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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, flotillins͞reggies, 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.lipid rafts ͉ microdomains ͉ lymphocyte signaling ͉ polarization ͉ cytokinesis A ccording to the hitherto accepted view, lymphocytes and monocytes are round, nonpolarized cells (1-3) that become polarized and recruit certain molecules to the polarized edge only upon activation (4, 5). Several recent studies (6-8) have emphasized the importance of certain proteins in activationdependent cellular polarization and subsequent immunological synapse formation. These studies also underscored the importance of lipid microdomains in the polarization and the activation processes (5, 7). Molecules of the ezrin, radixin, and moesin (ERM) family (6, 8-10), PKC-(7), several signaling molecules like lck (11), fyn (12), LAT, and vav, and several coreceptors like CD28 (11), CD21, and CD19 (13) (in the case of B cells) have been shown to be translocated upon activation to the supramolecular activation clusters in a polarized fashion. However, under nonactivated conditions none of these molecules showed any preferential localization that could act as a predetermined platform for activation.The existence of these lipid microdomains in live cells has been a disputatious subject because the demonstration of these microdomains in nontreated cells has been extremely difficult (14). The concept of rafts itself has been very controversial, and the definite proof of their existence in living cells has been very elusive. Crosslinking with bifunctional reagents (15) and fluorescence resonance energy transf...

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 Â 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 Â N model demonstrated key roles of autoantigenic immune complexes activating the alternative pathway of complement, the subsequent association with C5aR and FccRIII-mediated cell activation and production of the inflammatory cytokines IL-1 and TNF-a, finally leading to joint destruction. The presence of high amounts of inflammatory cytokines and matrix-degrading proteases at sites of inflammation obviously put the cytokineproducing 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 Â N serum-induced arthritis. Reconstituting clodronate liposometreated 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.

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Objective. Induction of arthritis with autoantibodies against glucose-6-phosphate isomerase (GPI) is entirely independent of T cells and B cells but isResults. Comparing wild-type mice, mast celldeficient Kit W /Kit W-v mice, and mast cell-reconstituted Kit W /Kit W-v mice, we first showed that intraarticular and intraperitoneal mast cell engraftment fully restores susceptibility to antibody-induced arthritis, angiogenesis, and ␣v␤3 integrin activation. Importantly, selective mast cell silencing with either salbutamol or cromolyn prevented ␣v␤3 integrin activation, angiogenesis, and joint destruction.Conclusion. Mast cell engraftment fully restores susceptibility to ␣v␤3 integrin activation, angiogenesis, and joint destruction in GPI antibody-induced arthritis. Importantly, selective mast cell stabilization prevents ␣v␤3 integrin activation, angiogenesis, and joint destruction.

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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 Ca 2+ ionophore, or by stimulation of the BCR with anti-IgM+anti-CD40.

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.