Epidermal growth factor (EGF) receptor kinase-independent signaling by EGF.

The ErbB family of receptors, which includes the epidermal growth factor receptor (EGFR), ErbB2, ErbB3, and ErbB4, mediate signaling by EGF-like polypeptides. To better understand the role of the EGFR tyrosine kinase, we analyzed signaling by a kinase-inactive EGFR (K721M) in ErbB-devoid 32D cells. K721M alone exhibited no detectable signaling capacity, whereas coexpression of K721M with ErbB2, but not ErbB3 or ErbB4, resulted in EGF-dependent mitogen-activated protein kinase (MAPK) activation. The kinase activity, but not tyrosine phosphorylation, of ErbB2 was required for EGF-induced MAPK activation. The presence of tyrosine phosphorylation sites in K721M was not a requisite for signaling, indicating that transphosphorylation of K721M by ErbB2 was not an essential mechanism of receptor activation. Conversely, the mutated kinase domain of K721M (residues 648-973) and tyrosine phosphorylation of at least one of the receptors were necessary. EGF was found to activate the pro-survival protein kinase Akt in stable cell lines expressing K721M and ErbB2 but, unlike cells expressing wild-type EGFR, was incapable of activating signal transducers and activators of transcription (STAT) or driving cell proliferation. These results demonstrate that EGFR-ErbB2 oligomers are potent activators of MAPK and Akt, and this signaling does not require EGFR kinase activity.

Differential effects of Cbl and 70Z/3 Cbl on T cell receptor-induced phospholipase Cgamma-1 activity.

We demonstrate that the differential effects Cbl and oncogenic 70Z/3 Cbl have on Ca(2+)/Ras-sensitive NF-AT reporters is partially due to their opposing ability to regulate phospholipase Cgamma1 (PLCgamma1) activation as demonstrated by analysis of the activation of an NF-AT reporter construct and PLCgamma1-mediated inositol phospholipid (PI) hydrolysis. Cbl over-expression resulted in reduced T cell receptor-induced PI hydrolysis, in the absence of any effect on PLCgamma1 tyrosine phosphorylation. In contrast, expression of 70Z/3 Cbl led to an increase in basal and OKT3-induced PLCgamma1 phosphorylation and PI hydrolysis. These data indicate that Cbl and 70Z/3 Cbl differentially regulate PLCgamma1 phosphorylation and activation. The implications of these data on the mechanism of Cbl-mediated signaling regulation are discussed.

Inefficient phospholipase C activation and reduced Lck expression characterize the signaling defect of umbilical cord T lymphocytes.

Adult and neonatal immunocompetent cells exhibit important functional distinctions, including differences in cytokine production and susceptibility to tolerance induction. We have investigated the molecular features that characterize the immune response of cord blood-derived T lymphocytes compared with that of adult T lymphocytes. Our findings demonstrate that phospholipase C (PLC) isozymes, which play a pivotal role in the control of protein kinase C activation and Ca2+ mobilization, are differently expressed in cord and adult T lymphocytes. PLCbeta1 and delta1 are expressed at higher levels in cord T cells, while PLCbeta2 and gamma1 expression is higher in adult T lymphocytes. PLCdelta2 and gamma2 appear to be equally expressed in both cell types. In addition, a functional defect in PLC activation via CD3 ligation or pervanadate treatment, stimuli that activate tyrosine kinases, was observed in cord blood T cells, whereas treatment with aluminum tetrafluoride (AlF4-), a G protein activator, demonstrated a similar degree of PLC activation in cord and adult T cells. The impaired PLC activation of cord blood-derived T cells was associated with a a very low expression of the Src kinase, Lck, along with a reduced level of ZAP70. No mitogenic response to CD3 ligation was observed in cord T cells. However, no signaling defect was apparent downstream of PLC activation, as demonstrated by the mitogenic response of cord T cells to the pharmacologic activation of protein kinase C and Ca2+ by treatment with PMA and ionomycin. Thus, neonatal cord blood-derived T cells show a signaling immaturity associated with inadequate PLCgamma activation and decreased Lck expression.

A role for neutral sphingomyelinase-mediated ceramide production in T cell receptor-induced apoptosis and mitogen-activated protein kinase-mediated signal transduction.

Studying apoptosis induced by T cell receptor (TCR) cross-linking in the T cell hybridoma, 3DO, we found both neutral sphingomyelinase activation and production of ceramide upon receptor engagement. Pharmacological inhibition of ceramide production by the fungal toxin, fumonisin B1, impaired TCR-induced interleukin (IL)-2 production and programmed cell death. Addition of either exogenous ceramide or bacterial sphingomyelinase reconstituted both responses. Moreover, specific inactivation of neutral sphingomyelinase by antisense RNA inhibited IL-2 production and mitogen-activated protein kinase activation after TCR triggering. These results suggest that ceramide production by activation of neutral sphingomyelinase is an essential component of the TCR signaling machinery.

Sequences surrounding the Src-homology 3 domain of phospholipase Cgamma-1 increase the domain's association with Cbl.

SH3 domains are protein modules that interact with proline-rich polypeptide fragments. Cbl is an adapter-like protein known to interact with several SH3 domains, including the PLCgamma1 SH3 domain and the Grb2 amino terminal SH3 domain. Here we explore whether sequences surrounding the PLCgamma1 SH3 domain core sequence (aa.796-851) can affect the binding to Cbl, a target used as a prototypical ligand. Consistent with previous reports, our results demonstrated a weak binding of Cbl to GST fusion proteins that strictly encompass the structural core of the PLCgamma1 SH3 domain but a high-avidity binding to the Grb2 amino-terminal SH3 domain. Inclusion of amino acids immediately flanking the PLCgamma1 SH3 core domain, however, substantially increased binding of Cbl to a level comparable to that of the Grb2 amino-terminal SH3 domain. The interaction of this extended PLCgamma1 SH3 domain fusion protein with Cbl was shown to depend entirely upon the interaction of the domain with a proline-rich motif in Cbl, ruling out the possibility that amino acids adjacent to the core SH3 domain of PLCgamma1 provide independent Cbl binding. These data suggest that sequences surrounding the SH3 domain of PLCgamma1 may contribute to or stabilize the association of the domain with the target protein, thus increasing its binding efficiency.

The amino-terminal Src homology 2 domain of phospholipase C gamma 1 is essential for TCR-induced tyrosine phosphorylation of phospholipase C gamma 1.

TCR engagement activates phospholipase C gamma 1 (PLC gamma 1) via a tyrosine phosphorylation-dependent mechanism. PLC gamma 1 contains a pair of Src homology 2 (SH2) domains whose function is that of promoting protein interactions by binding phosphorylated tyrosine and adjacent amino acids. The role of the PLC gamma 1 SH2 domains in PLC gamma 1 phosphorylation was explored by mutational analysis of an epitope-tagged protein transiently expressed in Jurkat T cells. Mutation of the amino-terminal SH2 domain (SH2(N) domain) resulted in defective tyrosine phosphorylation of PLC gamma 1 in response to TCR/CD3 perturbation. In addition, the PLC gamma 1 SH2(N) domain mutant failed to associate with Grb2 and a 36- to 38-kDa phosphoprotein (p36-38), which has previously been recognized to interact with PLC gamma 1, Grb2, and other molecules involved in TCR signal transduction. Conversely, mutation of the carboxyl-terminal SH2 domain (SH2(C) domain) did not affect TCR-induced tyrosine phosphorylation of PLC gamma 1. Furthermore, binding of p36-38 to PLC gamma 1 was not abrogated by mutations of the SH2(C) domain. In contrast to TCR/CD3 ligation, treatment of cells with pervanadate induced tyrosine phosphorylation of either PLC gamma 1 SH2(N) or SH2(C) domain mutants to a level comparable with that of the wild-type protein, indicating that pervanadate treatment induces an alternate mechanism of PLC gamma 1 phosphorylation. These data indicate that the SH2(N) domain is required for TCR-induced PLC gamma 1 phosphorylation, presumably by participating in the formation of a complex that promotes the association of PLC gamma 1 with a tyrosine kinase.

Cbl-mediated regulation of T cell receptor-induced AP1 activation. Implications for activation via the Ras signaling pathway.

The functional role of Cbl in regulating T cell receptor (TCR)-mediated signal transduction pathways is unknown. This study uses Cbl overexpression in conjunction with a Ras-sensitive AP1 reporter construct to examine its role in regulating TCR-mediated activation of the Ras pathway. Cbl overexpression in Jurkat T cells inhibited AP1 activity after TCR ligation. However, AP1 induction by 4beta-phorbol 12-myristate 13-acetate, which up-regulates Ras activity in a protein kinase C-dependent, TCR/tyrosine kinase-independent manner, was not affected by Cbl overexpression. Cbl overexpression also did not affect AP1 induction by an activated Ras protein or a membrane-bound form of the guanine nucleotide exchange factor Sos. In addition, activation of the mitogen-activated protein kinase Erk2 was decreased by Cbl overexpression. Therefore, Cbl regulates events that are required for full TCR-mediated Ras activation, and data are presented to support a model whereby Cbl regulates events required for Ras activation via its association with Grb2.

CD3-induced preferential hydrolysis of polyphosphoinositides and calcium regulation of inositol phosphate metabolism in a permeabilized murine T cell clone.

Murine T helper cloned cells permeabilized with the bacterial lysin, tetanolysin, were used to investigate the role of intracellular Ca2+ in regulating myo-[2-3H] inositol phospholipid (InsPL) hydrolysis triggered upon perturbation of the T cell receptor-CD3 complex. [Ca2+] was controlled by a calcium/magnesium/EGTA buffer. Antibody (mAb) aggregation of CD3 induced InsPL hydrolysis in the absence of added Ca2+. However, stimulated InsPL hydrolysis increased with the free [Ca2+], reaching a maximum at 100-300 nM [Ca2+]. Ca2+ increased the overall efficiency of hydrolysis without changes in EC50 of the anti-CD3 mAb. The response diminished at > 300 nM [Ca2+] due to a mixed type inhibition. Ca2+ alone had no effect on inositol phosphate levels. Polyphosphoinositides were preferentially cleaved, since no accumulation of Ins(1)P/Ins(3)P was detected, indicating that direct hydrolysis of phosphatidylinositol did not occur, irrespective of the Ca2+ concentration. [Ca2+] above 300 nM shifted the relative amounts of CD3-induced inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) in favor of the latter. Unlabeled permeabilized cells exposed to > or = 100 nM [Ca2+] showed enhanced conversion of [3H]Ins(1,4,5)P3 to [3H]Ins(1,3,4,5)P4. In conclusion, InsPL hydrolysis is optimally triggered by CD3 perturbation at intracellular Ca2+ levels approximating those observed in intact resting lymphocytes (100 nM). Ca2+ concentrations similar to those triggered by InsPL-derived metabolites may inhibit InsPL hydrolysis and promote Ins(1,3,4,5)P4 production, thus controlling the amounts of Ins(1,4,5)P3.

Stimulation of human monocytes by anti-CD3 monoclonal antibody: induction of inflammatory mediator release via immobilization of Fc receptor by adsorbed immunoglobulin and T-lymphocytes.

Human monocytes released superoxide anion, prostaglandin E2, leukotriene B4, IL-1, and TNF when exposed to plastic surfaces coated with murine anti-CD3 monoclonal antibody, OKT 3. Stimulation of mediator release by OKT 3 was dependent on the amount of antibody immobilized onto wells of plastic tissue culture plates. Soluble antibody or antibody adsorbed to monocytes and reacted with an aggregating ("cross-linking") second antibody failed to induce mediator release. Monocytes "armed" with OKT 3 formed rosettes with T cells in a fashion indistinguishable from that seen between monocytes and T cells sensitized with OKT 3. Monocytes with adsorbed OKT 3 antibodies released IL-1 beta and TNF-alpha when exposed to unsensitized T cells, although increased superoxide release could not be detected. OKT 4a, a murine IgG2a antibody that reacts with a different T cell epitope (CD4), failed to induce cytokine release from monocytes when cross-linked by T cells or a CD4+ T cell line, even in the presence of IL-2 or IFN-gamma. These data indicate that certain antibodies bound to Fc receptors (FcR) of monocytes may trigger monocyte function when reacting with cells bearing the appropriate target antigens. FcR-mediated signaling resulting in mediator release may be involved in initiating or regulating the immune response. Furthermore, systemically administered monoclonal antibodies may induce inflammatory responses and their attendant symptomatologies via their interaction with FcR-bearing inflammatory cells.

Characterization of the T cell antigen receptor--p60fyn protein tyrosine kinase association by chemical cross-linking.

Engagement of the TCR by specific antigen results in activation of a tyrosine kinase pathway. A candidate for the kinase responsible for the rapid tyrosine phosphorylation detected with T cell activation is p60fyn, a member of the src kinase family. In an earlier study [Samelson et al. (1990) Proc. Natl Acad. Sci. USA 87:4358] this enzyme was co-immunoprecipitated with the TCR from T cells solubilized in digitonin. In that study a sensitive in vitro kinase assay was used to detect the associated p60fyn. It was subsequently found that the reproducibility of the interaction depended on lot-to-lot variations in digitonin. To eliminate the possibility that the association of antigen receptor and kinase is an artifact of solubilization with ill-defined digitonin preparations, a cross-linking protocol was developed to stabilize the interaction between the TCR and p60fyn. T cells were permeabilized with tetanolysin and proteins were cross-linked with the water soluble chemical cross-linker, 3,3' dithiobis(sulfosuccinimidylpropionate). These experiments allowed the confirmation of the interaction between the TCR, p60fyn, and several additional proteins. The cross-linking studies also enabled the mapping of the interaction of p60fyn and associated proteins to the TCR zeta-chain. This technique should have a general use in stabilizing interactions between other receptors and molecules required for intracellular signaling.

Inflammatory mediator release from human monocytes via immobilized Fc receptors. Its potential role in adverse reactions to systemic monoclonal antibody therapy.

Human monocytes released superoxide anion, IL-1, and TNF subsequent to binding of their Fc receptor I to murine IgG2a or rabbit IgG. Fc receptor II binding to murine IgG2b or IgG1 had similar consequences. Immobilized murine monoclonal antibodies, IgG2a anti-CD3 (OKT3) or IgG1 anti-CD44 also induced superoxide anion and monokine production. Monocytes bound OKT3 via FcRI and responded to immobilized OKT3 by inflammatory mediator release in the absence of T cells. These results suggest that direct interaction of immunoglobulins with monocytes via FcR may represent an important phase of the pathophysiology of adverse reactions to systemic monoclonal antibodies.

Increased intracellular cyclic AMP inhibits inositol phospholipid hydrolysis induced by perturbation of the T cell receptor/CD3 complex but not by G-protein stimulation. Association with protein kinase A-mediated phosphorylation of phospholipase C-gamma 1.

Modulation of inositol phospholipid (InsPL) hydrolysis in response to increasing intracellular concentrations of cyclic AMP (cAMP) was studied in a murine T helper type II (Th2) lymphocyte clone, 8-5-5. Intact 8-5-5 cells produced maximal amounts of cAMP in response to prostaglandin E2 (PGE2), cholera toxin (CTx) or 7 beta-deacetyl-7 beta-(gamma-N-methylpiperazino)butyryl forskolin (dmpb-forskolin). cAMP generation reached a plateau after 5 min of treatment with dmpb-forskolin (300 microM) or PGE2 (1 microM), but required 60 min of treatment with CTx (1 microgram/ml). Preincubation of 8-5-5 cells with 1 microM-PGE2 or 300 microM-dmpb-forskolin (10 min at 37 degrees C) or with 1 microgram of CTx/ml (60 min at 37 degrees C) completely inhibited InsPL hydrolysis induced by perturbation of the T cell receptor (TCR)/CD3 complex with the monoclonal antibody 145.2C11. Preincubation with the cAMP analogue 8-bromo-cyclic AMP (8-Br-cAMP) also inhibited InsPL hydrolysis. Tetanolysin-permeabilized 8-5-5 cells produced cAMP in response to PGE2, dmpb-forskolin and guanosine 5'-[gamma-thio]triphosphate (GTP[S]), a non-cell-permeating, non-hydrolysable analogue of GTP that directly activates G-proteins. No inhibition of TCR/CD3-induced InsPL hydrolysis was observed under these conditions. InsPL hydrolysis was also unaffected when permeabilized cells were incubated with up to 10 mM-8-Br-cAMP, suggesting that permeabilized cells lost (a) soluble effector molecule(s) involved in mediating the inhibitory effect observed in intact cells. Treatment of 8-5-5 cells with dmpb-forskolin or CTx prior to permeabilization resulted in inhibition of TCR/CD3-induced InsPL hydrolysis, but did not affect InsPL hydrolysis induced via G-protein stimulation with GTP[S]. Treatment of permeabilized 8-5-5 cells with purified cAMP-dependent protein kinase (PKA) resulted in inhibition of TCR/CD3- but not GTP[S]-induced InsPL hydrolysis. This effect was associated with phosphorylation of phospholipase (PLC)-gamma 1 in the absence of phosphorylation of components of the TCR/CD3 complex. These results suggest that PKA-mediated phosphorylation of PLC may regulate TCR/CD3-induced InsPL hydrolysis.

Inhibition of release of arachidonic acid, superoxide, and IL-1 from human monocytes by monoclonal anti-HLA class II antibodies: effects at proximal and distal points of inositol phospholipid hydrolysis pathway.

Incubation of human elutriator-purified monocytes with anti-HLA-DR or DQ antibody inhibited the release of arachidonic acid induced by serum-treated zymosan (STZ), a phagocytic stimulus that is known to induce inositol phospholipid hydrolysis and Ca2+ influx. However, only anti-HLA-DR antibody partially inhibited STZ-induced inositol phospholipid hydrolysis and concanavalin-A-induced Ca2+ influx. Incubation with anti-HLA-DR or -DQ antibody inhibited phorbol ester-induced AA release as well as superoxide production and IL-1 release. Inhibition of monocyte function by anti-class II antibodies was not accompanied by cAMP elevation. Furthermore, addition of exogenous db-cAMP and other agents (forskolin, cholera toxin, or 3-isobutyl-1-methyl-xanthine) that increase cAMP levels through different mechanisms, alone or in combination with anti-HLA antibodies, had no inhibitory effect on factor release. Our results demonstrate that perturbation of class II molecules down-modulates cell activation at more than one point of the signal transduction pathway with dominant inhibition distal to inositol phospholipid hydrolysis. They also suggest that the inhibition by anti-HLA class II antibody is probably not mediated via cAMP elevation.

A role for guanine-nucleotide-binding proteins in mediating T-cell-receptor coupling to inositol phospholipid hydrolysis in a murine T-helper (type II) lymphocyte clone.

Perturbation of the T-cell receptor (TCR) complex is followed by the rapid hydrolysis of inositol phospholipids (InsPL) by phospholipase C (PLC), producing diacylglycerol and inositol phosphates, which act as second messengers in signal transduction. The mechanism coupling the TCR to InsPL hydrolysis is not clearly defined, and no information is available on this mechanism in the CD4+ helper subset of T-lymphocytes (Th). We have tested the hypothesis that guanine-nucleotide-binding proteins (G-proteins) may couple the TCR to PLC in a murine Th type II (Th2) cell clone. Cell permeabilization with streptolysin O (SLO) or tetanolysin (TL) was used to allow membrane-impermeable nucleotides access to intracellular sites of action. Exposure of permeabilized Th2 cells to guanosine 5'-[gamma-thio]triphosphate (GTP gamma S), a non-hydrolysable GTP analogue, resulted in a 2.1-2.5-fold increase in inositol phosphate generation. Similarly, perturbation of the TCR with the monoclonal antibody 145.2C11 (directed against the epsilon-chain of the CD3 component of the TCR) resulted in a 3.1-4.2-fold increase in InsPL hydrolysis by permeabilized cells. Both lysins were similarly effective in allowing GTP gamma S induction of InsPL hydrolysis, but TL-permeabilized cells responded better to TCR perturbation than SLO-treated cells. A role for G-proteins in TCR coupling to PLC was further supported by the inhibition of TCR-induced InsPL hydrolysis by guanosine 5'-[beta-thio]diphosphate (GDP beta S), a guanine nucleotide analogue that inhibits G-protein function. ATP was required for TCR-mediated InsPL hydrolysis, and potentiated GTP gamma S-induced hydrolysis. Other nucleotides (i.e. CTP, GDP, GTP, ITP) did not affect the response. These data indicate that G-proteins may contribute to the regulation of PLC activation in Th2 cells, coupling it to the TCR.

Differential changes in lipid metabolism of myeloid and lymphoid cell lines induced by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA).

Treatment of the myeloid cell lines, U-937 or HL-60, with 10 nM of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), for 24 h increased the rate of incorporation of [3H]glycerol into total chloroform extracts. A proportionally greater labeling of the non-polar lipid (NL) fraction compared to the polar, phospholipid (PL), fraction was observed. Chromatographic analysis showed a 6-fold increase in the labeling of triacylglycerols (TAG), a 2-fold increase in diacylglycerols, and no changes in monoacylglycerols. PL labeling showed a 3-fold increase in phosphatidylcholine (PC). The effect of TPA on TAG labeling was selectively observed in myeloid cell lines. No such a change was found in the lymphoid cell line. MOLT-3, which did respond to TPA with increased PC labeling. Incorporation of [3H]arachidonic acid (AA) into TAG by U-937 cells was selectively increased (2.5-fold) after treatment with TPA for 24 h. Treatment of U-937 cells with TPA in serum-free medium resulted in no increased labeling of TAG. These studies suggest that changes in TAG metabolism may be characteristic of myeloid differentiation and depend on the presence of serum factor(s).

High-performance liquid chromatographic separation of inositol phosphate isomers employing a reversed-phase column and a micellar mobile phase.

Surfactants have been employed in high-performance liquid chromatography (HPLC) for the separation of ionic and non-ionic compounds. We have developed a method employing a reversed-phase column and a mobile phase containing a surfactant, hexadecyltrimethylammonium hydroxide (HDTMA+OH-), for the separation of several inositol phosphate positional isomers. Various parameters were studied for their effect on the chromatographic capacity factor (k'). They included the concentration of HDTMA+OH-, the pH of the bulk micellar suspension and the addition of inorganic salts to the mobile phase. Resolution of the inositol monophosphates was controlled by a mixed mechanism, where the predominant elements were electrostatic forces and the formation of micelles. The elution of the inositol polyphosphate isomers was obtained by increasing the amount of a non-polar solvent, in agreement with an ion-pairing process. This method represents an alternative to ion-exchange HPLC. If offers a practical advantage when detection of radiolabeled samples by in-line radioactive flow detectors is required, because low-quenching solvents with good miscibility with scintillant fluids are employed. The analysis of various chromatographic conditions, the system reproducibility and its application to the analysis of biological samples are described.

Absence of demonstrable phospholipid turnover in B cells stimulated by low mitogenic concentrations of dextran-anti-immunoglobulin conjugates.

Previously we have demonstrated that when anti-immunoglobulin (Ig) is conjugated to high molecular weight dextran (Dex) it stimulates B cell activation at pg/ml concentrations in the absence of detectable phosphoinositide hydrolysis or increases in intracellular ionized calcium. To study carefully whether anti-Ig-Dex recruited a phosphoinositide-dependent pathway of activation, we stimulated B cells that were labeled with 32P and [3H]glycerol with anti-Ig-Dex conjugates at concentrations ranging from 1-1 x 10(-4) micrograms/ml. Thirty seconds to thirty minutes after stimulation lipids were extracted and analyzed by thin layer chromatography and spots correlating with known lipid standards were isolated and counted. There was a four- and tenfold increase in the ratio of 32P/3H incorporated into phosphatidic acid (a metabolite of diacylglycerol) and phosphatidylinositol, respectively, when cells were stimulated with 0.1-1.0 microgram/ml of anti-Ig-Dex for 30 min. Below 1 ng/ml there was no detectable increase in the turnover of these metabolites despite the fact that in parallel cultures B cells were stimulated to proliferate by this concentration of anti-Ig-Dex. To determine whether a cAMP-dependent pathway was recruited by low concentrations of conjugates, we evaluated cAMP levels from B cells that were stimulated with anti-Ig-Dex for 5-60 min using a radioimmunoassay. While cholera toxin stimulated a 50-100-fold increase in the levels of cAMP, we observed no alteration in cAMP in anti-Ig-stimulated cells. These results support and extend our previous findings by demonstrating that B cell activation that is induced by cross-linking of surface Ig may not stimulate phosphoinositide-dependent or cAMP-dependent pathways of activation. Possible alternative mechanisms of activation will be discussed.

Differential in vitro modulation of suppressor and antitumor functions of mouse macrophages by lymphokines and/or endotoxin.

Peritoneal macrophages of normal mice exhibited natural suppressor activity, as indicated by their ability to inhibit the proliferation of spleen cells in response to stimulation with phytohemagglutinin (PHA) or concanavalin A (Con A). Their suppressor function could be modulated in vitro with a variety of treatment regimens. High-dose lipopolysaccharide (LPS) (LPSH; 10 micrograms/ml) or lymphokines (supernatant from Con A-stimulated spleen cells) plus low-dose LPS (LPSL; 10 ng/ml) caused a reduction in the suppressor activity of adherent peritoneal macrophages. In contrast, these same treatments induced the macrophages to become tumoricidal and cytostatic for tumor cells, indicating a major dissociation between the regulation of suppressor and cytotoxic activities of macrophages. The lack of correlation between these activities was further demonstrated by macrophages that had been activated in vitro by Corynebacterium parvum: these cells expressed high tumoricidal and cytostatic activities, and also strong suppressor activity. The suppressor function could be selectively downregulated by in vitro pretreatment with LPSH.

Selective inhibition of 28S ribosomal RNA in macrophages activated by interferon-gamma or -beta.

We have investigated the metabolism of RNA in mouse peritoneal exudate macrophages activated by interferon (IFN)-gamma or -beta. Both species of IFN induce cytotoxic activity in macrophages. We observed a decrease in the incorporation of [3H]-uridine into total RNA in macrophages treated with doses of IFN that induce cytotoxic activity. IFN-gamma was 100-fold to 1000-fold more potent that IFN-beta in inhibiting RNA synthesis. [3H]Uridine-labeled RNA was purified from IFN-activated and control macrophages and was size fractionated on agarose gels. Macrophages activated by either IFN-gamma or IFN-beta had an imbalanced accumulation of 28S ribosomal RNA compared with their accumulation of 18S ribosomal RNA. Pulse-chase experiments suggested that IFN induced a selective inhibition of the processing of 28S ribosomal RNA. These results provide the first evidence that IFN can modulate ribosomal gene expression at the post-transcriptional level. Moreover, they indicate that inhibition of 28S ribosomal RNA accumulation in macrophages is a molecular event triggered by IFN-gamma, as well as IFN-beta.

Interleukin 2 rapidly stimulates synthesis and breakdown of polyphosphoinositides in interleukin 2-dependent, murine T-cell lines.

Several T-cell functions are controlled by the regulatory peptide interleukin 2 (IL-2). Binding of IL-2 with specific receptors has been well documented, but the molecular mechanism by which IL-2/IL-2 receptor interaction is transduced is not known. We have found that treatment of IL-2-dependent T-cell lines with IL-2 is followed by a rapid stimulation of inositol phospholipid metabolism, as determined by isotopic methodology employing myo-[1,2-3H]inositol. Increased incorporation of the metabolic precursor into phosphatidylinositol and phosphatidylinositol 4-monophosphate, together with the appearance of radiolabeled phosphatidylinositol 4,5-bisphosphate, occurred within minutes of treatment with IL-2 of factor-dependent CT6 cells. Analysis of labeled water-soluble compounds from prelabeled cells indicated a rapid (within 1 min) stimulation of inositol phospholipid hydrolysis following IL-2 treatment. Increased recovery of [3H] inositol phosphates and appearance of [3H]inositol trisphosphate were observed after treatment with IL-2 of CT6 cells, as well as of a second IL-2-dependent cell line, CTB6. These findings suggests that inositol phospholipid-derived metabolites (i.e. diacylglycerol and inositol trisphosphate) may be part of the mechanism by which certain IL-2 signals are transduced.