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Impaired up-regulation of CD86 in B cells of "type A" common variable immunodeficiency patients
(2000)
Autoantibodies in sera from patients with autoimmune diseases have long been known and have become diagnostic tools. Analysis of their functional role again became popular with the availability of mice mutant for several genes of the complement and Fcγ receptor (FcγR) systems. Evidence from different inflammatory models suggests that both systems are interconnected in a hierarchical way. The complement system mediators such as complement component 5a (C5a) might be crucial in the communication between the complement system and FcγR-expressing cells. The split complement protein C5a is known to inactivate cells by its G-protein-coupled receptor and to be involved in the transcriptional regulation of FcγRs, thereby contributing to the complex regulation of autoimmune disease.
A soluble form of the complement receptor CD21 (sCD21) is shed from the lymphocyte surface. The sCD21 is able to bind all known ligands such as CD23, sCD23, Epstein-Barr virus and C3d in immune complexes. Here, we show the serum levels of sCD21 in sera the of antiphospholipid syndrome (APS) patients. Antiphospholipid syndrome is an autoimmune disorder in which autoantibodies cause heart attack, stroke and miscarriage. Antiphospholipid syndrome may appear as primary or in association with systemic lupus erythromatosus (SLE) and other autoimmune diseases. Here, we ask whether APS patients have different sCD21 titers compared to healthy persons and whether sCD21 levels correlate with the presence of anti-β2-GPI autoantibodies. We show that autoimmune APS patients have significantly reduced amounts of sCD21 in their sera, irrespective of the presence of anti-β2-GPI autoantibodies. In our APS patients cohort additional SLE, vasculities, DVT (deep vein thrombosis), fetal loss or thrombosis did not correlate to the reduced level of sCD21.
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.
A soluble form of CD21 (sCD21) and CD23 (sCD23) is released from the surface of human white blood cells upon shedding of the extracellular domain. sCD21 circulates in a complex with cleavage fragments of C3 and sCD23, which were previously identified as ligands of membrane and soluble CD21. sCD21 seems to be a marker of chronic inflammatory disease. To assess the sCD21 and sCD23 status in patients with subsets of juvenile arthritis (JA), we determined plasma levels sCD21 and sCD23. Plasma sCD21 levels were significantly decreased in all JA subtypes (O-JA P < 0.0068; P- and S-JA P < 0.0001) compared to healthy controls. Plasma sCD23 levels were significantly decreased in P-JA and S-JA (both P < 0.0001), but not in O-JA (P < 0.3843) in comparison with healthy controls, and data statistically analyzed. Our results suggest that pathological mechanisms relevant to autoimmune disorders interfere with the regulation of both CD21 and CD23 shedding.
Rheumatoid arthritis (RA) is characterized by the interaction of multiple mediators, among the most important of which are cytokines. In recent years, extensive studies demonstrate a pivotal role for one cytokine, macrophage migration inhibitory factor (MIF), in fundamental events in innate and adaptive immunity. MIF has now been demonstrated to be involved in the pathogenesis of many diseases, but in the case of RA the evidence for a role of MIF is very strong. MIF is abundantly expressed in the sera of RA patients and in RA synovial tissue correlating with disease activity. MIF-deficient mice were used to induce arthritis by serum transfer from K/BxN mice. K/BxN serum transfer arthritis was markedly attenuated in MIF(-) mice, with reduction in clinical index and histological severity as well as decrease in synovial cytokines. Macrophage transfers were done to investigate the specific role of macrophage-derived MIF. We show that adoptive transfer of wild-type macrophages into MIF(-) mice restores the sensitivity of MIF(-) mice to arthritis development, and this affect was associated with a restoration in serum IL-1? and IL-6 production. These results indicate that MIF plays a critical role in inflammation and joint destruction in K/BxN serum-induced arthritis and that the systemic expression of MIF by a subpopulation of macrophages is necessary and sufficient for the full development of arthritis.
It is well established that arthritis depresses locomotion in humans as well as in animal disease models. The K/BxN mouse model resembles rheumatoid arthritis and is widely used for research. Here, we investigate the behavioral alterations of arthritic K/BxN mice during arthritis development with respect to horizontal locomotion. Locomotor activity measurements and the methodology of ankle thickness measurements are compared to demonstrate the feasibility of motion tracking in the K/BxN mouse model. Arthritic K/BxN mice show significantly decreased locomotion compared to their non-arthritis K/BxN littermates. We found an indirect correlation of ankle thickness and locomotor activity. However, both parameters are only partially interdependent resulting in temporal displacement of maximal ankle swelling and maximal depression of locomotion by 1 week. Assessing the impaired movement as a behavioral test appears to be a valuable multifactorial parameter for the evaluation of arthritis in the K/BxN mouse model and provides additional information on disease progression and severity.
TNF-related activation-induced cytokine (TRANCE), also known as receptor activator of NF-kappaB ligand (RANKL), is the key molecule responsible for the bone loss observed in osteoporosis. Passive administration of osteoprotegerin, the soluble decoy receptor of TRANCE/RANKL, is efficient in blocking disease progression, but may not find widespread clinical use due to patient compliance problems and the expected high costs. In this study, we describe an efficient, safe, and potentially cost-effective active immunization strategy against TRANCE/RANKL. We show in mice that immunization with TRANCE/RANKL covalently linked to virus-like particles can overcome the natural tolerance of the immune system toward self proteins and produce high levels of specific Abs without the addition of any adjuvant. Serum Abs of immunized mice neutralized TRANCE/RANKL activity in vitro and were highly active in preventing bone loss in a mouse model of osteoporosis. Active immunization against TRANCE/RANKL was essentially reversible and did not produce any measurable immunosuppressive side effects, underscoring its potential as a new therapeutic approach to the treatment of human bone-degenerative disorders.
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.
The complement receptor II (CD21) recognises the complement component C3d of immune complexes. Expression of the CD21 gene is tightly regulated during B lymphocyte differentiation. Only mature B lymphocytes express CD21 but not pro-, pre-, or plasma B lymphocytes. Previously we found that pro-, pre-, and intermediate B lymphocytes contain a methylated CpG island and do not express CD21. CD21-expressing mature B lymphocytes, plasma B lymphocytes, and nonlymphoid cells carried a demethylated CD21 CpG island. Furthermore, we found that synovial lymphocytes from patients with rheumatic disease show reduced expression of CD21. This observation tempted us to analyse the methylation status of the CD21 CpG island in peripheral blood mononuclear cells and synovial fluid mononuclear cells derived from these patients. While methylation is involved in silencing CD21 in early types of B lymphocytes, we found the CD21-CpG island to be demethylated in peripheral blood mononuclear cells and synovial fluid mononuclear cells of patient DNA.
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.
Soluble CD21 (sCD21) is the ectodomain of the CD21 glycoprotein released by shedding from the cellular membrane. The ectodomain of CD21 is capable of binding complement fragments, Epstein-Barr virus (EBV) and CD23. Functionally sCD21 can activate monocytes and abrogate B-cell/follicular dendritic cell interaction, thereby inhibiting antibody production by antigen primed B cells. Levels of sCD21 vary in several clinical conditions. Here we analyzed sCD21 in synovial fluids and sera in arthritic patients. sCD21 concentrations were consistently lower in synovial fluids compared to paired sera samples from the same patients. In contrast to healthy donors, sCD21 levels are significantly reduced in rheumatoid arthritis patient's sera. Potential causes and consequences of the data are discussed.
Lipid rafts are liquid ordered platforms that dynamically compartmentalize membranes. Caveolins and flotillins constitute a group of proteins that are enriched in these domains. Caveolin-1 has been shown to be an essential component of caveolae. Flotillins were also discovered as an integral component of caveolae and have since been suggested to interact with caveolins. However, flotillins are also expressed in non-caveolae-containing cells such as lymphocytes and neuronal cells. Hence, a discrepancy exists in the literature regarding the caveolin dependence of flotillin expression and their subcellular localization. To address this controversy, we used mouse embryonic fibroblasts (MEFs) from caveolin-1 knockout (Cav-1(-/-)) and wild-type mice to study flotillin expression and localization. Here we show that both membrane association and lipid raft partitioning of flotillins are not perturbed in Cav-1(-/-) MEFs, whereas membrane targeting and raft partitioning of caveolin-2, another caveolin family protein, is severely impaired. Moreover, we demonstrate that flotillin-1, but not flotillin-2, associates with lipid droplets upon oleic acid treatment and that this association is completely independent of caveolin. Taken together, our results show that flotillins are localized in lipid rafts independent of caveolin-1 and that translocation of flotillin-1 to lipid droplets is a caveolin-independent process.