Silvia Naus

PSGL‐1 function in immunity and steady state homeostasis

Summary:  The substantial importance of P‐selectin glycoprotein ligand 1 (PSGL‐1) in leukocyte trafficking has continued to emerge beyond its initial identification as a selectin ligand. PSGL‐1 seemed to be a relatively simple molecule with an extracellular mucin domain extended as a flexible rod, teleologically consistent with its primary role in tethering leukocytes to endothelial selectins. The rolling interaction between leukocyte and endothelium mediated by this selectin‐PSGL‐1 interaction requires branched O‐glycan extensions on specific PSGL‐1 amino acid residues. In some cells, such as neutrophils, the glycosyltransferases involved in formation of the O‐glycans are constitutively expressed, while in other cells, such as T cells, they are expressed only after appropriate activation. Thus, PSGL‐1 supports leukocyte recruitment in both innate and adaptive arms of the immune response. A complex array of amino acids within the selectins engage multiple sugar residues of the branched O‐glycans on PSGL‐1 and provide the molecular interactions responsible for the velcro‐like catch bonds that support leukocyte rolling. Such binding of PSGL‐1 can also induce signaling events that influence cell phenotype and function. Scrutiny of PSGL‐1 has revealed a better understanding of how it performs as a selectin ligand and yielded unexpected insights that extend its scope from supporting leukocyte rolling in inflammatory settings to homeostasis including stem cell homing to the thymus and mature T‐cell homing to secondary lymphoid organs. PSGL‐1 has been found to bind homeostatic chemokines CCL19 and CCL21 and to support the chemotactic response to these chemokines. Surprisingly, the O‐glycan modifications of PSGL‐1 that support rolling mediated by selectins in inflammatory conditions interfere with PSGL‐1 binding to homeostatic chemokines and thereby limit responsiveness to the chemotactic cues used in steady state T‐cell traffic. The multi‐level influence of PSGL‐1 on cell traffic in both inflammatory and steady state settings is therefore substantially determined by the orchestrated addition of O‐glycans. However, central as specific O‐glycosylation is to PSGL‐1 function, in vivo regulation of PSGL‐1 glycosylation in T cells remains poorly understood. It is our purpose herein to review what is known, and not known, of PSGL‐1 glycosylation and to update understanding of PSGL‐1 functional scope.

Metalloproteinase Disintegrins ADAM8 and ADAM19 Are Highly Regulated in Human Primary Brain Tumors and their Expression Levels and Activities Are Associated with Invasiveness

Patients with primary brain tumors have bleak prognoses and there is an urgent desire to identify new markers for sensitive diagnosis and new therapeutic targets for effective treatment. A family of proteins, the disintegrin and metalloproteinases (ADAMs or adamalysins), are cell surface and extracellular multidomain proteins implicated in cell-cell signaling, cell adhesion, and cell migration. Their putative biological and pathological roles make them candidates for promoting tumor growth and malignancy. We investigated the expression levels of 12 cerebrally expressed ADAM genes in human primary brain tumors (astrocytoma WHO grade I-III, glioblastoma WHO grade IV, oligoastrocytoma WHO grade II and III, oligodendroglioma WHO grade II and III, ependymoma WHO grade II and III, and primitive neuroectodermal tumor WHO grade IV) using real-time PCR. The mRNAs of the five ADAMs 8, 12, 15, 17, and 19 were significantly upregulated. The ADAM8 and ADAM19 proteins were mainly located in tumor cells and in some tumors in endothelia of blood vessels. In brain tumor tissue, ADAM8 and ADAM19 undergo activation by prodomain removal resulting in active proteases. By using specific peptide substrates for ADAM8 and ADAM19, respectively, we demonstrated that the proteases exert enhanced proteolytic activity in those tumor specimens with the highest expression levels. In addition, expression levels and the protease activities of ADAM8 and ADAM19 correlated with invasive activity of glioma cells, indicating that ADAM8 and ADAM19 may play a significant role in tumor invasion that may be detrimental to patients survival.

Identification of candidate substrates for ectodomain shedding by the metalloprotease-disintegrin ADAM8

Abstract ADAM proteases are type I transmembrane proteins with extracellular metalloprotease domains. As for most ADAM family members, ADAM8 (CD156a, MS2) is involved in ectodomain shedding of membrane proteins and is linked to inflammation and neurodegeneration. To identify potential substrates released under these pathologic conditions, we screened 10-mer peptides representing amino acid sequences from extracellular domains of various membrane proteins using the ProteaseSpot™ system. A soluble ADAM8 protease containing a pro- and metalloprotease domain was expressed in E. coli and purified as active protease owing to autocatalytic prodomain removal. From 34 peptides tested in the peptide cleavage assay, significant cleavage by soluble ADAM8 was observed for 14 peptides representing membrane proteins with functions in inflammation and neurodegeneration, among them the β-amyloid precursor protein (APP). The in vivo relevance of the ProteaseSpot™ method was confirmed by cleavage of full-length APP with ADAM8 in human embryonic kidney 293 cells expressing tagged APP. ADAM8 cleaved APP with similar efficiency as ADAM10, whereas the inactive ADAM8 mutant did not. Exchanging amino acids at defined positions in the cleavage sequence of myelin basic protein (MBP) revealed sequence criteria for ADAM8 cleavage. Taken together, the results allowed us to identify novel candidate substrates that could be cleaved by ADAM8 in vivo under pathologic conditions.

The Metalloprotease-Disintegrin ADAM8 Is Essential for the Development of Experimental Asthma

RATIONALE Expression of the metalloprotease ADAM8 is increased in patients with asthma, but the functional significance of elevated ADAM8 expression in the context of asthma pathogenesis remains elusive. OBJECTIVES To study development of asthma in ADAM8-deficient mice. METHODS Ovalbumin-induced asthma was studied in wild-type, ADAM8-deficient, and ADAM8-chimeric mice. Lung inflammation was assessed by histology, analysis of bronchoalveolar lavage, and airway hyperresponsiveness. MEASUREMENTS AND MAIN RESULTS ADAM8-deficient mice are highly resistant to the development of ovalbumin-induced airway inflammation and hyperresponsiveness. ADAM8 expression was induced in both hematopoietic cells and the nonhematopoietic microenvironment after induction of asthma, and ADAM8 expression in both cell populations was required for the full manifestation of asthma. Interestingly, loss of ADAM8 on T cells alone was sufficient to significantly decrease the asthma response. The attenuated response was not due to an intrinsic defect in antigen presentation or cytokine production but reflected an impaired migration of T cells, eosinophils, CD11b(+) CD11c(-), and CD11c(+) cells from blood vessels to the lung and alveolar space, suggesting a general hematopoietic cell deficiency in the absence of ADAM8. CONCLUSIONS The results show that ADAM8 plays a proinflammatory role in airway inflammation. The milder disease outcome in the absence of ADAM8 suggests that this protein might be an interesting new target in treatment of this, and potentially other, inflammatory diseases in which recruitment of inflammatory cells is an essential part of pathogenesis.

Tumor Necrosis Factor-α (TNF-α) Regulates Shedding of TNF-α Receptor 1 by the Metalloprotease-Disintegrin ADAM8: Evidence for a Protease-Regulated Feedback Loop in Neuroprotection

Tumor necrosis factor α (TNF-α) is a potent cytokine in neurodegenerative disorders, but its precise role in particular brain disorders is ambiguous. In motor neuron (MN) disease of the mouse, exemplified by the model wobbler (WR), TNF-α causes upregulation of the metalloprotease-disintegrin ADAM8 (A8) in affected brain regions, spinal cord, and brainstem. The functional role of A8 during MN degeneration in the wobbler CNS was investigated by crossing WR with A8-deficient mice: a severely aggravated neuropathology was observed for A8-deficient WR compared with WR A8+/− mice, judged by drastically reduced survival [7 vs 81% survival at postnatal day 50 (P50)], accelerated force loss in the forelimbs, and terminal akinesis. In vitro protease assays using soluble A8 indicated specific cleavage of a TNF-α receptor 1 (p55 TNF-R1) but not a TNF-R2 peptide. Cleavage of TNF-R1 was confirmed in situ, because levels of soluble TNF-R1 were increased in spinal cords of standard WR compared with wild-type mice but not in A8-deficient WR mice. In isolated primary neurons and microglia, TNF-α-induced TNF-R1 shedding was dependent on the A8 gene dosage. Furthermore, exogenous TNF-α showed higher toxicity for cultured neurons from A8-deficient than for those from wild-type mice, demonstrating that TNF-R1 shedding by A8 is neuroprotective. Our results indicate an essential role for ADAM8 in modulating TNF-α signaling in CNS diseases: a feedback loop integrating TNF-α, ADAM8, and TNF-R1 shedding as a plausible mechanism for TNF-α mediated neuroprotection in situ and a rationale for therapeutic intervention.

Deficiency of the Metalloproteinase-Disintegrin ADAM8 Is Associated with Thymic Hyper-Cellularity

Background Thymopoiesis requires thymocyte-stroma interactions and proteases that promote cell migration by degrading extracellular matrix and releasing essential cytokines and chemokines. A role for several members of the A Disintegrin and Metalloprotease (ADAM) family in T cell development has been reported in the past. Methodology/Principal Findings Here, we present data indicating that the family member ADAM8 plays a role in thymic T cell development. We used qrtPCR on FACS sorted thymic subsets together with immunofluorescene to analyze thymic ADAM8 expression. We found that ADAM8 was expressed in murine thymic stromal cells and at lower levels in thymocytes where its expression increased as cell matured, suggesting involvement of ADAM8 in thymopoiesis. Further flow cytometry analysis revealed that ADAM8 deficient mice showed normal development and expansion of immature thymocyte subsets. There was however an intrathymic accumulation of single positive CD4 and CD8 T cells which was most noticeable in the late mature T cell subsets. Accumulation of single positive T cells coincided with changes in the thymic architecture manifest in a decreased cortex/medulla ratio and an increase in medullary epithelial cells as determined by histology and flow cytometry. The increase in single positive T cells was thymus-intrinsic, independent of progenitor homing to the thymus or thymic exit rate of mature T cells. Chemotaxis assays revealed that ADAM8 deficiency was associated with reduced migration of single positive thymocytes towards CCL21. Conclusions/Significance Our results show that ADAM8 is involved in T cell maturation in the medulla and suggest a role for this protease in fine-tuning maturation of thymocytes in the medulla. In contrast to ADAM10 and ADAM17 lack of ADAM8 appears to have a relatively minor impact on T cell development, which was unexpected given that maturation of thymocytes is dependent on proper localization and timing of migration.

ADAM8/MS2/CD156a

From all ADAM family members known, interesting features of some members of this the family are is their distinct expression patterns. ADAM8 is such an example, as it was identified originally in monocytes and is expressed in many specialised cell types, among them macrophages, B-cells, granulocytes, follicle cells, glandular epithelial cells, osteoclasts, oligodendrocytes, microglia, neurons and astrocytes. ADAM8 is activated by autocatalytic prodomain removal and the substrates like the Close Homologue of L1 (CHL1) and CD23 identified so far are either involved in cell adhesion or immune responses. In turn, ADAM8 expression in some cell types such as macrophages, astrocytes and microglia is regulated by inflammatory mediators including tumor necrosis factor-α, lipopolysaccharides (LPS) and prostaglandins. Whereas embryonal development in ADAM8 deficient mice appears normal, its upregulation under inflammatory conditions like that seen in chronic neurodegeneration, after administration of LPS and in allergic asthma, seems to reflect a specific function of ADAM8 in cytokine response. From recent experiments it can be concluded that the ADAM8 induction by inflammatory cytokines serves protective functions, e.g. by shedding of receptors mediating inflammatory responses or by degrading immune mediators directly.