A LAD-III syndrome is associated with defective expression of the Rap-1 activator CalDAG-GEFI in lymphocytes, neutrophils, and platelets

Leukocyte and platelet integrins rapidly alter their affinity and adhesiveness in response to various activation (inside-out) signals. A rare leukocyte adhesion deficiency (LAD), LAD-III, is associated with severe defects in leukocyte and platelet integrin activation. We report two new LAD cases in which lymphocytes, neutrophils, and platelets share severe defects in β1, β2, and β3 integrin activation. Patients were both homozygous for a splice junction mutation in their CalDAG-GEFI gene, which is a key Rap-1/2 guanine exchange factor (GEF). Both mRNA and protein levels of the GEF were diminished in LAD lymphocytes, neutrophils, and platelets. Consequently, LAD-III platelets failed to aggregate because of an impaired αIIbβ3 activation by key agonists. β2 integrins on LAD-III neutrophils were unable to mediate leukocyte arrest on TNFα-stimulated endothelium, despite normal selectin-mediated rolling. In situ subsecond activation of neutrophil β2 integrin adhesiveness by surface-bound chemoattractants and of primary T lymphocyte LFA-1 by the CXCL12 chemokine was abolished. Chemokine inside-out signals also failed to stimulate lymphocyte LFA-1 extension and high affinity epitopes. Chemokine-triggered VLA-4 adhesiveness in T lymphocytes was partially defective as well. These studies identify CalDAG-GEFI as a critical regulator of inside-out integrin activation in human T lymphocytes, neutrophils, and platelets

α4β1-dependent adhesion strengthening under mechanical strain is regulated by paxillin association with the α4-cytoplasmic domain

The capacity of integrins to mediate adhesiveness is modulated by their cytoplasmic associations. In this study, we describe a novel mechanism by which α4-integrin adhesiveness is regulated by the cytoskeletal adaptor paxillin. A mutation of the α4 tail that disrupts paxillin binding, α4(Y991A), reduced talin association to the α4β1 heterodimer, impaired integrin anchorage to the cytoskeleton, and suppressed α4β1-dependent capture and adhesion strengthening of Jurkat T cells to VCAM-1 under shear stress. The mutant retained intrinsic avidity to soluble or bead-immobilized VCAM-1, supported normal cell spreading at short-lived contacts, had normal α4-microvillar distribution, and responded to inside-out signals. This is the first demonstration that cytoskeletal anchorage of an integrin enhances the mechanical stability of its adhesive bonds under strain and, thereby, promotes its ability to mediate leukocyte adhesion under physiological shear stress conditions

In the Hunt for Therapeutic Targets: Mimicking the Growth, Metastasis, and Stromal Associations of Early-Stage Lung Cancer Using a Novel Orthotopic Animal Model

BackgroundThe existing shortage of animal models that properly mimic the progression of early-stage human lung cancer from a solitary confined tumor to an invasive metastatic disease hinders accurate characterization of key interactions between lung cancer cells and their stroma. We herein describe a novel orthotopic animal model that addresses these concerns and consequently serves as an attractive platform to study tumor–stromal cell interactions under conditions that reflect early-stage lung cancer.MethodsUnlike previous methodologies, we directly injected small numbers of human or murine lung cancer cells into murine's left lung and longitudinally monitored disease progression. Next, we used green fluorescent protein-tagged tumor cells and immuno-fluorescent staining to determine the tumor's microanatomic distribution and to look for tumor-infiltrating immune cells and stromal cells. Finally, we compared chemokine gene expression patterns in the tumor and lung microenvironment.ResultsWe successfully generated a solitary pulmonary nodule surrounded by normal lung parenchyma that grew locally and spread distally over time. Notably, we found that both fibroblasts and leukocytes are recruited to the tumor's margins and that distinct myeloid cell attracting and CCR2-binding chemokines are specifically induced in the tumor microenvironment.ConclusionOur orthotopic lung cancer model closely mimics the pathologic sequence of events that characterizes early-stage human lung cancer propagation. It further introduces new means to monitor tumor–stromal cell interactions and offers unique opportunities to test therapeutic targets under conditions that reflect early-stage lung cancer. We argue that for such purposes our model is superior to lung cancer models that are based either on genetic induction of epithelial transformation or on ectopic transplantation of malignant cells

ICAMs support B cell interactions with T follicular helper cells and promote clonal selection.

The germinal center (GC) reaction begins with a diverse and expanded group of B cell clones bearing a wide range of antibody affinities. During GC colonization, B cells engage in long-lasting interactions with T follicular helper (Tfh) cells, a process that depends on antigen uptake and antigen presentation to the Tfh cells. How long-lasting T-B interactions and B cell clonal expansion are regulated by antigen presentation remains unclear. Here, we use in vivo B cell competition models and intravital imaging to examine the adhesive mechanisms governing B cell selection for GC colonization. We find that intercellular adhesion molecule 1 (ICAM-1) and ICAM-2 on B cells are essential for long-lasting cognate Tfh-B cell interactions and efficient selection of low-affinity B cell clones for proliferative clonal expansion. Thus, B cell ICAMs promote efficient antibody immune response by enhancement of T cell help to cognate B cells

Chemokine-triggered microtubule polymerization promotes neutrophil chemotaxis and invasion but not transendothelial migration.

Microtubules (MTs) are critically involved in the transport of material within cells, but their roles in chemotactic leukocyte motility and effector functions are still obscure. Resting neutrophils contain few MTs assembled in an MT organizing center (MTOC) behind their multilobular nuclei. Using a probe of real-time tubulin polymerization, SiR-tubulin, we found that neutrophils elongated their MTs within minutes in response to signals from the two prototypic chemotactic peptides, CXCL1 and fMLP. Taxol, a beta-tubulin binding and MT stabilizing drug, was found to abolish this CXCL1- and fMLP-stimulated MT polymerization. Nevertheless, taxol treatment as well as disruption of existing and de novo generated MTs did not impair neutrophil protrusion and squeezing through IL-1β-stimulated endothelial monolayers mediated by endothelial deposited CXCL1 and neutrophil CXCR2. Notably, CXCL1-dependent neutrophil TEM was not associated with neutrophil MT polymerization. Chemokinetic neutrophil motility on immobilized CXCL1 was also not associated with MT polymerization, and taxol treatment did not interfere with this motility. Nevertheless, and consistent with its ability to suppress MT polymerization induced by soluble CXCL1 and fMLP, taxol treatment inhibited neutrophil chemotaxis toward both chemotactic peptides. Taxol treatment also suppressed CXCL1- and fMLP-triggered elastase-dependent neutrophil invasion through collagen I barriers. Collectively, our results highlight de novo chemoattractant-triggered MT polymerization as key for neutrophil chemotaxis and elastase-dependent invasion but not for chemotactic neutrophil crossing of inflamed endothelial barriers

Lung ICAM-1 and ICAM-2 support spontaneous intravascular effector lymphocyte entrapment but are not required for neutrophil entrapment or emigration inside endotoxin-inflamed lungs.

The pulmonary vasculature constitutively expresses the integrin lymphocyte function-associated antigen-1 ligands intercellular adhesion molecule (ICAM)-1 and -2. In this study, effector T cells were temporarily entrapped by the lung vasculature on their way to inflamed lymph nodes, and this entrapment was strongly reduced in ICAM-1 and -2 double-deficient mice (79 and 86% reduction for CD8(+) and CD4(+) effectors, respectively, compared with wild-type mice). Although the pulmonary vasculature has been suggested to be masked by the heparan sulfate-containing glycocalyx, which is susceptible to heparanase-mediated shedding, lung and lymphocyte heparanase have been found to be unnecessary for this entrapment. Systemic LPS induced rapid neutrophil entrapment in the lung vasculature, but in contrast to T-cell entrapment, this sequestration was ICAM-1, ICAM-2, and heparanase independent. Furthermore, neutrophil migration into the bronchoalveolar space induced by LPS inhalation and LPS-induced leakage of red blood cells into this space were not dependent on lung ICAMs or heparanase activity. Nevertheless, heparanase was critical for neutrophil accumulation in smoke-exposed lungs. Our results indicate that, whereas T cells use ICAM-1 and -2 for temporary pulmonary entrapment, neutrophils get sequestered and extravasate into inflamed lungs independent of ICAMs. This is the first demonstration that the pulmonary vasculature is differentially recognized by T cells and neutrophils.-Petrovich, E., Feigelson, S. W., Stoler-Barak, L., Hatzav, M., Solomon, A., Bar-Shai, A., Ilan, N., Li, J.-P., Engelhardt, B., Vlodavsky, I., Alon, R. Lung ICAM-1 and ICAM-2 support spontaneous intravascular effector lymphocyte entrapment but are not required for neutrophil entrapment or emigration inside endotoxin-inflamed lungs