The effectiveness of different rat IgG subclasses as IgE-blocking antibodies in the rat basophil leukaemia cell model.
The degranulation of mast cells in an allergic response is initiated by the aggregation of high-affinity IgE receptors (Fc epsilon RI) by IgE and antigen. Recently it has been shown that such degranulation can be inhibited by cross-linking Fc epsilon RI and low-affinity IgG receptors (Fc gamma RII) which are also expressed by mast cells. The ability of various monoclonal antibodies to block the degranulation of rat basophil leukaemia (RBL) cells sensitized with IgE antidinitrophenyl (DNP) antibodies has been investigated. Sensitized cells were challenged with immune complexes formed using varying concentrations of antigen, and of both high- and low-valency antigen. It is reported here that rat IgG1 antibodies, which are associated in the rat with a Th1-type response, act as highly effective blocking antibodies over a wide concentration range. Rat IgG2a antibodies, which are associated with a Th2-type response, were able only to inhibit degranulation when immune complexes were formed with very low concentrations of high-valency antigen (DNP32-HSA). Under these conditions, some inhibitory activity was seen with high-affinity murine IgA anti-DNP but not with low-affinity rat IgG2b anti-DNP antibody-containing immune complexes. In addition to this inhibitory activity, IgG2a antibodies were shown to be capable of inducing degranulation of cells via unoccupied Fc epsilon RI. These results demonstrate that blocking activity may arise via both inhibitory receptors and by masking of antigen. (+info
Cutting edge: extracellular signal-regulated kinase activates syk: a new potential feedback regulation of Fc epsilon receptor signaling.
The protein tyrosine kinase Syk is an essential element in several cascades coupling Ag receptors to cell responses. Syk and the mitogen-activated protein kinase extracellular signal-regulated kinase 1 (ERK1) were found to form a tight complex in both resting and Ag-stimulated rat mucosal-type mast cells (rat basophilic leukemia 2H3 cell line RBL-2H3). A direct serine phosphorylation and activation of Syk by ERK was observed in in vitro experiments. Moreover the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK) kinase (MEK) inhibitors markedly decreased the Ag-induced phosphorylation of the tyrosyl residues of Syk and its activation as well as suppressed the degranulation of the cells. These results suggest a positive feedback regulation of Syk by ERK in the cascade coupling the type 1 Fc epsilon receptor to the secretory response of mast cells; hence, the existence of a novel type of cross-talk between protein serine/threonine kinases and protein tyrosine kinases is suggested. (+info
Phenotype of a recombinant store-operated channel: highly selective permeation of Ca2+.
1. Genes related to trp (transient receptor potential) are proposed to encode store-operated channels. We examined the ionic permeation of recombinant channels formed by stable and transient expression of the TRP homologue bCCE1 in Chinese hamster ovary (CHO) cells (CHO(CCE1)) and rat basophilic leukaemia (RBL) cells, respectively. 2. Store-operated currents were activated in CHO(CCE1) cells by internal dialysis of IP3 under strong buffering of intracellular Ca2+. The action of IP3 was mimicked by thapsigargin but not by IP4. 3. With extracellular Ca2+, Na+ and Mg2+, the store-operated currents of CHO(CCE1) rectified inwardly in the presence of internal Cs+. Outward currents were not detected below +80 mV. Identical currents were recorded with external Ba2+ and also with no external Na+ and Mg2+. In the absence of external Mg2+, the inward currents showed an anomalous mole fraction behaviour between Ca2+ and Na+. Half-maximal inhibition of Na+ currents was observed with approximately 100 nM and full block with 2-5 microM external Ca2+. 4. In the parental CHO(-) cells, IP3 dialysis evoked inward currents that also displayed anomalous mole fraction behaviour between Ca2+ and Na+. However, half-maximal block of Na+ currents required 5 times higher Ca2+ concentrations in CHO(-) cells. Additionally, the density of Ca2+ and Na+ currents at -80 mV was 5 and 2 times larger in CHO(CCE1) cells, respectively. 5. In RBL cells, dialysis of IP3 evoked store-operated currents that showed 1.4-fold larger densities at -80 mV in cells expressing bCCE1. 6. The enhanced density of store-operated currents in CHO(CCE1) cells and in bCCE1-transfected RBL cells probably reflects the phenotype of CCE1. These results suggest a highly selective permeation of Ca2+ through recombinant channels formed by CCE1 either alone or in combination with endogenous channel proteins. (+info
Cutting edge: CXCR4-Lo: molecular cloning and functional expression of a novel human CXCR4 splice variant.
Human CXCR4 is a specific receptor for the CXC chemokine stromal cell-derived factor-1 (SDF-1) and a coreceptor for T cell line tropic strains of HIV-1. Genetic knockouts of CXCR4 and SDF-1 have delineated their critical role during embryonic cardiogenesis, leukopoiesis, and vasculogenesis. Herein, we used bioinformatics and differential strategies like isoform-specific RT-PCR and Northern blots to identify and clone a novel unspliced isoform of human CXCR4, termed CXCR4-Lo. CXCR4-Lo corresponds to a larger approximately 4. 0-kb mRNA transcript and differs from the known human CXCR4 by the first 9 aa in the functionally important NH2-terminal extracellular domain of the receptor. CXCR4-Lo-transfected rat basophil leukemia-2H3 cells responded to SDF-1 with a transient rise of intracellular Ca2+ concentration and by undergoing chemotaxis. Expression of CXCR4-Lo is noteworthy, as it may have differential affinity as a coreceptor for HIV strains in comparison with CXCR4. Furthermore, CXCR4-Lo may also provide a functional backup to CXCR4 during embryogenesis. (+info
Direct neurite-mast cell communication in vitro occurs via the neuropeptide substance P.
Communication between nerves and mast cells is a prototypic demonstration of neuroimmune interaction. However, whether mast cell activation occurs as a direct response to neuronal activation or requires an intermediary cell is unclear. Addressing this issue, we used an in vitro coculture approach comprising cultured murine superior cervical ganglia and rat leukemia basophilic cells (RBLs; possesses properties of mucosal-type mast cells). Following loading with the calcium fluorophore, Fluo-3, neurite-RBL units (separated by <50 nm) were examined by confocal laser scanning microscopy. Addition of bradykinin, or scorpion venom, dose-dependently elicited neurite activation (i.e., Ca2+ mobilization) and, after a lag period, RBL Ca2+ mobilization. Neither bradykinin nor scorpion venom had any direct effect on the RBLs in the absence of neurites. Addition of a neutralizing substance P Ab or a neurokinin (NK)-1 receptor antagonist, but not an NK-2 receptor antagonist, dose-dependently prevented the RBL activation that resulted as a consequence of neural activation by either bradykinin or scorpion venom. These data illustrate that nerve-mast cell cross-talk can occur in the absence of an intermediary transducing cell and that the neuropeptide substance P, operating via NK-1 receptors, is an important mediator of this communication. Our findings have implications for the neuroimmune signaling cascades that are likely to occur during airways inflammation, intestinal hypersensitivity, and other conditions in which mast cells feature. (+info
CD45 is essential for Fc epsilon RI signaling by ZAP70, but not Syk, in Syk-negative mast cells.
The ZAP70/Syk family of protein tyrosine kinases plays an important role in Ag receptor signaling. Structural similarity of Syk and ZAP70 suggests their functional overlap. Previously, it was observed that expression of either ZAP70 or Syk reconstitutes Ag receptor signaling in Syk-negative B cells. However, in CD45-deficient T cells, Syk, but not ZAP70, restores T cell receptor-signaling pathway. To study the function of Syk, ZAP70, and CD45 in mast cells, a Syk/CD45 double-deficient variant of RBL-2H3 cells was characterized. After transfection, stable cell lines were isolated that expressed ZAP70, Syk, CD45, ZAP70 plus CD45, and Syk plus CD45. IgE stimulation did not induce degranulation in parental double-deficient cells, nor in the cells expressing only CD45. ZAP70 expression did not restore Fc epsilon RI signaling unless CD45 was coexpressed in the cells. However, Syk alone restored the IgE signal transduction pathway. The coexpression of CD45 with Syk had no significant effects on the responses to FcepsilonRI-aggregation. There was much better binding of Syk than ZAP70 to the phosphorylated Fc epsilon RI gamma-ITAM. Furthermore, unlike Syk, ZAP70 required CD45 to display receptor-induced increase in kinase activity. Therefore, in mast cells, ZAP70, but not Syk, requires CD45 for Ag receptor-induced signaling. (+info
On the characterisation of the mechanism underlying passive activation of the Ca2+ release-activated Ca2+ current ICRAC in rat basophilic leukaemia cells.
1. Tight-seal whole-cell patch clamp experiments were performed to investigate the mechanism whereby passive depletion of stores activates the Ca2+ release-activated Ca2+ current (ICRAC) in rat basophilic leukaemia (RBL) cells. 2. Passive depletion of stores was achieved by dialysing cells with different concentrations of Ca2+ chelators. Low concentrations generally evoked a submaximal ICRAC, which developed slowly and monophasically. Higher concentrations resulted in a biphasic current in which the initial slow monophasic component developed into a faster and bigger second phase. 3. The kinetics of ICRAC as well as its final amplitude were not affected by Ca2+ chelators that had different affinities or speeds of binding. 4. Exogenous Ca2+ binding ratios > or = 16,670 were necessary to fully activate ICRAC. Because the Ca2+ binding ratio within the stores is presumably low, this indicates that other factors like Ca2+ transport across the stores membrane are rate limiting for passive store depletion. 5. Heparin and Ruthenium Red both failed to affect passive Ca2+ leak from the intracellular stores. 6. Treatment with sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump blockers dramatically altered the kinetics of activation of biphasic currents, and increased the amplitude of monophasic ones. 7. Our results suggest that SERCA pumps are very effective in preventing ICRAC from activating passively, and are responsible for the phasic nature of the current, its time course of development and its overall extent. (+info
Protein tyrosine kinase p53/p56(lyn) forms complexes with gamma-tubulin in rat basophilic leukemia cells.
The aggregation of receptors with high affinity for IgE (FcepsilonRI) on the surface of mast cells and basophils initiates a chain of biochemical events culminating in the release of allergy mediators. Although microtubules have been implicated in the activation process, the molecular mechanism of their interactions with signal transduction molecules is poorly understood. Here we show that in rat basophilic leukemia cells large amounts of alphabeta-tubulin dimers ( approximately 70%) and gamma-tubulin ( approximately 85%) are found in a soluble pool which was released from the cells after permeabilization with saponin, or extraction with non-ionic detergents. Soluble tubulins were found in large complexes with other molecules. Complexes of soluble gamma-tubulin released from activated cells contained tyrosine-phosphorylated proteins of relative mol. wt approximately 25, 50, 53, 56, 60, 75, 80, 97, 115 and 200 kDa. Increased tyrosine phosphorylation of proteins associated with the cytoskeleton, i.e. around centrosomes, was detected by immunofluorescence microscopy. In vitro kinase assays revealed increased tyrosine phosphorylation of proteins in gamma-tubulin complexes isolated from activated cells. Two of the tyrosine phosphorylated proteins in these complexes were identified as the p53/56(lyn) kinase. Furthermore, gamma-tubulin bound to the N-terminal fragment of recombinant Lyn kinase and its binding was slightly enhanced in activated cells. Pretreatment of the cells with Src family-selective tyrosine kinase inhibitor, PP1, decreased the amount of tyrosine phosphorylated proteins in gamma-tubulin complexes, as well as the amount of gamma-tubulin in Lyn kinase immunocomplexes. The combined data suggest that gamma-tubulin is involved in early stages of mast cell activation. (+info