Glucocorticoids down-regulate dendritic cell function in vitro and in vivo

Exogenous glucocorticoid hormones are widely used as therapeutical agents, whereas endogenous glucocorticoids may act as physiological immunosuppressants involved in the control of immune and inflammatory responses. The optimal activation of T lymphocytes requires two distinct signals: the major histocompatibility complex-restricted presentation of the antigen and an additional co-stimulatory signal provided by the antigen-presenting cells. There is ample evidence that, among the cells able to present the antigen, the dendritic cells (DC) have the unique property to activate antigen-specific, naive T cells in vitro and in vivo, and are therefore required for the induction of primary immune responses. In this work, we tested whether glucocorticoids affected the capacity of DC to sensitize naive T cells. Our data show that, in vitro, the steroid hormone analog dexamethasone (Dex) affects the viability of DC, selectively downregulates the expression of co-stimulatory molecules on viable DC, and strongly reduces their immunostimulatory properties. In vivo, a single injection of Dex results in impaired antigen presenting function, a finding which correlates with reduced numbers of splenic DC. These results show that glucocorticoids regulate DC maturation and immune function in vitro and in vivo and suggest that this mechanism may play a role in preventing overstimulation of the immune system.     

CYFIP2 is highly abundant in CD4+ cells from multiple sclerosis patients and is involved in T cell adhesion

DNA microarray profiling of CD4(+) and CD8(+) cells from non-treated relapsing and remitting multiple sclerosis (MS) patients determined that the cytoplasmic binding partner of fragile X protein (CYFIP2, also called PIR121) was increased significantly compared to healthy controls. Western analysis confirmed that CYFIP2 protein was increased approximately fourfold in CD4(+) cells from MS compared to inflammatory bowel disorder (IBD) patients or healthy controls. Because CYFIP2 acts as part of a tetrameric complex that regulates WAVE1 activation we hypothesized that high levels of CYFIP2 facilitate T cell adhesion, which is elevated in MS patients. Several findings indicated that increased levels of CYFIP2 facilitated adhesion. First, adenoviral-mediated overexpression of CYFIP2 in Jurkat cells increased fibronectin-mediated adhesion. Secondly, CYFIP2 knock-down experiments using antisense oligodeoxynucleotides reduced fibronectin-mediated binding in Jurkat and CD4(+) cells. Thirdly, inhibition of Rac-1, a physical partner with CYFIP2 and regulator of WAVE1 activity, reduced fibronectin-mediated adhesion in Jurkat and CD4(+) cells. Finally, inhibition of Rac-1 or reduction of CYFIP2 protein decreased fibronectin-mediated adhesion in CD4(+) cells from MS patients to levels similar to controls. These studies suggest that overabundance of CYFIP2 protein facilitates increased adhesion properties of T cells from MS patients.     

Distinct pathways for constitutive endocytosis of fully conformed and non-conformed L(d) molecules

PROBLEM: To characterize the constitutive internalization of major histocompatibility complex (MHC) class I molecules, we have studied the expression of completely conformed (full) and unconformed (empty) L(d) molecules on non-polarized murine P815 cells. METHODS OF STUDY: Spontaneous endocytosis of L(d) molecules was induced by cycloheximide, an inhibitor of protein synthesis, and their disappearance from the cell surface was determined by flow cytometry. In order to investigate the mechanism of internalization, a palette of inhibitors of endocytosis and vesicular transport was used. RESULTS: Inhibitors of clathrine endocytosis did not influence the internalization of L(d) molecules. Inhibitors of caveolar endocytosis and inhibitors of endolysosomal degradation prevented down-regulation of empty, but not of full L(d) molecules. CONCLUSIONS: Empty L(d) molecules are internalized mostly by caveolar endocytosis and full L(d) molecules use a different pathway, neither clathrine-mediated nor caveolar. After internalization, full L(d) molecules are probably degraded and empty L(d) molecules recycle between endosomal compartment and the cell surface before they enter into the degradation compartment.     

Interaction of B7RP-1 with ICOS Negatively Regulates Antigen Presentation by B Cells

Stimulation of T cells through the T cell receptor is insufficient for optimal T cell activation. A second activation signal is necessary, being usually provided by the costimulatory molecule CD28. Recently, additional costimulatory pathways have been identified, including inducible costimulator (ICOS) and its ligand B7RP-1. We have examined the role of the B7RP-1/ICOS costimulatory pathway on antigen presentation by B cells, using the I-A^k and I-E^k-positive CH27 B cell line and several different T cell lines. We found that CH27 expressed B7RP-1 and PD-L1 whereas the T cell lines expressed ICOS and PD-1. In the presence of HEL, the T cell hybridomas C10 and 3A9 released IL-2, which is indicative of antigen-specific T cell activation by the CH27 cells. Unexpectedly, blocking antibodies for B7RP-1 and ICOS enhanced the IL-2 response in both T cells. As expected, an increase in the production of IL-2 was seen when blocking antibodies for PD-1 were used. Blocking with antibodies for I-A^k, CD28, B7.1, and B7.2 lead to a decrease in IL-2 production. Additionally we tested a Th1 and a Th2 T cell clone. Blockade of B7RP-1/ICOS lead to an increased IFN- response in Th1 cells (A.E7) and an increased IL-4 response in Th2 cells (D10.G4.1). Intracellular staining also showed an increase in cytokine production when the B7RP-1/ICOS pathway was blocked. In conclusion, the B7RP-1/ICOS pathway is negatively regulating T cell activation by B cells and may play a role similar to that of the PD-L1/PD-1 pathway.     

In the absence of major histocompatibility complex class II molecules,invariant chain is translocated to late endocytic compartments by autophagy

It has been suggested that the cytoplasmic amino-terminal tail of invariant chain (Ii) contains a sorting signal that directs trafficking of the major histocompatibility complex (MHC) class II: Ii oligomeric complex to endocytic compartments. This model is based, in part, on the observation that in the absence of MHC class II molecules, Ii is detectable in lysosomal structures, a phenotype that is dependent on an intact NH₂ terminus. However, the route by which Ii gains access to endosomal compartments in the absence of class II molecules remains uncertain. Here we report a mechanism that localizes Ii in lysosomal compartments independently of class II. We show that murine Ii can be detected by immunofluorescence within late endocytic compartments of stably transfected Ltk^? mouse fibroblasts. Immunochemical studies indicate that degradation of Ii in these cells is sensitive to the lysosomotropic agent ammonium chloride, yet the majority of Ii that undergoes this apparent lysosomal degradation is sensitive to the enzyme endoglycosidase H. This finding suggests that Ii may reach the lysosomal compartment by a route that bypasses the Golgi complex. Consistent with this possibility, we found that in contrast to Ii which is complexed to class II molecules, transport of free Ii to lysosomes is prevented by 3-methyladenine, an inhibitor of the autophagic pathway of protein degradation, a process which involves direct transport from the endoplasmic reticulum to lysosomes. These data suggest the route of transport that leads to endosomal localization of Ii in the absence of class II is distinct from that taken when expressed with class II. This forces a re-evaluation of the concept that the cytosolic tail of Ii contains a dominant Golgi-to-endosomal sorting signal.     

Endocytosis of horseradish peroxidase-poly-lysine conjugate by glomerular epithelial cells: an in vivo study

Native horseradish peroxidase (HRP) is known to pass rapidly through glomeruli when injected into rats. We have found that a conjugate of HRP with poly-lysine is readily endocytosed by glomerular epithelial cells (GEC). We have used this conjugate to study the GEC endocytotic process in male Wistar rats. The conjugate has an approximate molecular weight of 55-58,000, a pI of greater than 10.0, and almost the same secondary conformation as HRP; it does not increase urinary protein excretion significantly or alter the morphology of the renal glomeruli. After intravenous injection of the conjugate, it could be found in the GBM from 1 min to 4 h. At 1 min, it was evenly distributed on GEC foot processes and plasma membrane. GEC start to take up the conjugate from 1 min post-injection, by cellular membrane invagination. This reached a maximum at 4 h. Some of the endocytosed conjugate passed to lysosomes from the endosomal system. The amount of peroxidase demonstrable in the glomerular epithelial cells was considerably reduced by 24 h.     

Monoclonal antibodies to three distinct epitopes on human IgE: their use for determination of allergen-specific IgE

Three different monoclonal antibodies (MAb) against human immunoglobulin E have been obtained which specifically bind to human myeloma and polyclonal IgE. The antibodies showed high avidities for soluble IgE (0.7 X 10(9) to 3.3 X 10(9) M-1). These MAb defined three distinct epitopes on IgE. A mixture of these antibodies in combination with an 125I-labelled anti-mouse Kappa chain MAb has been used to measure allergen-specific IgE. This determination was performed by a solid-phase radioimmunoassay using allergen extracts coated to either chemically activated paper discs or to polyvinyl chloride wells. This method is 4-10 times more sensitive than other previously reported procedures. A similar technique has also been applied to detect individual allergens in immunoblots of allergen extracts.     

Immunolocalization of intercellular adhesion molecule-1 and leukocyte functional associated antigen-1 in schistosomal soluble egg antigen-induced granulomatous hyporesponsiveness

In this work, the changes in expression of the adhesion molecules ICAM-1/LFA-1 on inflammatory cells of the liver were studied by immunohistochemistry. Mice sensitized with SEA and infected with S. mansoni and S. mansoni-infected controls were examined from day 35 to day 56 postinfection. A significant upregulation of ICAM-1 and LFA-1 in both the SEA group and the infected control group started shortly after egg deposition at day 35 and persisted up to day 56 p.i. Notably, both ICAM-1 and LFA-1 expression peaks were shifted earlier to day 38 p.i. in the SEA group compared to day 40 in the infected control group. The distribution of ICAM-1 and LFA-1 in both groups was comparable. At the early phase of infection before granuloma formation, both ICAM-1 and LFA-1 were detected along the sinusoidal wall of small blood vessels. At the acute cellular granuloma phase, they were homogeneously distributed all over the inflammatory cells, while at the chronic fibrocellular stage a non-homogeneous staining of granuloma cells at the periphery of the granuloma was apparent. The present data suggest that adhesion molecules play a role in the initiation and maintenance of granuloma formation. Thus, the granulomatous hyporesponsiveness induced by sensitization with SEA was associated with reduced expression of adhesion molecules.     

Increased expression of adhesion molecules (ICAM-1 and LFA-1) on alveolar macrophages from asthmatic patients

In the airways inflammation observed in asthma, activated macrophages are present in increased numbers. Adhesion molecules are required for the cell: cell contacts between leukocytes and endothelial cells or other leukocytes, and they are induced by inflammatory stimuli. We studied the expression of two adhesion molecules (ICAM-1 and LFA-1) on alveolar macrophages recovered by bronchoalveolar lavage from 11 normal subjects and 13 asthmatic patients by using immunocytochemistry. Two specific monoclonal antibodies were used, and the reaction was revealed by the alkaline phosphatase-antialkaline phosphatase (APAAP) method. The percentage of cells expressing ICAM-1 or LFA-1 was significantly increased in asthmatic patients, as compared with normal subjects ( P < 0.001 and P < 0.002, respectively; Mann-Whitney U test), and there was a significant correlation with the percentage of cells expressing both markers in asthma ( P < 0.03, Spearman rank test). This study highlights the importance of macrophages in the inflammation of asthma and suggests that macrophage interactions with other cells play a role in this inflammation.     

Cadherins in the central nervous system

The central nervous system (CNS) is divided into diverse embryological and functional compartments. The early embryonic CNS consists of a series of transverse subdivisions (neuromeres) and longitudinal domains. These embryonic subdivisions represent histogenetic fields in which neurons are born and aggregate in distinct cell groups (brain nuclei and layers). Different subsets of these aggregates become selectively connected by nerve fiber tracts and, finally, by synapses, thus forming the neural circuits of the functional systems in the CNS. Recent work has shown that 30 or more members of the cadherin family of morphoregulatory molecules are differentially expressed in the developing and mature brain at almost all stages of development. In a regionally specific fashion, most cadherins studied to date are expressed by the embryonic subdivisions of the early embryonic brain, by developing brain nuclei, cortical layers and regions, and by fiber tracts, neural circuits and synapses. Each cadherin shows a unique expression pattern that is distinct from that of other cadherins. Experimental evidence suggests that cadherins contribute to CNS regionalization, morphogenesis and fiber tract formation, possibly by conferring preferentially homotypic adhesiveness (or other types of interactions) between the diverse structural elements of the CNS. Cadherin-mediated adhesive specificity may thus provide a molecular code for early embryonic CNS regionalization as well as for the development and maintenance of functional structures in the CNS, from embryonic subdivisions to brain nuclei, cortical layers and neural circuits, down to the level of individual synapses.