Duration and magnitude of nerve growth factor signaling depend on the ratio of p75LNTR to TrkA.

The role of the low affinity neurotrophin receptor p75LNTR in neurotrophin signal transduction remains open. Recent reports show that this receptor generates intracellular signals independent of Trk activity, and others imply that it collaborates with Trk(s) to enhance cellular responses to low neurotrophin concentrations. We have used the Cytosensor microphysiometer as a direct marker of intracellular metabolic activity to address the physiologic role of p75LNTR in nerve growth factor (NGF) signal transduction. NGF treatment of PC12 or TrkA-transfected Chinese hamster ovary (CHO) cells results in a rapid, transient increase in the extracellular acidification rate as measured by the Cytosensor; in both cell types, p75LNTR enhances this response. p75LNTR affects both the magnitude of and the duration of the extracellular acidification response to NGF. Moreover, it is not merely the presence of p75LNTR, but also the ratio of p75LNTR:TrkA which determines cellular responsiveness to NGF. In transiently transfected CHO cells, a 5:1 ratio of p75LNTR:trkA cDNAs produced the greatest change in NGF-induced acid secretion. Pretreatment of PC12 cells with anti-p75LNTR antibodies decreased the responsiveness to NGF. However, long-term NGF exposure to PC12 cells in which p75LNTR expression was decreased to approximately 10% of wild-type levels showed a longer duration of acid secretion compared to wild-type PC12 cells. Together, these data suggest that p75LNTR may play a dual role in modulating NGF signal transduction by enhancing and extending cellular responses to short-term ligand exposures while attenuating the metabolic response to long-term ligand exposures. With regard to potential Trk-independent p75LNTR signal transduction mechanisms, we detected no change in extracellular acidification response in 75LNTR-transfected CHO cells, PCNA-15 fibroblasts, or Schwann cells, all of which express large amounts of p75LNTR and no Trk. Thus, p75LNTR cannot produce any signal detected by microphysiometry in the absence of TrkA.

B-lymphoma cells are activated by peptide ligands of the antigen binding receptor or by anti-idiotypic antibody to induce extracellular acidification.

Synthetic peptide ligands specific for the surface immunoglobulin receptor of the human Burkitt's lymphoma cell line SUP-B8, previously identified using phage display libraries, induced apoptosis of the SUP-B8 cells in vitro when administered as dimers or tetramers. The use of synthetic peptide ligands is being explored for immunotherapy of B-cell lymphoma. It will be critical to identify which of the peptide ligands identified are the most active functionally. Using the Cytosensor microphysiometer, SUP-B8 cells and B-lymphoma cells obtained from patients were found to acidify their extracellular environment within minutes of specific activation by surrogate peptide ligands or by anti-idiotype antibodies. This signal was blocked by pretreatment of the lymphoma cells with the tyrosine kinase inhibitor genistein. Treatment of SUP-B8 cells with dimeric and tetrameric specific peptide ligands caused a rapid increase in extracellular acidification rate, which peaked after 10 min at approximately 15 and 20% above basal rates, respectively. These responses were blocked by excess monomeric peptide. To evaluate the ability of different peptide ligands to induce a signal directly on lymphoma cells, thereby establishing their relative affinity to the surface immunoglobulin receptor, acidification rate changes were measured at varying peptide concentrations. The microphysiometer signal correlated with the known relative affinities and antiproliferative potencies of the peptides. This approach is particularly useful for primary tumor cells that cannot be cultured. The signal may be predictive of the efficacy of treatment with synthetic peptide ligands and may be useful in the evaluation of ligands for other cell surface receptors with biological effects on B-lymphoma cells.

Heregulin activation of extracellular acidification in mammary carcinoma cells is associated with expression of HER2 and HER3.

HER2, the erbB-2/neu proto-oncogene product, is a 185-kDa transmembrane glycoprotein related to the epidermal growth factor receptor. Overexpression of HER2 was reported in several human adenocarcinomas, including mammary and ovarian carcinomas. A family of glycoproteins, the heregulin/neu differentiation factors, was characterized and implicated as the ligands for HER2. Recently, it has been shown that HER2 alone is not sufficient to reconstitute high affinity heregulin receptors and that HER3 or HER4 may be the required components of the heregulin receptors on mammary carcinoma cells (Sliwkowski, M.X., Schaefer, G., Akita, R.W., Lofgren, J.A., Fitzpatrick, V.D., Nuijens, A., Fendly, B.M., Cerione, R.A., Vandlen, R.L., and Carraway, K.L., III (1994) J. Biol. Chem. 269, 14661-14665; Plowman, G.D., Green, J.M., Culouscou, J.-M., Carlton, G.W., Rothwell, V.M., and Buckley, W. (1993) Nature 366, 473-475). Using the Cytosensor to measure the extracellular acidification rate, we have examined the effects of recombinant human heregulin-alpha on three mammary carcinoma cell lines expressing HER2 (MDA-MB-453, SK-BR-3, and MCF-7), an ovarian carcinoma cell line expressing HER2 (SK-OV-3), and CHO-K1 and 293-EBNA cells stably transfected with HER2. By reverse transcription polymerase chain reaction and Western blotting, we found that the breast cells also express HER3 and that the ovarian line co-expresses the HER4 message. A dramatic increase in the acidification rate was observed for the mammary carcinoma cells co-expressing high levels of HER2 and HER3. In contrast, the ovarian cells expressing high levels of HER2 and low levels of HER4 or CHO-K1 and 293-EBNA cells expressing HER2 alone were not responsive to heregulin. When these same transfected cells were exposed to monoclonal anti-HER2 antibody followed by anti-IgG to cause aggregation of the HER2 molecules, an increase in the acidification rate was observed, indicating coupling of transfected HER2 to the signal transduction pathway. Transfection of HER2 into MCF-7 cells, on the other hand, gave 4-fold enhanced acidification responses. These data, together with the previously reported high affinity heregulin binding and activation of tyrosine phosphorylation in HER2 and HER3 co-transfected cells support the role of HER2 and HER3 as components of the heregulin receptor in breast cells.

Stimulation of T cells by antigen-presenting cells is kinetically controlled by antigenic peptide binding to major histocompatibility complex class II molecules.

Activation of CD4+ T cells by antigenic peptide involves the interaction of major histocompatibility complex (MHC) class II-peptide complexes on the surface of antigen-presenting cells (APCs) with T-cell receptors. This report describes the kinetics of T-cell triggering by exogenous antigenic peptides in the presence of APCs. A rapid specific increase in extracellular acidification rate is observed within minutes upon exposure of A.E7 T cells (restricted for IEk and moth cytochrome c peptide containing residues 88-103) and 4R3.9 T cells (restricted for IAk and myelin basic protein peptide containing residues 1-14 [AcMBP-(1-14)]) to their cognate peptides in the presence of CH27 cells bearing both IAk and IEk MHC class II molecules. Pretreatment of cloned T cells, but not APCs, with herbimycin A resulted in complete inhibition of triggering events, indicating that the acidification response is mediated by T-cell second messenger pathways. This rapid assay for 4R3.9 T-cell stimulation showed increased T-cell triggering activity for AcMBP-(1-14)-A4 and MBP-(1-14)-M4 peptides compared to the native AcMBP-(1-14)-K4. By using the previously determined kinetic constants for MBP-(1-14)-A4 reactions with IAk, it is possible to show that at the lowest peptide concentrations the kinetics of T-cell triggering are limited by the kinetics of the peptide binding to MHC class II molecules.

Activation of TNF-R1 receptor in the presence of copper kills TNF resistant CEM leukemic T cells.

The cytotoxic effects of TNF on malignant cells are known to be mediated through high affinity surface receptors. The precise mechanism by which transformed cells are selectively killed by the activation of these receptors is yet unknown, but several intracellular signaling pathways are known to be involved. Phospholipase A2 activation by TNF-alpha has been shown to be important in the transduction of signals leading to cell death. We have used monitoring of extracellular acidification rate as a measure of cellular metabolism to follow the early time course of TNF effects on a human leukemic T cell line (CEM-SS cells). CEM-SS cells were relatively resistant to TNF cell killing but TNF caused an early stimulation of metabolism within 2-4 hr, followed by a suppression of metabolic activity occurring over 20 hr. In contrast, a TNF sensitive subclone of CEM cells (C1Ca) showed a rapid and dramatic decrease in metabolic activity corresponding to cytotoxicity within 18 hr. It was discovered that cupric o-phenanthroline markedly potentiated the effects of TNF on the resistant CEM-SS cells leading to cell death. This observation was specific for copper because ferric o-phenanthroline was without effect at the same concentration. The copper cytotoxic effect was shown to be mediated through the TNF-R1 receptor and independent of phospholipase A2 signaling.

Antigen-specific stimulation of T cell extracellular acidification by MHC class II-peptide complexes.

A specific T cell response to a preformed complex of detergent-solubilized MHC class II molecule and cognate antigenic peptide was observed by monitoring the extracellular acidification. An increase in this rate was observed when the resting 4R3.9 T cell clone specific for the peptide fragment MBP(1-14) of myelin basic protein was exposed to preformed detergent-solubilized IAk-MBP(1-14)A4 complexes. MBP peptide alone, IAk alone, or complexes of IAs-proteolipid protein(139-151) and IAd-OVA(323-339), did not cause significant increases in the acidification rates of the MBP(1-14)-restricted 4R3.9 T cell clone. In addition, BW 5147 T lymphoma cells, which lack TCR, did not show any increase in rate when exposed to IAk-MBP(1-14)A4 complexes. Similar increases in acidification rate were observed in the presence of IL-2, anti-CD3 and anti-TCR antibodies. The enhanced acidification responses were blocked by genistein, a tyrosine kinase inhibitor.