Harald Illges

Genetic CD21 deficiency is associated with hypogammaglobulinemia

BACKGROUND Complement receptor 2 (CR2/CD21) is part of the B-cell coreceptor and expressed by mature B cells and follicular dendritic cells. CD21 is a receptor for C3d-opsonized immune complexes and enhances antigen-specific B-cell responses. OBJECTIVE Genetic inactivation of the murine CR2 locus results in impaired humoral immune responses. Here we report the first case of a genetic CD21 deficiency in human subjects. METHODS CD21 protein expression was analyzed by means of flow cytometry and Western blotting. CD21 transcripts were quantified by using real-time PCR. The CD21 gene was sequenced. Wild-type and mutant CD21 cDNA expression was studied after transfection of 293T cells. Binding of EBV-gp350 or C3d-containing immune complexes and induction of calcium flux in CD21-deficient B cells were analyzed by means of flow cytometry. Antibody responses to protein and polysaccharide vaccines were measured. RESULTS A 28-year-old man presented with recurrent infections, reduced class-switched memory B cells, and hypogammaglobulinemia. CD21 receptor expression was undetectable. Binding of C3d-containing immune complexes and EBV-gp350 to B cells was severely reduced. Sequence analysis revealed a compound heterozygous deleterious mutation in the CD21 gene. Functional studies with anti-immunoglobulin- and C3d-containing immune complexes showed a complete loss of costimulatory activity of C3d in enhancing suboptimal B-cell receptor stimulation. Vaccination responses to protein antigens were normal, but the response to pneumococcal polysaccharide vaccination was moderately impaired. CONCLUSIONS Genetic CD21 deficiency adds to the molecular defects observed in human subjects with hypogammaglobulinemia.

A crucial role for macrophages in the pathology of K/B × N serum-induced arthritis

Autoantibodies in the form of immune complexes are known to be crucial mediators in initiating inflammation in a variety of autoimmune diseases. This has been well documented in the anti‐collagen II antibody‐induced arthritis animal model for a long time now. Recently, in the K/B × N mouse model (the F1 of the TCR‐transgenic KRN and the diabetic NOD mice), anti‐glucose‐6‐phosphate isomerase (GPI) autoantibodies have been shown to induce arthritis. Experimental work in the K/B × N model demonstrated key roles of autoantigenic immune complexes activating the alternative pathway of complement, the subsequent association with C5aR and FcγRIII‐mediated cell activation and production of the inflammatory cytokines IL‐1 and TNF‐α, finally leading to joint destruction. The presence of high amounts of inflammatory cytokines and matrix‐degrading proteases at sites of inflammation obviously put the cytokine‐producing macrophages as the next target for investigation in this model. Here, we show that mice depleted of macrophages by clodronate liposome treatment are completely resistant to K/B × N serum‐induced arthritis. Reconstituting clodronate liposome‐treated mice with macrophages from naive animals could reverse this resistance. Also, we found that deficiencies in the Wiskott‐Aldrich syndrome protein and CD40, which are both implicated in macrophage activation, chemotaxis and phagocytosis, are not essential in serum‐induced arthritis. Mast cell degranulation was seen in arthritogenic serum‐treated mice even in the absence of macrophages, possibly suggesting that mast cell degranulation/activation acts hierarchically before macrophages in the inflammatory cascade of anti‐GPI antibody‐induced arthritis.

Asymmetric localization of flotillins/reggies in preassembled platforms confers inherent polarity to hematopoietic cells

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.

Protection against Osteoporosis by Active Immunization with TRANCE/RANKL Displayed on Virus-Like Particles

TNF-related activation-induced cytokine (TRANCE), also known as receptor activator of NF-κB 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.

The Lipid Raft Microdomain-Associated Protein Reggie-1/ Flotillin-2 is Expressed in Human B Cells and Localized at the Plasma Membrane and Centrosome in PBMCs

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.

Transmission of antibody-induced arthritis is independent of complement component 4 (C4) and the complement receptors 1 and 2 (CD21/35)

The K/BxN murine model of rheumatoid arthritis (RA) is dependent on the specificity of the KRN α β‐TCR, to recognize glucose‐6‐phosphate‐isomerase (GPI) on the NOD MHC class II Ag7 allele and production of GPI‐specific autoantibodies. Transfer of K/BxN serum into MHC‐unrelated and lymphocyte‐deficient mice induces RA. To investigate whether K/BxN serum‐induced RA involves complement activation and/or the complement receptors (CR) 1 and 2, we analyzed the role of complement C4 and of CR1 and CR2. For this purpose we used C4–/– mice impaired in the classical and the lectin complement pathways; Cr2–/– mice lacking CR1 and CR2 and, as control strains, BALB/c, C57BL/6, KRN and NOD. RA was assessed by calliper measurement of ankle thickness, clinical index and joint histology. We found that all mouse strains except NOD developed RA. The lack of protection in C4–/– mice suggests that antibody‐mediated RA is independent of the classical as well as the lectin complement pathways and the split complement product C4b. The lack of protection in Cr2–/– mice suggests that absence of CR1 had no significant affect, considering its role in immune complex clearance, inhibition of C3 and C5 convertase and as receptor for C3b/C4b. Also, CR2 lacks a role in disease as analyzed here, in its possible functions as receptor for C3dg, germinal center reaction and activation of alternative pathway on binding iC3. Hence we conclude that the transmission of K/BxN serum‐induced RA is independentof the classical and the lectin complement pathways and CR1 and CR2. The crucial role of complement C5, while neither classical nor lectin pathway is necessary, indicates that the alternative complement pathway may have a role in the K/BxN serum‐induced RA model.

Raft association and lipid droplet targeting of flotillins are independent of caveolin.

Abstract 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.

B cell activation leads to shedding of complement receptor type II (CR2/CD21)

Complement receptor type II (CR2/CD21) is the major receptor for C3d fragments on immune complexes. CD21 also serves as the receptor for Epstein–Barr virus in humans. On mature B cells, CD21 reduces the threshold of BCR signaling together with CD81, Leu13 and CD19, but it also occurs on other cells of the immune system where it performs unknown functions. A soluble form of CD21 (sCD21) is shed from the cell surface and is found in human blood plasma. An as‐yet‐unknown protease is thought to be responsible for this shedding. Altered levels of sCD21 occur in plasma in certain clinical conditions. We show here by mass spectrometry that sCD21 in human plasma of healthy donors is predominantly a short form of CD21 without the exon‐11‐encoded sequences. Whereas the N terminus of sCD21 was found unmodified, the C terminus is truncated, implying that only the extracellular portion of CD21 is shed. Peripheral blood B cells, but not T cells, contribute to the plasma CD21‐pool. CD21 shedding is induced by stimulation with PMA plus Ca2+ ionophore, or by stimulation of the BCR with anti‐IgM+anti‐CD40.

D3 dopamine receptor mRNA is elevated in T cells of schizophrenic patients whereas D4 dopamine receptor mRNA is reduced in CD4+-T cells

The expression of dopamine receptors was examined in purified human neutrophils, monocytes, B cells, natural killer cells and CD4+ - and CD8+ -T lymphocytes by RT-PCR. In healthy subjects, D1 and D2 receptors were not expressed in leukocytes. Real Time PCR for dopamine receptors D3 and D4 disclosed that D3 receptors are expressed in T cells and natural killer cells and D4 receptors in CD4+ -T cells. The comparison of schizophrenic patients with sex- and age-matched controls revealed a significantly higher expression of D3 receptor mRNA in T cells of schizophrenic patients, whereas D4 receptor mRNA in CD4+ -T cells was downregulated.

The role of the complement and the FcγR system in the pathogenesis of arthritis

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.