrix stiffness induces CCN1 mediated upregulation of N-Cadherin in endothelial cells: Implications for cancer cell metastasis. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr PR11.

Critical Roles for the IQ‐Motif and Actin‐Bnding Domain of Endothelial Cell IQGAP1 During Leukocyte Transendothelial Migration

In order to mount an appropriate inflammatory response, leukocytes must cross the endothelium to gain access to the target tissue and carry out their essential roles. This process is referred to as transendothelial migration (TEM) and requires a novel endothelial cell membrane compartment, the Lateral Border Recycling Compartment (LBRC), which is delivered to the migrating leukocyte to supply it with several requisite membrane proteins. IQGAP1, a large soluble scaffolding protein, is associated with the LBRC and is required for its function during TEM. In a variety of other processes, IQGAP1 integrates signals from diverse pathways to regulate cytoskeletal remodeling, Rho family GTPase activity, and other downstream readouts, however its role in TEM remains unclear. Here we show that genetic ablation of endothelial IQGAP1 expression arrests leukocytes during TEM by preventing the targeted delivery of the LBRC. Expression of a full length GFP‐tagged IQGAP1 construct completely restores this defect and also shows that endothelial IQGAP1 is enriched around the leukocyte during TEM. Furthermore, using fluorescently tagged IQGAP1 truncation constructs, we show that the actin‐binding domain of IQGAP1 is required for its localization to the junction in resting endothelial cells and its recruitment and function during TEM. Interestingly, the IQ motif of IQGAP1, which facilitates its interaction with calmodulin, is not required for localization to the junction or recruitment during TEM, but is necessary for IQGAP1′s function during TEM. Together these data suggest that IQGAP1 integrates signals from various sources to facilitate the function and recruitment of the LBRC during TEM. Supported by R01 HL046849 and R37 HL064774.

Regulatory Role of Endothelial CD47 during Leukocyte Transmigration

The vascular endothelium plays a critical role in inflammation and leukocyte recruitment by expression of adhesion molecules and leukocyte chemoattractants. Adhesion molecules bind leukocytes and transmit signals to the endothelial cells (EC) required for transendothelial migration (TEM). An important unanswered question is how ECs integrate the signals arising from leukocyte engagement of multiple adhesion molecules. CD47, also known as Integrin Associated Protein, is a broadly expressed glycoprotein and binds thrombospondin‐1 and leukocyte expressed Signal Regulatory Proteins (SIRPα and SIRPg). Besides these “in trans” interactions, CD47 also interacts “in cis” (in the same plasma membrane) with multiple integrins. We previously reported that endothelial CD47 plays a role in TEM and transmits intracellular signals upon engagement. Interestingly, CD47 signals phenocopy those occurring after engagement of ICAM‐1. We hypothesized CD47 interacts with ICAM‐1 to form a protein complex in EC plasma membrane and that this complex binds leukocytes and transmits intracellular signals during leukocyte TEM. Here we report CD47 associates with ICAM‐1 in EC by Fluorescence Lifetime Imaging Microscopy of Förster Resonance Energy Transfer (FLIM‐FRET). Furthermore, ICAM‐1 crosslinking‐induced signaling through pyk2 and src kinases was impaired in CD47‐/‐ mouse heart ECs (MHEC) as compared to WT MHEC. This was also demonstrated in HUVEC silenced for CD47. Understanding how CD47 interacts with different molecules will clarify its role in leukocyte recruitment and EC dependent mechanisms that regulate TEM.Supported by grants from National Research Fund of Luxembourg (FNR, Award # 4800397) and NIH HL‐36028.

Role of cortactin homolog HS1 in transendothelial migration of natural killer cells.

Natural Killer (NK) cells perform many functions that depend on actin assembly, including adhesion, chemotaxis, lytic synapse assembly and cytolysis. HS1, the hematopoietic homolog of cortactin, binds to Arp2/3 complex and promotes actin assembly by helping to form and stabilize actin filament branches. We investigated the role of HS1 in transendothelial migration (TEM) by NK cells. Depletion of HS1 led to a decrease in the efficiency of TEM by NK cells, as measured by transwell assays with endothelial cell monolayers on porous filters. Transwell assays involve chemotaxis of NK cells across the filter, so to examine TEM more specifically, we imaged live-cell preparations and antibody-stained fixed preparations, with and without the chemoattractant SDF-1α. We found small to moderate effects of HS1 depletion on TEM, including whether the NK cells migrated via the transcellular or paracellular route. Expression of HS1 mutants indicated that phosphorylation of HS1 tyrosines at positions 222, 378 and 397 was required for rescue in the transwell assay, but HS1 mutations affecting interaction with Arp2/3 complex or SH3-domain ligands had no effect. The GEF Vav1, a ligand of HS1 phosphotyrosine, influenced NK cell transendothelial migration. HS1 and Vav1 also affected the speed of NK cells migrating across the surface of the endothelium. We conclude that HS1 has a role in transendothelial migration of NK cells and that HS1 tyrosine phosphorylation may signal through Vav1.

Abstract 3163: Transendothelial migration of uveal melanoma cells

Abstract Uveal melanoma, cancer arising from the pigmented uveal layer of the eye, is highly metastatic despite being limited to spreading only through the bloodstream. Almost half of patients develop distant metastatic disease, most often in the liver, even after the tumor-bearing eye is completely removed. The key to understanding and targeting uveal melanoma metastasis is in understanding how circulating tumors cells exit the bloodstream at distant sites of metastasis, and then invade and colonize distant organs. To address the first process, transendothelial migration, we modeled the blood vessel wall using primary human microvascular endothelial monolayers grown on polyacrylamide substrates that mimic the physiological stiffness of normal tissue. To these monolayers, we added 92.1 and OCM-1A uveal melanoma cells, and then collected time-lapse movies using spinning-disc confocal and differential interference contrast microscopy to follow the transmigration of the uveal melanoma cells. We found that both cell lines transmigrate through endothelial monolayers by a route distinctly different from the paracellular migration we see for trafficking immune cells. During this multistep process, uveal melanoma cells first identify paracellular junctions and then intercalate between endothelial cells, taking on a flattened morphology and maintaining contacts with their adjacent endothelial cells; reminiscent of vasculogenic mimicry. Tumor cells then extend invasive projections beneath adjacent endothelial cells, before eventually migrate under the endothelium. Immunofluorescence revealed cortical actin networks with strong cortactin staining within these invasive projections. To visualize these structures better, we transfected uveal melanoma cells with F-tractin, a fluorescent fusion protein that specifically binds filamentous actin without disrupting actin function, that allows imaging of actin dynamics in live cells. Time-lapse confocal images revealed complex actin-rich projections invading and sampling the interface between the endothelial monolayer and the substrate. Dynamic actin cytoskeletal reorganization was also apparent as cells migrated under the monolayer. Previous studies have linked inactivating mutations in BAP1 to metastatic spread of uveal melanoma in patients. To test whether BAP1 loss plays a role in transendothelial migration, we knocked down BAP1 expression in our uveal melanoma cell lines. We found that BAP1-depleted cells transmigrated significantly faster through endothelial monolayers in transwell assays than control knockdown cells. Time-lapse movies showed no significant differences in the first steps of initiation or intercalation between BAP1 knockdown and control cells. These studies identify a novel mechanism of transendothelial migration in uveal melanoma cells and suggest that BAP1 depletion specifically affects the ability of cells to invade under and exit the monolayer. Citation Format: Michael D. Onken, Olivia L. Mooren, Jinmei Li, John A. Cooper. Transendothelial migration of uveal melanoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3163. doi:10.1158/1538-7445.AM2014-3163

Blocking monocyte transmigration in in vitro system by a human antibody scFv anti-CD99. Efficient large scale purification from periplasmic inclusion bodies in E. coli expression system

Migration of leukocytes into site of inflammation involves several steps mediated by various families of adhesion molecules. CD99 play a significant role in transendothelial migration (TEM) of leukocytes. Inhibition of TEM by specific monoclonal antibody (mAb) can provide a potent therapeutic approach to treating inflammatory conditions. However, the therapeutic utilization of whole IgG can lead to an inappropriate activation of Fc receptor-expressing cells, inducing serious adverse side effects due to cytokine release. In this regard, specific recombinant antibody in single chain variable fragments (scFvs) originated by phage library may offer a solution by affecting TEM function in a safe clinical context. However, this consideration requires large scale production of functional scFv antibodies and the absence of toxic reagents utilized for solubilization and refolding step of inclusion bodies that may discourage industrial application of these antibody fragments. In order to apply the scFv anti-CD99 named C7A in a clinical setting, we herein describe an efficient and large scale production of the antibody fragments expressed in E. coli as periplasmic insoluble protein avoiding gel filtration chromatography approach, and laborious refolding step pre- and post-purification. Using differential salt elution which is a simple, reproducible and effective procedure we are able to separate scFv in monomer format from aggregates. The purified scFv antibody C7A exhibits inhibitory activity comparable to an antagonistic conventional mAb, thus providing an excellent agent for blocking CD99 signaling. This protocol can be useful for the successful purification of other monomeric scFvs which are expressed as periplasmic inclusion bodies in bacterial systems.

Characterisation of interleukin‐10 expression on different vascular structures in allergic nasal mucosa

BackgroundInterleukin-10 (IL-10) is a negative regulator of immune responses and was previously shown to be expressed by human nasal endothelial cells, while the adhesion molecule MECA-79 plays a role in trans-endothelial migration of immune competent cells. In this study we investigate the relationship between endothelial IL-10 and MECA-79 expression to address the question whether immune competent cells could be affected at the mucosal entry site.MethodsNasal turbinate biopsies were taken from house dust mite allergic patients, before and after nasal allergen provocation. Subsequent slides of biopsies were stained for IL10, MECA-79, CD34, and IL10-Receptor. Capillaries, arteries/veins, and sinusoids were evaluated separately.Results90% of sinusoids are IL-10 positive and all sinusoids are negative for MECA-79, while 4.8% of capillaries are positive for IL-10, and 2.2% are positive for MECA-79. Although about 47% of arteries/veins are positive for IL-10 and 57.1% are positive for MECA-79, only about 20% are positive for both markers. Furthermore, we showed that the myo-fibroblasts surrounding all sinusoids stain positive for IL10R.ConclusionsIL10 expression on vascular structures is not related to MECA expression for sinusoids and capillaries and only partly related on arteries/veins, however sinusoidal endothelial IL10 expression is always seen in combination with IL-10R expression of sinusoidal myo-fibroblasts.