Stefan Butz

Contribution of Proteasome-Mediated Proteolysis to the Hierarchy of Epitopes Presented by Major Histocompatibility Complex Class I Molecules

Major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL) recognize peptide epitopes of protein antigens in a hierarchical fashion. We investigated whether proteolytic cleavage, in particular by proteasomes, is important in determining epitope hierarchy. Using highly purified 20S proteasomes, we find preferred cleavage sites directly adjacent to the N- and C-terminal ends of the immunodominant epitope of chicken ovalbumin, Ova257-264, while most of the subdominant epitope, Ova55-62, is destroyed by a major cleavage site located within this epitope. Moreover, we show that variations in amino acid sequences flanking these epitopes influence proteasomal cleavage patterns in parallel with the efficacy of their presentation. The results suggest that proteasomal cleavage within and adjacent to class I-restricted epitopes contributes to their level of presentation.

GDF-15 is an inhibitor of leukocyte integrin activation required for survival after myocardial infarction in mice

Inflammatory cell recruitment after myocardial infarction needs to be tightly controlled to permit infarct healing while avoiding fatal complications such as cardiac rupture. Growth differentiation factor-15 (GDF-15), a transforming growth factor-β (TGF-β)–related cytokine, is induced in the infarcted heart of mice and humans. We show that coronary artery ligation in Gdf15-deficient mice led to enhanced recruitment of polymorphonuclear leukocytes (PMNs) into the infarcted myocardium and an increased incidence of cardiac rupture. Conversely, infusion of recombinant GDF-15 repressed PMN recruitment after myocardial infarction. In vitro, GDF-15 inhibited PMN adhesion, arrest under flow and transendothelial migration. Mechanistically, GDF-15 counteracted chemokine-triggered conformational activation and clustering of β2 integrins on PMNs by activating the small GTPase Cdc42 and inhibiting activation of the small GTPase Rap1. Intravital microscopy in vivo in Gdf15-deficient mice showed that Gdf-15 is required to prevent excessive chemokine-activated leukocyte arrest on the endothelium. Genetic ablation of β2 integrins in myeloid cells rescued the mortality of Gdf15-deficient mice after myocardial infarction. To our knowledge, GDF-15 is the first cytokine identified as an inhibitor of PMN recruitment by direct interference with chemokine signaling and integrin activation. Loss of this anti-inflammatory mechanism leads to fatal cardiac rupture after myocardial infarction.

The junctional adhesion molecule (JAM) family members JAM-2 and JAM-3 associate with the cell polarity protein PAR-3: a possible role for JAMs in endothelial cell polarity.

Tight junctions play a central role in the establishment of cell polarity in vertebrate endothelial and epithelial cells. A ternary protein complex consisting of the cell polarity proteins PAR-3 and PAR-6 and the atypical protein kinase C localizes at tight junctions and is crucial for tight junction formation. We have recently shown that PAR-3 directly associates with the junctional adhesion molecule (JAM), which suggests that the ternary complex is targeted to tight junctions of epithelial cells through PAR-3 binding to JAM. The expression of JAM-related proteins by endothelial cells prompted us to test whether recruitment of the ternary complex in endothelial cells can occur through binding to JAM-2, JAM-3, endothelial cell-selective adhesion molecule (ESAM) or coxsackie- and adenovirus receptor (CAR). Here we show that the two JAM-related proteins JAM-2 and JAM-3 directly associate with PAR-3. The association between PAR-3 and JAM-2/-3 is mediated through the first PDZ domain of PAR-3. In agreement with the predominant expression of JAM-2 and JAM-3 in endothelial cells, we found that PAR-3 is expressed by endothelial cells in vivo and is localized at cell contacts of cultured endothelial cells. PAR-3 associates with JAM-2/-3 but not with the JAM-related Ig-superfamily members ESAM or CAR. In addition, we show that the tight junction-associated protein ZO-1 associates with JAM-2/-3 in a PDZ domain-dependent manner. Using ectopic expression of JAM-2 in CHO cells, we show that the junctional localization of JAM-2 is regulated by serine phosphorylation and that its clustering at cell-cell contacts recruits endogenous PAR-3 and ZO-1. Our findings suggest that JAM-2 affects endothelial cell junctions by its regulated clustering at intercellular contacts, and they support a role for JAM-2, and possibly JAM-3, in tight junction formation of endothelial cells.

VE-PTP maintains the endothelial barrier via plakoglobin and becomes dissociated from VE-cadherin by leukocytes and by VEGF

We have shown recently that vascular endothelial protein tyrosine phosphatase (VE-PTP), an endothelial-specific membrane protein, associates with vascular endothelial (VE)–cadherin and enhances VE-cadherin function in transfected cells (Nawroth, R., G. Poell, A. Ranft, U. Samulowitz, G. Fachinger, M. Golding, D.T. Shima, U. Deutsch, and D. Vestweber. 2002. EMBO J. 21:4885–4895). We show that VE-PTP is indeed required for endothelial cell contact integrity, because down-regulation of its expression enhanced endothelial cell permeability, augmented leukocyte transmigration, and inhibited VE-cadherin–mediated adhesion. Binding of neutrophils as well as lymphocytes to endothelial cells triggered rapid (5 min) dissociation of VE-PTP from VE-cadherin. This dissociation was only seen with tumor necrosis factor α–activated, but not resting, endothelial cells. Besides leukocytes, vascular endothelial growth factor also rapidly dissociated VE-PTP from VE-cadherin, indicative of a more general role of VE-PTP in the regulation of endothelial cell contacts. Dissociation of VE-PTP and VE-cadherin in endothelial cells was accompanied by tyrosine phoshorylation of VE-cadherin, β-catenin, and plakoglobin. Surprisingly, only plakoglobin but not β-catenin was necessary for VE-PTP to support VE-cadherin adhesion in endothelial cells. In addition, inhibiting the expression of VE-PTP preferentially increased tyrosine phosphorylation of plakoglobin but not β-catenin. In conclusion, leukocytes interacting with endothelial cells rapidly dissociate VE-PTP from VE-cadherin, weakening endothelial cell contacts via a mechanism that requires plakoglobin but not β-catenin.

Distinct cadherin—catenin complexes in Ca2+ dependent cell—cell adhesion

Catenins are peripheral cytoplasmic proteins originally identified in association with the mouse epithelial cell adhesion molecule E‐cadherin. Molecular cloning and primary structure analysis demonstrated that α‐catenin is homologous to vinculin and that β‐catenin is homologous to human plakoglobin and the Drosophila gene product armadillo. With the use of peptide‐specific anti plakoglobin antibodies we confirm here that plakoglobin is a component of the cadherin‐catenin complex and that it is most likely identical to γ‐catenin. We show that plakoglobin binds directly to E‐cadherin. We consolidate the biochemical evidence for the existence of two distinct and separable E‐cadherin‐catenin complexes in the same cell. One complex is composed of E‐cadherin, α‐and β‐catenin, the other of E‐cadherin, α‐catenin and plakoglobin. A similar distinct association with catenins is also found for other cadherins. Comparison of different cell lines revealed that the relative amounts of the two complexes vary depending on cell types.

Coxsackievirus-adenovirus receptor (CAR) is essential for early embryonic cardiac development.

The coxsackievirus-adenovirus receptor (CAR) is a cell contact protein on various cell types with unknown physiological function. It belongs to a subfamily of the immunoglobulin-superfamily of which some members are junctional adhesion molecules on epithelial and/or endothelial cells. CAR is dominantly expressed in the hearts and brains of mice until the newborne phase after which it becomes mainly restricted to various epithelial cells. To understand more about the physiological function of CAR, we have generated CAR-deficient mice by gene targeting. We found that these mice die between E11.5 and E13.5 of embryonal development. Ultrastructural analysis of cardiomyocytes revealed that the density of myofibrils was reduced and that their orientation and bundling was disorganized. In addition, mitochondria were enlarged and glycogen storage strongly enriched. In line with these defects, we observed pericardial edema formation as a clear sign of insufficient heart function. Developmental abnormalities likely to be secondary effects of gene ablation were the persistent singular cardial atrio-ventricular canal and dilatations of larger blood vessels such as the cardinal veins. The secondary nature of these defects was supported by the fact that CAR was not expressed on vascular cells or on cells of the vascular wall. No obvious signs for alterations of the histological organization of the placenta were observed. We conclude that CAR is required for embryonal heart development, most likely due to its function during the organization of myofibrils in cardiomyocytes.

Expression and cell membrane localization of catenins during mouse preimplantation development.

We have studied transcription, expression, and membrane localization of components of the E‐cadherin‐catenin complex stage by stage during mouse preimplantation development. Maternal E‐cadherin and α‐ and β‐catenin are stored as mRNA and/or protein in unfertilized eggs and are already assembled into a protein complex at this stage. After fertilization, it is likely that they mediate adhesion of early‐stage blastomeres. Biosynthesis of plakoglobin is delayed relative to the other components. The temporal mRNA and protein expression patterns of the components of the cadherin‐catenin complex correlate with the presence or absence of potential cytoplasmic polyadenylation elements (CPEs) in the 3′‐UTRs of the respective cDNAs. Our results suggest that the components of the E‐cadherin‐catenin complex derived from both maternal and zygotic gene activity are increasingly accumulated and stored in a nonfunctional form during early cleavage stages and are ready to be used for compaction and the formation of the trophectodermal cell layer.

A Novel Gene Expression Profile in Lymphatics Associated with Tumor Growth and Nodal Metastasis

Invasion of lymphatic vessels is a key step in the metastasis of primary tumors to draining lymph nodes. Although the process is enhanced by tumor lymphangiogenesis, it is unclear whether this is a consequence of increased lymphatic vessel number, altered lymphatic vessel properties, or both. Here we have addressed the question by comparing the RNA profiles of primary lymphatic endothelial cells (LEC) isolated from the vasculature of normal tissue and from highly metastatic T-241/vascular endothelial growth factor (VEGF)-C fibrosarcomas implanted in C57BL/6 mice. Our findings reveal significant differences in expression of some 792 genes (i.e., >or=2-fold up- or down-regulated, P <or= 0.05) that code for a variety of proteins including components of endothelial junctions, subendothelial matrix, and vessel growth/patterning. The tumor LEC profile, validated by immunohistochemical staining, is distinct from that of normal, inflammatory cytokine, or mitogen-activated LEC, characterized by elevated expression of such functionally significant molecules as the tight junction regulatory protein endothelial specific adhesion molecule (ESAM), the transforming growth factor-beta coreceptor Endoglin (CD105), the angiogenesis-associated leptin receptor, and the immunoinhibitory receptor CD200, and reduced expression of subendothelial matrix proteins including collagens, fibrillin, and biglycan. Moreover, we show similar induction of ESAM, Endoglin, and leptin receptor within tumor lymphatics in a series of human head and neck and colorectal carcinomas, and uncover a dramatic correlation between ESAM expression and nodal metastasis that identifies this marker as a possible prognostic indicator. These findings reveal a remarkable degree of phenotypic plasticity in cancer lymphatics and provide new insight into the processes of lymphatic invasion and lymph node metastasis.

Expression of Catenins during Mouse Embryonic Development and in Adult Tissues

Classical cadherins are cell-surface glycoproteins that mediate calcium-dependent cell adhesion. The cytoplasmic domain of these glycoproteins is linked to the cytoskeleton through the catenins (alpha, beta and gamma). The catenins are intracellular polypeptides that are part of a complex sub-membranous network modulating the adhesive ability of the cells. One approach to elucidate the role of these molecules in the cell is to investigate their distribution during mouse development and in adult tissues. This study reports that catenins are widely expressed but in varying amounts in embryos and adult tissues. The expression of all three catenins is most prominent in the adult heart muscle and in epithelia of all developmental stages. In other embryonic and adult tissues, lower expression of catenins was detected, e.g., in smooth muscle or connective tissue. Catenins are coexpressed with various cadherins in different tissues. Gastrulation is the first time during embryogenesis when a discrepancy occurs between the expression of catenins and E-cadherin. E-cadherin expression is suppressed in mesodermal cells but not the expression of catenins. This discrepancy suggests that another cadherin may interact with catenins. Similarly, E-cadherin is generally expressed in adult liver but not in the regions surrounding the central veins. In contrast, catenins are uniformly expressed in the liver, suggesting that they are associated with other cadherins in E-cadherin negative cells. Finally, the three catenins are not always concurrently expressed. For example, in peripheral nerves, only beta-catenin is observable, and in smooth muscle plakoglobin is not detectable.

Endothelial LSP1 is involved in endothelial dome formation minimizing vascular permeability changes during neutrophil transmigration in vivo

The endothelium actively participates in neutrophil migration out of the vasculature via dynamic, cytoskeleton-dependent rearrangements leading to the formation of transmigratory cups in vitro, and to domes that completely surround the leukocyte in vivo. Leukocyte-specific protein 1 (LSP1), an F-actin-binding protein recently shown to be in the endothelium, is critical for effective transmigration, although the mechanism has remained elusive. Herein we show that endothelial LSP1 is expressed in the nucleus and cytosol of resting endothelial cells and associates with the cytoskeleton upon endothelial activation. Two-photon microscopy revealed that endothelial LSP1 was crucial for the formation of endothelial domes in vivo in response to neutrophil chemokine keratinocyte-derived chemokine (KC) as well as in response to endogenously produced chemokines stimulated by cytokines (tumor necrosis factor α [TNFα] or interleukin-1β [IL-1β]). Endothelial domes were significantly reduced in Lsp1(-/-) compared with wild-type (WT) mice. Lsp1(-/-) animals not only showed impaired neutrophil emigration after KC and TNFα stimulation, but also had disproportionate increases in vascular permeability. We demonstrate that endothelial LSP1 is recruited to the cytoskeleton in inflammation and plays an important role in forming endothelial domes thereby regulating neutrophil transendothelial migration. The permeability data may underscore the physiologic relevance of domes and the role for LSP1 in endothelial barrier integrity.