CC chemokine receptor (CCR)4 and the CCR10 ligand cutaneous T cell-attracting chemokine (CTACK) in lymphocyte trafficking to inflamed skin.

The chemokine thymus and activation-regulated chemokine (TARC; CCL17) is displayed by cutaneous (but not intestinal) venules, and is thought to trigger vascular arrest of circulating skin homing memory T cells, which uniformly express the TARC receptor CC chemokine receptor (CCR)4. Cutaneous T cell-attracting chemokine (CTACK; CCL27), expressed by skin keratinocytes, also attracts cutaneous memory T cells, and is hypothesized to assist in lymphocyte recruitment to skin as well. Here we show that chronic cutaneous inflammation induces CD4 T cells expressing E-selectin binding activity (a marker of skin homing memory cells) in draining lymph node, and that these E-selectin ligand+ T cells migrate efficiently to TARC and to CTACK. In 24 h in vivo homing assays, stimulated lymph node T cells from wild-type mice or, surprisingly, from CCR4-deficient donors migrate efficiently to inflamed skin; and an inhibitory anti-CTACK antibody has no effect on wild-type lymphocyte recruitment. However, inhibition with anti-CTACK monoclonal antibody abrogates skin recruitment of CCR4-deficient T cells. We conclude that CTACK and CCR4 can both support homing of T cells to skin, and that either one or the other is required for lymphocyte recruitment in cutaneous delayed type hypersensitivity.

A role for a novel luminal endoplasmic reticulum aminopeptidase in final trimming of 26 S proteasome-generated major histocompatability complex class I antigenic peptides.

Peptides presented to cytotoxic T lymphocytes by the class I major histocompatability complex are 8-11 residues long. Although proteasomal activity generates the precise C termini of antigenic epitopes, the mechanism(s) involved in generation of the precise N termini is largely unknown. To investigate the mechanism of N-terminal peptide processing, we used a cell-free system in which two recombinant ornithine decarboxylase (ODC) constructs, one expressing the native H2-K(b)-restricted ovalbumin (ova)-derived epitope SIINFEKL (ODC-ova) and the other expressing the extended epitope LESIINFEKL (ODC-LEova), were targeted to degradation by 26 S proteasomes followed by import into microsomes. We found that the cleavage specificity of the 26 S proteasome was influenced by the N-terminal flanking amino acids leading to significantly different yields of the final epitope SIINFEKL. Following incubation in the presence of purified 26 S proteasome, ODC-LEova generated largely ESIINFEKL that was efficiently converted to the final epitope SIINFEKL following translocation into microsomes. The conversion of ESIINFEKL to SIINFEKL was strictly dependent on the presence of H2-K(b) and was completely inhibited by the metalloaminopeptidase inhibitor 1,10-phenanthroline. Importantly, the converting activity was resistant to a stringent salt/EDTA wash of the microsomes and was only apparent when transport of TAP, the transporter associated with antigen processing, was facilitated. These results strongly suggest a crucial role for a luminal endoplasmic reticulum-resident metalloaminopeptidase in the N-terminal trimming of major histocompatability complex class I-associated peptides.

Efficient presentation of exogenous antigen by liver endothelial cells to CD8+ T cells results in antigen-specific T-cell tolerance.

Myeloid antigen-presenting cells (APC) are known to cross-present exogenous antigen on major histocompatibility class I molecules to CD8+ T cells and thereby induce protective immunity against infecting microorganisms. Here we report that liver sinusoidal endothelial cells (LSEC) are organ-resident, non-myeloid APC capable of cross-presenting soluble exogenous antigen to CD8+ T cells. Though LSEC employ similar molecular mechanisms for cross-presentation as dendritic cells, the outcome of cross-presentation by LSEC is CD8+ T cell tolerance rather than immunity. As uptake of circulating antigens into LSEC occurs efficiently in vivo, it is likely that cross-presentation by LSEC contributes to CD8+ T cell tolerance observed in situations where soluble antigen is present in the circulation.

T cell interaction with ICAM-1-deficient endothelium in vitro: transendothelial migration of different T cell populations is mediated by endothelial ICAM-1 and ICAM-2.

The trafficking of T lymphocytes is carefully regulated by adhesive interactions with the vascular endothelium. Depending on their maturation and activation stage, T lymphocytes exhibit distinctive patterns of homing and recirculation, which is at least partly due to the selective expression of cell adhesion molecules (CAM) on the T cell surface. In order to define whether the differential usage of CAM during the steps of transendothelial migration is involved in organ-specific recirculation of different T cell subsets we compared the interaction of three different T cell populations with mouse endothelioma cell lines in vitro. Using a novel approach, where we directly compared T cell interaction with ICAM-1-deficient endothelium to wild-type endothelium, we recently demonstrated that endothelial ICAM-1 and ICAM-2 play a key role in mediating the transendothelial migration of CD4(+) memory T cells. Here we show that endothelial ICAM-1 and ICAM-2 are equally required for the transendothelial migration of other T cell populations such as thymocytes and T lymphoma cells, which differ from CD4(+) memory T cells in their maturation and activation stage, as well as in their surface expression of adhesion molecules. Our data therefore demonstrate that transendothelial migration of different T cell populations is mediated by the same endothelial CAM, i.e. ICAM-1 and ICAM-2, and thus subset-specific interaction of T cells with endothelial cells must be regulated prior to transendothelial migration.

26 S proteasome-mediated production of an authentic major histocompatibility class I-restricted epitope from an intact protein substrate.

Peptides displayed on the cell surface by major histocompatibility class I molecules (MHC class I) are generated by proteolytic processing of protein-antigens in the cytoplasm. Initially, antigens are degraded by the 26 S proteasome, most probably following ubiquitination. However, it is unclear whether this proteolysis results in the generation of MHC class I ligands or if further processing is required. To investigate the role of the 26 S proteasome in antigen presentation, we analyzed the processing of an intact antigen by purified 26 S proteasome. A recombinant ornithine decarboxylase was produced harboring the H-2K(b)-restricted peptide epitope, derived from ovalbumin SIINFEKL (termed ODC-ova). Utilizing recombinant antizyme to target the antigen to the 26 S proteasome, we found that proteolysis of ODC-ova by the 26 S proteasome resulted in the generation of the K(b)-ligand. Mass spectrometry analysis indicated that in addition to SIINFEKL, the N-terminally extended ligand, HSIINFEKL, was also generated. Production of SIINFEKL was linear with time and directly proportional to the rate of ODC-ova degradation. The overall yield of SIINFEKL was approximately 5% of the amount of ODC-ova degraded. The addition of PA28, the 20 S, or the 20 S-PA28 complex to the 26 S proteasome did not significantly affect the yield of the antigenic peptide. These findings demonstrate that the 26 S proteasome can efficiently digest an intact physiological substrate and generate an authentic MHC class I-restricted epitope.

Prolonged eosinophil accumulation in allergic lung interstitium of ICAM-2 deficient mice results in extended hyperresponsiveness.

ICAM-2-deficient mice exhibit prolonged accumulation of eosinophils in lung interstitium concomitant with a delayed increase in eosinophil numbers in the airway lumen during the development of allergic lung inflammation. The ICAM-2-dependent increased and prolonged accumulation of eosinophils in lung interstitium results in prolonged, heightened airway hyperresponsiveness. These findings reveal an essential role for ICAM-2 in the development of the inflammatory and respiratory components of allergic lung disease. This phenotype is caused by the lack of ICAM-2 expression on non-hematopoietic cells. ICAM-2 deficiency on endothelial cells causes reduced eosinophil transmigration in vitro. ICAM-2 is not essential for lymphocyte homing or the development of leukocytes, with the exception of megakaryocyte progenitors, which are significantly reduced.

Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1.

Receptor desensitization is accomplished by accelerated endocytosis and degradation of ligand-receptor complexes. An in vitro reconstituted system indicates that Cbl adaptor proteins directly control downregulation of the receptor for the epidermal growth factor (EGFR) by recruiting ubiquitin-activating and -conjugating enzymes. We infer a sequential process initiated by autophosphorylation of EGFR at a previously identified lysosome-targeting motif that subsequently recruits Cbl. This is followed by tyrosine phosphorylation of c-Cbl at a site flanking its RING finger, which enables receptor ubiquitination and degradation. Whereas all three members of the Cbl family can enhance ubiquitination, two oncogenic Cbl variants, whose RING fingers are defective and phosphorylation sites are missing, are unable to desensitize EGFR. Our study identifies Cbl proteins as components of the ubiquitin ligation machinery and implies that they similarly suppress many other signaling pathways.

T cell interaction with ICAM-1-deficient endothelium in vitro: essential role for ICAM-1 and ICAM-2 in transendothelial migration of T cells.

Transendothelial migration is a crucial step in the complex process of lymphocyte extravasation during lymphocyte homing, immunosurveillance and inflammation. However, little is known about the precise role of cell adhesion molecules (CAM) involved in this particular event. To define the CAM involved in T cell adhesion versus transendothelial migration, we have previously established an in vitro transendothelial migration system using mouse T cells and mouse endothelioma cells. We demonstrate here that, using ICAM-1-deficient endothelioma cells derived from ICAM-1 mutant mice, transendothelial migration of T cells was inhibited to a much greater extent when compared to migration across wild-type cells treated with a blocking anti-ICAM-1 monoclonal antibody. This unexpected result was confirmed by a rescue experiment using retroviral transfer of wild-type ICAM-1 into ICAM-1-deficient endothelial cells. Additional experiments showed that, in the absence of functional ICAM-1, only ICAM-2 was involved in transendothelial migration, but not PECAM-1, VCAM-1, or E-selectin. Taking this novel approach, we show that ICAM-1 and ICAM-2 are essential for transendothelial migration of T cells.

Enhanced phosphorylation of p53 by ATM in response to DNA damage.

The ATM protein, encoded by the gene responsible for the human genetic disorder ataxia telangiectasia (A-T), regulates several cellular responses to DNA breaks. ATM shares a phosphoinositide 3-kinase-related domain with several proteins, some of them protein kinases. A wortmannin-sensitive protein kinase activity was associated with endogenous or recombinant ATM and was abolished by structural ATM mutations. In vitro substrates included the translation repressor PHAS-I and the p53 protein. ATM phosphorylated p53 in vitro on a single residue, serine-15, which is phosphorylated in vivo in response to DNA damage. This activity was markedly enhanced within minutes after treatment of cells with a radiomimetic drug; the total amount of ATM remained unchanged. Various damage-induced responses may be activated by enhancement of the protein kinase activity of ATM.

The mouse and human genes encoding the recognition component of the N-end rule pathway.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The N-end rule pathway is one proteolytic pathway of the ubiquitin system. The recognition component of this pathway, called N-recognin or E3, binds to a destabilizing N-terminal residue of a substrate protein and participates in the formation of a substrate-linked multiubiquitin chain. We report the cloning of the mouse and human Ubr1 cDNAs and genes that encode a mammalian N-recognin called E3alpha. Mouse UBR1p (E3alpha) is a 1,757-residue (200-kDa) protein that contains regions of sequence similarity to the 225-kDa Ubr1p of the yeast Saccharomyces cerevisiae. Mouse and human UBR1p have apparent homologs in other eukaryotes as well, thus defining a distinct family of proteins, the UBR family. The residues essential for substrate recognition by the yeast Ubr1p are conserved in the mouse UBR1p. The regions of similarity among the UBR family members include a putative zinc finger and RING-H2 finger, another zinc-binding domain. Ubr1 is located in the middle of mouse chromosome 2 and in the syntenic 15q15-q21.1 region of human chromosome 15. Mouse Ubr1 spans approximately 120 kilobases of genomic DNA and contains approximately 50 exons. Ubr1 is ubiquitously expressed in adults, with skeletal muscle and heart being the sites of highest expression. In mouse embryos, the Ubr1 expression is highest in the branchial arches and in the tail and limb buds. The cloning of Ubr1 makes possible the construction of Ubr1-lacking mouse strains, a prerequisite for the functional understanding of the mammalian N-end rule pathway.

Production of a specific major histocompatibility complex class I-restricted epitope by ubiquitin-dependent degradation of modified ovalbumin in lymphocyte lysate.

Peptide epitopes presented through class I major histocompatability complex (MHC class I) on the cell surface, are generated by proteolytic processing of protein-antigens in the cytoplasm. The length and amino acid sequence determine whether a given peptide can fit into the peptide binding groove of class I heavy chain molecules and subsequently be presented to the immune system. The mode of action of the processing pathway is therefore of great interest. To study the processing mechanism of MHC class I-restricted intracellular antigens, we reconstituted the proteolytic processing of a model antigen in a cell-free system. Incubation of oxidized and urea-treated OVA in lymphocyte lysate resulted in partial degradation of the antigen. Degradation of the antigen depended on the presence of ATP. Addition of methylated ubiquitin abolished the reaction which was then restored by addition of an excess of native ubiquitin, indicating that the breakdown of the antigen in lymphocyte lysate is mediated by the ubiquitin proteolytic system. Upon incubation of modified OVA in lymphocyte lysate, a specific antigenic peptide was generated. The peptide was recognized by cytotoxic T lymphocytes directed against OVA-derived, H-2Kb-restricted peptide (SIINFEKL), and by a monoclonal antibody that recognizes cell-bound Kb-SIINFEKL complexes. Formation of the peptide epitope depended on the presence of ATP and ubiquitin. These results indicate that proteolytic processing of modified OVA is carried out by the ubiquitin-mediated degradation system. The experimental system described provides a tool to analyze the molecular mechanisms underlying the generation of specific, MHC class I-restricted peptide epitopes.

Butyrylcholinesterase-positive cells of the developing chicken retina that are non-cholinergic and GABA-positive.

Butyrylcholinesterase (BChE) is closely related to acetylcholinesterase (AChE), but its function in nervous system development or physiology is unclear. Here, the distribution of BChE was investigated by immunohistochemical methods in the developing chick retina. Using a specific anti-BChE antibody, we detected immunoreactivity associated with different cell types in two nuclear layers and in plexiform layers of the retina. At embryonic day 10 (E10), a transient BChE staining is detected in the inner plexiform layer (IPL) and in radial cells, the latter possibly representing Müller glia. At E12, a subpopulation of amacrine cells appeared, followed by cells in the middle and outer half of the inner nuclear layer. These cells at locations of amacrine, bipolar and horizontal cells represented the predominant three cell types persisting until hatching. The BChE+ amacrine cells were studied in more detail. Their distribution was not significantly different in the central and peripheral retina. Double labelling experiments revealed that BChE+ amacrine cells did not express choline acetyltransferase (ChAT), and, thus, are non-cholinergic. Only a minority of them coexpressed AChE. On the other hand, the majority of them colocalized with anti-GABA immunoreactivity. Taken together, these data support a hitherto unsuspected role of BChE in non-cholinergic cells, possibly in conjunction with GABA.

Immunosurveillance modelled in vitro: naive and memory T cells spontaneously migrate across unstimulated microvascular endothelium.

As a model for T cell immigration into non-lymphoid tissue we set up an in vitro assay that would allow us to investigate the phenotype of T lymphocytes from peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN) or peripheral blood (PBL) of mice, which were able to spontaneously migrate across unstimulated microvascular endothelium. The transendothelial migrating T cell population was enriched for T lymphocytes expressing a "recently activated/memory' phenotype: LFA-1/CD44/ICAM-1high, but also contained CD45RBhigh and LFA-1low T cells, which in the case of MLN T cells were phenotyped as CD4+ and thus characterized as naive T cells. Transmigrated T cells could be further distinguished from their original populations and from each other by their distinct but heterogeneous expression patterns for L-selectin, alpha 4 beta 7-integrin and PECAM-1. This observation suggests the presence of phenotypically different migratory T cells among MLN, PLN and PBL. Additional studies provided evidence that the capacity to migrate across unstimulated microvascular endothelium was a characteristic of a T cell population that could phenotypically be differentiated from activated T cells. The endothelial cells were found to play an active role in selecting the traversing T cell population, as they controlled the number and phenotype of spontaneously transmigrating T cells. Our studies suggest that the capacity to transmigrate across unstimulated microvascular endothelium and hence to immigrate into non-lymphoid tissue is owned by a phenotypically heterogeneous T cell population, which is enriched for memory T cells but not devoid of naive T cells.

Extracellular and asymmetric forms of acetylcholinesterase are expressed on cholinergic and noncholinergic terminal neuropil of the developing chick retina.

Only two out of four major acetylcholinesterase (AChE) subbands in the inner plexiform layer (IPL) of vertebrate retinae correspond to sites of cholinergic synaptic transmission, as has been shown by the co-distribution of AChE and choline acetyltransferase (ChAT) staining. The function and molecular identity of AChE in non-cholinergic subbands is unknown. We have used immunocytochemical methods to compare the development of asymmetric or extracellularly localized AChE with that of total AChE and ChAT in embryonic and adult chicken retinae. After injection of the AChE-specific monoclonal antibody 3D10 into the vitreous body of live embryos, a method that labels only extracellular AChE, five subbands in the IPL were labelled, whereas cell somata or their radial processes remained unstained. In contrast, the entire cell including processes was immunoreactive, when the 3D10 antibody was applied to permeabilized cryosections, suggesting that in cell bodies the enzyme is exclusively localized intracellularly. Compared with total AChE, detection of asymmetric AChE with the monoclonal antibody 6B6 was delayed, first being seen in cells of the inner nuclear layer and finally appearing on all subbands, reflecting more closely the course of synaptogenesis. Thus, extracellular and asymmetric forms of AChE are predominantly found on the terminal arbor neuropil of both cholinergic and non-cholinergic IPL subbands. These data show a differential distribution of extra- and intracellular AChE and suggest novel roles for the AChE in non-cholinergic IPL subbands.

Divalent cation and prenyl pyrophosphate specificities of the protein farnesyltransferase from rat brain, a zinc metalloenzyme.

The separate catalytic roles of Zn2+ and Mg2+ and the specificity of the prenyl pyrophosphate-binding site of the rat brain protein farnesyltransferase were explored using a purified enzyme preparation. The binding of p21Hras to the enzyme was abolished by dialysis against EDTA and restored by addition of ZnCl2, as demonstrated by chemical cross-linking. The binding of the other substrate, farnesyl pyrophosphate, was independent of divalent cations, as demonstrated by gel filtration. Transfer of the enzyme-bound farnesyl group to the bound p21Hras required Mg2+. Geranylgeranyl pyrophosphate bound to the prenyl pyrophosphate-binding site with an affinity equal to that of farnesyl pyrophosphate, but the geranylgeranyl group was not transferred efficiently to p21Hras. It also was not transferred to a modified p21Hras containing COOH-terminal leucine, a protein that was shown previously to be a good substrate for a rat brain geranylgeranyltransferase. We conclude that the protein farnesyltransferase is a metalloenzyme that most likely contains Zn2+ at the peptide-binding site. It thus resembles certain metallopeptidases, including carboxypeptidase A and the angiotensin-converting enzyme. Strategies previously developed to screen for inhibitors of those enzymes may aid in the search for inhibitors of the protein farnesyltransferase.

Nonfarnesylated tetrapeptide inhibitors of protein farnesyltransferase.

The protein farnesyltransferase from rat brain was previously shown to be inhibited competitively by tetrapeptides that conform to the consensus Cys-A1-A2-X, where A1 and A2 are aliphatic amino acids and X is methionine, serine, or phenylalanine. In the current studies we use a thin layer chromatography assay to show that most of these tetrapeptides are themselves farnesylated by the purified enzyme. Two classes of tetrapeptides are not farnesylated and therefore act as true inhibitors: 1) those that contain an aromatic residue at the A2 position and 2) those that contain penicillamine (beta,beta-dimethylcysteine) in place of cysteine. The most potent of these pure inhibitors was Cys-Val-Phe-Met, which inhibited farnesyltransferase activity by 50% at less than 0.1 microM. These data indicate that the inclusion of bulky aromatic or methyl residues in a tetrapeptide can abolish prenyl group transfer without blocking binding to the enzyme. This information should be useful in the design of peptides or peptidomimetics that inhibit farnesylation and thus block the action of p21ras proteins in animal cells.

Nonidentical subunits of p21H-ras farnesyltransferase. Peptide binding and farnesyl pyrophosphate carrier functions.

The protein farnesyltransferase purified from rat brain contains two nonidentical subunits, alpha and beta. The holoenzyme forms a stable complex with [3H]farnesyl pyrophosphate (FPP) that can be isolated by gel filtration. The [3H]FPP is not covalently bound to the enzyme; it is released unaltered when the enzyme is denatured. When incubated with an acceptor such as p21H-ras, the complex transfers [3H]farnesyl from the bound [3H]FPP to the ras protein. This transfer is not sensitive to dilution by unbound FPP, suggesting that the [3H]FPP is bound at a site that leads to direct transfer to the p21H-ras acceptor. Cross-linking studies show that the p21H-ras binds to the lower molecular weight subunit (beta-subunit), raising the possibility that the [3H]FPP binds to the alpha-subunit. If this suggestion can be confirmed, it would invoke a reaction mechanism in which the alpha-subunit acts as a prenyl pyrophosphate carrier that delivers FPP to p21H-ras which is bound to the beta-subunit.

Protein farnesyltransferase and geranylgeranyltransferase share a common alpha subunit.

Mammalian farnesyltransferase, which attaches a 15 carbon isoprenoid, farnesyl, to a cysteine in p21ras proteins, contains two subunits, alpha and beta. The beta subunit is known to bind p21ras proteins. We show here that the alpha subunit is shared with another prenyltransferase that attaches 20 carbon geranylgeranyl to Ras-related proteins. Farnesyltransferase and geranylgeranyltransferase have similar molecular weights on gel filtration, but are separated by ion exchange chromatography. Both enzymes are precipitated and immunoblotted by multiple antibodies directed against the alpha subunit of farnesyltransferase. The two transferases have different specificities for the protein acceptor; farnesyltransferase prefers methionine or serine at the COOH-terminus and geranylgeranyltransferase prefers leucine. The current data indicate that both prenyltransferases are heterodimers that share a common alpha subunit with different beta subunits.