In vivo lymphocyte activation and apoptosis by lectins of the Diocleinae subtribe

This paper reports the overall effects of three lectins, extracted from Canavalia brasiliensis, Dioclea violacea, and D. grandiflora, on BALB/c mice popliteal draining lymph nodes. These lectins have presented high stimulatory capacity on lymph node T cells. Additionally, they were able to induce apoptosis and inflammation (frequently associated with high endothelial venule necrosis). The data presented here suggest that the Diocleinae lectins studied can stimulate in vivo T cell activation and apoptosis, as well as present important side effects.

High tidal volume ventilation affects neuronal activation in mechanically ventilated rats

Els malalts crítics presenten sovint seqüeles cognitives a llarg termini, l’aplicació de ventilació mecànica (VM) pot contribuir al seu desenvolupament. El principal objectiu del nostre estudi fou investigar l’efecte de dos patrons de ventilació (volum corrent elevat/baix) en l’activació neuronal (expressió de c-fos) en determinades àrees cerebrals en un model en rates. Després de 3 hores sota VM, es va trobar activació neuronal; la seva intensitat va ser superior al grup de volum corrent elevat, suggerint un efecte iatrogènic de la VM al cervell. Aquests resultats suggereixen que cal aprofundir en l’estudi del crosstalk cervell-pulmó en malalts crítics sota VM.

CARMA1 is a critical lipid raft-associated regulator of TCR-induced NF-kappa B activation.

CARMA1 is a lymphocyte-specific member of the membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins, which coordinate signaling pathways emanating from the plasma membrane. CARMA1 interacts with Bcl10 via its caspase-recruitment domain (CARD). Here we investigated the role of CARMA1 in T cell activation and found that T cell receptor (TCR) stimulation induced a physical association of CARMA1 with the TCR and Bcl10. We found that CARMA1 was constitutively associated with lipid rafts, whereas cytoplasmic Bcl10 translocated into lipid rafts upon TCR engagement. A CARMA1 mutant, defective for Bcl10 binding, had a dominant-negative (DN) effect on TCR-induced NF-kappa B activation and IL-2 production and on the c-Jun NH(2)-terminal kinase (Jnk) pathway when the TCR was coengaged with CD28. Together, our data show that CARMA1 is a critical lipid raft-associated regulator of TCR-induced NF-kappa B activation and CD28 costimulation-dependent Jnk activation.

Role of glutathione in macrophage activation: effect of cellular glutathione depletion on nitrite production and leishmanicidal activity.

We have examined the effects of two agents depleting the intracellular pool of glutathione (GSH) on macrophage activation induced by IFN-gamma + LPS, as measured by nitrite production and leishmanicidal activity. Diethylmaleate (DEM), which depletes intracellular GSH by conjugation via a reaction catalyzed by the GSH-S-transferase, strongly inhibited nitrite secretion and leishmanicidal activity when added before or at the time of addition of IFN-gamma + LPS; this inhibition was progressively lost when addition of DEM was delayed up to 10 hr. A close correlation was observed between levels of intracellular soluble GSH during activation and nitrite secretion. Inhibition was partially reversed by the addition of glutathione ethyl ester (GSH-Et). Buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase, also inhibited macrophage activation, although to a lesser extent than DEM despite a more pronounced soluble GSH depletion. This inhibition was completely reversed by the addition of GSH-Et. DEM and BSO did not alter cell viability or PMA-triggered O2- production by activated macrophages, suggesting that the inhibitory effects observed on nitrite secretion and leishmanicidal activity were not related to a general impairment of macrophage function. DEM and BSO treatment reduced iNOS specific activity and iNOS protein in cytosolic extracts. DEM also decreased iNOS mRNA expression while BSO had no effect. Although commonly used as a GSH-depleting agent, DEM may have additional effects because it can also act as a sulhydryl reagent; BSO, on the other hand, which depletes GSH by enzymatic inhibition, has no effect on protein-bound GSH. Our results suggest that both soluble and protein-bound GSH may be important for the induction of NO synthase in IFN-gamma + LPS-activated macrophages.

Protease-activated receptor 2 signalling promotes dendritic cell antigen transport and T-cell activation in vivo.

Deficiency of protease-activated receptor-2 (PAR2) modulates inflammation in several models of inflammatory and autoimmune disease, although the underlying mechanism(s) are not understood. PAR2 is expressed on endothelial and immune cells, and is implicated in dendritic cell (DC) differentiation. We investigated in vivo the impact of PAR2 activation on DCs and T cells in PAR2 wild-type (WT) and knockout (KO) mice using a specific PAR2 agonist peptide (AP2). PAR2 activation significantly increased the frequency of mature CD11c(high) DCs in draining lymph nodes 24 hr after AP2 administration. Furthermore, these DCs exhibited increased expression of major histocompatibility complex (MHC) class II and CD86. A significant increase in activated (CD44(+) CD62(-)) CD4(+) and CD8(+) T-cell frequencies was also observed in draining lymph nodes 48 hr after AP2 injection. No detectable change in DC or T-cell activation profiles was observed in the spleen. The influence of PAR2 signalling on antigen transport to draining lymph nodes was assessed in the context of delayed-type hypersensitivity. PAR2 WT mice that were sensitized by skin-painting with fluorescein isothiocyanate (FITC) to induce delayed-type hypersensitivity possessed elevated proportion of FITC(+) DCs in draining lymph nodes 24 hr after FITC painting when compared with PAR2 KO mice (0.95% versus 0.47% of total lymph node cells). Collectively, these results demonstrate that PAR2 signalling promotes DC trafficking to the lymph nodes and subsequent T-cell activation, and thus provides an explanation for the pro-inflammatory effect of PAR2 in animal models of inflammation.

Arthritis is linked to local and systemic activation of coagulation and fibrinolysis pathways.

BACKGROUND: Activation of coagulation and fibrinolysis play a role in the pathophysiology of experimental arthritis. Objective: To determine the extent of activation of the coagulation and fibrinolytic pathways in different joint diseases in humans and to ascertain the factors that may influence fibrin deposition within the joint. METHODS: Plasma from normal subjects (controls, n= 21) and plasma and synovial fluid samples from patients with rheumatoid arthritis (RA; n = 64), osteoarthritis (OA; n = 29), spondyloarthropathy (SpA; n = 22) and crystal arthritis (CA; n = 25) were analyzed for the levels of TF (tissue factor) and tissue factor pathway inhibitor (TFPI) activities, thrombin-antithrombin III (TAT) complexes, and F1 + 2 (thrombin fragment), fibrin d-dimer and thrombin-activated fibrinolysis inhibitor (TAFI) antigenic levels. The measurements were analyzed by pairwise correlation with each other as well as with standard parameters of inflammation [C-reactive protein (CRP), joint leukocyte count]. Inter-group comparisons were performed to look for disease-specific differences. RESULTS: Compared with healthy controls, patients with joint diseases had higher levels of TAT, F1 + 2 and d-dimers in their plasma. In the synovial fluid, TF activity, TAT, d-dimers, and TAFI were significantly higher in inflammatory arthritides than in OA. The levels were highest in RA patients. In the plasma, TF activity was correlated with TAT and d-dimer levels with CRP, TFPI, and TAT. In the synovial fluid, TF activity correlated with plasma CRP levels, synovial fluid leukocyte count, and synovial TAT and TAFI levels. In addition, synovial d-dimers correlated with CRP, and synovial TAFI levels were correlated with synovial F1 + 2 and TAT. CONCLUSIONS: Activation of the coagulation and fibrinolytic cascades in the joint and in the circulation is evident in both inflammatory and degenerative joint diseases. Within the joint, inflammatory mechanisms leading to TF-mediated activation of the coagulation pathway and subsequent fibrin deposition is the most likely explanation for the observed findings. In the plasma, the link between inflammation (CRP increase) and TF activation is weak, and a non-TF-mediated mechanism of coagulation activation could explain these findings. RA is characterized by significantly higher levels of TAT in the synovial fluid and plasma than other arthritides. Although fibrinolytic activity is linked to inflammation, the increased amounts of TAFI in the joint, particularly in RA, may explain why fibrin formation is so prominent in this condition compared with other joint diseases.

Kinetics of T cell-activation molecules in response to Mycobacterium tuberculosis antigens

The phenotypic features acquired subsequent to antigen-specific stimulation in vitro were evaluated by means of the kinetic expressions of CD69 and CD25 activation molecules on T lymphocytes and assayed by flow cytometry in response to PPD, Ag85B, and ferritin in PPD-positive healthy control individuals. In response to PHA, CD69 staining on both CD4+ and CD8+ T cells became initially marked after 4 h, peaked at 24 h, and quickly decreased after 120 h. For CD25, a latter expression was detected around 8 h, having increased after 96 h. As expected, the response rate to the mycobacterial antigens was much lower than that to the mitogen. Positive staining was high after 96 h for CD25 and after 24 h for CD69. CD69 expression was significantly enhanced (p < 0.05) on CD8+ as compared to CD4+ T cells. High levels were also found between 96-120 h. Regarding Ag85B, CD25+ cells were mostly CD4+ instead of CD8+ T cells. Moreover, in response to ferritin, a lower CD25 expression was noted. The present data will allow further characterization of the immune response to new mycobacterial-specific antigens and their evaluation for possible inclusion in developing new diagnostic techniques for tuberculosis as well in a new vaccine to prevent the disease.

E2F activation of S phase promoters via association with HCF-1 and the MLL family of histone H3K4 methyltransferases.

E2F transcriptional regulators control human-cell proliferation by repressing and activating the transcription of genes required for cell-cycle progression, particularly the S phase. E2F proteins repress transcription in association with retinoblastoma pocket proteins, but less is known about how they activate transcription. Here, we show that the human G1 phase regulator HCF-1 associates with both activator (E2F1 and E2F3a) and repressor (E2F4) E2F proteins, properties that are conserved in insect cells. Human HCF-1-E2F interactions are versatile: their associations and binding to E2F-responsive promoters are cell-cycle selective, and HCF-1 displays coactivator properties when bound to the E2F1 activator and corepressor properties when bound to the E2F4 repressor. During the G1-to-S phase transition, HCF-1 recruits the mixed-lineage leukemia (MLL) and Set-1 histone H3 lysine 4 methyltransferases to E2F-responsive promoters and induces histone methylation and transcriptional activation. These results suggest that HCF-1 induces cell-cycle-specific transcriptional activation by E2F proteins to promote cell proliferation.

Disease-driven T cell activation predicts immune responses to vaccination against melanoma.

Tumor vaccines may induce activation and expansion of specific CD8 T cells which can subsequently destroy tumor cells in cancer patients. This phenomenon can be observed in approximately 5-20% of vaccinated melanoma patients. We searched for factors associated with T cell responsiveness to peptide vaccines. Peptide antigen-specific T cells were quantified and characterized ex vivo before and after vaccination. T cell responses occurred primarily in patients with T cells that were already pre-activated before vaccination. Thus, peptide vaccines can efficiently boost CD8 T cells that are pre-activated by endogenous tumor antigen. Our results identify a new state of T cell responsiveness and help to explain and predict tumor vaccine efficacy.

Zymogen activation and subcellular activity of subtilisin kexin isozyme 1/site 1 protease.

The proprotein convertase subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P) plays crucial roles in cellular homeostatic functions and is hijacked by pathogenic viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P involves sequential autocatalytic processing of its N-terminal prodomain at sites B'/B followed by the herein newly identified C'/C sites. We found that SKI-1/S1P autoprocessing results in intermediates whose catalytic domain remains associated with prodomain fragments of different lengths. In contrast to other zymogen proprotein convertases, all incompletely matured intermediates of SKI-1/S1P showed full catalytic activity toward cellular substrates, whereas optimal cleavage of viral glycoproteins depended on B'/B processing. Incompletely matured forms of SKI-1/S1P further process cellular and viral substrates in distinct subcellular compartments. Using a cell-based sensor for SKI-1/S1P activity, we found that 9 amino acid residues at the cleavage site (P1-P8) and P1' are necessary and sufficient to define the subcellular location of processing and to determine to what extent processing of a substrate depends on SKI-1/S1P maturation. In sum, our study reveals novel and unexpected features of SKI-1/S1P zymogen activation and subcellular specificity of activity toward cellular and pathogen-derived substrates.

The role of APRIL and BAFF in lymphocyte activation.

The TNF family ligands BAFF (also called BLyS) and APRIL regulate lymphocyte survival and activation. BAFF binds to three receptors, BAFF-R, TACI and BCMA, whereas APRIL interacts with TACI, BCMA and proteoglycans. The contribution of BAFF and APRIL to B-cell and plasma-cell survival, CD154 (CD40L)-independent antibody isotype switching, germinal center maintenance, T-dependent and T-independent antibody responses, and T cell co-stimulation are relatively well understood. Constitutive BAFF produced by stromal cells determines the size of the peripheral B cell pool, whereas inducible BAFF produced by myeloid and other cells supports local survival of B lymphocytes and can be associated with development of autoimmunity when deregulated.

CTL activation is induced by cross-linking of TCR/MHC-peptide-CD8/p56lck adducts in rafts.

To investigate the role of the coreceptor CD8 and lipid rafts in cytotoxic T lymphocyte (CTL) activation, we used soluble mono-and multimeric H-2Kd-peptide complexes and cloned S14 CTL specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite (PbCS) peptide 252-260 [PbCS(ABA)]. We report that activation of CTL in suspension requires multimeric Kd-PbCS(ABA) complexes co-engaging TCR and CD8. Using TCR ligand photo-cross-linking, we find that monomeric Kd-PbCS(ABA) complexes promote association of TCR/CD3 with CD8/p56lck. Dimerization of these adducts results in activation of p56lck in lipid rafts, where phosphatases are excluded. Additional cross-linking further increases p56lck kinase activity, induces translocation of TCR/CD3 and other signaling molecules to lipid rafts and intracellular calcium mobilization. These events are prevented by blocking Src kinases or CD8 binding to TCR-associated Kd molecules, indicating that CTL activation is initiated by cross-linking of CD8-associated p56lck. They are also inhibited by methyl-beta-cyclodextrin, which disrupts rafts and by dipalmitoyl phosphatidylethanolamine, which interferes with TCR signaling. Because efficient association of CD8 and p56lck takes place in rafts, both reagents, though in different ways, impair coupling of p56lck to TCR, thereby inhibiting the initial and essential activation of p56lck induced by cross-linking of engaged TCR.

Glucose and leptin induce apoptosis in human beta-cells and impair glucose-stimulated insulin secretion through activation of c-Jun N-terminal kinases.

c-Jun N-terminal kinases (SAPK/JNKs) are activated by inflammatory cytokines, and JNK signaling is involved in insulin resistance and beta-cell secretory function and survival. Chronic high glucose concentrations and leptin induce interleukin-1beta (IL-1beta) secretion from pancreatic islets, an event that is possibly causal in promoting beta-cell dysfunction and death. The present study provides evidence that chronically elevated concentrations of leptin and glucose induce beta-cell apoptosis through activation of the JNK pathway in human islets and in insulinoma (INS 832/13) cells. JNK inhibition by the dominant inhibitor JNK-binding domain of IB1/JIP-1 (JNKi) reduced JNK activity and apoptosis induced by leptin and glucose. Exposure of human islets to leptin and high glucose concentrations leads to a decrease of glucose-induced insulin secretion, which was partly restored by JNKi. We detected an interplay between the JNK cascade and the caspase 1/IL-1beta-converting enzyme in human islets. The caspase 1 gene, which contains a potential activating protein-1 binding site, was up-regulated in pancreatic sections and in isolated islets from type 2 diabetic patients. Similarly, cultured human islets exposed to high glucose- and leptin-induced caspase 1 and JNK inhibition prevented this up-regulation. Therefore, JNK inhibition may protect beta-cells from the deleterious effects of high glucose and leptin in diabetes.

Ezrin-Radixin-Moesin-binding Sequence of PSGL-1 Glycoprotein Regulates Leukocyte Rolling on Selectins and Activation of Extracellular Signal-regulated Kinases.

P-selectin glycoprotein ligand-1 (PSGL-1) mediates the capture (tethering) of free-flowing leukocytes and subsequent rolling on selectins. PSGL-1 interactions with endothelial selectins activate Src kinases and spleen tyrosine kinase (Syk), leading to α(L)β(2) integrin-dependent leukocyte slow rolling, which promotes leukocyte recruitment into tissues. In addition, but through a distinct pathway, PSGL-1 engagement activates ERK. Because ezrin, radixin and moesin proteins (ERMs) link PSGL-1 to actin cytoskeleton and because they serve as adaptor molecules between PSGL-1 and Syk, we examined the role of PSGL-1 ERM-binding sequence (EBS) on cell capture, rolling, and signaling through Syk and MAPK pathways. We carried out mutational analysis and observed that deletion of EBS severely reduced 32D leukocyte tethering and rolling on L-, P-, and E-selectin and slightly increased rolling velocity. Alanine substitution of Arg-337 and Lys-338 showed that these residues play a key role in supporting leukocyte tethering and rolling on selectins. Importantly, EBS deletion or Arg-337 and Lys-338 mutations abrogated PSGL-1-induced ERK activation, whereas they did not prevent Syk phosphorylation or E-selectin-induced leukocyte slow rolling. These studies demonstrate that PSGL-1 EBS plays a critical role in recruiting leukocytes on selectins and in activating the MAPK pathway, whereas it is dispensable to phosphorylate Syk and to lead to α(L)β(2)-dependent leukocyte slow rolling.

Nitric oxide induces the expression of the monocarboxylate transporter MCT4 in cultured astrocytes by a cGMP-independent transcriptional activation.

The monocarboxylate transporter MCT4 is a proton-linked carrier particularly important for lactate release from highly glycolytic cells. In the central nervous system, MCT4 is exclusively expressed by astrocytes. Surprisingly, MCT4 expression in primary cultures of mouse cortical astrocytes is conspicuously low, suggesting that an external, nonastrocytic signal is necessary to obtain the observed pattern of expression in vivo. Here, we demonstrate that nitric oxide (NO), delivered by various NO donors, time- and dose-dependently induces MCT4 expression in cultured cortical astrocytes both at the mRNA and protein levels. In contrast, NO does not enhance the expression of MCT1, the other astrocytic monocarboxylate transporter. The transcriptional effect of NO is not mediated by a cGMP-dependent mechanism as shown by the absence of effect of a cGMP analog or of a selective guanylate cyclase inhibitor. NO causes an increase in astrocytic lactate transport capacity which requires the enhancement of MCT4 expression as both are prevented by the use of a specific siRNA against MCT4. In addition, cumulated lactate release by astrocytes over a period of 24 h was also enhanced by NO treatment. Our data suggest that NO represents a putative intercellular signal to control MCT4 expression in astrocytes and in doing so, to facilitate lactate transfer to other surrounding cell types in the central nervous system. © 2011 Wiley-Liss, Inc.