In vivo induction of specific cytotoxic T lymphocytes in mice and rhesus macaques immunized with DNA vector encoding an HIV epitope fused with hepatitis B surface antigen

BACKGROUND: In human blood basophils, cross-linking the high-affinity IgE receptor Fc epsilonRI with multivalent antigen activates a signaling pathway leading to Ca2+ mobilization, actin polymerization, shape changes, secretion, and cytokine production. METHODS AND RESULTS: The role of tyrosine kinases in human Fc epsilonRI signaling was explored by using human basophils isolated by Percoll gradient centrifugation followed by negative and/or positive selection with antibody-coated magnetic beads. Fc epsilonRI cross-linking of more than 95% pure basophil preparations activates the protein-tyrosine kinases Lyn and Syk, previously linked to Fc epsilonRI-coupled rodent mast cell activation, as well as Zap-70, previously implicated in T-cell receptor signaling, and causes the tyrosine phosphorylation of multiple proteins. The presence of Lyn, Syk, and Zap-70 in basophils was confirmed by Western blotting in lysates of highly purified basophils and independently by confocal fluorescence microscopy in cells labeled simultaneously with kinase-specific antibodies and with the basophil-specific antibody 2D7. Comparable amounts of Lyn and Syk were found in basophils and B cells, whereas T cells appear to have greater amounts of Zap-70 than basophils. The tyrosine kinase inhibitor piceatannol spares IgE-mediated Lyn activation but inhibits IgE-induced Syk and Zap-70 activation as well as overall protein tyrosine phosphorylation and secretion. Overall protein-tyrosine phosphorylation increases steadily over a range of anti-IgE concentrations that are low to optimal for secretion. However, tyrosine phosphorylation continues to increase at high anti-IgE concentrations that elicit very little secretion (the characteristic high-dose inhibition of secretion). CONCLUSIONS: Our data demonstrate the association of anti-IgE-stimulated, protein-tyrosine phosphorylation by a cascade of tyrosine kinases, including Zap-70 as well as Lyn and Syk, with the initiation of Fc epsilonRI-mediated signaling in human basophils.     

Critical role for the tyrosine kinase Syk in signalling through the high affinity IgE receptor of mast cells

Activation of the high affinity IgE receptor (Fc epsilon RI) of mast cells, a member of the antigen receptor family, leads to the release of allergic mediators, a critical event in the onset of immediate hypersensitivity. Stimulation of Fc epsilon RI results in the rapid association and activation of the Syk tyrosine kinase. Using Syk-deficient mast cells we show that they fail to degranulate, synthesize leukotrienes and secrete cytokines when stimulated through Fc epsilon RI, conclusively demonstrating an essential role for Syk in Fc epsilon RI signalling. Furthermore, our data strongly supports a model of Fc epsilon RI engagement leading to the sequential activation of the tyrosine kinases Lyn and then Syk. A similar mechanism is likely to apply to signal transduction through all members of the antigen receptor family.     

Metabolic activation of halothane to neoantigens in C57Bl/10 mice: immunochemical studies

Previous studies have shown that protein-serine/threonine kinases and protein-tyrosine kinase(s) are activated by cross-linking of the high-affinity receptor for IgE, Fc epsilon RI, on mast cells and basophils. In vitro kinase assays (ISDR kinase assays) on cellular proteins immobilized on polyvinylidene difluoride membrane after denaturation and renaturation were employed to estimate the complexity of protein kinases expressed in mouse mast cells. The results demonstrated that a large number (more than 60) of both serine/threonine- and tyrosine-specific kinases are present in a mouse mast cell line, PT-18. Cross-linking of Fc epsilon RI-induced activation of a subset of both serine/threonine kinases and tyrosine kinases in PT-18 as well as bone marrow-derived mouse mast cells, as revealed by the ISDR kinase assay. Among them, MAP kinase (or ERK2) was shown to be tyrosine phosphorylated and activated transiently upon Fc epsilon RI cross-linking, suggesting its potential role in mast cell signal transduction.     

Effects of an antiallergic drug, pemirolast potassium on tyrosine phosphorylation and map kinase activation in antigen-stimulated rat basophilic leukemia (RBL-2H3) cells

Aggregation of high affinity IgE Fc receptors (Fc epsilon RI) on RBL-2H3 cells results in tyrosine phosphorylation of 33-, 42-, 44-, 72-, 80-, 90-, 125-kDa proteins. The 42 and 44 kDa proteins were identified as mitogen-activated protein (MAP) kinases with immunoblotting of anti-MAP kinase antibody. The effects of an antiallergic drug, pemirolast potassium (TBX) on Ag-induced protein tyrosine phosphorylation and MAP kinase activation were investigated. When RBL-2H3 cells were stimulated with Ag in the presence of TBX, tyrosine phosphorylation of three proteins (33, 42 and 44 kDa) was inhibited concentration-dependently (0.1-10 micrograms/ml). Inhibition of Ag-induced tyrosine phosphorylation of 33 kDa protein, which could be a beta subunit of Fc epsilon RI, suggests that TBX may prevent the activation of Fc epsilon RI. TBX suppressed activation of MAP kinases (42 and 44 kDa) in response to Ag as well as phorbol myristate acetate (100 nM) or calcium ionophore A23187 (500 nM), implying that the drug acts on signal transduction component(s) between the second messengers and MAP kinases. However, TBX had no effects on protein tyrosine phosphorylation and MAP kinase activation in MC3T3-E1 osteoblastic cells. These results indicate that TBX may affect Fc epsilon RI and also may act as a step distal of Ca2+ mobilization and protein kinase C activation leading to MAP kinase activation in RBL-2H3 cells.     

Presence of IgM antibodies to Trypanosoma cruzi urinary antigen in sera from patients with acute Chagas'' disease

We have investigated the ability of an antisense immunoglobulin E (IgE) receptor alpha-subunit oligodeoxynucleotide (Fc epsilon RI alpha ODN) specifically to inhibit IgE-mediated allergic reactions in the mouse. Synthetic antisense Fc epsilon RI alpha ODN dose-dependently inhibited passive cutaneous anaphylaxis and histamine release from the mouse peritoneal mast cells (MPMC) activated by anti-dinitrophenyl (DNP) IgE. Northern blot analysis showed that the mast cells treated with antisense Fc epsilon RI alpha ODN exhibited no detectable levels of L-histidine decarboxylase mRNA after anti-DNP IgE stimulation, whereas the cells treated with sense Fc epsilon RI alpha ODN possessed significant amounts of this mRNA. Examination of the elevation of cAMP levels in MPMC following the activation with anti-DNP IgE demonstrated a significant rise in activated cells, but not in the antisense Fc epsilon RI alpha ODN-treated cells. Moreover, antisense Fc epsilon RI alpha ODN had a significant inhibitory effect on anti-DNP IgE-induced tumour necrosis factor-alpha production. Our results demonstrated that antisense Fc epsilon RI alpha ODN inhibited the IgE-mediated allergic reaction in vivo and in vitro.     

Endogenous superallergen protein Fv induces IL-4 secretion from human Fc epsilon RI+ cells through interaction with the VH3 region of IgE

We investigated the mechanism whereby protein Fv (pFv), a human sialoprotein found in normal liver and largely released in the intestinal tract in patients with viral hepatitis, induces mediator release from basophils and mast cells and evaluated whether it also induces IL-4 synthesis and secretion in basophils. pFv is a potent stimulus for histamine and IL-4 release from purified basophils. Histamine and IL-4 secretion from basophils activated by pFv was significantly correlated (rs = 0.70; p < 0.001). There was also a correlation (rs = 0.58; p < 0.01) between the maximum pFv- and anti-IgE-induced IL-4 release from basophils. The average t1/2 for pFv-induced histamine release was lower (3.5+/-1.5 min) than for IL-4 release (79.5+/-8.5 min; p < 0.01). IL-4 mRNA, constitutively present in basophils, was increased after stimulation by pFv and was inhibited by cyclosporin A and tacrolimus. Basophils from which IgE had been dissociated by brief exposure to lactic acid no longer released IL-4 in response to pFv and anti-IgE. The response to an mAb cross-linking the alpha-chain of Fc epsilon RI was unaffected by this treatment. Three human VH3+ monoclonal IgM concentration-dependently inhibited pFv-induced secretion of IL-4 and histamine from basophils and of histamine from human lung mast cells. In contrast, VH6+ monoclonal IgM did not inhibit the release of IL-4 and histamine induced by pFv. These results indicate that pFv, which acts as an endogenous superallergen, interacts with the VH3 domain of IgE to induce the synthesis and release of IL-4 from human Fc epsilon RI+ cells.     

Src family tyrosine kinase Lyn binds several proteins including paxillin in rat basophilic leukemia cells

Aggregation of the high affinity IgE receptors on rat basophilic leukemia (RBL-2H3) cells results in protein tyrosine phosphorylation although the receptor has no intrinsic enzymatic activity. The Src related protein tyrosine kinase p53/56lyn present in RBL-2H3 cells could play a role in this reaction. Here we have isolated the cDNA for rat Lyn and found it to be very homologous at the amino acid level to both the human and mouse proteins. A bacterially expressed maltose binding protein-Lyn (MBP-Lyn) fusion protein was already tyrosine phosphorylated and had tyrosine kinase activity. In a filter-binding assay, MBP-Lyn fusion protein (at 0.1 microM) specifically bound to several proteins of RBL-2H3 cells. In lysates of IgE receptor-activated cells, there was increased binding of MBP-Lyn to 65, 72, 78 and 110 kDa tyrosine phosphorylated proteins. The 72, 78 and 110 kDa tyrosine phosphorylated proteins were precipitated by a fusion protein containing the Lyn Src Homology 2 (SH2) domain. The 72 kDa Lyn binding protein was different from p72syk. Furthermore, paxillin, a cytoskeletal protein, was identified as one of the Lyn binding proteins. Thus Fc epsilon RI mediated signal transduction in RBL-2H3 cells may result from the interaction of p53/56lyn with paxillin, pp72, pp110 and other proteins.     

IgE antibody up-regulates high affinity IgE binding on murine bone marrow-derived mast cells

We have examined 3-week-old alcian blue positive cells (putatively mast cells) derived from mouse bone marrow for their expression of Fc epsilon RI. Using an indirect method of sensitizing the cells with immunoglobulin E (IgE) antibody (anti-DNP IgE) and detecting the level of bound IgE antibody by flow cytometry, we found that prolonged culture (1-5 days) with IgE, but not IgG, increased the total receptor density 6 +/- 1.9 fold. During the same period, histamine release in response to antigen (DNP-HSA) increased approximately 6-fold while the cell's response to either thrombin or ionomycin remained constant. The greatest up-regulation occurred in the first 2 days of culture. Using 2.4G2 to detect Fc epsilon RII RIII, we could not detect any up-regulation of this receptor. Culturing the cells for 1 h after sensitization did not result in any loss of cell surface IgE, suggesting a reasonably high affinity binding similar to that expected for Fc epsilon RI. This up-regulation was completely inhibited by co-culture with 2 micrograms/ml cycloheximide. These data suggest that IgE is capable of inducing a significant, protein synthesis, dependent up-regulation of its own high affinity receptor on mast cells/basophils.     

Endogenous hematopoietic reconstitution induced by human umbilical cord blood cells in immunocompromised mice: implications for adoptive therapy

Cross-linking the heterotrimeric (alpha beta gamma 2) IgE receptor, Fc epsilon RI, of mast cells activates two tyrosine kinases: Lyn, which phosphorylates beta and gamma subunit immunoreceptor tyrosine-based activation motifs, and Syk, which binds gamma-phospho-immunoreceptor tyrosine-based activation motifs and initiates cellular responses. We studied three Fc epsilon RI-dimerizing mAbs that maintain similar dispersed distributions over the surface of RBL-2H3 mast cells but elicit very different signaling responses. Specifically, mAb H10 receptor dimers induce very little inositol 1,4,5-trisphosphate synthesis, Ca2+ mobilization, secretion, spreading, ruffling, and actin plaque assembly, whereas dimers generated with the other anti-Fc epsilon RI mAbs induce responses that are only modestly lower than that to multivalent Ag. H10 receptor dimers activate Lyn and support Fc epsilon RI beta and gamma subunit phosphorylation but are poor Syk activators compared with Ag and the other anti-Fc epsilon RI mAbs. H10 receptor dimers have two other distinguishing features. First, they induce stable complexes between activated Lyn and receptor subunits. Second, the predominant Lyn-binding phospho-beta isoform found in mAb H10-treated cells is a less tyrosine phosphorylated, more electrophoretically mobile species than the predominant isoform in Ag-treated cells that does not coprecipitate with Lyn. These studies implicate Lyn dissociation from highly phosphorylated receptor subunits as a new regulatory step in the Fc epsilon RI signaling cascade required for Syk activation and signal progression.     

Translocation of glucokinase in pancreatic beta-cells during acute and chronic hyperglycemia

We recently reported that Fc mu R on NK cells is a signal transducing protein that stimulates a rapid increase in the level of cytoplasmic free calcium upon binding of IgM. This study was designed to examine signal transduction via the Fc mu R on NK cells and to characterize intracellular second messengers activated by IgM. Immunoprecipitation of IgM-bound Fc mu R by IgM-specific Ab coimmunoprecipitated the zeta- and Fc epsilon RI gamma-chains. Furthermore, engagement and clustering of Fc mu R by polyclonal IgM induced tyrosine phosphorylation of the zeta- and Fc epsilon RI gamma-chains, indicating their functional association with the Fc mu R-induced signal transduction cascade. Ligand-induced clustering of the Fc mu R also induced activity of src family kinases, Lck, Fyn, Lyn, and Src, as well as their physical interaction with the receptor. Triggering via Fc mu R also induced the activity of Syk and Zap-70, tyrosine kinases demonstrated to associate with zeta and Lck. Phospholipase C-gamma 1 and phosphatidylinositol 3-kinase were identified as substrates phosphorylated on tyrosine, as down-stream components of the signaling pathway activated in NK cells by polyclonal IgM. Although the Fc mu R on NK cells has not yet been biochemically characterized, our results suggest that the zeta- and Fc epsilon RI gamma-chains are functional subunits of this as well as other important cell surface receptors and that the Fc mu R is coupled either directly or indirectly to nonreceptor tyrosine kinases, which phosphorylate and thereby activate regulatory enzymes such as phospholipase C-gamma 1 and phosphatidylinositol 3-kinase.     

CGP 41251, a novel protein kinase inhibitor with in vitro selectivity for protein kinase C, strongly inhibits immunological activation of human skin mast cells and human basophils

The process of high-affinity IgE receptor (Fc epsilon RI)-mediated signal transduction in human basophils and mast cells is accompanied by activation of protein kinase C (PKC). The present study investigated the effects of a novel protein kinase inhibitor with in vitro selectivity for PKC (CGP 41251) in comparison with the potent but non-selective PKC inhibitor staurosporine on the activation of human peripheral basophilic leukocytes and enzymatically isolated human skin mast cells. CGP 41251 exerted strong concentration-dependent inhibitory effects on Fc epsilon RI-mediated histamine release from both cell populations. In addition, the IgE-mediated generation of arachidonic acid metabolites (leukotriene C4/D4 and prostaglandin E2) from human basophils was also significantly inhibited by this compound. Its action was not significantly different from the action of staurosporine. Direct activation of cellular PKC by the phorbol ester 12-o-tetradecanoyl-phorbol-13-acetate and subsequent histamine release from basophils was also inhibited by both compounds. CGP 41251 did not suppress N-formyl-met-leu-phe- or A23187-induced activation of basophils, whereas A23187-induced mediator release from human skin mast cells was inhibited in a concentration-dependent fashion. We conclude that an increase of in vitro selectivity for PKC does not significantly enhance inhibitory effects on immunological activation of histamine-containing cells. Moreover, nonimmunological pathways of signal transduction in basophils and mast cells appear to be mediated by distinct biochemical events.     

Anaphylaxis mediated through a humanized high affinity IgE receptor

Mast cells and basophils, which are activated by IgE and allergens through the high affinity IgE receptor (Fc epsilon RI), play a prominent role in anaphylaxis in the mouse. Mice deficient in this receptor become resistant to passive anaphylaxis. As a first step in developing an in vivo model that more closely mimics the IgE-mediated responses in man, we used a combination of transgenic and embryonic stem cell technology to generate a mouse line in which the murine Fc epsilon RI alpha-chain has been replaced with its human homologue. We demonstrate here that these mice express a tetrameric high affinity IgE receptor, in which the human alpha-chain associates with the murine beta- and gamma-chains, and that upon triggering with relevant Ag, this receptor mediates the initiation of the expected intracellular events. In addition, we show that the human alpha-chain restores an anaphylactic response to the nonresponsive alpha-deficient parental mouse line. This "humanized" mouse represents a potentially important model system, not only for studying the role of IgE in human immune responses, but also for testing potential therapeutic reagents that can interfere with responses mediated through the human Fc epsilon RI receptor.     

Quantitative differences between the optic nerve head and peripapillary retina in low-tension and high-tension primary open-angle glaucoma

Anti-IgE antibodies directed against the Fc epsilon RI-binding region on IgE inhibit binding of IgE to IgE receptors without inducing mediator release from IgE sensitized cells. In mice these antibodies selectively reduce serum IgE, inhibit antigen induced skin reactions, cytokine production by lung Th2 cells, and pulmonary eosinophil infiltration. Clinical trials in humans reveal that such antibodies are well tolerated and reduce rhinitis symptoms and early and late phase bronchoconstriction responses. Thus interruption of the allergic cascade at the IgE antibody level with non-anaphylactogenic anti-IgE antibodies is effective and represents an attractive intervention for the treatment of allergic diseases.     

Activation and interaction with protein kinase C of a cytoplasmic tyrosine kinase, Itk/Tsk/Emt, on Fc epsilon RI cross-linking on mast cells

Cross-linking of the high-affinity IgE receptor (Fc epsilon RI) on mast cells induces rapid phosphorylation on serine, threonine, and tyrosine residues and increases the enzymatic activity, of a Tec subfamily tyrosine kinase, Itk/Tsk/Emt (Emt). The pleckstrin homology domain of Emt at its amino-terminal interacts directly with multiple isoforms of protein kinase C (PKC) in vitro. In addition, a portion of Emt is physically associated with multiple isoforms of PKC in intact mast cells. PKC phosphorylates a bacterial fusion protein containing the pleckstrin homology domain of Emt in vitro. Coexpression of Emt in COS-7 cells with Ca(2+)-dependent PKC isoforms (alpha, beta I, or beta II) induces an enhancement in tyrosine phosphorylation of Emt. In vivo inhibition of PKC expression or activity attenuates tyrosine phosphorylation and enzymatic activity of Emt induced upon Fc epsilon RI cross-linking. These data collectively suggest that PKC phosphorylates Emt and activates its autophosphorylating activity. Alternatively, PKC could activate another tyrosine kinase that phosphorylates Emt, or PKC-mediated phosphorylation of Emt may render it a target for another tyrosine kinase. In any case, PKC appears to play a major role in the activation of Emt induced upon Fc epsilon RI cross-linking.     

Thermodynamics of the interaction of human immunoglobulin E with its high-affinity receptor Fc epsilon RI

We have employed isothermal titration calorimetry (ITC) and circular dichroism (CD) spectroscopy to characterize the binding of soluble fragments of IgE (IgE-Fc and Fc epsilon 3-4) to a soluble fragment of the high-affinity receptor Fc epsilon RI alpha-chain (sFc epsilon RI alpha). The thermodynamic parameters for the interaction of IgE-Fc and Fc epsilon 3-4 with sFc epsilon RI alpha, determined using ITC, confirm the earlier conclusion that the C epsilon 2 domain is not involved in the interaction and that the stoichiometry of both complexes is 1:1. For both IgE-Fc and Fc epsilon 3-4, the value of Delta H degrees is -36.9 +/- 4.6 kcal mol-1 at 37.3 degreesC and Delta Cp degrees is -820 +/- 120 cal mol-1 K-1. The temperature at which DeltaS degrees is zero is 284 +/- 1 K, indicating that the entropy contribution to the thermodynamics of association is unfavorable at physiological temperature. Of particular interest is the large value of Delta Cp degrees. The large surface area of IgE and Fc epsilon RI alpha that is implicated in complex formation from previous mutagenesis studies on the two proteins may account in part for the magnitude of Delta Cp degrees. Additional contributions may arise from hydration within the binding site and changes in tertiary structure of the individual components of the complex. However, the CD spectra of IgE, IgE-Fc, and Fc epsilon 3-4 complexes with sFc epsilon RI alpha are merely the sum of the spectra of their individual components, indicating that the secondary structure of the immunoglobulin domain folds are preserved on complex formation. Thus, any change in tertiary structure must be limited to the relative disposition of the immunoglobulin domains C epsilon 3 and C epsilon 4 in IgE and the two immunoglobulin-like domains in the alpha-chain of Fc epsilon RI.     

Amino acid residues that influence Fc epsilon RI-mediated effector functions of human immunoglobulin E

Immunoglobulin E (IgE) mediates its effector functions via the Fc region of the molecule. IgE binding to and subsequent aggregation of the high-affinity receptor (Fc epsilon RI) by allergen plays a pivotal role in type I hypersensitivity responses. Earlier studies implicated the C epsilon 2 and 3 interface and the A-B loop in C epsilon 3 in the IgE-Fc epsilon RI interaction. These regions and glycosylation sites in C epsilon 3 were now targeted by site-specific mutagenesis. IgE binding to Fc epsilon RI was compared with surface plasmon resonance (SPR) measurements, which assessed the binding of the soluble extracellular domain of Fc epsilon RI to IgE. Kinetic analysis based on a pseudo-first-order model agrees with previous determinations. A more refined SPR-based kinetic analysis suggests a biphasic interaction. A model-free empirical analysis, comparing the binding strength and kinetics of native and mutant forms of IgE, identified changes in the kinetics of IgE-Fc epsilon RI interaction. Conservative substitutions introduced into the A-B loop have a small effect on binding, suggesting that the overall conformation of the loop is important for the complementary interaction, but multiple sites across the C epsilon 3 domain may influence IgE-Fc epsilon RI interactions. Asn394 is essential for the generation of a functional IgE molecule in mammalian cells. A role of Pro333 in the maintenance of a constrained conformation at the interface between C epsilon 2-3 emerged by studying the functional consequences of replacing this residue by Ala and Gly. These substitutions cause a dramatic decrease in the ability of the ligand to mediate stimulus secretion coupling, although only small changes in the association and dissociation rates are observed. Understanding the molecular basis of this phenomenon may provide important information for the design of inhibitors of mast cell degranulation.     

Non-pharmacological management of hypertension: results from interviews with 100 general practitioners

The high-affinity receptor for IgE, Fc epsilon RI, expressed on antigen-presenting cells such as monocytes and Langerhans' cells exhibits profound differences to its homologue expressed on mast cells and basophils. The lack of the beta chain, the presence of an intracellular pool of preformed alpha chains, highly variable surface expression, and its function (to provide antigen focusing for T cells) are some of the main issues which have led us to consider the functional role of this receptor in a new light.     

Fc epsilon RI-mediated recruitment of p53/56lyn to detergent-resistant membrane domains accompanies cellular signaling

Detergent-resistant plasma membrane structures, such as caveolae, have been implicated in signalling, transport, and vesicle trafficking functions. Using sucrose gradient ultracentrifugation, we have isolated low-density, Triton X-100-insoluble membrane domains from RBL-2H3 mucosal mast cells that contain several markers common to caveolae, including a src-family tyrosine kinase, p53/56lyn. Aggregation of Fc epsilon RI, the high-affinity IgE receptor, causes a significant increase in the amount of p53/56lyn associated with these low-density membrane domains. Under our standard conditions for lysis, IgE-Fc epsilon RI fractionates with the majority of the solubilized proteins, whereas aggregated receptor complexes are found at a higher density in the gradient. Stimulated translocation of p53/56lyn is accompanied by increased tyrosine phosphorylation of several proteins in the low-density membrane domains as well as enhanced in vitro tyrosine kinase activity toward these proteins and an exogenous substrate. With a lower detergent-to-cell ratio during lysis, significant Fc epsilon RI remains associated with these membrane domains, consistent with the ability to coimmunoprecipitate tyrosine kinase activity with Fc epsilon RI under similar lysis conditions [Pribluda, V. S., Pribluda, C. & Metzger, H. (1994) Proc. Natl. Acad. Sci. USA 91, 11246-11250]. These results indicate that specialized membrane domains may be directly involved in the coupling of receptor aggregation to the activation of signaling events.     

Differential expression of ZAP-70 and Syk protein tyrosine kinases, and the role of this family of protein tyrosine kinases in TCR signaling

TCR stimulation results in the tyrosine phosphorylation of a number of cellular substrates. We have recently identified a 70-kDa protein tyrosine kinase, ZAP-70, which associates with the human TCR zeta-chain after TCR stimulation. We report here the isolation and sequence of a cDNA clone that encodes murine ZAP-70. Murine and human ZAP-70 share 93% amino acid identity and are homologous to the 72-kDa protein tyrosine kinase Syk. Syk has been implicated in the signal transduction pathways of the B cell membrane Ig and high affinity IgE receptors, Fc epsilon RI. In addition, we examined the tissue distribution of ZAP-70 and Syk in human and murine thymocyte subsets, B cells, and peripheral T cell subsets. ZAP-70 protein is expressed in all major thymocyte populations, with the level of expression being comparable to that found in both CD4+ and CD8+ peripheral T cells. Although Syk protein is also present in all thymocyte subsets, expression of Syk protein is down-regulated threefold to fourfold in peripheral T cells. In contrast to ZAP-70, expression of Syk is 12- to 15-fold higher in peripheral B cells when compared with peripheral T cells. In addition, whereas T cell stimulation results in down-regulation of Lck, no significant change in ZAP-70 or Syk protein is detected. Finally, we provide evidence that both ZAP-70 and Syk can associate with the TCR after TCR stimulation. With the use of a heterologous expression system, we show that, like ZAP-70, Syk is dependent upon a Src-family protein tyrosine kinase for association with the phosphorylated zeta-chain. Thus, the differential expression of these kinases suggests the possibility of different roles for ZAP-70 and Syk in TCR signaling and thymic development.