Upregulation Of Adhesion Research Articles

Munc13-4 Is a Rab11-binding Protein That Regulates Rab11-positive Vesicle Trafficking and Docking at the Plasma Membrane

The small GTPase Rab11 and its effectors control trafficking of recycling endosomes, receptor replenishment and the up-regulation of adhesion and adaptor molecules at the plasma membrane. Despite recent advances in the understanding of Rab11-regulated mechanisms, the final steps mediating docking and fusion of Rab11-positive vesicles at the plasma membrane are not fully understood. Munc13-4 is a docking factor proposed to regulate fusion through interactions with SNAREs. In hematopoietic cells, including neutrophils, Munc13-4 regulates exocytosis in a Rab27a-dependent manner, but its possible regulation of other GTPases has not been explored in detail. Here, we show that Munc13-4 binds to Rab11 and regulates the trafficking of Rab11-containing vesicles. Using a novel Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay, we demonstrate that Munc13-4 binds to Rab11a but not to dominant negative Rab11a. Immunoprecipitation analysis confirmed the specificity of the interaction between Munc13-4 and Rab11, and super-resolution microscopy studies support the interaction of endogenous Munc13-4 with Rab11 at the single molecule level in neutrophils. Vesicular dynamic analysis shows the common spatio-temporal distribution of Munc13-4 and Rab11, while expression of a calcium binding-deficient mutant of Munc13-4 significantly affected Rab11 trafficking. Munc13-4-deficient neutrophils showed normal endocytosis, but the trafficking, up-regulation, and retention of Rab11-positive vesicles at the plasma membrane was significantly impaired. This correlated with deficient NADPH oxidase activation at the plasma membrane in response to Rab11 interference. Our data demonstrate that Munc13-4 is a Rab11-binding partner that regulates the final steps of Rab11-positive vesicle docking at the plasma membrane.

Murid Gammaherpesvirus Latency-Associated Protein M2 Promotes the Formation of Conjugates between Transformed B Lymphoma Cells and T Helper Cells.

Establishment of persistent infection in memory B cells by murid herpesvirus-4 (MuHV-4) depends on the proliferation of latently infected germinal center B cells, for which T cell help is essential. Whether the virus is capable of modulating B-T helper cell interaction for its own benefit is still unknown. Here, we investigate if the MuHV-4 latency associated M2 protein, which assembles multiprotein complexes with B cell signaling proteins, plays a role. We observed that M2 led to the upregulation of adhesion and co-stimulatory molecules in transduced B cell lines. In an MHC-II restricted OVA peptide-specific system, M2 polarized to the B-T helper contact zone. Furthermore, it promoted B cell polarization, as demonstrated by the increased proximity of the B cell microtubule organizing center to the interface. Consistent with these data, M2 promoted the formation of B-T helper cell conjugates. In an in vitro competition assay, this translated into a competitive advantage, as T cells preferentially conjugated with M2-expressing B cells. However, expression of M2 alone in B cells was not sufficient to lead to T cell activation, as it only occurred in the presence of specific peptide. Taken together, these findings support that M2 promotes the formation of B-T helper cell conjugates. In an in vivo context this may confer a competitive advantage to the infected B cell in acquisition of T cell help and initiation of a germinal center reaction, hence host colonization.

Elotuzumab enhances natural killer cell activation and myeloma cell killing through interleukin-2 and TNF-α pathways.

Elotuzumab is a humanized monoclonal antibody specific for signaling lymphocytic activation molecule-F7 (SLAMF7, also known as CS1, CD319, or CRACC) that enhances natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) of SLAMF7-expressing myeloma cells. This study explored the mechanisms underlying enhanced myeloma cell killing with elotuzumab as a single agent and in combination with lenalidomide, to support ongoing phase III trials in patients with relapsed/refractory or newly-diagnosed multiple myeloma (MM). An in vitro peripheral blood lymphocyte (PBL)/myeloma cell co-culture model was developed to evaluate the combination of elotuzumab and lenalidomide. Expression of activation markers and adhesion receptors was evaluated by flow cytometry, cytokine expression by Luminex and ELISPOT assays, and cytotoxicity by myeloma cell counts. Elotuzumab activated NK cells and promoted myeloma cell death in PBL/myeloma cell co-cultures. The combination of elotuzumab plus lenalidomide demonstrated superior anti-myeloma activity on established MM xenografts in vivo and in PBL/myeloma cell co-cultures in vitro than either agent alone. The combination enhanced myeloma cell killing by modulating NK cell function that coincided with the upregulation of adhesion and activation markers, including interleukin (IL)-2Rα expression, IL-2 production by CD3+CD56+ lymphocytes, and tumor necrosis factor (TNF)-α production. In co-culture assays, TNF-α directly increased NK cell activation and myeloma cell death with elotuzumab or elotuzumab plus lenalidomide, and neutralizing TNF-α decreased NK cell activation and myeloma cell death with elotuzumab. These results demonstrate that elotuzumab activates NK cells and induces myeloma cell death via NK cell-mediated ADCC, which is further enhanced when combined with lenalidomide.

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes. This method focuses on neutrophil transmigration across human endothelial cells. One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC). Neutrophil isolation has been described visually elsewhere; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-α results in increased E-selectin and IL-8 expression. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature. The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass. Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi μslide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

The Role of Tissue Transglutaminase 2 in the Modulation of Polymorphonuclear Leukocyte Function

Inflammatory Bowel Disease is mostly represented by Crohn's Disease and Ulcerative Colitis. The disease presents as a series of inflammatory disorders that involves dysregulated neutrophil (PMN) migration across the intestinal epithelium. Tissue Transglutaminase (TGM) 2 is an enzyme that catalyzes calcium‐dependent crosslinks of amino groups and has been implicated in a variety of cellular functions including cell growth and wound healing, which involves GTPase mediated activities (shown to affect PMN adhesion). TGM 2 antibodies are present in the small intestine of patients with Celiac Disease (diet induced inflammation in the small intestine) and a majority of Crohn's Disease patients also have Celiac Disease. Despite these observations, the role of TGM 2 in PMN activities has not been widely explored. We determined that TGM 2 has an extracellular and intracellular localization in PMN and appears to inhibit translocation of CD11b (activating integrin of PMN) to the surface of PMN. TGM 2 can inhibit PMN attachment to fibrinogen (a ligand for CD11b) by approximately 50% and can inhibit fMLP‐directed PMN migration by approximately 50%, which strongly suggests that TGM 2 may be connected to CD11b mediated events. The fMLP receptor is a G‐protein coupled (7TM spanning) receptor that regulates PMN response by an up‐regulation of adhesion and migration receptors. Using Flow Cytometry, we determined that TGM 2 affects PMN adhesion and migration through the PMN fMLP receptor. These data highlight TGM 2 as a potential target in the treatment of mucosal inflammation involving aberrant PMN migration.

17 and 23

The discovery of the cytokine IL-17 more than 10 yr ago1 paved the way for the recent identification of a distinct subset of helper T cells producing IL-17 (Th17 cells). This novel proinflammatory class of Th cells2 disrupts the Th1/Th2 paradigm that had shaped our view of the immune system and immune-mediated disease for more than 20 yr.3 Th1 cells, regulated by the transcription factor T-bet and characterized by the production of IFN-γ and IL-12, provide defense against viruses and intracellular pathogens and are associated with autoimmunity, whereas Th2 cells, modulated by GATA-3 and producing IL-4 and IL-13, provide immunity against extracellular parasites and play a role in the promotion of allergy. The novel Th17 cells represent a third distinct lineage of helper CD4+ T cells that are regulated by a specific transcription factor, RoR-γt,4 and produce IL-17. IL-17 possesses pleiotropic effects,5 including release of proinflammatory cytokines (TNF-α, IL-6, and IL-8), upregulation of adhesion and MHC molecules, and recruitment of monocytes and neutrophils. Th17 cells in mice develop from naive CD4+ cells in the presence of TGF-β and IL-6; however, in humans, unraveling their pathway to differentiation is more complex. Initially, Th17 differentiation was thought to be driven by IL-1 and enhanced by IL-6 and IL-23.6,7 More recently, TGF-β and IL-21 also seem integral for their differentiation in humans.8 Significantly, IL-23 is a cytokine …

Hydroxyurea Therapy Increases Expression of BCAM/LU and Adhesion to Laminin in Children with Sickle Cell Disease

Vaso-occlusion is the major cause of morbidity and mortality in sickle cell disease. The tendency of red blood cells (RBCs) to adhere to extracellular matrix molecules and the vascular endothelium is believed to be a significant contributor to the vaso-occlusive process. Some published studies have shown that hydroxyurea decreases sickle (SS) RBC adhesion to some ligands, although the mechanism by which this occurs is not completely understood. SS RBCs demonstrate increased expression of several adhesion molecules, especially BCAM/LU, and also conserve functional signaling pathways that are associated with upregulation of adhesion. BCAM/LU mediates adhesion to the extracellular matrix protein laminin. We hypothesized that patients responsive to hydroxyurea (HU) therapy would exhibit reduced adhesion to laminin as well as a decrease in adhesion molecule expression. Our subjects included patients with Hb SS between the ages of 5 to 18. They were divided into three groups:children not receiving HU therapy (n = 3);children receiving HU therapy for over 6 months (n = 5), andchildren initially not receiving HU but who were initiating therapy at the time of study enrollment (n = 5).Adhesion to laminin was examined using a graduated height flow chamber to quantitate the adhesion of SS RBCs. Expression of adhesion molecules was analyzed by western blot and densitometry, using monoclonal antibody to BCAM/LU. We found that HU therapy was associated with significantly increased expression of BCAM/LU (HU: 145.8 ± 14.0 SEM; no HU: 60.8 ± 11.0 SEM densitometry units, p = .0014). This somewhat unexpected finding confirms results published earlier this year by Odievre et al. (2008). Adhesion to laminin was also increased for patients on HU (HU: 9.3 ± 5.9; no HU: .3 ± .3, p=.2), although this increase was not significant, given the variability in adhesion seen among patients and the small number of subjects. Nevertheless, the increase in adhesion corresponded to the increase in BCAM/LU expression. In contrast, adhesion to endothelial cells was decreased, although not significantly, in patients on HU (HU: 38.1 ± 38; no HU: 127.2 ± 122.5, p=.6). Our findings thus confirm earlier published data showing that HU increases the expression of BCAM/LU measured by flow cytometry and further shows that this increased expression is associated with increased adhesion to laminin but not to endothelial cells. Potential mechanisms by which HU affects adhesion molecule expression and activity merit further investigation, as does the physiologic role of these alterations. Comparison of results from patient-matched pre-treatment and post-treatment samples should also help define the effects of HU. [Display omitted]

Activation of Immature Monocyte-Derived Dendritic Cells After Transduction with High Doses of Lentiviral Vectors

Dendritic cells (DCs) are an attractive tool for immunomodulation, targeting mature DCs (mDCs) for immunization or immature/semimature DCs (iDCs) for tolerization. Therefore, introducing antigens into DCs has become a prime topic in various immunological disciplines. Numerous studies have shown that lentiviruses are an efficient vehicle for this purpose. This study evaluates the effects of lentiviral transduction on iDC activation. Immature DCs are efficiently transduced with increasing doses of lentivirus without affecting cell viability. Transduction at low multiplicities of infection (MOIs) did not result in phenotypical or functional maturation. Higher doses of lentivirus, however, resulted in upregulation of adhesion, costimulatory, and HLA molecules, as well as in increased allostimulatory capacity and secretion of interleukin (IL)-6, IL-8, and tumor necrosis factor-alpha. Production of IL-12 p70, IL-10, and interferon-alpha was observed only at extremely high doses. Protein kinase R phosphorylation on transduction at an MOI of 150 was demonstrated by Western blotting. A Toll-like receptor (TLR)-driven luciferase reporter assay showed dose-dependent activation of TLR2, TLR3, and TLR8, which was independent of the pseudotype, production, or transduction protocol and was abrogated on heat inactivation. These data show that lentiviral vectors provide not only the antigen but also appropriate activation signals to iDCs, favoring their use for immunotherapy and vaccine development.

Trophoblast adhesion of the peri-implantation mouse blastocyst is regulated by integrin signaling that targets phospholipase C

Integrin signaling modulates trophoblast adhesion to extracellular matrices during blastocyst implantation. Fibronectin (FN)-binding activity on the apical surface of trophoblast cells is strengthened after elevation of intracellular Ca2+ downstream of integrin ligation by FN. We report here that phosphoinositide-specific phospholipase C (PLC) mediates Ca2+ signaling in response to FN. Pharmacological agents used to antagonize PLC (U73122) or the inositol phosphate receptor (Xestospongin C) inhibited FN-induced elevation of intracellular Ca2+ and prevented the upregulation of FN-binding activity. In contrast, inhibitors of Ca2+ influx through either voltage-gated or non-voltage-gated Ca2+ channels were without effect. Inhibition of protein tyrosine kinase activity by genistein, but not G-protein inhibition by suramin, blocked FN-induced intracellular Ca2+ signaling and upregulation of adhesion, consistent with involvement of PLC-γ. Confocal immunofluorescence imaging of peri-implantation blastocysts demonstrated that PLC-γ2, but not PLC-γ1 nor PLC-β1, accumulated near the outer surface of the embryo. Phosphotyrosine site-directed antibodies revealed phosphorylation of PLC-γ2, but not PLC-γ1, upon integrin ligation by FN. These data suggest that integrin-mediated activation of PLC-γ to initiate phosphoinositide signaling and intracellular Ca2+ mobilization is required for blastocyst adhesion to FN. Signaling cascades regulating PLC-γ could, therefore, control a critical feature of trophoblast differentiation during peri-implantation development.

Incipient Alzheimer's disease: microarray correlation analyses reveal major transcriptional and tumor suppressor responses.

The pathogenesis of incipient Alzheimer's disease (AD) has been resistant to analysis because of the complexity of AD and the overlap of its early-stage markers with normal aging. Gene microarrays provide new tools for addressing complexity because they allow overviews of the simultaneous activity of multiple cellular pathways. However, microarray data interpretation is often hindered by low statistical power, high false positives or false negatives, and by uncertain relevance to functional endpoints. Here, we analyzed hippocampal gene expression of nine control and 22 AD subjects of varying severity on 31 separate microarrays. We then tested the correlation of each gene's expression with MiniMental Status Examination (MMSE) and neurofibrillary tangle (NFT) scores across all 31 subjects regardless of diagnosis. These well powered tests revealed a major transcriptional response comprising thousands of genes significantly correlated with AD markers. Several hundred of these genes were also correlated with AD markers across only control and incipient AD subjects (MMSE > 20). Biological process categories associated with incipient AD-correlated genes were identified statistically (ease program) and revealed up-regulation of many transcription factor/signaling genes regulating proliferation and differentiation, including tumor suppressors, oligodendrocyte growth factors, and protein kinase A modulators. In addition, up-regulation of adhesion, apoptosis, lipid metabolism, and initial inflammation processes occurred, and down-regulation of protein folding/metabolism/transport and some energy metabolism and signaling pathways took place. These findings suggest a new model of AD pathogenesis in which a genomically orchestrated up-regulation of tumor suppressor-mediated differentiation and involution processes induces the spread of pathology along myelinated axons.

All-trans retinoic acid skews monocyte differentiation into interleukin-12-secreting dendritic-like cells.

All-trans retinoic acid (ATRA) and retinoid derivatives are essential agents for multiple biological processes. Numerous immune system dysfunctions can occur in the case of retinoid deficiency. Because of the central role of dendritic cells (DCs) in controlling immunity and the wide effects of retinoids on the immune system homeostasis, we investigated the ability of ATRA to influence the differentiation of DCs from circulating peripheral blood monocytes. Human peripheral blood monocytes were cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and various concentrations of ATRA. Differentiated cells were assayed for their morphology, phenotype, antigen uptake, allostimulatory capacity and cytokine secretion profile. ATRA (10(-12) mol/l) and GM-CSF drove the differentiation of monocytes into dendritic-like cells (ATRA-DC). ATRA-DCs exhibited DC morphology, had a phenotype of immature DCs, with the expression of CD1a, and upregulation of adhesion and co-stimulatory molecules. ATRA-DCs could induce a proliferative response in naive CD4+ T cells. Although ATRA-DCs retained their antigen-capture capacity, they secreted interleukin (IL)-12p70 without the need for any maturation agent. In addition, ATRA-DCs could drive T cells towards an IL-12-dependent T-helper cell type 1 response with secretion of interferon-gamma. DCs appear to be potential targets for ATRA, giving new insights into the immunomodulatory function of retinoids, with implications potentially related to immunotherapy.

Regulation of the very late antigen-4-mediated adhesive activity of normal and nonreleaser basophils: roles for Src, Syk, and phosphatidylinositol 3-kinase

Normal human basophils express the integrin, VLA-4, and cross-linking their high-affinity IgE receptor, FcepsilonRI, increases their VLA-4-dependent adhesion to VCAM-1-transfected Chinese hamster ovary (CHO) cells. Here we show that the FcepsilonRI-mediated up-regulation of normal basophil VLA-4 adhesion is abolished by the Src inhibitor, PP1, the Syk inhibitor, ER-27319, and the phosphatidylinositol 3-kinase inhibitor, wortmannin. PP1, but not ER-27319 or wortmannin, also reduces basal adhesion and adhesion stimulated by chemotactic peptide, by Ca(++) ionophores, and by phorbol myristate acetate (PMA). Nonreleaser basophils (the consistently Syk-deficient, variably Lyn-deficient, severely degranulation-impaired cells found in about 10% of donors) share the PP1 phenotype of lowered basal adhesion, no FcepsilonRI-mediated adhesion up-regulation, and reduced adhesive responses to chemoattractant ionophores and PMA. These results implicate Src kinases in the control of basal VLA-4 activity and place Syk and phosphatidylinositol 3-kinase in the pathway linking FcepsilonRI cross-linking to VLA-4 up-regulation. Both Src and Syk-regulated components of adhesion may be impaired in nonreleaser basophils.

The TNF superfamily members LIGHT and CD154 (CD40 ligand) costimulate induction of dendritic cell maturation and elicit specific CTL activity.

LIGHT is a recently identified member of the TNF superfamily that is up-regulated upon activation of T cells. Herpesvirus entry mediator, one of its receptors, is constitutively expressed on immature dendritic cells (DCs). In this report, we demonstrate that LIGHT induces partial DC maturation as demonstrated by Ag presentation and up-regulation of adhesion and costimulatory molecules. LIGHT-stimulated DCs show reduced macropinocytosis and enhanced allogeneic stimulatory capacity but fail to produce significant amounts of IL-12, IL-6, IL-1beta, or TNF-alpha compared with unstimulated DCs. However, LIGHT cooperates with CD154 (CD40 ligand) in DC maturation, with particular potentiation of allogeneic T cell proliferation and cytokine secretion of IL-12, IL-6, and TNF-alpha. Moreover, LIGHT costimulation allows DCs to prime in vitro-enhanced specific CTL responses. Our results suggest that LIGHT plays an important role in DC-mediated immune responses by regulating CD154 signals and represents a potential tool for DC-based cancer immunotherapy.

CD40 cross-linking enhances the immunogenicity of Burkitt's-lymphoma cell lines.

Epstein-Barr-virus (EBV)-positive Burkitt's-lymphoma (BL) cell lines are not recognized by EBV-specific T cells, due to their non-immunogenic phenotype and restricted expression of latent EBV genes. We tested whether triggering of CD40 can alter the phenotype of the tumor cells with regard to: (i) expression of surface markers, (ii) expression of viral antigens, (iii) presentation of endogenous antigens to MHC-class-1 restricted cytotoxic T lymphocytes (CTLs), (iv) stimulatory capacity in allogeneic mixed-lymphocyte cultures (MLCs), (v) sensitivity to natural-killer (NK)-cell-mediated lysis. Co-culture of EBV-positive BL cells with CD40-ligand-transfected L cells induced up-regulation of CD54 and CD80 but did not affect the expression of viral genes. In spite of significant up-regulation of TAP1 and TAP2, and increased expression of MHC class 1, the BL cells remained unable to present endogenously expressed viral antigens to EBV-specific CTL. However, the up-regulation of adhesion and co-stimulatory molecules was associated with increased stimulatory capacity in MLC and enhanced sensitivity to NK cells. These findings indicate that, while inducing only a modest phenotype shift, cross-linking of CD40 under physiologic conditions may selectively enhance the sensitivity of BL cells to anti-tumor immune responses.

Up-regulation of adhesion and MHC molecules on splenic monocyte/macrophages in adult haemophagocytic syndrome.

Haemophagocytic syndrome (HPS) has been associated with the abnormal activation of mono/macrophages and increased cytokine production. However, neither the phenotype of haemophagocytic monomacrophages nor the cellular origin of cytokine production have been described. We studied splenic monomacrophages and lymphocytes from five patients with HPS (two HIV- and three HIV+) and from controls without HPS (three normal HIV and two pathological HIV+). Using flow-cytometry, we observed a marked increase in the expression of MHC class I and II, M-CSF-receptor and adhesion molecules LFA-1, LFA-3, ICAM-1 (P<0.05) on HPS+ splenic monomacrophages compared to HPS-, which was independent of their HIV status. A high percentage of CD8+ lymphocytes from 4/5 HPS+ patients produced TNF alpha and IFNgamma, but no IL-6 upon in-vitro activation. In a fifth patient CD4+ but not CD8+ lymphocytes produced these cytokines. Although other cytokines might be involved in the pathophysiology of HPS as suggested by the high expression of M-CSF-receptor, these results suggest that TNF alpha and IFNgamma secretion by T cells might play a role in the up-regulation of adhesion and MHC molecules on monomacrophages from HPS.

Adenosine diphosphate (ADP)-ribosylation of the guanosine triphosphatase (GTPase) rho in resting peripheral blood human T lymphocytes results in pseudopodial extension and the inhibition of T cell activation.

Scrape loading Clostridium botulinum C3 exoenzyme into primary peripheral blood human T lymphocytes (PB T cells) efficiently adenosine diphosphate (ADP)-ribosylates and thus inactivates the guanosine triphosphatase (GTPase) Rho. Basal adhesion of PB T cells to the beta1 integrin substrate fibronectin (Fn) was not inhibited by inactivation of Rho, nor was upregulation of adhesion using phorbol myristate acetate (PMA; 10 ng/ml) or Mn++ (1 mM) affected. Whereas untreated PB T cells adherent to Fn remain spherical, C3-treated PB T cells extend F-actin-containing pseudopodia. Inactivation of Rho delayed the kinetics of PMA-dependent PB T cell homotypic aggregation, a process involving integrin alphaLbeta2. Although C3 treatment of PB T cells did not prevent adhesion to the beta1 integrin substrate Fn, it did inhibit beta1 integrin/CD3-mediated costimulation of proliferation. Analysis of intracellular cytokine production at the single cell level demonstrated that ADP-ribosylation of Rho inhibited beta1 integrin/ CD3 and CD28/CD3 costimulation of IL-2 production within 6 h of activation. Strikingly, IL-2 production induced by PMA and ionomycin was unaffected by C3 treatment. Thus, the GTPase Rho is a novel regulator of T lymphocyte cytoarchitecture, and functional Rho is required for very early events regulating costimulation of IL-2 production in PB T cells.

Cytokines and thyroid function

Cytokines play a crucial role in autoimmune thyroid disease (ATD) through various mechanisms. They are produced in the thyroid by intrathyroidal inflammatory cells, in particular lymphocytes, as well as by the thyroid follicular cells (TFC) themselves and may thus act in a cascade to enhance the autoimmune process (Fig. 1). Cytokines upregulate the inflammatory reaction through stimulation of both T and B cells, resulting in antibody production and tissue injury. In addition, intrathyroidal cytokines induce immunological changes in TFC including enhancement of both major histocompatibility complex (MHC) class I and class II molecule expression, and upregulation of adhesion and complement regulatory molecule expression. Cytokines can also modulate both growth and function of TFC and have a role in extrathyroidal complications of ATD, most importantly thyroid-associated ophthalmopathy (TAO), where they induce fibroblast proliferation and enhance the production of glycosaminoglycans (GAG), resulting in proptosis and the other clinical features of the disease. In addition to these effects, exogenous administration of cytokines has been associated with impairment of thyroid function ranging from the appearance of autoantibodies alone to the development of frank thyroid dysfunction. Cytokines have also been implicated in subacute thyroiditis (SAT) and amiodarone-induced thyroid dysfunction, as well as in thyroid function abnormalities occurring in patients with non-thyroidal illnesses (NTI). Genetic variations in cytokine genes represent potential risk factors for ATD, and disease associations have been described for polymorphisms in IL-1ra and TNF beta genes. Recent experimental evidence suggests the possibility of novel cytokine-based therapeutic approaches for ATD and its complications, in particular TAO.

Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products.

We have previously demonstrated that human peripheral blood low density mononuclear cells cultured in granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 develop into dendritic cells (DCs) that are extremely efficient in presenting soluble antigens to T cells. To identify the mechanisms responsible for efficient antigen capture, we studied the endocytic capacity of DCs using fluorescein isothiocyanate-dextran, horseradish peroxidase, and lucifer yellow. We found that DCs use two distinct mechanisms for antigen capture. The first is a high level of fluid phase uptake via macropinocytosis. In contrast to what has been found with other cell types, macropinocytosis in DCs is constitutive and allows continuous internalization of large volumes of fluid. The second mechanism of capture is mediated via the mannose receptor (MR), which is expressed at high levels on DCs. At low ligand concentrations, the MR can deliver a large number of ligands to the cell in successive rounds. Thus, while macropinocytosis endows DCs with a high capacity, nonsaturable mechanism for capture of any soluble antigen, the MR gives an extra capacity for antigen capture with some degree of selectivity for non-self molecules. In addition to their high endocytic capacity, DCs from GM-CSF + IL-4-dependent cultures are characterized by the presence of a large intracellular compartment that contains high levels of class II molecules, cathepsin D, and lysosomal-associated membrane protein-1, and is rapidly accessible to endocytic markers. We investigated whether the capacity of DCs to capture and process antigen could be modulated by exogenous stimuli. We found that DCs respond to tumor necrosis factor alpha, CD40 ligand, IL-1, and lipopolysaccharide with a coordinate series of changes that include downregulation of macropinocytosis and Fc receptors, disappearance of the class II compartment, and upregulation of adhesion and costimulatory molecules. These changes occur within 1-2 d and are irreversible, since neither pinocytosis nor the class II compartment are recovered when the maturation-inducing stimulus is removed. The specificity of the MR and the capacity to respond to inflammatory stimuli maximize the capacity of DCs to present infectious non-self antigens to T cells.

Monocyte adhesion to cerebromicrovascular endothelial cells derived from hypertensive and normotensive rats.

The stroke risk factor hypertension may function as a predisposing agent by increasing the vulnerability of blood vessels to thrombosis or hemorrhage. The research here demonstrates that cerebrovascular endothelial cells (EC) from spontaneously hypertensive (SHR) and Wistar-Kyoto normotensive (WKY) rats exhibit similar levels of adhesiveness for syngeneic peripheral blood monocytes (e.g., 22.53 +/- 1.32 and 24.35 +/- 1.16%, respectively). Monocyte adhesion to SHR EC was dramatically increased by treatment of EC with lipopolysaccharide, interferon-gamma, or interleukin-1 beta and tumor necrosis factor-alpha (e.g., 106, 68, and 171%, respectively). Identical treatment of WKY EC also increased adhesion albeit at significantly lower levels than observed on concomitantly tested SHR EC (e.g., 47.8, 12.7, and 60.7%, respectively). Allogeneic combinations of monocytes and EC again demonstrated significantly more upregulation of adhesion by treatment of SHR EC than WKY EC. Characterization of these adhesive interactions revealed the interplay of adhesion pathways, which include lymphocyte functional antigen-1/intercellular adhesion molecule-1 (ICAM-1), Mac-1/ICAM-1, and very late activation antigen-4/vascular adhesion molecule-1 as well as other undetermined mechanisms. In summary, these findings indicate hypertension may enhance responsiveness of endothelium to factors that promote monocyte adhesion.