CD8+ alpha beta+ T cells that lack surface CD5 antigen expression are a major lymphotactin (XCL1) source in peripheral blood lymphocytes

To better characterize the cellular source of lymphotactin (XCL1), we compared XCL1 expression in different lymphocyte subsets by real-time PCR. XCL1 was constitutively expressed in both PBMC and CD4(+) cells, but its expression was almost 2 log higher in CD8(+) cells. In vitro activation was associated with a substantial increase in XCL1 expression in both PBMC and CD8(+) cells, but not in CD4(+) lymphocytes. The preferential expression of XCL1 in CD8(+) cells was confirmed by measuring XCL1 production in culture supernatants, and a good correlation was found between figures obtained by real-time PCR and XCL1 contents. XCL1 expression was mostly confined to a CD3(+)CD8(+) subset not expressing CD5, where XCL1 expression equaled that shown by gammadelta(+) T cells. Compared with the CD5(+) counterpart, CD3(+)CD8(+)CD5(-) cells, which did not express CD5 following in vitro activation, showed preferential expression of the alphaalpha form of CD8 and a lower expression of molecules associated with a noncommitted/naive phenotype, such as CD62L. CD3(+)CD8(+)CD5(-) cells also expressed higher levels of the XCL1 receptor; in addition, although not differing from CD3(+)CD8(+)CD5(+) cells in terms of the expression of most alpha- and beta-chemokines, they showed higher expression of CCL3/macrophage inflammatory protein-1alpha. These data show that TCR alphabeta-expressing lymphocytes that lack CD5 expression are a major XCL1 source, and that the contribution to its synthesis by different TCR alphabeta-expressing T cell subsets, namely CD4(+) lymphocytes, is negligible. In addition, they point to the CD3(+)CD8(+)CD5(-) population as a particular T cell subset within the CD8(+) compartment, whose functional properties deserve further attention.     

Inhibitory action of a new lectin from Xerocomus chrysenteron on cell-substrate adhesion

Lectins are carbohydrate-binding proteins which potentially link to cell surface glycoconjugates and affect cell proliferation. We investigated the effect of a new lectin from the mushroom Xerocomus chrysenteron (XCL) on cell proliferation using adherent and suspension cell lines. XCL caused a dose-dependent inhibition of proliferation of the adherent cell lines NIH-3T3 and HeLa. Several experiments suggest that disruption of cell-substrate adhesion is the main factor affecting cell growth inhibition. (i) No antiproliferative effect was observed on the SF9 cell line, which does not require to be attached to grow. (ii) XCL was shown to affect the adherence of cells following their suspension by trypsin treatment. (iii) XCL was localized on the cell surface where it would act as a coating agent. (iv) XCL induced morphological changes from well spread to rounded cells and disrupted the actin cytoskeleton. By contrast, flow cytometric analysis showed that XCL does not interfere with the cell cycle, and does not induce apoptosis.     

CXCL10/XCL1 fusokine elicits in vitro and in vivo chemotaxis

Fusokines are proteins formed by the fusion of two cytokines. They have greater bioavailability and therapeutic potential than individual cytokines or a combination of different cytokines. Interferon-gamma-inducible protein 10 (CXCL10) and lymphotactin (XCL1) are members of the chemotactic family of cytokines, which induce tumor regression by eliciting immune-system cell chemotaxis. We engineered a replication-deficient adenoviral system expressing CXCL10/XCL1 fusokine (Ad FIL) and assessed its chemotactic response in vitro and in vivo. The CXCL10/XCL1 fusokine elicited a greater chemotactic effect in IL-2 stimulated lymphocytes than individual or combined cytokines in vitro. CXCL10/XCL1 fusokine biological activity was demonstrated in vivo by intratumoral chemoattraction of CXCR3+ cells. Thus, this novel CXCL10/XCL1 fusokine may represent a potential tool for gene therapy treatment of cancer and other illnesses that require triggering immune-system cell recruitment.     

Conservation of a chemokine system, XCR1 and its ligand, XCL1, between human and mice

Understanding dendritic cell (DC) subset functions should lead to the development of novel types of vaccine. Here we characterized expression of XC chemokine receptor 1 (XCR1) and its ligand, XCL1. Murine XCR1 was the only chemokine receptor selectively expressed in CD8α⁺ conventional DCs. XCL1 was constitutively expressed in NK cells, which contribute to serum XCL1 levels. NK and CD8⁺ T cells increased XCL1 production upon activation. These expression patterns were conserved in human blood cells, including the BDCA3⁺ DC subset. Thus, in human and mice, certain DC subsets should be chemotactic towards NK or activated CD8⁺ T cells through XCR1.     

Activity and expression of Xenopus laevis matrix metalloproteinases: identification of a novel role for the hormone prolactin in regulating collagenolysis in both amphibians and mammals

Prolactin (PRL) has long been implicated in Xenopus metamorphosis as an anti-metamorphic and/or juvenilizing hormone. Numerous studies showed that PRL could prevent effects of either endogenous or exogenous thyroid hormone (TH; T(3)). It has been shown that expression of matrix metalloproteinases (MMPs) is induced by TH during Xenopus metamorphosis. Direct in vivo evidence, however, for such anti-TH effects by PRL with respect to MMPs has not been available for the early phase of Xenopus development or metamorphosis. To understand the functional role of PRL, we investigated effects of PRL on Xenopus collagenase-3 (XCL3) and collagenase-4 (XCL4) expression in a cultured Xenopus laevis cell line, XL-177. Northern blot analysis demonstrated that XCL3 and XCL4 expression were not detected in control or T(3)-treated cells, but were differentially induced by PRL in a dose- and time-dependent fashion. Moreover, treatment with IL-1alpha as well as phorbol myristate acetate (PMA), a protein kinase C (PKC) activator, or H8, a protein kinase A (PKA) inhibitor, augmented PRL-induced collagenase expression, suggesting that multiple protein kinase pathways and cytokines may participate in PRL-induced collagenase expression. Interestingly, XCL3 expression could be induced in XL-177 cells by T(3), but only when co-cultured with prometamorphic Xenopus tadpole tails (stage 54/55), suggesting that the tails secrete a required intermediate signaling molecule(s) for T(3)-induced XCL3 expression. Taken together, these data demonstrate that XCL3 and XCL4 can be differentially induced by PRL and T(3) and further suggest that PRL is a candidate regulator of TH-independent collagenase expression during the organ/tissue remodeling which occurs in Xenopus development.     

XCL1 enhances regulatory activities of CD4+ CD25(high) CD127(low/-) T cells in human allergic asthma

Chemokine-mediated recruitment of regulatory cell subsets to the airway during inflammation and enhancement of their activities are potential strategies for therapeutic development in allergic asthma (AA). In this study, we aim to explore the role of XCL1, a chemokine associated with immune suppression and allergy, on CD4(+)CD25(high)CD127(low/-) regulatory T cell (Treg) function in AA. Flow cytometry and PCR analysis showed a reduction in XCL1 and XCR1 expression in AA Treg compared with healthy control and nonallergic asthmatic counterparts. This reduction in XCL1 expression was associated with the suboptimal regulatory function of Treg in AA. Interestingly, incubation with recombinant human XCL1 significantly increased Treg-mediated suppression and cytotoxicity by up-regulating expression of XCL1 and chief effector molecules of Treg function. Altogether, these results suggest an association between dysregulated XCL1 expression and reduced Treg activities in AA, as well as a potential role of XCL1 in reversing defective Treg function in the disease.     

Endocytosis in Drosophila: progress, possibilities, prognostications.

By many outside the field, endocytosis is often perceived as a "house-keeping" function performed via identical mechanisms in yeast and man. Recent discoveries have done much to reduce this misperception. (1) Endocytosis occurs via different mechanisms and different pathways in different cellular contexts. (2) Molecular mechanisms that regulate homologous pathways in unicellular and multicellular organisms show considerable variance. (3) Temporally controlled endocytosis of specific regulatory molecules underlies several important and intricate biological processes including synapse formation, synaptic plasticity, cell fate determination, and morphogen gradient formation. Interactions between endocytosis and cytoskeletal and signaling pathways have been particularly revealing. In this intellectual context, Drosophila has become invaluable as a metazoan genetic model in which to understand the many faces of endocytosis. This review discusses two aspects of work in Drosophila: (a) its contributions toward understanding fundamental mechanisms that underlie the operation of endocytic pathways; (b) how analyses in Drosophila provide insights into varied biological processes regulated by endocytosis. In addition, while offering our commentary on merits and limitations of Drosophila work, we speculate on likely areas for contributions and future research on endocytosis in Drosophila.     

Mammalian Abp1, a signal-responsive F-actin-binding protein, links the actin cytoskeleton to endocytosis via the GTPase dynamin

The actin cytoskeleton has been implicated in endocytosis, yet few molecular links to the endocytic machinery have been established. Here we show that the mammalian F-actin-binding protein Abp1 (SH3P7/HIP-55) can functionally link the actin cytoskeleton to dynamin, a GTPase that functions in endocytosis. Abp1 binds directly to dynamin in vitro through its SH3 domain. Coimmunoprecipitation and colocalization studies demonstrated the in vivo relevance of this interaction. In neurons, mammalian Abp1 and dynamin colocalized at actin-rich sites proximal to the cell body during synaptogenesis. In fibroblasts, mAbp1 appeared at dynamin-rich sites of endocytosis upon growth factor stimulation. To test whether Abp1 functions in endocytosis, we overexpressed several Abp1 constructs in Cos-7 cells and assayed receptor-mediated endocytosis. While overexpression of Abp1's actin-binding modules did not interfere with endocytosis, overexpression of the SH3 domain led to a potent block of transferrin uptake. This implicates the Abp1/dynamin interaction in endocytic function. The endocytosis block was rescued by cooverexpression of dynamin. Since the addition of the actin-binding modules of Abp1 to the SH3 domain construct also fully restored endocytosis, Abp1 may support endocytosis by combining its SH3 domain interactions with cytoskeletal functions in response to signaling cascades converging on this linker protein.     

Endocytosis of antigen, anti-idiotype, and anti-immunoglobulin antibodies and receptor re-expression by murine B cells

The endocytosis of Ag mediated by membrane-associated Ig (mIg) molecules has been spectrophotometrically monitored using a cell line (2C3) specific for the hapten phthalate (Xmp) and employing conjugates of Xmp and horseradish peroxidase (HRP) as the labeled ligand. Approximately 50% of both Xmp-HRP, or the larger ligand, Xmp-keyhole limpet hemocyanin-HRP, are internalized rapidly, reaching an initial plateau by 30 min. The rate of endocytosis of anti-idiotype-HRP is similar to the rates that were observed for the hapten-bearing ligands, while a slower rate of endocytosis of anti-Ig-HRP was observed. The percent of ligand bound that is internalized and the rate of endocytosis appear to be largely independent of the size and amount of ligand bound per cell. However, mIg-mediated endocytosis is markedly reduced when mIg-ligand complexes are more extensively cross-linked by the binding of a second antibody. In addition to the initial rapid phase of endocytosis, there is a prolonged phase during which more of the bound ligand is internalized, and up to 90% of the internalized ligand is degraded. Re-expression of Ag-binding receptors by the 2C3 cells is independent of new protein synthesis and is accomplished in part by the translocation of a presynthesized pool of mIg molecules from the cytoplasm to the plasma membrane of the cell. The kinetics of endocytosis of HRP-labeled anti-Ig antibodies by BALB/c splenic B-lymphocytes and other B-lymphocyte cell lines is very similar to the endocytosis of Ag and anti-idiotype observed with the 2C3 cell line. Light and electron microscopy are also performed to visually confirm that the HRP-labeled ligands are being internalized and to determine the percentage of cells involved in this process. Finally it was determined that the transmembrane and cytoplasmic domains of the mIg molecules are required for endocytosis since the secreted form of the molecule (which lacks these domains) fails to mediate the internalization of bound ligand.     

Inhibitory role of endophilin 3 in receptor-mediated endocytosis

Endophilin 1 (Endo1) participates in synaptic vesicle biogenesis through interactions of its Src homology 3 domain with the polyphosphoinositide phosphatase Synaptojanin and the GTPase Dynamin. Endo1 has also been reported to affect endocytosis by converting membrane curvature via its lysophosphatidic acid acyltransferase activity. Here we report that a closely related isoform of Endo1, Endo3, inhibits clathrin-mediated endocytosis. Mutational analyses showed that the variable region of Endo3 is important in regulating transferrin endocytosis. In the brain, Endo3 is co-localized with dopamine D2 receptor in olfactory nerve terminals and inhibits its clathrin-mediated endocytosis in COS-7 cells. Furthermore, overexpression of Endo3 in an olfactory epithelium-derived cell line suppressed dopamine D2 receptor-mediated endocytosis and therefore accelerated its dopamine-induced differentiation. These results indicate that Endo3 may act as a negative regulator of clathrin-mediated endocytosis in brain neurons.     

XCL1 and XCR1 in the immune system

XCL1, a C class chemokine also known as lymphotactin, is produced by T, NK, and NKT cells during infectious and inflammatory responses, whereas XCR1, the receptor of XCL1, is expressed by a dendritic cell subpopulation. The XCL1-XCR1 axis plays an important role in dendritic-cell-mediated cytotoxic immune response. It has been also shown that XCL1 and XCR1 are constitutively expressed in the thymus and regulate the thymic establishment of self-tolerance and the generation of regulatory T cells. This review summarizes the expression and function of XCL1 and XCR1 in the immune system.     

The CD8-Derived Chemokine XCL1/Lymphotactin Is a Conformation-Dependent,Broad-Spectrum Inhibitor of HIV-1

CD8+ T cells play a key role in the in vivo control of HIV-1 replication via their cytolytic activity as well as their ability to secrete non-lytic soluble suppressive factors. Although the chemokines that naturally bind CCR5 (CCL3/MIP-1α, CCL4/MIP- 1β, CCL5/RANTES) are major components of the CD8-derived anti-HIV activity, evidence indicates the existence of additional, still undefined, CD8-derived HIV-suppressive factors. Here, we report the characterization of a novel anti-HIV chemokine, XCL1/lymphotactin, a member of the C-chemokine family that is produced primarily by activated CD8+ T cells and behaves as a metamorphic protein, interconverting between two structurally distinct conformations (classic and alternative). We found that XCL1 inhibits a broad spectrum of HIV-1 isolates, irrespective of their coreceptor-usage phenotype. Experiments with stabilized variants of XCL1 demonstrated that HIV-1 inhibition requires access to the alternative, all-β conformation, which interacts with proteoglycans but does not bind/activate the specific XCR1 receptor, while the classic XCL1 conformation is inactive. HIV-1 inhibition by XCL1 was shown to occur at an early stage of infection, via blockade of viral attachment and entry into host cells. Analogous to the recently described anti-HIV effect of the CXC chemokine CXCL4/PF4, XCL1-mediated inhibition is associated with direct interaction of the chemokine with the HIV-1 envelope. These results may open new perspectives for understanding the mechanisms of HIV-1 control and reveal new molecular targets for the design of effective therapeutic and preventive strategies against HIV-1.     

The YXXL motif, but not the two NPXY motifs, serves as the dominant endocytosis signal for low density lipoprotein receptor-related protein

All members of the low density lipoprotein (LDL) receptor family contain at least one copy of the NPXY sequence within their cytoplasmic tails. For the LDL receptor, it has been demonstrated that the NPXY motif serves as a signal for rapid endocytosis through coated pits. Thus, it is generally believed that the NPXY sequences function as endocytosis signals for all the LDL receptor family members. The primary aim of this study is to define the endocytosis signal(s) within the cytoplasmic tail of LDL receptor-related protein (LRP). By using LRP minireceptors, which mimic the function and trafficking of full-length endogenous LRP, we demonstrate that the YXXL motif, but not the two NPXY motifs, serves as the dominant signal for LRP endocytosis. We also found that the distal di-leucine motif within the LRP tail contributes to its endocytosis, and its function is independent of the YXXL motif. Although the proximal NPXY motif and the proximal di-leucine motif each play a limited role in LRP endocytosis in the context of the full-length tail, these motifs were functional within the truncated receptor tail. In addition, we show that LRP minireceptor mutants defective in endocytosis signal(s) accumulate at the cell surface and are less efficient in delivery of ligand for degradation.     

JosD1, a Membrane-targeted Deubiquitinating Enzyme,Is Activated by Ubiquitination and Regulates Membrane Dynamics,Cell Motility,and Endocytosis

The functional diversity of deubiquitinating enzymes (DUBs) is not well understood. The MJD family of DUBs consists of four cysteine proteases that share a catalytic “Josephin” domain. The family is named after the DUB ATXN3, which causes the neurodegenerative disease Machado-Joseph disease. The two closely related Josephin domain-containing (JosD) proteins 1 and 2 consist of little more than the Josephin domain. To gain insight into the properties of Josephin domains, we investigated JosD1 and JosD2. JosD1 and JosD2 were found to differ fundamentally in many respects. In vitro, only JosD2 can cleave ubiquitin chains. In contrast, JosD1 cleaves ubiquitin chains only after it is monoubiquitinated, a form of posttranslational-dependent regulation shared with ATXN3. A significant fraction of JosD1 is monoubiquitinated in diverse mouse tissues. In cell-based studies, JosD2 localizes to the cytoplasm whereas JosD1 preferentially localizes to the plasma membrane, particularly when ubiquitinated. The membrane occupancy by JosD1 suggests that it could participate in membrane-dependent events such as cell motility and endocytosis. Indeed, time-lapse imaging revealed that JosD1 enhances membrane dynamics and cell motility. JosD1 also influences endocytosis in cultured cells by increasing the uptake of endocytic markers of macropinocytosis while decreasing those for clathrin- and caveolae-mediated endocytosis. Our results establish that two closely related DUBs differ markedly in activity and function and that JosD1, a membrane-associated DUB whose activity is regulated by ubiquitination, helps regulate membrane dynamics, cell motility, and endocytosis.     

A Large PEST-like Sequence Directs the Ubiquitination,Endocytosis, and Vacuolar Degradation of the Yeast a-Factor Receptor

The yeast a-factor receptor (encoded by STE3) is subject to two modes of endocytosis, a ligand-dependent endocytosis as well as a constitutive, ligand-independent mode. Both modes are associated with receptor ubiquitination (Roth, A.F., and N.G. Davis. 1996. J. Cell Biol. 134:661–674) and both depend on sequence elements within the receptor''s regulatory, cytoplasmically disposed, COOH-terminal domain (CTD). Here, we concentrate on the Ste3p sequences required for constitutive endocytosis. Constitutive endocytosis is rapid. Receptor is synthesized, delivered to the cell surface, endocytosed, and then delivered to the vacuole where it is degraded, all with a t _1/2 of 15 min. Deletion analysis has defined a 36-residue-long sequence mapping near the COOH-terminal end of the Ste3p CTD that is the minimal sequence required for this rapid turnover. Deletions intruding into this interval block or severely slow the rate of endocytic turnover. Moreover, the same 36-residue sequence directs receptor ubiquitination. Mutants deleted for this sequence show undetectable levels of ubiquitination, and mutants having intermediate endocytosis defects show a correlated reduced level of ubiquitination. Not only necessary for ubiquitination and endocytosis, this sequence also is sufficient. When transplanted to a stable cell surface protein, the plasma membrane ATPase Pma1p, the 36-residue STE3 signal directs both ubiquitination of the PMA1-STE3 fusion protein as well as its endocytosis and consequent vacuolar degradation. Alanine scanning mutagenesis across the 36-residue-long interval highlights its overall complexity—no singular sequence motif or signal is found, instead required sequence elements distribute throughout the entire interval. The high proportion of acidic and hydroxylated amino acid residues in this interval suggests a similarity to PEST sequences—a broad class of sequences which have been shown to direct the ubiquitination and subsequent proteosomal degradation of short-lived nuclear and cytoplasmic proteins. A likely possibility, therefore, is that this sequence, responsible for both endocytosis and ubiquitination, may be first and foremost a ubiquitination signal. Finally, we present evidence suggesting that the true signal in the wild-type receptor extends beyond the 36-residue-long sequence defined as a minimal signal to include contiguous PEST-like sequences which extend another 21 residues to the COOH terminus of Ste3p. Together with sequences identified in two other yeast plasma membrane proteins, the STE3 sequence defines a new class of ubiquitination/endocytosis signal.     

Signaling, Actin Dynamics and Endocytosis

Endocytosis is an essential process for normal function of all living cells. Cells get nutrients, and control the surface-expressional level of proteins as well as membrane hemostats through the endocytosis. Endocytosis process is regulated in response to functional status of a particular cell. Signaling events and the endocytosis process go hand in hand to fulfill cellular functions. Although our understanding of the endocytosis process has grown rapidly during the last decade, little is known about how it is interconnected functionally with the signaling status of cells. During endocytosis, vesicles are formed from the plasma membrane through complex molecular machinery. The location where the vesicles are formed is rich in cortical actin cytoskeleton that supports the plasma membrane. To enter cells, vesicles have to diffuse through the cortical actin cytoskeleton. The actin cytoskeleton has a very dynamic structure and actively participates a wide variety of cellular functions. In addition to its central role in cytokinesis, cell shape, cell motility, and cell polarity, a connection between the endocytosis process and the actin cytoskeleton has been implicated in both yeast and mammalian system. In recent years the knowledge on how the actin cytoskeleton participates in the generation of coordinated cellular responses to external stimuli is grown rapidly. In this review, we focus on the potential roles of the actin cytoskeleton in regulating the endocytosis process in response to signaling events.     

The complement inhibitor, CRIT, undergoes clathrin-dependent endocytosis

Complement C2 receptor inhibitor trispanning (CRIT) is a receptor for the second component of complement and is found in various tissues and hemopoietic cells. On binding to CRIT, C2 cannot be activated to potentially form a variant-C3 convertase as it is rendered non-cleavable by C1s. CRIT thus limits the amount of C3 convertase formed on the cell surface. In this study we have shown, using flow cytometry and immunofluorescence microscopy, that human CRIT undergoes endocytosis from the plasma membrane. The endocytosis, possibly ligand mediated, occurs via clathrin-coated pits as it can be inhibited by prior incubation of cells in hypertonic medium or with chlorpromazine, at 37 degrees C. However, inhibition of endocytosis was not possible after treatment with nystatin, or filipin, inhibitors of caveolae/raft-dependent endocytosis. In the presence of C2 alone, CRIT associates with the adapter protein, beta-arrestin-2, and whether in association with C2 or not, then appears in the perinuclear region, but does not appear to be translocated into the nucleus. Apart from the C3aR and C5aR that internalize the anaphylatoxic peptides, this is the first report of the internalization via the clathrin pathway of a receptor for a complement serum protein.