Molecular, cellular, and antigen requirements for development of age-associated T cell clonal expansions in vivo

T cell aging manifests itself both at the cellular (cell-autonomous defects in signaling) and at the population (age-related dysregulation of T cell homeostasis) levels. A prominent contributor to the latter is the appearance of T cell clonal expansions (TCE), with a potential to impair immune defense. In this study, we investigated molecular, cellular, and Ag requirements for TCE development. Of the mutant mice tested, old animals lacking MHC class I exhibited 7-fold fewer TCE than controls, with a 7-fold reduction in TCE. By contrast, animals lacking only one of the MHC class I molecules (Kb or Db), or IL-7R, or devoid of T cell renewal via adult thymectomy, all exhibited significant increases in TCE incidence. This increase directly correlated to lymphopenia, increased CD8 T cell turnover and an accumulation of memory-phenotype T cells. These data suggested that homeostatic cell division in the CD8 compartment enhances the formation of TCE. Repeated immunization with peptide/adjuvant did not result in an increase in Ag-specific TCE; however, adjuvant alone increased TCE incidence. In these experiments, therefore, nonspecific and/or homeostatic proliferation was more efficient in generating TCE in mice than repeated Ag-driven stimulation, suggesting that many, if not most, TCE in specific pathogen-free laboratory mice may be Ag-independent.     

West Nile Virus Capsid Degradation of Claudin Proteins Disrupts Epithelial Barrier Function

During acute infection, West Nile virus (WNV) has been reported to infect a variety of cell types in various tissues of both experimentally and naturally infected hosts. Virus infects epithelial cells in the skin, kidney, intestine, and testes, although the importance of these findings is unclear. In the current study, we have observed that WNV infection of kidney tubules in mice coincides with the loss of expression of several members of the claudin family. Proteins of this family are often involved in epithelial barrier formation and function. WNV infection of epithelial cells in culture resulted in a decrease in the transepithelial electrical resistance, an increase in the efflux of mannitol across the monolayer, and a loss of intracellular levels of claudin-1 to -4. WNV capsid alone was sufficient for the degradation event, which was mediated through lysosomal proteases. Since epithelial cells are frequent sites of WNV infection, these observations imply a potential mechanism for virus dissemination and extraneural pathogenesis.West Nile Virus (WNV) is a mosquito-borne flavivirus that first appeared in the United States in 1999. Since that time, the virus has spread across the continental United States, causing significant morbidity and mortality. Roughly 20% of WNV infections are symptomatic, and approximately 1 out of every 150 infections progresses to encephalitis and/or meningitis (39, 42). Following the bite of a carrier mosquito, WNV infection of the host is thought to initiate in the Langerhans cells of the skin (13). Viral replication continues in the regional tissue and lymph nodes, which results in the dissemination of the virus into the bloodstream. Replication then proceeds at several sites throughout the host, including the kidneys, heart, connective tissue, smooth muscle, spleen, and ultimately the brain (46). Infection of the nervous system is characteristic of the most severe cases of WNV disease, often resulting in death or long-term neurologic sequelae (26). Pathologies associated with extraneural sites of infection have also been reported, including acute renal failure (11, 24).Epithelial cells are major targets of WNV infection in vivo in both humans and experimentally infected rodents. In a hamster model of WNV infection, virus can be detected in the epithelial cells of the kidney up to 60 days postinfection, suggesting that this tissue may be a site of viral persistence (53). Infection of kidney epithelium has also been found in WNV-infected mice (20) and dogs (12). In WNV-infected humans, epithelial cell infection has also been demonstrated in several organs, including the kidney, lung, pancreas, thyroid, intestine, and testes (4). These studies suggest that the epithelial cells may play an important role in WNV pathogenesis. However, the growth characteristics of WNV in epithelial cells and the effect of WNV on the polarity and permeability functions of polarized epithelial cells have not been investigated. One important feature of epithelial cells is the presence of tight junctions (TJ). TJ are intercellular contacts between endothelial or epithelial cells which allow the formation of polarized cells with discrete apical and basolateral plasma membrane domains and separate physiologically distinct apical and basolateral fluid compartments (22). Macromolecules larger than 30 Å in diameter are generally excluded from the TJ, but permeability to small ions varies depending on tissue-specific requirements and local physiological stimuli (2, 23). TJ are thus not static seals but dynamic structures subject to transient changes in permeability (30). The TJ is composed of the transmembrane proteins occludin, claudin (a family of more than 20 members), and junctional adhesion molecule (a family of four known members). These proteins mediate cell-cell interactions and regulate junctional permeability. The transmembrane proteins interact on their cytoplasmic side with several other components of the TJ. These include zonula occludens 1, 2, and 3 (ZO-1 to -3, which belong to a family of membrane-associated guanylate kinase homologues). ZO-1 is thought to nucleate the initial formation of the TJ and to provide a scaffold for TJ assembly. This process is thought to require the interaction of ZO-1 with the adherens junction (1, 7).In this study, we show that WNV infection of epithelial cells in vivo and in vitro results in a loss of claudin protein expression. The loss of claudin expression coincides with perturbation of the permeability functions of cultured epithelial cells as measured by a reduction in transepithelial electrical resistance (TER) and an increase in the efflux of [¹⁴C]mannitol across the cell monolayer. Expression of WNV capsid alone was sufficient to mediate these events. Epithelial cells are likely to be targets for WNV infection from the earliest times of exposure (e.g., the keratinocytes at the site of the mosquito bite) and also may be sites of long-term persistence. Therefore, the effect of the virus on the TJ, a crucial component of epithelial function, suggests a possible mechanism of virus spread, as well as potentially contributing to pathogenesis.     

The role of cathepsin X in cell signaling

Cathepsin X is a lysosomal cysteine protease, found predominantly in cells of monocyte/macrophage lineage. It acts as a monocarboxypepidase and has a strict positional and narrower substrate specificity relative to the other human cathepsins. In our recent studies we identified—β₂ subunit of integrin receptors and α and γ enolase as possible substrates for cathepsin X carboxypeptidase activity. In both cases cathepsin X is capable to cleave regulatory motifs at C-terminus affecting the function of targeted molecules. We demonstrated that via activation of β₂ integrin receptor Mac-1 (CD11b/CD18) active cathepsin X enhances adhesion of monocytes/macrophages to fibrinogen and regulates the phagocytosis. By activation of Mac-1 receptor cathepsin X may regulate also the maturation of dendritic cells, a process, which is crucial in the initiation of adaptive immunity. Cathepsin X activates also the other β₂ integrin receptor, LFA-1 (CD11a/CD18) which is involved in the proliferation of T lymphocytes. By modulating the activity of LFA-1 cathepsin X causes cytoskeletal rearrangements and morphological changes of T lymphocytes enhancing ameboid-like migration in 2-D and 3-D barriers and increasing homotypic aggregation. The cleavage of C-terminal amino acids of α and γ enolase by cathepsin X abolishes their neurotrophic activity affecting neuronal cell survival and neuritogenesis.Key words: cathepsin X, integrin, enolase, T lymphocyte, macrophage, dendritic cell, adhesion, migration, neuritogenesisProteases comprise a group of enzymes that catalyse the cleavage of a peptide bond in a polypeptide chain by nucleophilic attack on the carbonyl carbon. The proteases are either exopeptidases cleaving one or a few amino acids at the N- or C-terminus of polypeptide chain or endopeptidases that cleave the peptide bond internally. According to the catalytic mechanism the endopeptidases are divided into aspartic, cysteine, serine, threonine and metallo endoproteases—see MEROPS database.¹ To date, 561 genes encoding for proteases have been identified in human genome. Among them 148 genes encode for cysteine proteases including a group of eleven lysosomal cysteine proteases (members of C1 family) also called cathepsins. They exhibit different expression patterns, levels and specificities, all of which contribute to their differential physiological roles. Some of them, like cathepsins B, H, L and C are ubiquitously present in tissues, whereas others (cathepsins S, V, X, O, K, F and W) are expressed by specific cell types. Cysteine cathepsins were long believed to be responsible for the terminal protein degradation in the lysosomes, however, this view has changed dramatically when they have been found to be involved in a number of important cellular processes and pathologies.^2,3In contrast to other cathepsins, cathepsin X was discovered only recently. Its gene,^4,5 structure^6,7 and activity properties^8,9 show several unique features that distinguish it clearly from other human cysteine proteases. It has a very short pro-region⁷ and a three residue insertion motif which forms a characteristic “mini loop.”⁶ Cathepsin X exhibits carboxypeptidase activity⁶ and, in contrast to cathepsin B, the other carboxypeptidase, it does not act as an endopeptidase. Contrary to the first reports,⁴ cathepsin X is not widely expressed in cells and tissues, but is restricted to the cells of the immune system, predominantly monocytes, macrophages and dendritic cells.¹⁰ Higher levels of cathepsin X were also found in tumor and immune cells of prostate¹¹ and gastric¹² carcinomas and in macrophages of gastric mucosa, especially after infection by Helicobacter pylori.¹³ Recently it was shown that cathepsin X is abundantly expressed in mouse brain cells, in particular glial cells. Its upregulation was also detected in the brains of patients with Alzheimer disease.¹⁴The involvement of cathepsin X in signal transduction is implied by the integrin-binding motifs, present in its pro-form (RGD: Arg-Gly-Asp) and mature form (ECD: Glu-Cys-Asp).^4,5 Moreover, cathepsin X binds cell surface heparan sulfate proteoglycans¹⁵ which are also involved in integrin regulation. A strong co-localization of pro-cathepsin X with β₃ integrin subunit was demonstrated in our study in pro-monocytic U-937 cells.¹⁶ Further, it was reported that the pro-form of cathepsin X interacts with α_vβ₃ integrin through the RGD motif in lamellipodia of human umbilical vein endothelial cells (HUVECs).¹⁷ However, we showed that the active form of cathepsin X co-localized predominantly with β₂ integrin subunit in various cells of monocytes/macrophage lineage. Active cathepsin X was shown to regulate β₂ integrin-dependent adhesion, phagocytosis and T lymphocyte activation by interaction with macrophage antigen-1 (CD11b/CD18, Mac-1). We showed that inhibitors and monoclonal antibodies, capable to impair cathepsin X enzymatic activity, reduced the binding of differentiated U-937 cells to fibrinogen and polystyrene surface in a dose dependent manner. The co-localization of active cathepsin X with β₂ integrin chain was particularly enhanced in interactions of monocyte/macrophages with endothelial and tumor cells.Besides in monocytes and macrophages the active cathepsin X plays a role in β₂ integrin activation also in dendritic cells (DC), which are crucial for effective antigen presentation and initiation of T cell dependent immune response. Maturation of dendritic cells is accompanied by a range of morphological and cytoskeleton structure changes. In response to maturation stimuli in vitro, DCs rapidly adhere, develop polarity and assemble actin rich structures at the leading edge, known as podosomes.¹⁸ The adhesion of immature DCs to the extracellular matrix, is accompanied by recruitment of Mac-1 integrin receptor, which can be activated by cathepsin X. We have shown that, during maturation, cathepsin X translocates to the plasma membrane of maturing DCs, enabling Mac-1 activation and, consequently, cell adhesion.¹⁹ In mature DCs cathepsin X redistributes from the membrane to the perinuclear region, which coincides with the de-adhesion of DCs, formation of cell clusters and acquisition of the mature phenotype. Again, the inhibition of cathepsin X activity during DC differentiation and maturation reduced the capacity of DCs to stimulate T lymphocytes.β₂ integrin receptors are important also in T lymphocyte functions, such as migration and invasion across the endothelium and tissues. Lymphocyte function-associated antigen-1 (CD11a/CD18, LFA-1), the predominant β₂ integrin receptor in T lymphocytes enables cell-cell interactions and homotypic aggregation via LFA-1-ICAM-1 (intracellular adhesion molecule-1) interactions. LFA-1 can act also as a true signaling receptor, causing F-actin reorganization that leads to cytoskeletal changes of the cell²⁰ and a switch from a spherical to a polarized shape.²¹ Although the concentration of cathepsin X in T lymphocytes is lower compared to monocytes and macrophages, we showed that it interacts with LFA-1 promoting cytoskeleton-dependent morphological changes and migration across 2D and 3D models of ICAM-1 and Matrigel.^22,23 Its co-localization with LFA-1 was particularly evident at the trailing edge protrusion, the uropod, which plays an important role in T lymphocyte migration and cell-cell interactions (Fig. 1). Uropodal active cathepsin X cleaves C-terminal amino acids of β chain in LFA-1 promoting its high affinity conformation and the binding of the cytoskeletal protein talin. This interaction stabilizes the uropod and promotes its elongation (Jevnikar, et al. submitted).Open in a separate windowFigure 1Activation of LFA-1 integrin receptor by cathepsin X at the uropod of T lymphocyte promotes cytoskeleton-dependent morphological changes and cell migration.³⁰We demonstrated that uropods of cathepsin X upregulated T lymphocytes elongate to extreme length and form cell-to-cell connections, the nanotubes (Obermajer, et al. in press). Membrane (or tunneling) nanotubes were recently found as a new principle of cell-to-cell communication enabling transmission of complex and specific messages to distant cells through a physically connected network. Calcium fluxes, vesicles and cell-surface components can all traffic between cells connected by nanotubes. In immune system nanotubes integrate communities of cells for a better coordination of their action in various stages of immune response. We showed that nanotubes of cathepsin X upregulated T lymphocytes could readily transfer cellular organelles such as mitochondria and lysosomes and proposed that nanotube mediated transfer makes possible T lymphocyte activation without the need for direct contact with antigen presenting cells.The exact mechanism of cathepsin X translocation towards plasma membrane and degradation of C-terminal amino acids of β chain remains unclear. In lysosomes cathepsin X can be found as a pro- and active form. After cell activation cathepsin X containing vesicles translocate towards the plasma membrane,¹⁶ as observed also for some other lysosomal proteases.²⁴ During this process it is possible that pro-cathepsin X is activated by the other cysteine protease cathepsin L, as shown already in vitro.⁸ Both proteases were strongly co-localized with β₂ integrin chain at plasma membrane of activated monocytes/macrophages and at uropodes of T lymphocytes. Simultaneous co-localization with the lysosomal markers demonstrates that at least the initial translocation of cathepsin X towards cytoplasmic tail of β₂ integrin chain is vesicular. The interaction of cathepsin X with β₂ integrin subunit was confirmed by immunoprecipitation and FRET.²² According to in vitro experiments we propose that cathepsin X cleaves sequentially C-terminal aminoacids F⁷⁶⁶, A⁷⁶⁷, E⁷⁶⁸ and S⁷⁶⁹ of β₂ integrin subunit (Fig. 2) until reaching proline in penultimate position, confirming previous observation that the proline in S2 position leads to resistance to cathepsin X proteolysis.²⁵ Also, our results are in agreement with the previously mentioned monocarboxypeptidase activity of cathepsin X.^26,27 Since the signaling to and from the integrins is mainly regulated by the short cytoplasmic tail of β₂ subunit,²⁸ cathepsin X mediated β₂ integrin truncation leads to regulation of the receptor signaling. The interaction of cytoplasmic tail with different cytoskeletal and regulatory proteins, such as talin, filamin, radixin and α-actinin is crucial for signal transduction and modulation of cytoskeleton.²⁹Open in a separate windowFigure 2Cathepsin X activates LFA-1 by sequential cleavage of C-terminal amino acids of β₂ integrin subunit.Besides β₂ integrin chain we recently identified isozymes α and γ enolases as another molecular target for cathepsin X carboxypeptidase activity (Obermajer, et al. submitted). We demonstrated that cathepsin X sequentially cleaves C-terminal amino acids of both isozymes, abolishing their neurotrophic activity. On this way the neuronal cell survival and neuritogenesis can be regulated. Inhibition of cathepsin X activity increases the generation of plasmin, essential for neuronal differentiation and changes the length distribution of neurites, especially in the early phase of neurite outgrowth. Moreover, cathepsin X inhibition increases neuronal survival and reduces serum deprivation induced apoptosis, particularly in the absence of nerve growth factor.     

Ensheathing glia promote increased lifespan and healthy brain aging

Glia have an emergent role in brain aging and disease. In the Drosophila melanogaster brain, ensheathing glia function as phagocytic cells and respond to acute neuronal damage, analogous to mammalian microglia. We previously reported changes in glia composition over the life of ants and fruit flies, including a decline in the relative proportion of ensheathing glia with time. How these changes influence brain health and life expectancy is unknown. Here, we show that ensheathing glia but not astrocytes decrease in number during Drosophila melanogaster brain aging. The remaining ensheathing glia display dysregulated expression of genes involved in lipid metabolism and apoptosis, which may lead to lipid droplet accumulation, cellular dysfunction, and death. Inhibition of apoptosis rescued the decline of ensheathing glia with age, improved the neuromotor performance of aged flies, and extended lifespan. Furthermore, an expanded ensheathing glia population prevented amyloid-beta accumulation in a fly model of Alzheimer's disease and delayed the premature death of the diseased animals. These findings suggest that ensheathing glia play a vital role in regulating brain health and animal longevity.     

Ion channels formed by chemical analogs of gramicidin A.

Channel-forming peptides such as gramicidin A offer the opportunity to study the relationship between chemical structure and transport properties of an ion channel. This article summarizes a number of recent experiments with chemical analogs and derivatives of gramicidin A using artificial lipid bilayer membranes. The introduction of negative charges near the channel mouth leads to an increase in the cation transport rate. Hybrid channels consisting of a neutral and a negatively charged or of a positively and a negatively charged half-channel may be formed. The current-voltage characteristic of these hybrid channels exhibits a pronounced asymmetry.Experiments with charged derivatives of gramicidin A have been used in order to distinguish between different structural models of the dimeric channel; these studies strongly support Urry's model of a single-stranded, head-to-head associated helical dimer. In a further set of experiments gramicidin analogs with modified amino acid sequence were studied. It was found that a single substitution (tryptophan replaced by phenylalanine) leads to marked changes in the conductance of the channel. Analogs with a simplified amino acid sequence such as (L-Trp-D-Leu)7-L-Trp or L-Trp-Gly-(L-Trp-D-Leu)6-L-Trp are able to form cation permeable channels with similar properties as gramicidin A.     

Structural and membrane modifying properties of suzukacillin,a peptide antibiotic related to alamethicin: Part A. Sequence and conformation

The primary structure and conformation of the polypeptide antibiotic suzukacillin A are investigated. Suzukacillin A isolated from the Trichoderma viride strain 1037 and exhibits membrane modifying and lysing properties similar to those of alamethicin.A combined gas chromatographic mass spectrometric analysis of the trifluoroacetylated peptide methyl esters of partial hydrolysates revealed a tentative sequence of 23 residues including 10 2-methylalanines and one phenylalaninol, which shows many fragments known from alamethicin: Ac-Aib-Pro-Val-Aib-Val-Ala-Aib-Ala-Aib-Aib-Gln-Aib-Leu-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Glu(Pheol)-Gln-OH. All chiral amino acids and phenylalaninol have l-configuration. Ultraviolet and infrared spectroscopy, circular dichroism in various solvents and in particular ¹³C nuclear magnetic resonance have been used for a comparative study of suzukacillin with alamethicin. Suzukacillin has a partially α-helical structure and the helix content increases largely from polar to lipophilic solvents. Suzukacillin aggregates more strongly than alamethicin in aqueous media due to a longer α-helical part and higher number of aliphatic residues. A part of the α-helix is exceptionally stabilized due to 2-methylalanine residues shielding the peptide bonds from interactions with polar solvents. In lipophilic solvents and lecithin vesicles particularly large temperature induced reductions of the high α-helix content are found for alamethicin. Suzukacillin shows similar temperature coefficients in lipophilic media, however, in contrast to alamethicin a more linear change in intensity of the Cotton effects is observed.     

Structural and membrane modifying properties of suzukacillin, a peptide antibiotic related to alamethicin. Part A. Sequence and conformation.

The primary structure and conformation of the polypeptide antibiotic suzukacillin A are investigated. Suzukacillin A is isolated from the Trichoderma viride strain 1037 and exhibits membrane modifying and lysing properties similar to those of alamethicin. A combined gas chromatographic mass spectrometric analysis of the trifluoroacetylated peptide methyl esters of partial hydrolysates revealed a tentative sequence of 23 residues including 10 2-methylalanines and one phenylalaninol, which shows many fragments known from alamethicin: Ac-Aib-Pro-Val-Aib-Val-Ala-Aib-Ala-Aib-Aib-Gln-Aib-Leu-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Glu(Pheol)-Gln-OH. All chiral amino acids and phenylalainol have L-configuration. Ultraviolet and infrared spectroscopy, circular dichroism in various solvents and in particular 13C nuclear magnetic resonance have been used for a comparative study of suzukacillin with alamethicin. Suzukacillin has a partially alpha-helical structure and the helix content increases largely from polar to lipophilic solvents. Suzukacillin aggregates more strongly than alamethicin in aqueous medis due to a longer alpha-helical part and higher number of aliphatic residues. A part of the alpha-helix is exceptionally stabilized due to 2-methylalanine residues shielding the peptide bonds from interactions with polar solvents. In lipophilic solvents and lecithin vesicles particularly large temperature induced reductions of the high alpha-helix content are found for alamethicin. Suzukacillin shows similar temperature coefficients in lipophilic media, however, in contrast to alamethicin a more linear change in intensity of the Cotton effects is observed.     

Inhibitory properties of cystatin F and its localization in U937 promonocyte cells

Cystatin F is a recently discovered type II cystatin expressed almost exclusively in immune cells. It is present intracellularly in lysosome-like vesicles, which suggests a potential role in regulating papain-like cathepsins involved in antigen presentation. Therefore, interactions of cystatin F with several of its potential targets, cathepsins F, K, V, S, H, X and C, were studied in vitro. Cystatin F tightly inhibited cathepsins F, K and V with Ki values ranging from 0.17 nM to 0.35 nM, whereas cathepsins S and H were inhibited with 100-fold lower affinities (Ki approximately 30 nM). The exopeptidases, cathepsins C and X were not inhibited by cystatin F. In order to investigate the biological significance of the inhibition data, the intracellular localization of cystatin F and its potential targets, cathepsins B, H, L, S, C and K, were studied by confocal microscopy in U937 promonocyte cells. Although vesicular staining was observed for all the enzymes, only cathepsins H and X were found to be colocalized with the inhibitor. This suggests that cystatin F in U937 cells may function as a regulatory inhibitor of proteolytic activity of cathepsin H or, more likely, as a protection against cathepsins misdirected to specific cystatin F containing endosomal/lysosomal vesicles. The finding that cystatin F was not colocalized with cystatin C suggests distinct functions for these two cysteine protease inhibitors in U937 cells.     

EPR study of a copper center in a single crystal of cytosine monohydrate

Copper is found incorporated into the crystal structure of cytosine monohydrate grown from aqueous solution of commercially available cytosine. Upon ionizing irradiation, the crystals exhibited the electron paramagnetic resonance (EPR) spectra characteristic of Cu(II) complex. Planar coordination bonding to the cupric ion, having three nitrogen atoms and an oxygen as ligands, is interpreted to bridge two cytosine molecules, replacing the two cytosine-cytosine hydrogen bonds present in pure crystals. The EPR signals are much stronger for crystals grown from the solutions to which small amount of copper powder were added.