Abraham, Robert T.

Abstract The condensed tannin present in cotton mill dust profoundly alters the functional capabilities of resident alveolar macrophages. Previous studies from this laboratory have shown that in vitro exposure of rabbit resident alveolar macrophages to condensed tannin significantly inhibits the ability of these cells to produce reactive oxygen intermediates or to ingest particles. In the present study, we demonstrate that condensed tannin also alters arachidonic acid (C20:4) metabolism in these cells. Exposure of rabbit resident alveolar macrophages to condensed tannin results in the time- and dose-dependent release of C20:4 from the membrane phospholipids. The release of C20:4 occurred only at tannin concentrations > 25 µg/ml and was maximal 90 min after the onset of exposure. The EC50 for release was 75 µg/ml. Exposure to 100 µg/ml tannin resulted in the release of 20 ± 3% of the [14C]C20:4 incorporated in the cell membrane. In comparison, exposure to 160 µg/ml zymosan resulted in the release of 14 ± 4% of the [14C]C20:4. For both tannin and zymosan, phosphatidylcholine and phosphatidylinositol were the principal sources of the released C20:4. Approximately 63% of the C20:4 released after zymosan stimulation was further metabolized, mainly via the cyclooxygenase pathway. The major metabolites were 6-ketoprostaglandin F1α, prostaglandin F2α, and prostaglandin E2. In contrast, only 24% of the C20:4 released by tannin was subsequently further metabolized. The metabolites formed were essentially evenly distributed between products of the cyclooxygenase pathway and the lipoxygenase pathway. Exposure of alveolar macrophages to 50 µg/ml tannin for 30 min reduced the ability of the cells to subsequently incorporate C20:4 by 50 to 70%. In contrast, exposure of the cells to 160 mg/ml zymosan for 30 min had only a minimal effect on the subsequent ability of these cells to incorporate C20:4. These results indicate that tannin promotes C20:4 release, at least in part, by inhibiting its reacylation back into phospholipids, a mechanism that differs from that of zymosan.

Abstract Evidence suggests that cellular adhesion is critical for eosinophil effector functions. Here, we tested the hypothesis that an adhesion molecule, specifically β2 integrin, participates in intracellular signaling events of eosinophils. Eosinophils stimulated with interleukin (IL)-5 and adherent to protein-coated tissue culture plates via β2 integrin (CD18) showed tyrosine phosphorylation of a number of proteins. Among these proteins, tyrosine phosphorylation of the 105 kD and 115 kD proteins and the product of the c-cbl protooncogene, Cbl, was specifically inhibited using soluble anti-CD18 monoclonal antibody (mAb) to block eosinophil cell adhesion. Furthermore, phosphoinositide turnover of IL-5-stimulated adherent eosinophils was also inhibited by anti-CD18 mAb, suggesting that cellular adhesion plays important roles in eosinophil signal transduction. αMβ2 (Mac-1, CD11b/18) was one of the β2 integrins involved in eosinophil adhesion to protein-coated plates. We found that direct ligation of eosinophil αMβ2 with anti-CD11b mAb coupled to polystyrene microbeads induced tyrosine phosphorylation of a 115 kD protein and Cbl. Furthermore, anti-CD11b mAb microbeads induced increases in both phosphoinositide hydrolysis and the eosinophil degranulation response. Control antibodies, such as mouse myeloma IgG1 and anti-HLA class I antigen mAb, did not induce these cellular responses. These results suggest that engagement of β2 integrin either by cell adhesion or by anti-CD11b mAb triggers activation of an intracellular signaling cascade, including protein tyrosine phosphorylation and phosphoinositide turnover, and subsequent cellular degranulation in human eosinophils. Tyrosine phosphorylation of a 115 kD protein and Cbl may play important roles in adhesion-dependent cellular functions of eosinophils.

Natural killer (NK) cells are a unique subpopulation of lymphocytes with the capability to kill malignant cells via one of two alternative mechanisms: (i) Fc receptor-dependent cytotoxicity against antibody-coated targets or (ii) direct cell-mediated cytotoxicity. However, the molecular mechanisms that trigger and subsequently regulate NK cell cytotoxicity are incompletely understood. We have therefore investigated the role of protein tyrosine phosphorylation in the transmembrane signaling initiated after Fc receptor stimulation or direct tumor cell contact in clonal CD16+/CD3- human NK cells. We report that stimulation of the Fc receptor rapidly induced the tyrosine phosphorylation of a number of NK cell proteins. These effects occurred within 2 min, were maximal at 10 min, and declined toward baseline after 60 min. In addition, Fc receptor ligation increased the in vitro protein kinase activity of NK cell phosphotyrosyl proteins. We have also demonstrated that direct contact of NK cells with K562 tumor cells induced the rapid phosphorylation of distinct NK cell phosphotyrosyl proteins. Furthermore, the protein-tyrosine kinase inhibitor herbimycin A blocked NK cell cytotoxic function in a concentration-dependent manner. Our results suggest that protein tyrosine phosphorylation is an obligatory early proximal signal in activating the cytotoxic function of NK cells.

Although diverse signaling events are initiated by stimulation of multichain immune recognition receptors on lymphocytes, it remains unclear as to which specific signal transduction pathways are functionally linked to granule exocytosis and cellular cytotoxicity. In the case of natural killer (NK) cells, it has been presumed that the rapid activation of protein kinase C (PKC) enables them to mediate antibody-dependent cellular cytotoxicity (ADCC) and "natural" cytotoxicity toward tumor cells. However, using cloned human NK cells, we determined here that Fc receptor stimulation triggers granule release and ADCC through a PKC-independent pathway. Specifically, pretreatment of NK cells with the selective PKC inhibitor, GF109203X (using concentrations that fully blocked phorbol myristate acetate/ionomycin-induced secretion) had no effect on FcR-initiated granule release or ADCC. In contrast, FcR ligation led to the rapid activation of phosphatidylinositol 3-kinase (PI 3-kinase), and inhibition of this enzyme with the selective inhibitor, wortmannin, blocked FcR-induced granule release and ADCC. Additional experiments showed that, whereas FcR-initiated killing was wortmannin sensitive and GF109203X insensitive, natural cytotoxic activity toward the tumor cell line K562 was wortmannin insensitive and GF109203X sensitive. Taken together, these results suggest that: (a) PI 3-kinase activation induced by FcR ligation is functionally coupled to granule exocytosis and ADCC; and (b) the signaling pathways involved in ADCC vs natural cytotoxicity are distinct.

The proliferation of antigen-activated T cells is mediated by the T cell-derived growth factor, interleukin 2 (IL-2). The biochemical signaling cascades initiating IL-2-induced growth are dependent upon protein tyrosine kinase (PTK) activity. One IL-2-regulated PTK implicated in this cascade is the Src-family kinase, Fyn. Previous studies have described a physical association between Fyn and a potential downstream substrate, phosphatidylinositol 3-kinase (PI3-kinase) as well as the IL-2-dependent activation of PI3-kinase in T cells; however, the role of Fyn in IL-2-induced PI3-kinase activation remains unclear. In this report, we demonstrate that IL-2 stimulation triggers tyrosine phosphorylation of the p85 subunit of PI3-kinase in the murine T cell line, CTLL-2. Lysates prepared from growth factor-deprived and IL-2-stimulated T cells reconstituted both the binding of CTLL-2 cell-derived Fyn to and the IL-2-inducible tyrosine phosphorylation of exogenously added recombinant p85. Furthermore, overexpression of wild-type Fyn in these cells enhanced both the basal and IL-2-mediated activation of PI3-kinase. Additional studies of the Fyn-PI3-kinase interaction demonstrated that the Src homology 3 (SH3) domain of Fyn constitutes a direct binding site for the p85 subunit of PI3-kinase. These results support the notion that Fyn may be directly involved in the activation of the downstream signaling enzyme, PI3-kinase, in IL-2-stimulated T cells.

Crosslinking of the low affinity immunoglobulin G (IgG) Fc receptor (Fc gamma R type III) on natural killer (NK) cells initiates antibody-dependent cellular cytotoxicity. During this process, Fc gamma R stimulation results in the rapid activation of phospholipase C (PLC), which hydrolyzes membrane phosphoinositides, generating inositol-1,4,5-trisphosphate and sn-1,2-diacylglycerol as second messengers. We have recently reported that PLC activation after Fc gamma R stimulation can be inhibited by a protein tyrosine kinase (PTK) inhibitor. Based on the paradigm provided by the receptor tyrosine kinases, we investigated whether PLC-gamma 1 and/or PLC-gamma 2 are expressed in NK cells, and whether the PLC-gamma isoforms are tyrosine phosphorylated in response to Fc gamma R stimulation. Immunoblotting analyses with PLC-gamma 1- and PLC-gamma 2-specific antisera demonstrate that both isoforms are expressed in human NK cells. Furthermore, Fc gamma R crosslinking triggers the tyrosine phosphorylation of both PLC-gamma 1 and PLC-gamma 2 in these cells. Phosphorylation of both isoforms is detectable within 1 min, and returns to basal level within 30 min. Pretreatment with herbimycin A, a PTK inhibitor, blocked the Fc gamma R-induced tyrosine phosphorylation of PLC-gamma 1 and PLC-gamma 2, and the subsequent release of inositol phosphates. These results suggest that Fc gamma R-initiated phosphoinositide turnover in human NK cells is regulated by the tyrosine phosphorylation of PLC-gamma. More broadly, these observations demonstrate that nonreceptor PTK(s) activated by crosslinkage of a multisubunit receptor can phosphorylate both PLC-gamma isoforms.

Although all three Vav family members are expressed in T lymphocytes, the role that Vav3 plays in T cell activation is poorly defined. Here we show that, like Vav1, Vav3 undergoes rapid tyrosine phosphorylation after T cell receptor (TCR) cross-linkage and interacts with the adaptor molecules SLP76 and 3BP2 in a SH2-dependent manner. However, depletion of Vav1 but not Vav3 protein by RNA interference affects TCR-mediated IL-2 promoter activity. In contrast, Vav3 function is specifically required for coupling TCR stimulation to serum response element–mediated gene transcription. These data indicate that, although both Vav proteins are biochemically coupled to the TCR, they regulate distinct molecular pathways leading to defined gene transcriptional events.

▪ Abstract The potent immunosuppressive drugs FK506 and rapamycin interfere with signal transduction pathways required for T cell activation and growth. The distinct inhibitory effects of these drugs on the T cell activation program are mediated through the formation of pharmacologically active complexes with members of a family of intracellular receptors termed the FK506 binding proteins (FKBPs). The FKBP12 · FK506 complex specifically binds to and inhibits calcineurin, a signaling protein required for transcriptional activation of the interleukin (IL)-2 gene in response to T cell antigen receptor engagement. The FKBP12 · rapamycin complex interacts with a recently defined target protein termed the mammalian target of rapamycin (mTOR). Accumulating data suggest that mTOR functions in a previously unrecognized signal transduction pathway required for the progression of IL-2-stimulated T cells from G1into the S phase of the cell cycle. Here we review the immunopharmacology of rapamycin, with particular emphasis on the characterization of mTOR.

The immunosuppressant rapamycin interferes with G 1 -phase progression in lymphoid and other cell types by inhibiting the function of the mammalian target of rapamycin (mTOR). mTOR was determined to be a terminal kinase in a signaling pathway that couples mitogenic stimulation to the phosphorylation of the eukaryotic initiation factor (eIF)-4E–binding protein, PHAS-I. The rapamycin-sensitive protein kinase activity of mTOR was required for phosphorylation of PHAS-I in insulin-stimulated human embryonic kidney cells. mTOR phosphorylated PHAS-I on serine and threonine residues in vitro, and these modifications inhibited the binding of PHAS-I to eIF-4E. These studies define a role for mTOR in translational control and offer further insights into the mechanism whereby rapamycin inhibits G 1 -phase progression in mammalian cells.