β2 Integrin CD11d/CD18: From Expression to an Emerging Role in Staged Leukocyte Migration

CD11d/CD18 is the most recently discovered and least understood β2 integrin. Known CD11d adhesive mechanisms contribute to both extravasation and mesenchymal migration – two key aspects for localizing peripheral leukocytes to sites of inflammation. Differential expression of CD11d induces differences in monocyte/macrophage mesenchymal migration including impacts on macrophage sub-set migration. The participation of CD11d/CD18 in leukocyte localization during atherosclerosis and following neurotrauma has sparked interest in the development of CD11d-targeted therapeutic agents. Whereas the adhesive properties of CD11d have undergone investigation, the signalling pathways induced by ligand binding remain largely undefined. Underlining each adhesive and signalling function, CD11d is under unique transcriptional control and expressed on a sub-set of predominately tissue-differentiated innate leukocytes. The following review is the first to capture the nearly three decades of CD11d research and discusses the emerging role of CD11d in leukocyte migration and retention during the progression of a staged immune response.

Gα13-Mediated β2 Integrin Outside-in Signaling in Neutrophil Migration

Transendothelial migration of neutrophils requires chemoattractant signals and also integrin family of adhesion receptors, particularly the β 2 family of integrins, including Mac-1 and LFA-1. Signals transmitted by G protein-coupled receptors (GPCR) for chemoattractants and cytokines induces inside-out signaling activating the ligand binding function of integrins. Conversely ligand binding to integrins stimulates outside-in signaling, leading to cell spreading, retraction and migration. The heterotrimeric G protein subunit, Gα13, is important for GPCR signaling leading to RhoA activation but also binds to integrins, including β2 integrins to stimulate outside-in signaling. To study the roles of Gα13 in neutrophil migration, we tested the effect of Gα13 knockout on transendothelial migration of neutrophils stimulated by chemoattractant fMIVIL. We demonstrate that transendothelial migration of Gα13 knockout neutrophils was significantly but partially reduced as compared with wild type mice. Transendothelial migration of Gα13 knockout neutrophils is similar to wild type neutrophil migration neutralized with an anti-Mac1 (anti-αm) antibody, and was not further inhibited by the anti-Mac1 antibody, suggesting that transendothelial migration mediated by integrin αmβ2 was predominantly Gα13-dependent. Interestingly, either anti-β2 antibody or anti-LFA1 (anti-αL) antibody appeared to inhibit transendothelial migration of not only wild type neutrophils, but also to a degree, Gα13-knockout neutrophils, suggesting a minor LFA1-dependent but Gα13-independent component of transendothelial migration in addition to the Gα13-dependent transendothlial migration. Furthermore, even though the fibrinogen and ICAM-1 are both β2 ligands, we show that more neutrophils migrated through ICAM-1-coated transwells than fibrinogen-coated transwells, and only ICAM-1-mediated neutrophil migration is Gα13 dependent, suggesting that Gα13-dependent neutrophil migration is selective for certain β2 integrin ligand (ICAM-1). Importantly, Gα13 knockout selectively inhibited the velocity of neutrophil migration on integrin ligand ICAM-1, but had no effect on the directionality of neutrophil migration which requires GPCR-dependent chemoattactant signaling. To understand whether and how Gα13 regulate integrin signaling, we show that Gα13 knockout did not affect the static adhesion of neutrophils to ICAM1, but significantly inhibited neutrophil spreading on ICAM-1. Furthermore, Gα13 bound to β2 integrins in neutrophils adherent on ICAM-1, and this binding was inhibited by the ExE motif peptide MB2mP6 derived from the Gα13 binding site of β2 integrin cytoplasmic domain. MB2mP6 also inhibited transendothelial cell migration similarly as Gα13 knockout. These data suggest that Gα13 plays an important role in promoting β2-integrin dependent neutrophil transendothelial migration mainly by mediating integrin outside-in signaling. Consistent with previous findings of the role of Gα13-dependent outside-in signaling in negative regulation of RhoA in other integrin subtypes, both Gα13 knockout and MB2mP6 abolished the transient inhibition in RhoA during adhesion of neutrophils on ICAM-1. These data suggest that Gα13 mediates outside-in signaling and transient inhibition of RhoA, and thus promotes neutrophil spreading and migration on integrin ligands. To test the role of Gα13 in neutrophil migration in vivo, we showed that neutrophil infiltration in vivo was reduced in leukocyte-selective Gα13 knockout mice using both thioglycolate-induced peritoneal neutrophil infiltration model and LPS-induced neutrophil lung infiltration model in vivo. Furthermore, MB2mP6 inhibited neutrophil infiltration in cardiac tissues in the cardiac ischemia-reperfusion injury model in mice. These data suggest that Gα13-integrin interaction plays an essential role in the integrin-dependent transendothelial migration and is likely to be important in neutrophils' immune and inflammatory functions. Disclosures No relevant conflicts of interest to declare.

ADHESION CONCEPT IN CANCER BIOLOGY: LOCAL AND CENTRAL MECHANISMS (PART 1)

The review presents the concept the key mechanism of the tumor process is a violation of adhesion interactions involving local and central mechanisms. Local features of adhesive dysregulation are demonstrated in the first part. The lack of histospecific adhesion molecules expression resulting from stress or genetic mutation damages an important mechanism of antitumor protection of the tissue disrupting the processes of proliferation and differentiation. The deficiency of histone-specific homotypic adhesion molecules which occurs later exacerbates the disorders. This leads to a decrease in the expression of leukocyte integrins (LFA-1, Mac-1) ligands of the β2 family on the surface of immune effectors and to an increase also in the expression of adhesion molecules to the substrate-antigens VLA (very late activation) family of β1 -integrins on tumor cells. The first restricts the interaction of ICAM family molecules with their contra-receptors from the β2 -integrin family reducing the elimination of target cells by immune effectors which contributes to the screening of the tumor from antitumor surveillance. The second promotes the invasion of the tumor into the surrounding tissues, the formation of blood vessels as well as its heterotypic adhesion with other tissues which further stimulates the proliferation and suppression processes of tumor cells apoptosis. So, the adhesion molecules can be compared to the Phoenix bird: disappearing at the beginning of the process (between the similar cells), they reappear in a new quality (increasing adhesion to cells of other tissues), increasing the totalysm of the tumor. It should be taken into account that tumor cells due to adhesion dysregulation “isolate themselves from society”, lose their differentiation, their maturity and “fall into childhood”, being unable to perform specific, “adult” functions. So, cancer can be considered as a manifestation of the cells aging. Therefore, the anti-stress, endogenous geroprotective mechanisms activation based on the adhesion correction can be effective for preventing and treatment the oncological process.

P02.09 Heteromerization of uPA and PAI-1 enforces pro-tumorigenic neutrophil trafficking to malignant tumors in breast cancer via VLDLr-dependent β2 integrin clustering

BackgroundHigh tumor levels of urokinase-type plasminogen activator (uPA)-plasminogen activator inhibitor-1 (PAI-1) heteromers independently predict poor survival in early breast cancer. The pathogenetic role of this protein complex, however, remains largely obscure.Material and MethodsNeutrophil trafficking was analyzed in orthotopic (multi-channel flow cytometry) and heterotopic (ear; multi-channel in vivo microscopy) mouse models of 4T1 breast cancer, in a mouse peritonitis assay (multi-channel flow cytometry), as well as in the mouse cremaster muscle (multi-channel in vivo microscopy). Cytokine expression in tumors was determined by multiplex ELISA. Phenotypic and functional properties of primary mouse neutrophils, microvascular endothelial cells (cell line bEnd.3), macrophages (cell line RAW 264.7), and breast cancer cells (cell line 4T1) were characterized in different in vitro assays. uPA/PAI-1 expression and neutrophil infiltration in human breast cancer samples were assessed by RNA sequencing, immunhistochemistry, and ELISA.Results and DiscussionHere, we demonstrate that uPA-PAI-1 heteromerization multiplies the potential of the single proteins to attract pro-tumorigenic neutrophils. To this end, tumor-released uPA-PAI-1 activates peritumoral macrophages (VLDL receptor- and ERK/MAPK-pathway). This promotes neutrophil trafficking to cancerous lesions (enhanced β2 integrin activation and clustering) and primes these immune cells towards a pro-tumorigenic phenotype (elevated neutrophil elastase expression), thus supporting tumor growth and metastasis. Blockade of uPA-PAI-1 heteromerization by a novel inhibitor effectively interfered with these events and prevented tumor progression.ConclusionsHere, we identified an already therapeutically targetable interplay between hemostasis and innate immunity that drives advanced stages of breast cancer as well as characterized the underlying mechanisms of this process. As a personalized immunotherapeutic strategy, blockade of uPA-PAI-1 heteromerization might be particularly beneficial for patients with highly aggressive uPA-PAI-1high tumors. This study was supported by Deutsche Forschungsgemeinschaft (DFG), Sonderforschungsbereich (SFB) 914Disclosure InformationB. Uhl: None. L. Mittmann: None. J. Dominik: None. R. Hennel: None. B. Smiljanov: None. F. Haring: None. J. Schaubächer: None. C. Braun: None. L. Padovan: None. R. Pick: None. M. Canis: None. C. Schulz: None. M. Mack: None. E. Gutjahr: None. P. Sinn: None. J. Heil: None. K. Steiger: None. S.M. Kanse: None. W. Weichert: None. M. Sperandio: None. K. Lauber: None. F. Krombach: None. C. Reichel: None.

Binding of Rap1 and Riam to Talin1 Fine-Tune β2 Integrin Activity During Leukocyte Trafficking

β2 integrins mediate key processes during leukocyte trafficking. Upon leukocyte activation, the structurally bent β2 integrins change their conformation towards an extended, intermediate and eventually high affinity conformation, which mediate slow leukocyte rolling and firm arrest, respectively. Translocation of talin1 to integrin adhesion sites by interactions with the small GTPase Rap1 and the Rap1 effector Riam precede these processes. Using Rap1 binding mutant talin1 and Riam deficient mice we show a strong Riam-dependent T cell homing process to lymph nodes in adoptive transfer experiments and by intravital microscopy. Moreover, neutrophils from compound mutant mice exhibit strongly increased rolling velocities to inflamed cremaster muscle venules compared to single mutants. Using Hoxb8 cell derived neutrophils generated from the mutant mouse strains, we show that both pathways regulate leukocyte rolling and adhesion synergistically by inducing conformational changes of the β2 integrin ectodomain. Importantly, a simultaneous loss of both pathways results in a rolling phenotype similar to talin1 deficient neutrophils suggesting that β2 integrin regulation primarily occurs via these two pathways.

Immune cells fold and damage fungal hyphae

Innate immunity provides essential protection against life-threatening fungal infections. However, the outcomes of individual skirmishes between immune cells and fungal pathogens are not a foregone conclusion because some pathogens have evolved mechanisms to evade phagocytic recognition, engulfment, and killing. For example, Candida albicans can escape phagocytosis by activating cellular morphogenesis to form lengthy hyphae that are challenging to engulf. Through live imaging of C. albicans–macrophage interactions, we discovered that macrophages can counteract this by folding fungal hyphae. The folding of fungal hyphae is promoted by Dectin-1, β2-integrin, VASP, actin–myosin polymerization, and cell motility. Folding facilitates the complete engulfment of long hyphae in some cases and it inhibits hyphal growth, presumably tipping the balance toward successful fungal clearance.