Expression of human DEC-205 (CD205) multilectin receptor on leukocytes

DEC-205 (CD205) belongs to the macrophage mannose receptor family of C-type lectin endocytic receptors and behaves as an antigen uptake/processing receptor for dendritic cells (DC). To investigate DEC-205 tissue distribution in human leukocytes, we generated a series of anti-human DEC-205 monoclonal antibodies (MMRI-5, 6 and 7), which recognized epitopes within the C-type lectin-like domains 1 and 2, and the MMRI-7 immunoprecipitated a single similar to 200 kDa band, identified as DEC-205 by mass spectrometry. MMRI-7 and another DEC-205 mAb (MG38), which recognized the epitope within the DEC-205 cysteine-rich and fibronectin type II domain, were used to examine DEC-205 expression by human leukocytes. Unlike mouse DEC-205, which is reported to have predominant expression on DC, human DEC-205 was detected by flow cytometry at relatively high levels on myeloid blood DC and monocytes, at moderate levels on B lymphocytes and at low levels on NK cells, plasmacytoid blood DC and T lymphocytes. MMRI-7 F(ab')(2) also labeled monocytes, B lymphocytes and NK cells similarly excluding reactivity due to non-specific binding of the mAb to Fc gamma R. Tonsil mononuclear cells showed a similar distribution of DEC-205 staining on the leukocytes. DEC-205-specific semiquantitative immunoprecipitation/western blot and quantitative reverse transcriptase-PCR analysis established that these leukocyte populations expressed DEC-205 protein and the cognate mRNA. Thus, human DEC-205 is expressed on more leukocyte populations than that were previously assumed based on mouse DEC-205 tissue localization studies. The broader DEC-205 tissue expression in man is relevant to clinical DC targeting strategies and DEC-205 functional studies.

Quantitative analysis of MC1R gene expression in human skin cell cultures

To address the issue of melanocortin-1 receptor (MC1R) expression in non-melanocytic cells, we have quantitatively evaluated the relative expression levels of both MC1R mRNA and protein in a subset of different cell types. Using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) at high cycle numbers, we detected MC1R mRNA in all cell types examined, including human embryonic kidney-293 (HEK 293) cells, a cell type widely used as a negative control in melanocortin expression studies. Quantitative real-time PCR revealed the highest levels of MC1R transcripts were in melanocytic cells, whereas the keratinocyte and fibroblast cell cultures examined had only a low level of expression, similar to that of HEK 293 cells. Antibody mediated detection of MC1R protein in membrane extracts demonstrated exogenous receptor in MC1R transfected cell lines, as well as endogenous MC1R in melanoma cells. However, radioligand binding procedures were required to detect MC1R protein of normal human melanocytes and no surface expression of MC1R was detected in any of the non-melanocytic cells examined. This was consistent with their low level of mRNA, and suggests that, if present, the levels of surface receptor are significantly lower than that in melanocytes. The capacity of such limited levels of MC1R protein to influence non-melanocytic skin cell biology would likely be severely compromised. Indeed, the MC1R agonist [NIe(4), D-Phe(7)] alpha-melanocyte stimulating hormone (NDP-MSH) was unable to elevate intracellular cyclic adenosine monophosphate (cAMP) levels in the keratinocyte and fibroblast cells examined, whereas a robust increase was elicited in melanocytes. Although there are a variety of cell types with detectable MC1R mRNA, the expression of physiologically significant levels of the receptor may be more restricted than the current literature indicates, and within epidermal tissue may be limited to the melanocyte

Effects of pathophysiological concentrations of albumin on NHE3 activity and cell proliferation in primary cultures of human proximal tubule cells

The progression of renal disease correlates strongly with hypertension and the degree of proteinuria, suggesting a link between excessive Na+ reabsorption and exposure of the proximal tubule to protein. The present study investigated the effects of albumin on cell growth and Na+ uptake in primary cultures of human proximal tubule cells (PTC). Albumin (1.0 mg/ml) increased cell proliferation to 134.1 +/- 11.8% (P < 0.001) of control levels with no change in levels of apoptosis. Exposure to 0.1 and 1.0 mg/ml albumin increased total Na-22(+) uptake to 119.1 &PLUSMN; 6.3% (P = 0.005) and 115.6 &PLUSMN; 5.3% (P < 0.006) of control levels, respectively, because of an increase in Na+/H+ exchanger isoform 3 (NHE3) activity. This was associated with an increase in NHE3 mRNA to 161.1 +/- 15.1% (P < 0.005) of control levels in response to 0.1 mg/ml albumin. Using confocal microscopy with a novel antibody raised against the predicted extracellular NH2 terminus of human NHE3, we observed in nonpermeabilized cells that exposure of PTC to albumin (0.1 and 1.0 mg/ml) increased NHE3 at the cell surface to 115.4 &PLUSMN; 2.7% (P < 0.0005) and 122.4 +/- 3.7% (P < 0.0001) of control levels, respectively. This effect was paralleled by significant increases in NHE3 in the subplasmalemmal region as measured in permeabilized cells. These albumin-induced increases in expression and activity of NHE3 in PTC suggest a possible mechanism for Na+ retention in response to proteinuria.

Lectin-induced haemocyte inactivation in insects

Most multimeric lectins are adhesion molecules, promoting attachment and spreading on surface glycodeterminants. In addition, some lectins have counter-adhesion properties, detaching already spread cells which then acquire round or spindle-formed cell shapes. Since lectin-mediated adhesion and detachment is observed in haemocyte-like Drosophila cells, which have haemomucin as the major lectin-binding glycoprotein, the two opposite cell behaviours may be the result of lectin-mediated receptor rearrangements on the cell surface. To investigate oligomeric lectins as a possible extracellular driving force affecting cell shape changes, we examined lectin-mediated reactions in lepidopteran haemocytes after cytochalasin D-treatment and observed that while cell-spreading was dependent on F-actin, lectin-uptake was less dependent on F-actin. We propose a model of cell shape changes involving a dynamic balance between adhesion and uptake reactions. (C) 2004 Elsevier Ltd. All rights reserved.

CCR5-Delta 32 mutation is strongly associated with primary sclerosing cholangitis

CCR5 plays a key role in the distribution of CD45RO+ T cells and contributes to generation of a T helper 1 immune response. CCR5-Delta32 is a 32-bp deletion associated with significant reduction in cell surface expression of the receptor. We investigated the role of CCR5-Delta32 on susceptibility to ulcerative colitis (UC), Crohn's disease ( CD) and primary sclerosing cholangitis (PSC). Genotype and allelic association analyses were performed in 162 patients with UC, 131 with CD, 71 with PSC and 419 matched controls. There was a significant difference in CCR5 genotype (OR 2.27, P = 0.003) between patients with sclerosing cholangitis and controls. Similarly, CCR5-Delta32 allele frequency was significantly higher in sclerosing cholangitis (17.6%) compared to controls (9.9%, OR 2.47, P = 0.007) and inflammatory bowel disease patients without sclerosing cholangitis ( 11.3%, OR 1.9, P = 0.027). There were no significant differences in CCR5 genotype or allele frequency between those with either UC or CD and controls. Genotypes with the CCR5-Delta32 variant were increased in patients with severe liver disease defined by portal hypertension and/or transplantation (45%) compared to those with mild liver disease (21%, OR 3.17, P = 0.03). The CCR5-Delta32 mutation may influence disease susceptibility and severity in patients with PSC.

Investigation of the immunocompetent cells that bind early pregnancy factor and preliminary studies of the early pregnancy factor target molecule

Early pregnancy factor (EPF) is a secreted protein with immunosuppressive and growth factor properties. It has been shown to suppress the delayed-type hypersensitivity response in mice as well as acute and chronic forms of experimental autommume encephalomyelitis in rats and mice, respectively. In previous studies, we have demonstrated that EPF binds to a population of lymphocytes and we hypothesized that it mediates its suppressive effects by binding to CD4(+) T cells. In the present study, we isolated monocytes and subpopulations of lymphocytes and labelled them with fluoresceinated EPF in order to determine which populations bind EPF. We demonstrated that EPF binds specifically to CD4(+), CD8(+), CD14(+) (monocytes) and CD56(+) NK cells but not to CD19(+) B cells. The identity of the molecule(s) on the cell surface that is targeted by EPF is unknown, but as EPF is an extracellular homologue of the intracellular protein chaperonin 10 (Cpn 10), we examined the possibility that the EPF receptor is a membrane-associated form of chaperonin 60 (Cpn60), the functional associate of Cpn 10 within the cell. The EPF target molecule on lymphocytes was visualized by chemical cross-linking of exogenous iodinated Cpn10 to cells and probed with anti-Cpn60. The effect of anti-Cpn60 on activity in the EPF bioassay, the rosette inhibition test, was also examined. In both instances, no specific interaction of this antibody and the putative receptor was observed. It was concluded that the cell surface molecule targeted by EPF is unlikely to be a homologue of Cpn60.

B cell chronic lymphocytic leukaemia cells have reduced capacity to upregulate expression of MHC class I in response to interferon-gamma

Aims: An important consideration in the design of a tumour vaccine is the ability of tumour-specific cytotoxic T lymphocytes (CTL) to recognise unmanipulated tumour cells in vivo. To determine whether B-CLL might use an escape strategy, the current studies compared B-CLL and normal B cell MHC class I expression. Methods: Flow cytometry, TAP allele PCR and MHC class I PCR were used. Results: While baseline expression of MHC class I did not differ, upregulation of MHC class I expression by B-CLL cells in response to IFN-gamma was reduced. No deletions or mutations of TAP 1 or 2 genes were detected. B-CLL cells upregulated TAP protein expression in response to IFN-gamma. Responsiveness of B-CLL MHC class I mRNA to IFN-gamma was not impaired. Conclusions: The data suggest that MHC class I molecules might be less stable at the cell surface in B-CLL than normal B cells, as a result of the described release of beta(2)m and beta(2)m-free class I heavy chains from the membrane. This relative MHC class I expression defect of B-CLL cells may reduce their susceptibility to CTL lysis in response to immunotherapeutic approaches.

Identifying the molecular phenotype of renal progenitor cells

Although many of the molecular interactions in kidney development are now well understood, the molecules involved in the specification of the metanephric mesenchyme from surrounding intermediate mesoderm and, hence, the formation of the renal progenitor population are poorly characterized. In this study, cDNA microarrays were used to identify genes enriched in the murine embryonic day 10.5 (E10.5) uninduced metanephric mesenchyme, the renal progenitor population, in comparison with more rostral derivatives of the intermediate mesoderm. Microarray data were analyzed using R statistical software to determine accurately genes differentially expressed between these populations. Microarray outliers were biologically verified, and the spatial expression pattern of these genes at E10.5 and subsequent stages of early kidney development was determined by RNA in situ hybridization. This approach identified 21 genes preferentially expressed by the E10.5 metanephric mesenchyme, including Ewing sarcoma homolog, 14-3-3 theta, retinoic acid receptor-alpha, stearoyl-CoA desaturase 2, CD24, and cadherin-11, that may be important in formation of renal progenitor cells. Cell surface proteins such as CD24 and cadherin-11 that were strongly and specifically expressed in the uninduced metanephric mesenchyme and mark the renal progenitor population may prove useful in the purification of renal progenitor cells by FACS. These findings may assist in the isolation and characterization of potential renal stem cells for use in cellular therapies for kidney disease.

PlexinA4 is necessary as a downstream target of Islet2 to mediate Slit signaling for promotion of sensory axon branching

Slit is a secreted protein known to repulse the growth cones of commissural neurons. By contrast, Slit also promotes elongation and branching of axons of sensory neurons. The reason why different neurons respond to Slit in different ways is largely unknown. Islet2 is a LIM/homeodomaintype transcription factor that specifically regulates elongation and branching of the peripheral axons of the primary sensory neurons in zebrafish embryos. We found that PlexinA4, a transmembrane protein known to be a coreceptor for class III semaphorins, acts downstream of Islet2 to promote branching of the peripheral axons of the primary sensory neurons. Intriguingly, repression of PlexinA4 function by injection of the antisense morpholino oligonucleotide specific to PlexinA4 or by overexpression of the dominant-negative variant of PlexinA4 counteracted the effects of overexpression of Slit2 to induce branching of the peripheral axons of the primary sensory neurons in zebrafish embryos, suggesting involvement of PlexinA4 in the Slit signaling cascades for promotion of axonal branching of the sensory neurons. Colocalized expression of Robo, a receptor for Slit2, and PlexinA4 is observed not only in the primary sensory neurons of zebrafish embryos but also in the dendrites of the pyramidal neurons of the cortex of the mammals, and may be important for promoting the branching of either axons or dendrites in response to Slit, as opposed to the growth cone collapse.

N4WBP5A (Ndfip2), a Nedd4-interacting protein, localizes to multivesicular bodies and the Golgi, and has a potential role in protein trafficking

N4WBP5A (Ndfip2) belongs to an evolutionarily conserved group of Nedd4-interacting proteins with two homologues in mammalian species. We have previously shown that N4WBP5A expression in Xenopus oocytes results in increased cell-surface expression of the epithelial sodium channel. N4WBPs are characterized by one or two amino terminal PPxY motifs and three transmembrane domains. Here we show that both PPxY motifs of N4WBP5A mediate interaction with WW domains of Nedd4 and that N4WBP5A can physically interact with the WW domains of several Nedd4-family proteins. N4WBP5A is ubiquitinated and ubiquitination does not significantly affect the turnover of N4WBP5A protein. Ubiquitination of N4WBP5A is enhanced by Nedd4 and Nedd4-2 expression. N4WBP5A localizes to the Golgi, vesicles associated with the Golgi complex and to multivesicular bodies. We show that the ectopic expression of N4WBP5A inhibits receptor-mediated endocytosis of labelled epidermal growth factor. N4WBP5A overexpression inhibits accumulation of EGF in large endocytic/lysosomal vesicles suggestive of a role for N4WBP5A in protein trafficking. We propose that N4WBP5A acts as an adaptor to recruit Nedd4 family ubiquitin-protein ligases to the protein trafficking machinery.

Sorting nexin 5 is localized to a subdomain of the early endosomes and is recruited to the plasma imembrane following EGF stimulation

Sorting nexins are a large family of proteins that contain the phosphoinositide-binding Phox homology (PX) domain. A number of sorting nexins are known to bind to PtdIns(3)P, which mediates their localization to membranes of the endocytic pathway. We show here that sorting nexin 5 (SNX5) can be recruited to two distinct membrane compartments. In non-stimulated cells, the PX domain was independently targeted to endosomal structures and colocalized with full-length SNX5. The membrane binding of the PX domain was inhibited by the PI 3-kinase inhibitor, wortmannin. Although SNX5 colocalized with a fluid-phase marker and was found predominantly within a PtdIns(3)P-rich endosomal domain, very little colocalization was observed between SNX5 and the PtdIns(3)P-binding protein, EEA1. Using liposome-based binding assays, we have shown that the PX domain of SNX5 interacts not only with PtdIns(3)P but also with PtdIns(3,4)P-2. In response to EGF stimulation, either the SNX5-PX domain or full-length SNX5 was rapidly recruited to the plasma membrane. The localization of SNX1, which does not bind PtdIns(3,4)P-2, was unaffected by EGF signalling. Therefore, SNX5 is localized to a subdomain of the early endosome distinct from EEA1 and, following EGF stimulation and elevation of PtdIns(3,4)P-2, is also transiently recruited to the plasma membrane. These results indicate that SNX5 may have functions not only associated with endosomal sorting but also with the phosphoinositide-signalling pathway.

Carboxy terminus of glucose transporter 3 contains an apical membrane targeting domain

We previously demonstrated that distinct facilitative glucose transporter isoforms display differential sorting in polarized epithelial cells. In Madin-Darby canine kidney (MDCK) cells, glucose transporter 1 and 2 (GLUT1 and GLUT2) are localized to the basolateral cell surface whereas GLUTs 3 and 5 are targeted to the apical membrane. To explore the molecular mechanisms underlying this asymmetric distribution, we analyzed the targeting of chimeric glucose transporter proteins in MDCK cells. Replacement of the carboxy-terminal cytosolic tail of GLUT1, GLUT2, or GLUT4 with that from GLUT3 resulted in apical targeting. Conversely, a GLUT3 chimera containing the cytosolic carboxy terminus of GLUT2 was sorted to the basolateral membrane. These findings are not attributable to the presence of a basolateral signal in the tails of GLUTs 1, 2, and 4 because the basolateral targeting of GLUT1 was retained in a GLUT1 chimera containing the carboxy terminus of GLUT5. In addition, we were unable to demonstrate the presence of an autonomous basolateral sorting signal in the GLUT1 tail using the low-density lipoprotein receptor as a reporter. By examining the targeting of a series of more defined GLUT1/3 chimeras, we found evidence of an apical targeting signal involving residues 473 - 484 (DRSGKDGVMEMN) in the carboxy tail. We conclude that the targeting of GLUT3 to the apical cell surface in MDCK cells is regulated by a unique cytosolic sorting motif.

Common E protein determinants for attenuation of glycosaminoglycan-binding variants of Japanese encephalitis and West Nile viruses

Natural isolates and laboratory strains of West Nile virus (WNV) and Japanese encephalitis virus (JEV) were attenuated for neuroinvasiveness in mouse models for flavivirus encephalitis by serial passage in human adenocarcinoma (SW13) cells. The passage variants displayed a small-plaque phenotype, augmented affinity for heparin-Sepharose, and a marked increase in specific infectivity for SW13 cells relative to the respective parental viruses, while the specific infectivity for Vero cells was not altered. Therefore, host cell adaptation of passage variants was most likely a consequence of altered receptor usage for virus attachment-entry with the involvement of cell surface glycosaminoglycans (GAG) in this process. In vivo blood clearance kinetics of the passage variants was markedly faster and viremia was reduced relative to the parental viruses, suggesting that affinity for GAG (ubiquitously present on cell surfaces and extracellular matrices) is a key determinant for the neuroinvasiveness of encephalitic flaviviruses. A difference in pathogenesis between WNV and JEV, which was reflected in more efficient growth in the spleen and liver of the WNV parent and passage variants, accounted for a less pronounced loss of neuroinvasiveness of GAG binding variants of WNV than JEV. Single gain-of-net-positive-charge amino acid changes at E protein residue 49, 138, 306, or 389/390, putatively positioned in two clusters on the virion surface, define molecular determinants for GAG binding and concomitant virulence attenuation that are shared by the JEV serotype flaviviruses.

Classical cadherin adhesion molecules: coordinating cell adhesion, signaling and the cytoskeleton

Classical cadherin adhesion molecules are fundamental determinants of tissue organization in both health and disease. Recent advances in understanding the molecular and cellular basis of cadherin function have revealed that these adhesion molecules serve as molecular couplers, linking cell surface adhesion and recognition to both the actin cytoskeleton and cell signalling pathways. We will review some of these developments. to provide an overview of progress in this rapidly-developing area of cell and developmental biology.

Nedd4-2 functionally interacts with ClC-5 - Involvement in constitutive albumin endocytosis in proximal tubule cells

Constitutive albumin uptake by the proximal tubule is achieved by a receptor-mediated process in which the Cl- channel, ClC-5, plays an obligate role. Here we investigated the functional interaction between ClC-5 and ubiquitin ligases Nedd4 and Nedd4-2 and their role in albumin uptake in opossum kidney proximal tubule (OK) cells. In vivo immunoprecipitation using an anti-HECT antibody demonstrated that ClC-5 bound to ubiquitin ligases, whereas glutathione S-transferase pull-downs confirmed that the C terminus of ClC-5 bound both Nedd4 and Nedd4-2. Nedd4-2 alone was able to alter ClC-5 currents in Xenopus oocytes by decreasing cell surface expression of ClC-5. In OK cells, a physiological concentration of albumin (10 mug/ml) rapidly increased cell surface expression of ClC-5, which was also accompanied by the ubiquitination of ClC-5. Albumin uptake was reduced by inhibiting either the lysosome or proteasome. Total levels of Nedd4-2 and proteasome activity also increased rapidly in response to albumin. Overexpression of ligase defective Nedd4-2 or knockdown of endogenous Nedd4-2 with small interfering RNA resulted in significant decreases in albumin uptake. In contrast, pathophysiological concentrations of albumin (100 and 1000 mug/ml) reduced the levels of ClC-5 and Nedd4-2 and the activity of the proteasome to the levels seen in the absence of albumin. These data demonstrate that normal constitutive uptake of albumin by the proximal tubule requires Nedd4-2, which may act via ubiquitination to shunt ClC-5 into the endocytic pathway.