Real-time imaging of vascular endothelial-cadherin during leukocyte transmigration across endothelium

Vascular endothelial-cadherin (VE-cadherin) is a component of the adherens junctions of endothelial cells whose role in endothelial transmigration of leukocytes has been controversial. Using a VE-cadherin/green fluorescent protein fusion construct (VEcadGFP) that mimics the native molecule, we visualized alterations in endothelial junctional structure in real time during transmigration of human neutrophils and monocytes in an in vitro flow model. We observed abundant transmigration occurring exclusively at the cell borders (paracellularly). Surprisingly, transmigration occurred both through de novo formation of transient gaps in VEcadGFP junctional distribution, and also through preexisting gaps. De novo gaps 4-6 microm in size were formed after a leukocyte arrived at a junction, whereas preexisting gaps were present even before the leukocyte had interacted with the endothelial cells contributing to a junction. Gaps rapidly resealed within 5 min after leukocyte transmigration. Migrating leukocytes appeared to push aside VEcadGFP in the plane of the junction, and this displaced material subsequently diffused back to refill the junction. To our knowledge, this is the first example where molecular events at the lateral junction have been tracked in real time during transmigration.     

Peptide-mediated inhibition of neutrophil transmigration by blocking CD47 interactions with signal regulatory protein alpha

CD47, a cell surface transmembrane Ig superfamily member, is an extracellular ligand for signal regulatory protein (SIRPalpha). Interactions between CD47 and SIRPalpha regulate many important immune cell functions including neutrophil (PMN) transmigration. Here we report identification of a novel function-blocking peptide, CERVIGTGWVRC, that structurally mimics an epitope on CD47 and binds to SIRPalpha. The CERVIGTGWVRC sequence was identified by panning phage display libraries on the inhibitory CD47 mAb, C5D5. In vitro PMN migration assays demonstrated that peptide CERVIGTGWVRC specifically inhibited PMN migration across intestinal epithelial monolayers and matrix in a dose-dependent fashion. Further studies using recombinant proteins indicated that the peptide specifically blocks CD47 and SIRPalpha binding in a dose-dependent fashion. Protein binding assays using SIRPalpha domain-specific recombinant proteins demonstrated that this peptide directly bound to the distal-most Ig loop of SIRPalpha, the same loop where CD47 binds. In summary, these findings support the relevance of CD47-SIRPalpha interactions in regulation of PMN transmigration and provide structural data predicting the key residues involved on the surface of CD47. Such peptide reagents may be useful for studies on experimental models of inflammation and provide a template for the design of anti-inflammatory agents.     

Non-bilayer lipids are required for efficient protein transport across the plasma membrane of Escherichia coli.

The construction of a mutant Escherichia coli strain which cannot synthesize phosphatidylethanolamine provides a tool to study the involvement of non-bilayer lipids in membrane function. This strain produces phosphatidylglycerol and cardiolipin (CL) as major membrane constituents and requires millimolar concentrations of divalent cations for growth. In this strain, the lipid phase behaviour is tightly regulated by adjustment of the level of CL which favours a nonbilayer organization in the presence of specific divalent cations. We have used an in vitro system of inverted membrane vesicles to study the involvement of non-bilayer lipids in protein translocation in the secretion pathway. In this system, protein translocation is very low in the absence of divalent cations but can be enhanced by inclusion of Mg2+, Ca2+ or Sr2+ but not by Ba2+ which is unable to sustain growth of the mutant strain and cannot induce a non-bilayer phase in E. coli CL dispersions. Alternatively, translocation in cation depleted vesicles could be increased by incorporation of the non-bilayer lipid DOPE (18:1) but not by DMPE (14:0) or DOPC (18:1), both of which are bilayer lipids under physiological conditions. We conclude that non-bilayer lipids are essential for efficient protein transport across the plasma membrane of E. coli.     

CD47 mediates post-adhesive events required for neutrophil migration across polarized intestinal epithelia

Transepithelial migration of neutrophils (PMN) is a defining characteristic of active inflammatory states of mucosal surfaces. The process of PMN transepithelial migration, while dependent on the neutrophil beta 2 integrin CD11b/CD18, remains poorly understood. In these studies, we define a monoclonal antibody, C5/D5, raised against epithelial membrane preparations, which markedly inhibits PMN migration across polarized monolayers of the human intestinal epithelial cell line T84 in a bidirectional fashion. In T84 cells, the antigen defined by C5/D5 is upregulated by epithelial exposure to IFN-gamma, and represents a membrane glycoprotein of approximately 60 kD that is expressed on the basolateral membrane. While transepithelial migration of PMN was markedly inhibited by either C5/D5 IgG or C5/D5 Fab fragments, the antibody failed to inhibit both adhesion of PMN to T84 monolayers and adhesion of isolated T84 cells to the purified PMN integrin, CD11b/CD18. Thus, epithelial-PMN interactions blocked by C5/D5 appear to be downstream from initial CD11b/CD18-mediated adhesion of PMN to epithelial cells. Purification, microsequence analysis, and cross-blotting experiments indicate that the C5/D5 antigen represents CD47, a previously cloned integral membrane glycoprotein with homology to the immunoglobulin superfamily. Expression of the CD47 epitope was confirmed on PMN and was also localized to the basolateral membrane of normal human colonic epithelial cells. While C5/D5 IgG inhibited PMN migration even in the absence of epithelial, preincubation of T84 monolayers with C5/D5 IgG followed by antibody washout also resulted in inhibition of transmigration. These results suggest the presence of both neutrophil and epithelial components to CD47-mediated transepithelial migration. Thus, CD47 represents a potential new therapeutic target for downregulating active inflammatory disease of mucosal surfaces.     

Intra-endodermal interactions are required for pancreatic beta cell induction

The cellular origin of signals that regulate pancreatic beta cell induction is not clearly defined. Here, we investigate the seeming paradox that Hedgehog/Smoothened signaling functions during gastrulation to promote pancreatic beta cell development in zebrafish, yet has an inhibitory role during later stages of pancreas development in amniotes. Our cell transplantation experiments reveal that in zebrafish, Smoothened function is not required in beta cell precursors. At early somitogenesis stages, when the zebrafish endoderm first forms a sheet, pancreatic beta cell precursors lie closest to the midline; however, the requirement for Smoothened lies in their lateral neighbors, which ultimately give rise to the exocrine pancreas and intestine. Thus, pancreatic beta cell induction requires Smoothened function cell-nonautonomously during gastrulation, to allow subsequent intra-endodermal interactions. These results clarify the function of Hedgehog signaling in pancreas development, identify an unexpected cellular source of factors that regulate beta cell specification, and uncover complex patterning and signaling interactions within the endoderm.     

Common lymphatic endothelial and vascular endothelial receptor-1 mediates the transmigration of regulatory T cells across human hepatic sinusoidal endothelium

The common lymphatic endothelial and vascular endothelial receptor (CLEVER-1; also known as FEEL-1 and stabilin-1) is a recycling and intracellular trafficking receptor with multifunctional properties. In this study, we demonstrate increased endothelial expression of CLEVER-1/stabilin-1 at sites of leukocyte recruitment to the inflamed human liver including sinusoids, septal vessels, and lymphoid follicles in inflammatory liver disease and tumor-associated vessels in hepatocellular carcinoma. We used primary cultures of human hepatic sinusoidal endothelial cells (HSEC) to demonstrate that CLEVER-1/stabilin-1 expression is enhanced by hepatocyte growth factor but not by classical proinflammatory cytokines. We then showed that CLEVER-1/stabilin-1 supports T cell transendothelial migration across HSEC under conditions of flow with strong preferential activity for CD4 FoxP3(+) regulatory T cells (Tregs). CLEVER-1/stabilin-1 inhibition reduced Treg transendothelial migration by 40% and when combined with blockade of ICAM-1 and vascular adhesion protein-1 (VAP-1) reduced it by >80%. Confocal microscopy demonstrated that 60% of transmigrating Tregs underwent transcellular migration through HSEC via ICAM-1- and VAP-1-rich transcellular pores in close association with CLEVER-1/stabilin-1. Thus, CLEVER-1/stabilin-1 and VAP-1 may provide an organ-specific signal for Treg recruitment to the inflamed liver and to hepatocellular carcinoma.     

Signal regulatory protein (SIRPalpha), a cellular ligand for CD47, regulates neutrophil transmigration.

Recent studies have demonstrated that CD47 plays an important role in regulating human neutrophil (PMN) chemotaxis. Two ligands for CD47, thrombospondin and SIRPalpha, have been described. However, it is not known if SIRP-CD47 interactions play a role in regulating PMN migration. In this study, we show that SIRPalpha1 directly binds to the immunoglobulin variable domain loop of purified human CD47 and that such SIRP-CD47 interactions regulate PMN transmigration. Specifically, PMN migration across both human epithelial monolayers and collagen-coated filters was partially inhibited by anti-SIRP monoclonal antibodies. Similar kinetics of inhibition were observed for PMN transmigration in the presence of soluble, recombinant CD47 consisting of the SIRP-binding loop. In contrast, anti-CD47 monoclonal antibodies inhibited PMN transmigration by markedly different kinetics. Results of signal transduction experiments suggested differential regulation of PMN migration by SIRP versus CD47 by phosphatidylinositol 3-kinase and tyrosine kinases, respectively. Immunoprecipitation followed by Western blotting after SDS-PAGE under nonreducing conditions suggested that several SIRP protein species may be present in PMN. Stimulation of PMN with fMLP resulted in increased surface expression of these SIRP proteins, consistent with the existence of intracellular pools. Taken together, these results demonstrate that PMN migration is regulated by CD47 through SIRPalpha-dependent and SIRPalpha-independent mechanisms.     

Duffy antigen facilitates movement of chemokine across the endothelium in vitro and promotes neutrophil transmigration in vitro and in vivo

The Duffy Ag expressed on RBCs, capillaries, and postcapillary venular endothelial cells binds selective CXC and CC chemokines with high affinity. Cells transfected with the Duffy Ag internalize but do not degrade chemokine ligand. It has been proposed that Duffy Ag transports chemokines across the endothelium. We hypothesized that Duffy Ag participates in the movement of chemokines across the endothelium and, by doing so, modifies neutrophil transmigration. We found that the Duffy Ag transfected into human endothelial cells facilitates movement of the radiolabeled CXC chemokine, growth related oncogene-alpha/CXC chemokine ligand 1 (GRO-alpha/CXCL1), across an endothelial monolayer. In addition, neutrophil migration toward GRO-alpha/CXCL1 and IL-8 (IL-8/CXCL8) was enhanced across an endothelial monolayer expressing the Duffy Ag. Furthermore, GRO-alpha/CXCL1 stimulation of endothelial cells expressing the Duffy Ag did not affect gene expression by oligonucleotide microarray analysis. These in vitro observations are supported by the finding that IL-8/CXCL8-driven neutrophil recruitment into the lungs was markedly attenuated in transgenic mice lacking the Duffy Ag. We conclude that Duffy Ag has a role in enhancing leukocyte recruitment to sites of inflammation by facilitating movement of chemokines across the endothelium.     

Nuclear cytoplasmic shuttling by thyroid hormone receptors. multiple protein interactions are required for nuclear retention

In this report, we have studied the intracellular dynamics and distribution of the thyroid hormone receptor-beta (TRbeta) in living cells, utilizing fusions to the green fluorescent protein. Wild-type TRbeta was mostly nuclear in both the absence and presence of triiodothyronine; however, triiodothyronine induced a nuclear reorganization of TRbeta. By mutating defined regions of TRbeta, we found that both nuclear corepressor and retinoid X receptor are involved in maintaining the unliganded receptor within the nucleus. A TRbeta mutant defective in DNA binding had only a slightly altered nuclear/cytoplasmic distribution compared with wild-type TRbeta; thus, site-specific DNA binding is not essential for maintaining TRbeta within the nucleus. Both ATP depletion studies and heterokaryon analysis demonstrated that TRbeta rapidly shuttles between the nuclear and the cytoplasmic compartments. Cotransfection of nuclear corepressor and retinoid X receptor markedly decreased the shuttling by maintaining unliganded TRbeta within the nucleus. In summary, our findings demonstrate that TRbeta rapidly shuttles between the nucleus and the cytoplasm and that protein-protein interactions of TRbeta with various cofactors, rather than specific DNA interactions, play the predominant role in determining the intracellular distribution of the receptor.     

Effect of endothelial cell polarity on beta-amyloid-induced migration of monocytes across normal and AD endothelium

During normal aging and amyloid beta-peptide (Abeta) disorders such as Alzheimer's disease (AD), one finds increased deposition of Abeta and activated monocytes/microglial cells in the brain. Our previous studies show that Abeta interaction with a monolayer of normal human brain microvascular endothelial cells results in increased adherence and transmigration of monocytes. Relatively little is known of the role of Abeta accumulated in the AD brain in mediating trafficking of peripheral blood monocytes (PBM) across the blood-brain barrier (BBB) and concomitant accumulation of monocytes/microglia in the AD brain. In this study, we showed that interaction of Abeta(1--40) with apical surface of monolayer of brain endothelial cells (BEC), derived either from normal or AD individuals, resulted in increased transendothelial migration of monocytic cells (HL-60 and THP-1) and PBM. However, transmigration of monocytes across the BEC monolayer cultivated in a Transwell chamber was increased 2.5-fold when Abeta was added to the basolateral side of AD compared with normal individual BEC. The Abeta-induced transmigration of monocytes was inhibited in both normal and AD-BEC by antibodies to the putative Abeta receptor, receptor for advanced glycation end products (RAGE), and to the endothelial cell junction molecule, platelet-endothelial cell adhesion molecule-1 (PECAM-1). We conclude that interaction of Abeta with the basolateral surface of AD-BEC induces cellular signaling, promoting transmigration of monocytes from the apical to basolateral direction. We suggest that Abeta in the AD brain parenchyma or cerebrovasculature initiates cellular signaling that induces PBM to transmigrate across the BBB and accumulate in the brain.     

Amino-terminal regions of polyomavirus middle T antigen are required for interactions with protein phosphatase 2A.

Polyomavirus middle T antigen (MT) is the major transforming protein of the virus. It functions through interactions with a number of cellular proteins involved in cell proliferation. MT forms complexes with protein phosphatase 2A (PP2A), pp60c-src, phosphatidylinositol 3-kinase, and Shc. We introduced both deletion and point mutations into three regions of MT and examined their ability to associate with PP2A and pp60c-src. The first 25 amino acid residues of MT are required for association with PP2A and pp60c-src. Amino acids 105 to 111, comprising the sequence Cys-Arg-Met-Pro-Leu-Thr-Cys, is also required for complex formation between MT and PP2A. However, the sequence Asp-Lys-Gly-Gly (amino acids 44 to 47), also found in the B subunit of PP2A, is dispensable for complex formation between MT and PP2A. We find a strict correlation between the ability of MT to associate with PP2A and the ability of MT to associate with pp60c-src. One mutant, L5E, associates with a phosphatase other than PP2A, pp60c-src, and phosphatidylinositol 3-kinase in a manner similar to that of wild-type MT yet is reduced in its transforming ability on NIH 3T3 cells.     

Two catalytic domains are required for protein deacetylation

Histone deacetylase (HDAC)-6 was recently identified as a dual substrate, possibly multisubstrate, deacetylase that can act both on acetylated histone tails and on alpha-tubulin acetylated on Lys40. HDAC-6 is unique among deacetylases in having two hdac domains, and we have used this enzyme as a useful model to dissect the structural requirements for the deacetylation reaction. In this report, we show that both hdac domains are required for the intact deacetylase activity of HDAC-6 in vitro and in vivo. The spatial arrangement of these two domains in HDAC-6 is essential and alteration of the linker region between the two domains severely affects the catalytic activity. Artificial chimeric HDACs, made by replacing the hdac domains in HDAC-6 with corresponding domains from other class II HDACs, show de novo deacetylase activity. Taken together, our results demonstrate for the first time that the spatial arrangement of hdac domains is critical for in vivo deacetylation reaction and may provide a useful model for the development of novel HDAC inhibitors.     

Evidence for functional protein interactions required for poliovirus RNA replication

Poliovirus protein 2C contains a predicted N-terminal amphipathic helix that mediates association of the protein with the membranes of the viral RNA replication complex. A chimeric virus that contains sequences encoding the 18-residue core from the orthologous amphipathic helix from human rhinovirus type 14 (HRV14) was constructed. The chimeric virus exhibited defects in viral RNA replication and produced minute plaques on HeLa cell monolayers. Large plaque variants that contained mutations within the 2C-encoding region were generated upon subsequent passage. However, the majority of viruses that emerged with improved growth properties contained no changes in the region encoding 2C. Sequence analysis and reconstruction of genomes with individual mutations revealed changes in 3A or 2B sequences that compensated for the HRV14 amphipathic helix in the polio 2C-containing proteins, implying functional interactions among these proteins during the replication process. Direct binding between these viral proteins was confirmed by mammalian cell two-hybrid analysis.     

Protein kinase C delta is required for p47phox phosphorylation and translocation in activated human monocytes

Our laboratory is interested in understanding the regulation of NADPH oxidase activity in human monocyte/macrophages. Protein kinase C (PKC) is reported to be involved in regulating the phosphorylation of NADPH oxidase components in human neutrophils; however, the regulatory roles of specific isoforms of PKC in phosphorylating particular oxidase components have not been determined. In this study calphostin C, an inhibitor for both novel PKC (including PKCdelta, -epsilon, -theta;, and -eta) and conventional PKC (including PKCalpha and -beta), inhibited both phosphorylation and translocation of p47phox, an essential component of the monocyte NADPH oxidase. In contrast, GF109203X, a selective inhibitor of classical PKC and PKCepsilon, did not affect the phosphorylation or translocation of p47phox, suggesting that PKCdelta, -theta;, or -eta is required. Furthermore, rottlerin (at doses that inhibit PKCdelta activity) inhibited the phosphorylation and translocation of p47phox. Rottlerin also inhibited O2 production at similar doses. In addition to pharmacological inhibitors, PKCdelta-specific antisense oligodeoxyribonucleotides were used. PKCdelta antisense oligodeoxyribonucleotides inhibited the phosphorylation and translocation of p47phox in activated human monocytes. We also show, using the recombinant p47phox-GST fusion protein, that p47phox can serve as a substrate for PKCdelta in vitro. Furthermore, lysate-derived PKCdelta from activated monocytes phosphorylated p47phox in a rottlerin-sensitive manner. Together, these data suggest that PKCdelta plays a pivotal role in stimulating monocyte NADPH oxidase activity through its regulation of the phosphorylation and translocation of p47phox.     

Temperature dependence of protein transport across lymphatic endothelium in vitro

The purpose of the work was to develop an in vitro model for the study of lymphatic endothelium and to determine, using this model, whether or not a cytoplasmic process may be involved in transendothelial transport. Segments of canine renal hilar lymphatics were dissected clean, cannulated at both ends, and transferred to a perfusion chamber for measurement of transendothelial protein transport and for ultrastructural tracer studies. The segments were subsequently processed for light and electron microscopy. By both structural and functional criteria the lymphatics were judged to have retained their integrity. At 37 degrees C, 36 lymphatics showed a mean rate of protein transport of 3.51 +/- 0.45 (SEM) micrograms/min per cm2 of lymphatic endothelium. The rate was influenced by the temperature of the system, being significantly reduced by 49% +/- 4.8, 31% +/- 5.3, and 29% +/- 3.9 when the temperature was lowered to 4 degrees, 24 degrees, and 30 degrees C, respectively. When the temperature was raised to 40 degrees C, the rate was significantly increased by 48% +/- 12.2. The vesicular system and the intercellular regions in vessels with increased or reduced rates of transport were analyzed quantitatively to ascertain whether the rate changes could be correlated with ultrastructurally demonstrable changes in either of these postulated pathways. No significant changes in junctional or vesicular parameters were found between the control lymphatics and those perfused at 24 degrees, 30 degrees, and 40 degrees C. At 4 degrees C, the temperature at which the rate of protein transport was maximally reduced, vesicular size decreased, and the number of free cytoplasmic vesicles increased, whereas the number associated with the abluminal and luminal surfaces decreased. We concluded that isolated perfused lymphatic segments transport protein at a relatively constant rate under control conditions, and that this transendothelial transport comprises both temperature-dependent and temperature-independent mechanisms. The findings were considered in terms of the different theories of lymph formation and were interpreted as providing support for the vesicular theory.     

An ATP-binding membrane protein is required for protein translocation across the endoplasmic reticulum membrane.

The role of nucleotides in providing energy for polypeptide transfer across the endoplasmic reticulum (ER) membrane is still unknown. To address this question, we treated ER-derived mammalian microsomal vesicles with a photoactivatable analogue of ATP, 8-N3ATP. This treatment resulted in a progressive inhibition of translocation activity. Approximately 20 microsomal membrane proteins were labeled by [alpha 32P]8-N3ATP. Two of these were identified as proteins with putative roles in translocation, alpha signal sequence receptor (SSR), the 35-kDa subunit of the signal sequence receptor complex, and ER-p180, a putative ribosome receptor. We found that there was a positive correlation between inactivation of translocation activity and photolabeling of alpha SSR. In contrast, our data demonstrate that the ATP-binding domain of ER-p180 is dispensable for translocation activity and does not contribute to the observed 8-N3ATP sensitivity of the microsomal vesicles.     

Multiple protein phosphatases are required for mitosis in Drosophila

BACKGROUND: Approximately one-third of the Drosophila kinome has been ascribed some cell-cycle function. However, little is known about which of its 117 protein phosphatases (PPs) or subunits have counteracting roles. RESULTS: We investigated mitotic roles of PPs through systematic RNAi. We found that G(2)-M progression requires Puckered, the JNK MAP-kinase inhibitory phosphatase and PP2C in addition to string (Cdc25). Strong mitotic arrest and chromosome congression failure occurred after Pp1-87B downregulation. Chromosome alignment and segregation defects also occurred after knockdown of PP1-Flapwing, not previously thought to have a mitotic role. Reduction of several nonreceptor tyrosine phosphatases produced spindle and chromosome behavior defects, and for corkscrew, premature chromatid separation. RNAi of the dual-specificity phosphatase, Myotubularin, or the related Sbf "antiphosphatase" resulted in aberrant mitotic chromosome behavior. Finally, for PP2A, knockdown of the catalytic or A subunits led to bipolar monoastral spindles, knockdown of the Twins B subunit led to bridged and lagging chromosomes, and knockdown of the B' Widerborst subunit led to scattering of all mitotic chromosomes. Widerborst was associated with MEI-S332 (Shugoshin) and required for its kinetochore localization. CONCLUSIONS: We identify cell-cycle roles for 22 of 117 Drosophila PPs. Involvement of several PPs in G(2) suggests multiple points for its regulation. Major mitotic roles are played by PP1 with tyrosine PPs and Myotubularin-related PPs having significant roles in regulating chromosome behavior. Finally, depending upon its regulatory subunits, PP2A regulates spindle bipolarity, kinetochore function, and progression into anaphase. Discovery of several novel cell-cycle PPs identifies a need for further studies of protein dephosphorylation.     

Spermatogenic cell-somatic cell interactions are required for maintenance of spermatogenic cell glutathione

Sertoli cells play a major role in the regulation of spermatogenic cell energy metabolism and differentiation. This study demonstrates that Sertoli cells are essential for the maintenance of spermatogenic cell glutathione (GSH), an important intracellular reductant and detoxicant. Primary spermatocytes and round spermatids isolated from Xenopus laevis contained 1.5 +/- 0.1 mM GSH, but sperm lacked detectable GSH. During a 5-day culture period, isolated spermatocytes and spermatids lost 80% of the initial GSH (t 1/2 = 55 h). The levels of GSH were unaffected by L-buthionine-SR-sulfoximine (BSO), a selective inhibitor of GSH synthesis. Cultures of testicular lobules and spermatocysts (composed of germ cells and Sertoli cells) depleted of interstitial tissue lost only 30% of their initial GSH in 4.5 days; the GSH levels decreased during treatment with BSO. Spermatogenic cells in cultured testes maintained their GSH levels for 7 days by a BSO-sensitive mechanism. These results demonstrate that the intracellular GSH levels of spermatogenic cells are dependent upon germ cell-somatic cell interactions. Spermatogenic cells were shown to possess gamma-glutamyl transpeptidase, glutathione synthetase, 5-oxoprolinase, and gamma-glutamylcysteine synthetase activities. [35S] Cysteine incorporation and distribution as analyzed by high performance liquid chromatography (HPLC) showed that isolated spermatogenic cells are capable of GSH synthesis. The rate of GSH synthesis, however, was insufficient to compensate for GSH turnover. These results demonstrate that production of spermatogenic cell GSH is dependent upon Sertoli cells. To our knowledge, this is the first evidence that interactions between different cell types may be of significance in GSH metabolism.