Ulrich Blank

Soluble NSF Attachment Protein Receptors (SNAREs) in RBL-2H3 Mast Cells: Functional Role of Syntaxin 4 in Exocytosis and Identification of a Vesicle-Associated Membrane Protein 8-Containing Secretory Compartment

Mast cells upon stimulation through high affinity IgE receptors massively release inflammatory mediators by the fusion of specialized secretory granules (related to lysosomes) with the plasma membrane. Using the RBL-2H3 rat mast cell line, we investigated whether granule secretion involves components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery. Several isoforms of each family of SNARE proteins were expressed. Among those, synaptosome-associated protein of 23 kDa (SNAP23) was central in SNARE complex formation. Within the syntaxin family, syntaxin 4 interacted with SNAP23 and all vesicle-associated membrane proteins (VAMPs) examined, except tetanus neurotoxin insensitive VAMP (TI-VAMP). Overexpression of syntaxin 4, but not of syntaxin 2 nor syntaxin 3, caused inhibition of FcεRI-dependent exocytosis. Four VAMP proteins, i.e., VAMP2, cellubrevin, TI-VAMP, and VAMP8, were present on intracellular membrane structures, with VAMP8 residing mainly on mediator-containing secretory granules. We suggest that syntaxin 4, SNAP23, and VAMP8 may be involved in regulation of mast cell exocytosis. Furthermore, these results are the first demonstration that the nonneuronal VAMP8 isoform, originally localized on early endosomes, is present in a regulated secretory compartment.

Inhibitory ITAMs as novel regulators of immunity

Summary:u2002 Immune homeostasis is regulated by a finely tuned network of positive–negative regulatory mechanisms that guarantees proper surveillance avoiding hyperactivity that would lead to autoimmunity and inflammatory diseases. Immune responses involve the activation of immunoreceptors that contain tyrosine‐based activation motifs (ITAMs). One arm of control involves immunoreceptor tyrosine‐based inhibitory motif (ITIM)‐bearing receptors, which upon co‐aggregation initiate an inhibitory signal through recruitment of signal‐aborting phosphatases. Recently, a new immunoregulatory function has been ascribed to ITAMs, which represent in fact dual function modules that, under specific configurations termed inhibitory ITAM (ITAMi), can propagate inhibitory signals. One paradigm is the immunoglobulin A (IgA) Fc receptor (FcαRI), which, upon interaction with IgA monomers in the absence of antigen, initiates a powerful inhibitory signal involving Src homology 2 domain‐containing phosphatase 1 (SHP‐1) recruitment that suppresses cell activation launched by a whole variety of heterologous receptors without co‐aggregation. This explains the long known function of IgA as an anti‐inflammatory isotype. The importance of this control mechanism in immune homeostasis is underlined by the high incidence of autoimmune and allergic diseases in IgA‐deficient patients. ITAMi is now described for an increasing number of immunoreceptors with multiple roles in immunity. ITAMi signaling is also exploited by Escherichia coli to achieve immune evasion during sepsis. Here, we review our current understanding of ITAMi regulatory mechanisms in immune responses and discuss its role in immune homeostasis.

VAMP-8 segregates mast cell preformed mediator exocytosis from cytokine trafficking pathways

Inflammatory responses by mast cells are characterized by massive exocytosis of prestored granular mediators followed by cytokine/chemokine release. The vesicular trafficking mechanisms involved remain poorly understood. Vesicular-associated membrane protein-8 (VAMP-8), a member of the soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) family of fusion proteins initially characterized in endosomal and endosomal-lysosomal fusion, may also function in regulated exocytosis. Here we show that in bone marrow-derived mast cells (BMMCs) VAMP-8 partially colocalized with secretory granules and redistributed upon stimulation. This was associated with increased SNARE complex formation with the target t-SNAREs, SNAP-23 and syntaxin-4. VAMP-8-deficient BMMCs exhibited a markedly reduced degranulation response after IgE+ antigen-, thapsigargin-, or ionomycin-induced stimulation. VAMP-8-deficient mice also showed reduced plasma histamine levels in passive systemic anaphylaxis experiments, while cytokine/chemokine release was not affected. Unprocessed TNF accumulated at the plasma membrane where it colocalized with a VAMP-3-positive vesicular compartment but not with VAMP-8. The findings demonstrate that VAMP-8 segregates secretory lysosomal granule exocytosis in mast cells from cytokine/chemokine molecular trafficking pathways.

Evidence of a role for Munc18-2 and microtubules in mast cell granule exocytosis

Compound exocytosis of inflammatory mediators from mast cells requires SNARE and a series of accessory proteins. However, the molecular steps that regulate secretory granule movement and membrane fusion as well as the role of the cytoskeleton are still poorly understood. Here, we report on our investigation of the role of syntaxin-binding Munc18 isoforms and the microtubule network in this process. We found that mast cells express Munc18-2, which interacts with target SNAREs syntaxin 2 or 3, as well as Munc18-3, which interacts with syntaxin 4. Munc18-2 was localised to secretory granules, whereas Munc18-3 was found on the plasma membrane. Increased expression of Munc18-2 and derived peptides containing an interfering effector loop inhibited IgE-triggered exocytosis, while increased expression of Munc18-3 showed no effect. Munc18-2 localisation on granules is polarised; however, upon stimulation Munc18-2 redistributed into forming lamellipodia and persisted on granules that were aligned along microtubules, but was excluded from F-actin ruffles. Disruption of the microtubule network with nocodazole provoked Munc18-2 redistribution and affected mediator release. These findings suggest a role for Munc18-2 and the microtubule network in the regulation of secretory granule dynamics in mast cells.

Tyrosine phosphorylation-dependent and -independent associations of protein kinase C-Δ with Src family kinases in the RBL-2H3 mast cell line: regulation of Src family kinase activity by protein kinase C-δ

Src kinases and protein kinase C (PKC) have been well studied for their role in oncogenic and normal cellular processes. Herein we report on a novel regulatory pathway mediated by the interaction of PKC-δ with p53/56Lyn (Lyn) and with p60Src (Src) that results in the phosphorylation and increased activity of Lyn and Src. In the RBL-2H3 mast cell line, the interaction of PKC-δ with Lyn required the activation of the high affinity receptor for IgE (FcεRI) while the interaction with Src was constitutive. Increased complex formation of PKC-δ with Lyn or Src led to increased serine phosphorylation and activity of the Src family kinases. Conversely, Lyn was found to phosphorylate Lyn-associated and recombinant PKC-δ in vitro and the tyrosine 52 phosphorylated PKC-δ was recruited to associate with the Lyn SH2 domain. The constitutive association of PKC-δ with Src did not result in the tyrosine phosphorylation of PKC-δ prior to or after FcεRI engagement. However in cells over-expressing PKC-δ, FcεRI engagement resulted in the dramatic inhibition of Src activity and some inhibition of Lyn activity. Thus, the interaction and cross-talk of PKC-δ with Src family kinases suggests a novel and inter-dependent mechanism for regulation of enzymatic activity that may serve an important role in cellular responses.

Immunologic Stimulation of Mast Cells Leads to the Reversible Exposure of Phosphatidylserine in the Absence of Apoptosis

Background: Loss of phospholipid asymmetry represents one of the hallmarks of apoptosis and results in the surface exposure of phosphatidylserine (PS) which can be indirectly monitored by the calcium-dependent binding of annexin V. Methods and Results: Here, we provide evidence that the IgE-dependent stimulation of a rat mast cell line, as well as murine and human nontransformed mast cells, leads to the exposure of PS at the plasma membrane. The appearance of PS was quantitatively related to allergic mediator release. Pharmacological agents that prevent stimulus-secretion coupling blocked PS cell surface exposure and calcium ionophore-induced PS appearance, suggesting that it is a direct consequence of exocytosis rather than early signaling events initiated by the aggregation of the high-affinity IgE receptor (FcεRI). The surface exposure of PS in mast cells was reversible even in the continuous presence of stimulus and was not associated with the appearance of apoptotic nuclei, demonstrating that it was independent of physiological cell death. Conclusions: In addition to providing a means of monitoring exocytosis at the single cell level, our results indicate that PS externalization in mast cells is not necessarily related to apoptosis but could be an important feature of the degranulation process.

Alternative Endocytic Pathway for Immunoglobulin A Fc Receptors (CD89) Depends on the Lack of FcRγ Association and Protects against Degradation of Bound Ligand

IgA is the most abundant immunoglobulin in mucosal areas but is only the second most common antibody isotype in serum because it is catabolized faster than IgG. IgA exists in monomeric and polymeric forms that function through receptors expressed on effector cells. Here, we show that IgA Fc receptor(s) (FcαR) are expressed with or without the γ chain on monocytes and neutrophils. γ-less FcαR represent a significant fraction of surface FcαR molecules even on cells overexpressing the γ chain. The FcαR-γ2 association is up-regulated by phorbol esters and interferon-γ. To characterize γ-less FcαR functionally, we generated mast cell transfectants expressing wild-type human FcαR or a receptor with a point mutation (Arg → Leu at position 209) which was unable to associate with the γ chain. Mutant γ-less FcαR bound monomeric and polymeric human IgA1 or IgA2 but failed to induce exocytosis after receptor clustering. The two types of transfectant showed similar kinetics of FcαR-mediated endocytosis; however, the endocytosis pathways of the two types of receptor differed. Whereas mutant FcαR were localized mainly in early endosomes, those containing FcαR-γ2 were found in endo-lysosomal compartments. Mutant γ-less FcαR recycled the internalized IgA toward the cell surface and protected against IgA degradation. Cells expressing the two forms of FcαR, associated or unassociated with γ chains, may thus have differential functions either by degrading IgA antibody complexes or by recycling serum IgA.

Vesicle associated membrane protein (VAMP)‐7 and VAMP‐8, but not VAMP‐2 or VAMP‐3, are required for activation‐induced degranulation of mature human mast cells

Mediator release from mast cells (MC) is a crucial step in allergic and non‐allergic inflammatory disorders. However, the final events in response to activation leading to membrane fusion and thereby facilitating degranulation have hitherto not been analyzed in human MC. Soluble N‐ethyl‐maleimide‐sensitive factor attachment protein receptors (SNARE) represent a highly conserved family of proteins that have been shown to mediate intracellular membrane fusion events. Here, we show that mature MC isolated from human intestinal tissue express soluble N‐ethylmaleide sensitive factor attachment protein (SNAP)‐23, Syntaxin (STX)‐1B, STX‐2, STX‐3, STX‐4, and STX‐6 but not SNAP‐25. Furthermore, we found that primary human MC express substantial amounts of vesicle associated membrane protein (VAMP)‐3, VAMP‐7 and VAMP‐8 and, in contrast to previous reports about rodent MC, only low levels of VAMP‐2. Furthermore, VAMP‐7 and VAMP‐8 were found to translocate to the plasma membrane and interact with SNAP‐23 and STX‐4 upon activation. Inhibition of SNAP‐23, STX‐4, VAMP‐7 or VAMP‐8, but not VAMP‐2 or VAMP‐3, resulted in a markedly reduced high‐affinity IgE receptor‐mediated histamine release. In summary, our data show that mature human MC express a specific pattern of SNARE and that VAMP‐7 and VAMP‐8, but not VAMP‐2, are required for rapid degranulation.

Inhibitory ITAM Signaling by FcαRI-FcRγ Chain Controls Multiple Activating Responses and Prevents Renal Inflammation

Inhibitory signaling is an emerging function of ITAM-bearing immunoreceptors in the maintenance of homeostasis. Monovalent targeting of the IgA Fc receptor (FcαRI or CD89) by anti-FcαRI Fab triggers potent inhibitory ITAM (ITAMi) signaling through the associated FcRγ chain (FcαRI-FcRγ ITAMi) that prevents IgG phagocytosis and IgE-mediated asthma. It is not known whether FcαRI-FcRγ ITAMi signaling controls receptors that do not function through an ITAM and whether this inhibition requires Src homology protein 1 phosphatase. We show in this study that FcαRI-Fcγ ITAMi signals depend on Src homology protein 1 phosphatase to target multiple non-ITAM-bearing receptors such as chemotactic receptors, cytokine receptors, and TLRs. We found that anti-FcαRI Fab treatment in vivo reduced kidney inflammation in models of immune-mediated glomerulonephritis and nonimmune obstructive nephropathy by a mechanism that involved decreased inflammatory cell infiltration and fibrosis development. This treatment also prevented ex vivo LPS activation of monocytes from patients with lupus nephritis or vasculitis, as well as receptor activation through serum IgA complexes from IgA nephropathy patients. These findings point to a crucial role of FcαRI-FcRγ ITAMi signaling in the control of multiple heterologous or autologous inflammatory responses. They also identify anti-FcαRI Fab as a new potential therapeutic tool for preventing progression of renal inflammatory diseases.

Vav1 Is a Component of Transcriptionally Active Complexes

The importance of the hematopoietic protooncogene Vav1 in immune cell function is widely recognized, although its regulatory mechanisms are not completely understood. Here, we examined whether Vav1 has a nuclear function, as past studies have reported its nuclear localization. Our findings provide a definitive demonstration of Vav1 nuclear localization in a receptor stimulation–dependent manner and reveal a critical role for the COOH-terminal Src homology 3 (SH3) domain and a nuclear localization sequence within the pleckstrin homology domain. Analysis of DNA-bound transcription factor complexes revealed nuclear Vav1 as an integral component of transcriptionally active nuclear factor of activated T cells (NFAT)- and nuclear factor (NF)κB-like complexes, and the COOH-terminal SH3 domain as being critical in their formation. Thus, we describe a novel nuclear role for Vav1 as a component and facilitator of NFAT and NFκB-like transcriptional activity.