Binding of c-Rel to STAT5 target sequences in HTLV-I-transformed T cells.
(1/3171)
The type I human T-cell leukemia virus (HTLV-I) induces abnormal growth and subsequent transformation of T cells, which is associated with the development of an acute T-cell malignancy termed adult T-cell leukemia. A characteristic of HTLV-I-transformed T cells is the constitutive nuclear expression of NF-kappaB/Rel family of transcription factors, which appears to be essential for the growth of these transformed cells. Although NF-kappaB/Rel factors are known to induce the expression of T-cell growth factor interleukin (IL)-2, it is unclear how they participate in the IL-2-independent growth of HTLV-I-transformed cells. In this study, we show that certain NF-kappaB/Rel members, predominantly c-Rel, interact with enhancer sequences for STAT5, a key transcription factor mediating IL-2-induced T-cell proliferation. Reporter gene assays reveal that the binding of c-Rel to the STAT5 site present in the Fc gammaR1 gene leads to potent transactivation of this enhancer. Binding of c-Rel to the Fc gammaR1 STAT site also occurs in human peripheral blood T cells immortalized with HTLV-I in vitro and is correlated with enhanced levels of proliferation of these cells. These results raise the possibility that NF-kappaB/Rel may participate in the growth control of HTLV-I-transformed T cells by regulating genes driven by both kappaB and certain STAT enhancers. (+info)
In situ detection of activated Bruton's tyrosine kinase in the Ig signaling complex by phosphopeptide-specific monoclonal antibodies.
(2/3171)
Bruton's tyrosine kinase (Btk) is a critical transducer of signals originating from the B cell antigen receptor (BCR). Dosage, sequential phosphorylation, and protein interactions are interdependent mechanisms influencing Btk function. Phosphopeptide-specific mAbs recognizing two distinct phosphotyrosine modifications were used to quantify Btk activation by immunofluorescent techniques during B cell stimulation. In a population of cultured B cells stimulated by BCR crosslinking and analyzed by flow cytometry, transient phosphorylation of the regulatory Btk tyrosine residues (551Y and 223Y) was detected. The kinetics of phosphorylation of the residues were temporally distinct. Tyrosine 551, a transactivating substrate site for Src-family kinases, was maximally phosphorylated within approximately 30 seconds of stimulation as monitored by flow cytometry. Tyrosine 223, an autophosphorylation site within the SH3 domain, was maximally phosphorylated at approximately 5 minutes. Btk returned to a low tyrosine phosphorylation level within 30 minutes, despite persistent elevation of global tyrosine phosphorylation. Colocalization of activated Btk molecules with the crosslinked BCR signaling complex was observed to coincide with the period of maximal Btk tyrosine phosphorylation when stimulated B cells were analyzed with confocal microscopy. The results of these in situ temporal and spatial analyses imply that Btk signaling occurs in the region of the Ig receptor signaling complex, suggesting a similar location for downstream targets of its activity. (+info)
Efficient IgG-mediated suppression of primary antibody responses in Fcgamma receptor-deficient mice.
(3/3171)
IgG antibodies can suppress more than 99% of the antibody response against the antigen to which they bind. This is used clinically to prevent rhesus-negative (Rh-) women from becoming immunized against Rh+ erythrocytes from their fetuses. The suppressive mechanism is poorly understood, but it has been proposed that IgG/erythrocyte complexes bind to the inhibitory Fc receptor for IgG (FcgammaRIIB) on the B cell surface, thereby triggering negative signals that turn off the B cell. We show that IgG induces the same degree of suppression of the response to sheep erythrocytes in animals lacking the known IgG-binding receptors FcgammaRIIB, FcgammaRI + III, FcgammaRI + IIB + III, and FcRn (the neonatal Fc receptor) as in wild-type animals. Reinvestigation of the ability of F(ab')2 fragments to suppress antibody responses demonstrated that they were nearly as efficient as intact IgG. In addition, monoclonal IgE also was shown to be suppressive. These findings suggest that IgG inhibits antibody responses through Fc-independent mechanisms, most likely by masking of antigenic epitopes, thereby preventing B cells from binding and responding to antigen. In agreement with this, we show that T cell priming is not abolished by passively administered IgG. The results have implications for the understanding of in vivo regulation of antibody responses and Rh prophylaxis. (+info)
Crystal structure of the soluble form of the human fcgamma-receptor IIb: a new member of the immunoglobulin superfamily at 1.7 A resolution.
(4/3171)
Fcgamma-receptors (FcgammaRs) represent the link between the humoral and cellular immune responses. Via the binding to FcgammaR-positive cells, immunocomplexes trigger several functions such as endocytosis, antibody-dependent cell-mediated cytotoxity (ADCC) and the release of mediators, making them a valuable target for the modulation of the immune system. We solved the crystal structure of the soluble human Fcgamma-receptor IIb (sFcgammaRIIb) to 1.7 A resolution. The structure reveals two typical immunoglobulin (Ig)-like domains enclosing an angle of approximately 70 degrees, leading to a heart-shaped overall structure. In contrast to the observed flexible arrangement of the domains in other members of the Ig superfamily, the two domains are anchored by several hydrogen bonds. The structure reveals that the residues relevant for IgG binding, which were already partially characterized by mutagenesis studies, are located within the BC, C'E and FG loops between the beta-strands of the second domain. Moreover, we discuss a model for the sFcgammaRIIb:IgG complex. In this model, two FcgammaR molecules bind one IgG molecule with their second domains, while the first domain points away from the complex and is therefore available for binding other cell surface molecules, by which potential immunosuppressing functions could be mediated. (+info)
The SH2 domain-containing inositol 5'-phosphatase (SHIP) recruits the p85 subunit of phosphoinositide 3-kinase during FcgammaRIIb1-mediated inhibition of B cell receptor signaling.
(5/3171)
Coligation of FcgammaRIIb1 with the B cell receptor (BCR) or FcepsilonRI on mast cells inhibits B cell or mast cell activation. Activity of the inositol phosphatase SHIP is required for this negative signal. In vitro, SHIP catalyzes the conversion of the phosphoinositide 3-kinase (PI3K) product phosphatidylinositol 3,4, 5-trisphosphate (PIP3) into phosphatidylinositol 3,4-bisphosphate. Recent data demonstrate that coligation of FcgammaRIIb1 with BCR inhibits PIP3-dependent Btk (Bruton's tyrosine kinase) activation and the Btk-dependent generation of inositol trisphosphate that regulates sustained calcium influx. In this study, we provide evidence that coligation of FcgammaRIIb1 with BCR induces binding of PI3K to SHIP. This interaction is mediated by the binding of the SH2 domains of the p85 subunit of PI3K to a tyrosine-based motif in the C-terminal region of SHIP. Furthermore, the generation of phosphatidylinositol 3,4-bisphosphate was only partially reduced during coligation of BCR with FcgammaRIIb1 despite a drastic reduction in PIP3. In contrast to the complete inhibition of Tec kinase-dependent calcium signaling, activation of the serine/threonine kinase Akt was partially preserved during BCR and FcgammaRIIb1 coligation. The association of PI3K with SHIP may serve to activate PI3K and to regulate downstream events such as B cell activation-induced apoptosis. (+info)
Differential effects of manipulating signaling in early T cell development in intestinal intraepithelial lymphocytes and thymocytes.
(6/3171)
A pre-TCR-CD3 signal is required for the efficient maturation of CD4- CD8- thymocytes to the CD4+ CD8+ stage. This study addressed whether a similar signal is required for maturation of intestinal intraepithelial lymphocytes (IEL) that may develop extrathymically. We have shown previously that IEL from mice deficient for CD3- associated zeta chains include an immature population of CD3- CD8alphaalpha+ cells expressing cytoplasmic TCR beta chains but lacking detectable surface TCRalphabeta, CD16 and B220. Here we stimulated the appearance of such IEL in epsilon+/- zeta-/- mice by expression of an activated Lck transgene or in vivo treatment with anti-CD3epsilon. Anti-CD3epsilon treatment of RAG-deficient animals also yielded CD16- B220- IEL. In contrast, expression of a TCRbeta transgene in rag-1(-/-) mice did not stimulate the appearance of CD3- CD8alphaalpha+ CD16- B220- cells. Taken together these data indicate that although anti-CD3epsilon treatment and LckF505 assist in catalyzing a CD16+ B220+ --> CD16- B220- transition, these manipulations are not equivalent to a pre-TCR signal in IEL lymphocytes. (+info)
Fc receptor beta subunit is required for full activation of mast cells through Fc receptor engagement.
(7/3171)
The high-affinity IgE receptor (Fc epsilonRI) and the low-affinity IgG receptor (Fc gammaRIII) on mast cells are the key molecules involved in triggering the allergic reaction. These receptors share the common beta subunit (FcRbeta) which contains an immunoreceptor tyrosine-based activation motif and transduces the signals of these receptors' aggregation. In rodents, FcRbeta is essential for the cell surface expression of the Fc epsilonRI. In humans, the FcRbeta gene was reported to be one of the candidate genes causing atopic diseases. However, the role of FcRbeta in vivo still remains ambiguous. To elucidate the functions of FcRbeta, we developed the mice lacking FcRbeta [FcRbeta(-/-)]. The FcRbeta(-/-) mice lacked the expression of the Fc epsilonRI on mast cells and IgE-mediated passive cutaneous anaphylaxis (PCA) was not induced in FcRbeta(-/-) mice as was expected. In these mice, the expression of IgG receptors on mast cells was augmented but the IgG-mediated PCA reaction was attenuated. Although with bone marrow-derived cultured mast cells from FcRbeta(-/-), adhesion to fibronectin and Ca2+ flux upon aggregation of IgG receptors were enhanced, mast cells co-cultured with 3T3 fibroblasts exhibited impaired degranulation on receptor aggregation. These observations indicate that FcRbeta accelerates the degranulation of mature mast cells via the IgG receptor in connective tissues. (+info)
Regulation of B cell receptor-mediated MHC class II antigen processing by FcgammaRIIB1.
(8/3171)
The processing and presentation of Ag by Ag-specific B cells is highly efficient due to the dual function of the B cell Ag receptor (BCR) in both signaling for enhanced processing and endocytosing bound Ag. The BCR for IgG (FcgammaRIIB1) is a potent negative coreceptor of the BCR that blocks Ag-induced B cell proliferation. Here we investigate the influence of the FcgammaRIIB1 on BCR-mediated Ag processing and show that coligating the FcgammaRIIB1 and the BCR negatively regulates both BCR signaling for enhanced Ag processing and BCR-mediated Ag internalization. Treatment of splenic B cells with F(ab')2 anti-Ig significantly enhances APC function compared with the effect of whole anti-Ig; however, whole anti-Ig treatment is effective when binding to the FcgammaRIIB1 was blocked by a FcgammaRII-specific mAb. Processing and presentation of Ag covalently coupled to anti-Ig were significantly decreased compared with Ag coupled to F(ab')2anti-Ig; however, the processing of the two Ag-Ab conjugates was similar in cells that did not express FcgammaRIIB1 and in splenic B cells treated with a FcgammaRII-specific mAb to block Fc binding. Internalization of monovalent Ag by B cells was reduced in the presence of whole anti-Ig as compared with F(ab')2 anti-Ig, but the internalized Ag was correctly targeted to the class II peptide loading compartment. Taken together, these results indicate that the FcgammaRIIB1 is a negative regulator of the BCR-mediated Ag-processing function. (+info)