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Department, Institute
Keywords
- B cells (1)
- CD21 (1)
- Cell activation (1)
- Complement receptor (1)
- Complement receptor 2 /CD21 (1)
- Diselenide bridge (1)
- Pregnancy (1)
- Redox potential (1)
- Selenocysteine (1)
- Shedding (1)
Hematopoietic cells have long been defined as round, nonpolar cells that show uniform distribution of cell surface-associated molecules. However, recent analyses of the immunological synapse and the importance of lipid microdomains in signaling have shed new light on the aspect of lymphocyte polarization during the activation processes, but none of the molecules implicated so far in either the activation process or the microdomain residency are known to have a preferential localization in nonactivated cells. Chemical crosslinking and fluorescence resonance energy transfer methods have allowed the visualization of certain glycosylphosphatidylinositol-anchored proteins in lipid rafts but so far no microdomain resident protein has been shown to exist as visible stable platforms in the membrane. We report here that two lipid microdomain resident proteins, flotillins/reggies, form preassembled platforms in hematopoietic cells. These platforms recruit signaling molecules upon activation through lipid rafts. The preassembled platforms significantly differ from the canonical cholesterol-dependent "lipid rafts," as they are resistant to cholesterol-disrupting agents. Most evidence for the functional relevance of microdomains in living cells remains indirect. Using laser scanning confocal microscopy, we show that these proteins exist as stable, microscopically patent domains localizing asymmetrically to one pole of the cell. We present evidence that the asymmetric concentration of these microdomain resident proteins is built up during cytokinesis.
Using single cell suspensions from synovial fluid cells of arthritis patients, we observed differentiation of three-dimensional tissues in vitro. This new model of pannus-like tissue (PLT) might be useful to study pannus tissue formation and differentiation. In the PLT cultures, we observed two cell types, fibroblast-like and macrophage-like cells, defined by their distinct morphology and major histocompatibility complex (MHC) by human leukocyte antigen (HLA) class II expression. We could discriminate several intermediate steps of differentiation which finally led to 3D villi-like structures. Secretion of interferon gamma, interleukin-10, and tumor necrosis factor alpha was measured in the culture supernatants. Using methotrexate at various concentrations, the growth of PLT could be inhibited. We describe definite intermediate steps of differentiation. The present approach could be a suitable model for the in vitro study of pannus tissue formation.
Reggie-1/flotillin-2 is a plasma membrane-associated cytoplasmic protein, which defines non-caveolar raft microdomains. Reggie-1/flotillin-2 is enriched in detergent insoluble (TX100) membrane fractions (DIG), co-localizes with activated GPI-linked proteins and the fyn-kinase in neurons and T cells, and thus apparently participates in the assembly of protein complexes essential for signal transduction. In T cells activated by crosslinking the GPI-linked protein Thy-1 or by crosslinking the ganglioside GM1, reggie-1/flotillin-2 co-localizes with the T cell receptor. To determine whether reggie-1/flotillin-2 is also expressed in B cells, primary B cells from human blood and cell lines representing the developmental stages of pro, pre, mature and plasma B cells were analyzed by Western blotting, RT-PCR and immunofluorescence. Here, we show that reggie-1/flotillin-2 is expressed throughout B cell development, as well as in primary B cells, purified by cell sorting. On non-activated mature B cell Raji cell line we found reggie-1/flotillin-2 are exclusively in the detergent (TX100) insoluble membrane fractions that are staining positive for the raft marker GM1. Immunofluorescence microscopy showed that reggie-1/flotillin-2 is localized at the plasma membrane and marks intracellular spots in PBMCs. Confocal co-localization studies showed that reggie-1/flotillin-2 is associated with the plasma membrane, and the centrosomes (microtubule organizing centers) in these PBMCs. Comparison of reggie-1/flotillin-2 cDNA sequences with the genomic sequence database allowed us to determine the exon/intron structures in mouse and human. The gene organizations are highly conserved suggesting an important function of reggie-1/flotillin-2. Since reggie/flotillin proteins co-cluster with the T cell receptor and fyn kinases upon T cell stimulation, our findings of reggie-1/flotillin-2 in B cells suggest a similar role in B cell function.
Soluble CD21 (sCD21), released from the plasma membrane by proteolytic cleavage (shedding) of its extracellular domain (ectodomain) blocks B cell/follicular dendritic cell interaction and activates monocytes. We show here that both serine- and metalloproteases are involved in CD21 shedding. Using the oxidant pervanadate to mimic B cell receptor activation and thiol antioxidants such as N-acetylcysteine (NAC) and glutathione (GSH) we show that CD21 shedding is a redox-regulated process inducible by oxidation presumably through activation of a tyrosine kinase-mediated signal pathway involving protein kinase C (PKC), and by reducing agents that either directly activate the metalloprotease and/or modify intramolecular disulfide bridges within CD21 and thereby facilitate access to the cleavage site. Lack of short consensus repeat 16 (SCR16) abolishes CD21 shedding, and opening of the disulfide bridge between cys-2 (Cys941) and cys-4 (Cys968) of SCR16 is a prerequisite for CD21 shedding. Replacing these cysteines with selenocysteines (thereby changing the redox potential from -180 to -381 mV) results in a loss of inducible CD21 shedding, and removing this bridge by exchanging these cysteines with methionines increases CD21 shedding.
Complement receptor type II/CD21 is the functional receptor for complement fragments such as C3d, iC3b and the Epstein Barr Virus. A soluble form of CD21 (sCD21) is shed from lymphocytes surface and is able to bind to its ligands found in the plasma. The amount of sCD21 in serum may modulate immunity as the plasma levels are correlated with autoimmune conditions, such as Systemic Lupus Erythematosus, Rheumatoid Arthritis and Sjoegren’s Syndrome. Because of the fact that pregnancy may lead to remission of autoimmune diseases we determined the serum levels of sCD21 during pregnancy and postpartum. The serum sCD21 levels during pregnancy are significantly lower as compared to that of the healthy controls. There were no significant differences in sCD21 levels between the mother and the cord blood also immediately after parturition. Restoration of sCD21 levels to normal values takes between 6 weeks and 1 year after childbirth. Our study indicates that CD21-shedding is affected during pregnancy comparable to that of autoimmunity.
