Panagiotis S. Kabouridis

Altered lipid raft–associated signaling and ganglioside expression in T lymphocytes from patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is characterized by abnormalities in T lymphocyte receptor-mediated signal transduction pathways. Our previous studies have established that lymphocyte-specific protein tyrosine kinase (LCK) is reduced in T lymphocytes from patients with SLE and that this reduction is associated with disease activity and parallels an increase in LCK ubiquitination independent of T cell activation. This study investigated the expression of molecules that regulate LCK homeostasis, such as CD45, C-terminal Src kinase (CSK), and c-Cbl, in lipid raft domains from SLE T cells and investigated the localization of these proteins during T cell receptor (TCR) triggering. Our results indicate that the expression of raft-associated ganglioside, GM1, is increased in T cells from SLE patients and LCK may be differentially regulated due to an alteration in the association of CD45 with lipid raft domains. CD45 tyrosine phosphatase, which regulates LCK activity, was differentially expressed and its localization into lipid rafts was increased in T cells from patients with SLE. Furthermore, T cells allowed to rest in vitro showed a reversal of the changes in LCK, CD45, and GM1 expression. The results also revealed that alterations in the level of GM1 expression and lipid raft occupancy cannot be induced by serum factors from patients with SLE but indicated that cell-cell contact, activating aberrant proximal signaling pathways, may be important in influencing abnormalities in T cell signaling and, therefore, function in patients with SLE.

Lipid rafts in T cell receptor signalling (Review)

The molecular events and the protein components that are involved in signalling by the T cell receptor (TCR) for antigen have been extensively studied. Activation of signalling cascades following TCR stimulation depends on the phosphorylation of the receptor by the tyrosine kinase Lck, which localizes to the cytoplasmic face of the plasma membrane by virtue of its post-translational modification. However, the precise order of events during TCR phosphorylation at the plasma membrane, remains to be defined. A current theory that describes early signalling events incorporates the function of lipid rafts, microdomains at the plasma membrane with distinct lipid and protein composition. Lipid rafts have been implicated in diverse biological functions in mammalian cells. In T cells, molecules with a key role in TCR signalling, including Lck, localize to these domains. Importantly, mutant versions of these proteins which fail to localise to raft domains were unable to support signalling by the TCR. Biochemical studies using purified detergent-resistant membranes (DRM) and confocal microscopy have suggested that upon stimulation, the TCR and Lck-containing lipid rafts may come into proximity allowing phosphorylation of the receptor. Further, there are data suggesting that phosphorylation of the TCR could depend on a transient increase in Lck activity that takes place within lipid rafts to initiate signalling. Current results and a model of how lipid rafts may regulate TCR signalling are discussed.

Microbiota Controls the Homeostasis of Glial Cells in the Gut Lamina Propria

Summary The intrinsic neural networks of the gastrointestinal tract are derived from dedicated neural crest progenitors that colonize the gut during embryogenesis and give rise to enteric neurons and glia. Here, we study how an essential subpopulation of enteric glial cells (EGCs) residing within the intestinal mucosa is integrated into the dynamic microenvironment of the alimentary tract. We find that under normal conditions colonization of the lamina propria by glial cells commences during early postnatal stages but reaches steady-state levels after weaning. By employing genetic lineage tracing, we provide evidence that in adult mice the network of mucosal EGCs is continuously renewed by incoming glial cells originating in the plexi of the gut wall. Finally, we demonstrate that both the initial colonization and homeostasis of glial cells in the intestinal mucosa are regulated by the indigenous gut microbiota.

Lipid rafts and T-lymphocyte function: Implications for autoimmunity

Experimental evidence indicates that the mammalian cell membrane is compartmentalized. A structural feature that supports membrane segmentation implicates assemblies of selected lipids broadly referred to as lipid rafts. In T‐lymphocytes, lipid rafts are implicated in signalling from the T‐cell antigen receptor (TCR) and in localization and function of proteins residing proximal to the receptor. This review summarizes the current literature that deals with lipid raft involvement in T‐cell activation and places particular emphasis in recent studies investigating lipid rafts in autoimmunity. The potential of lipid rafts as targets for the development of a new class of immune‐modulating compounds is discussed.

Inhibition of NF-κB Activity by a Membrane-Transducing Mutant of IκBα

The transcription factor NF-κB is regulated by the IκB family of proteins. The nonphosphorylatable, nondegradable superrepressor IκBα (srIκBα) mutant is a potent inhibitor of NF-κB activity when expressed in cells. We generated a form of srIκBα in which its N terminus is fused to the protein transduction domain of HIV TAT (TAT-srIκBα). Purified TAT-srIκBα protein rapidly and efficiently entered HeLa or Jurkat T cells. TAT-srIκBα, when exogenously added to HeLa cells, inhibited in a dose-dependent manner TNF-α- or IL-1β-induced NF-κB activation and binding of NF-κB to its consensus DNA sequence. TAT-srIκBα was coimmunoprecipitated with the p65 subunit of NF-κB, and this interaction was resistant to stimulation with IL-1β. Therefore, TAT-srIκBα-mediated inhibition could result from its nonreversible binding and sequestration of endogenous NF-κB. In contrast, exogenously added TAT-srIκBα did not inhibit IL-1β-induced activation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, or p38 mitogen-activated protein kinases or the phosphorylation and degradation of endogenous IκBα. These results identify a novel way for direct regulation of NF-κB activity in diverse cell types that may be useful for therapeutic purposes.

Emerging roles of gut microbiota and the immune system in the development of the enteric nervous system

The enteric nervous system (ENS) consists of neurons and glial cells that differentiate from neural crest progenitors. During embryogenesis, development of the ENS is controlled by the interplay of neural crest cell-intrinsic factors and instructive cues from the surrounding gut mesenchyme. However, postnatal ENS development occurs in a different context, which is characterized by the presence of microbiota and an extensive immune system, suggesting an important role of these factors on enteric neural circuit formation and function. Initial reports confirm this idea while further studies in this area promise new insights into ENS physiology and pathophysiology.

Selective interaction of LAT (linker of activated T cells) with the open-active form of Lck in lipid rafts reveals a new mechanism for the regulation of Lck in T cells.

In T cells, the lipid raft-associated Lck is strongly tyrosine phosphorylated and has reduced enzymic activity in contrast with the detergent-soluble pool, which has substantial activity. Lck tagged at the C-terminus (Lck/V5-His) was efficiently captured by epitope-specific reagents from the detergent-soluble fraction but not from lipid rafts. Binding was restored following urea denaturation, suggesting that Lck/V5-His is in a closed conformation in these domains. In agreement with this hypothesis, the Tyr(505) --> Phe/V5-His and Arg(154) --> Lys/V5-His mutants, which disrupt the SH2-Tyr(505) intramolecular interaction, were efficiently precipitated from lipid rafts. In contrast to Lck, Fyn/V5-His was precipitated equally well from both fractions. In the LAT(linker of activated T cells)-deficient J.CaM2 cells, Tyr(505) phosphorylation of raft-associated Lck was reduced whereas its enzymic activity was elevated. This correlated with decreased levels of raft-localized Csk (C-terminal Src kinase) kinase. Increased tyrosine phosphorylation of Lck was restored in LAT-reconstituted J.CaM2 cells suggesting that LAT negatively regulates Lck activity in lipid rafts. Co-immunoprecipitation experiments from Tyr(505) --> Phe/V5-His-expressing cells revealed that LAT preferentially interacts with the open form of Lck in T cell raft domains. These results demonstrate that, unlike the non-raft pool, Lck in lipid rafts has a closed-inactive structure, and that LAT plays a role in maintaining this conformation, possibly by facilitating critical associations within lipid rafts via its capacity to interact with the open form of the kinase.

Secretion and uptake of TAT-fusion proteins produced by engineered mammalian cells.

BACKGROUNDnIntracellular signaling can be regulated by the exogenous addition of physiological protein inhibitors coupled to the TAT protein transduction domain. Thus far experiments have been performed with purified inhibitors added exogenously to cells in vitro or administered in vivo. Production of secretable TAT-fusion proteins by engineered mammalian cells, their uptake, and route of entry has not been thoroughly investigated. Such methodology, if established, could be useful for transplantation purposes.nnnMETHODSnSecretion of TAT-fusion proteins from transfected mammalian cells was achieved by means of a signal peptide. Cell uptake and subcellular localization of TAT-fusion proteins were determined by immunoblotting and confocal microscopy.nnnRESULTSnEngineered TAT-fusion proteins were secreted with variable efficiency depending on the nature of the protein fused to the TAT peptide. Secreted proteins were able to transduce unmanipulated cells. Their mechanism of entry into cells partly involves lipid rafts and a portion of the internalised protein is directed to the Golgi.nnnCONCLUSIONSnGeneration of secretable TAT-coupled inhibitors of signaling pathways, able to transduce other cells can be achieved.nnnGENERAL SIGNIFICANCEnThese results provide key information that will assist in the design of TAT-inhibitors and engineered cells in order to regulate cell function within tissues.

Single chain antibodies specific for fatty acids derived from a semi-synthetic phage display library.

The biological activities of many acylated molecules are lipid dependent. Lipids, however, are poorly immunogenic or non-immunogenic. We employed a phage display semi-synthetic human antibody library to isolate anti-lipid antibodies. Selection was done against methyl palmitate, a 16 carbon aliphatic chain, and a major component of bacterial glycolipids and lipoproteins in animal cells. The selected single chain variable fragment (scFv) bound specifically to a 16 carbon aliphatic chain and to a lesser extent to a 14 or 18 carbon aliphatic chain and poorly to either 12, 22 or 8 carbon aliphatic chains. Furthermore, the scFv prevented micelle formation of lipoteichoic acid from Gram-positive bacteria; inhibited lipopolysaccharide-induced tumor necrosis factor alpha release in mononuclear cells; bound to hydrophobic bacterial surfaces, especially those of Gram-positive bacteria, and bound to Lck, a mammalian palmitated lipoprotein. Our data suggest that the phage antibody library can be successfully employed to obtain human anti-aliphatic scFv human antibody fragment with potential therapeutic applications in neutralizing the deleterious effects of bacterial toxins as well as in structure--function analysis of lipoproteins in animal cells.

Agrin Signalling Contributes to Cell Activation and Is Overexpressed in T Lymphocytes from Lupus Patients

It is shown in this study that the heparan sulfate proteoglycan agrin is overexpressed in T cells isolated from patients with the autoimmune disease systemic lupus erythematosus (SLE). Freshly isolated CD4+ and CD8+ subpopulations both exhibited higher expression over healthy controls, which however, gradually declined when cells were cultured in vitro. Agrin expression was induced following in vitro activation of cells via their Ag receptor, or after treatment with IFN-α, a cytokine shown to be pathogenic in lupus. Furthermore, serum from SLE patients with active disease was able to induce agrin expression when added to T cells from healthy donors, an increase that was partially blocked by neutralizing anti-IFN-α Abs. Cross-linking agrin with mAbs resulted in rapid reorganization of the actin cytoskeleton, activation of the ERK MAPK cascade, and augmentation of anti-CD3-induced proliferation and IL-10 production, indicating that agrin is a functional receptor in T cells. These results demonstrate that agrin expression in human T cells is regulated by cell activation and IFN-α, and may have an important function during cell activation with potential implications for autoimmunity.