Interactions of Lyn with the antigen receptor during B cell activation.

Signaling through the B cell antigen receptor requires a complex set of interactions involving transmembrane components of the IgM receptor complex and cytosolic protein-tyrosine kinases. We have focused on the nature of these protein-protein interactions, the requirements for their occurrence, as well as the temporal sequence of events during the activation process. We found that cross-linking B cell antigen receptors at 0 degree C resulted in the rapid association of the Src-family protein-tyrosine kinase, Lyn, with the antigen receptor complex as judged by the presence of Lyn in anti-IgM and anti-phosphotyrosine immune complexes and the presence of MB-1 in anti-Lyn immune complexes. Receptor engagement also resulted in the rapid association of Lyn with the phosphotyrosine phosphatase, CD45. This association of Lyn with receptor components was stable in the detergent Brij 96, but was readily disrupted by Nonidet P-40, suggesting the involvement of hydrophobic interactions in stabilizing formation of the Lyn-receptor complex. The protein-tyrosine kinase, Syk, was also found associated with activated receptor complexes. This association of Syk with components of the antigen receptor complex was stable to Nonidet P-40. Antibodies directed against the carboxyl teminus of Syk, but not against the amino-terminal SH2 domain, co-immunoprecipitated MB-1 from activated cells, consistent with the binding of Syk through an SH2 domain-phosphotyrosine interaction.

Inhibition of mast cell Fc epsilon R1-mediated signaling and effector function by the Syk-selective inhibitor, piceatannol.

In RBL-2H3 rat tumor mast cells, antigens that cross-link the high affinity cell surface IgE receptor, Fc epsilon R1, activate at least two receptor-associated protein-tyrosine kinases, Lyn and Syk, and cause the tyrosine phosphorylation of the receptor beta and gamma subunits, PLC gamma 1, Vav, and other proteins. Cross-linking antigens also induce increased phosphatidylinositol turnover, Ca2+ mobilization, secretion, actin polymerization, spreading, and membrane ruffling. We have used the protein-tyrosine kinase inhibitor, piceatannol (3,4,3',5'-tetrahydroxy-trans-stilbene), to explore the coupling of specific kinases to cellular responses. Piceatannol preferentially inhibits the activity of Syk as compared with Lyn when added to in vitro assays with isolated enzymes. Treatment of RBL-2H3 cells with piceatannol strongly inhibits the antigen-stimulated activation of Syk measured in anti-Syk and anti-phosphotyrosine immune complex kinase assays at concentrations that have no effect on receptor subunit phosphorylation and maintain or increase the activity of Lyn in anti-phosphotyrosine immune complex kinase assays. In cells metabolically labeled with [32P]orthophosphate, piceatannol inhibits the antigen-stimulated tyrosine phosphorylation of Syk and most other proteins. However, receptor subunit phosphorylation is unchanged. Selective inhibition of Syk in this manner blocks receptor-mediated down-stream cellular responses (inositol 1,4,5-trisphosphate production, secretion, ruffling, and spreading) while having only minor effects when these responses are induced with drugs that bypass the receptor complex. These results reveal receptor-mediated Lyn activation as a relatively piceatannol-insensitive event that may contribute to receptor subunit phosphorylation and Syk activation but does not per se elicit cellular responses. Receptor-mediated Syk activation on the other hand is highly sensitive to piceatannol, is essential to Fc epsilon R1-mediated cellular responses, and, based on the increased phosphorylation of Lyn in piceatannol-treated cells, may be involved in terminating Lyn activity.

Phosphorylation of human erythrocyte band 3 by endogenous p72syk.

The anion transporter, band 3, is the major tyrosine kinase substrate in both red cell membranes and intact human erythrocytes. Using antibodies to various protein-tyrosine kinases, we found that p72syk and p56/53lyn are present in human red cells, while p56lck, pp60src, p59fyn, and p55blk are absent. Treatment of intact red cells with a combination of vanadate and hydrogen peroxide dramatically increased the tyrosine phosphorylation of band 3. This treatment increased the tyrosine kinase activity of p72syk and decreased the activity of p56/53lyn in immune complex kinase assays. Band 3 was found to be associated in immune complexes with p72syk but not with p56/53lyn. These findings suggest that p72syk is responsible, at least in part, for the tyrosine phosphorylation of band 3 in human erythrocytes.

p72syk tyrosine kinase is activated by oxidizing conditions that induce lymphocyte tyrosine phosphorylation and Ca2+ signals.

We have used H2O2 as a pharmacologic agent to examine the effects of oxidizing conditions on lymphocyte signal pathways. Treatment of Ramos cells with 5-10 mM H2O2 gave rapid and strong tyrosine phosphorylation of multiple cellular proteins and activation of p72syk to levels equal to or greater than that observed upon surface Ig cross-linking. Strong Ca2+ signals that could be blocked by the tyrosine kinase inhibitor herbimycin A were also observed under these conditions. However, there was no increase in activity for the Src family kinases p56lck, p59fyn, or p56/p53lyn. Our findings that the p72syk tyrosine kinase responds to H2O2 treatment of cells suggest that this kinase is likely to contribute to cellular tyrosine phosphorylation and calcium signaling induced by oxidizing conditions. Furthermore, H2O2 may be useful as a pharmacologic agent to distinguish the effects of p72syk-related kinases from those of Src family kinases.

Cell cycle-specific activation of the PTK72 protein-tyrosine kinase in B lymphocytes.

The engagement of cell-surface antigen receptors on B lymphocytes by anti-IgM antibodies leads to the rapid tyrosine phosphorylation and activation of the protein-tyrosine kinase, PTK72. High concentrations of anti-IgM, which promote cell cycle entry and progression through G1, result in a biphasic change in the state of tyrosine phosphorylation of PTK72. An initial, rapid increase is seen within 5 min, which slowly declines to the level found in resting cells over a period of 9 h. A second increase is then observed 18-30 h following the initial stimulation. Low concentrations of anti-IgM, which promote cell cycle entry but not progression through G1, result only in the initial, rapid phosphorylation. The polyclonal mitogens lipopolysaccharide and dextran sulfate, which stimulate both cell cycle entry and progression to late G1, result only in the second, late phase of tyrosine phosphorylation. This second phase of elevated tyrosine phosphorylation is cell cycle-dependent, as demonstrated by its appearance in cells blocked at G1/S and absence in cells blocked at G2/M. The increase in the tyrosine phosphorylation of PTK72 is accompanied by an increase in its activity with no change in its concentration. These data suggest a possible second role for PTK72 in the commitment of activated B cells to proliferation.

Synthesis and protein-tyrosine kinase inhibitory activities of flavonoid analogues.

Treatment of o-hydroxyacetophenones 2a-e with excess lithium bis(trimethylsilyl)amide followed by dialkyl carbonates gave alkyl 3-(2-hydroxyaryl)-3-oxopropanoates 3a-e. The latter substances were transformed through the reaction of their magnesium chelates with benzoyl chlorides into a series of 3-(alkoxycarbonyl)-2-arylflavones, which were subsequently elaborated into a variety of flavonoids. These compounds were tested for their abilities to inhibit the in vitro protein-tyrosine kinase activity of p56lck, an enzyme which is thought to play a key role in mediating signal transduction from the CD4 receptor during lymphocyte activation. All of the active compounds had either an amino or a hydroxyl substituent at the 4'-position of the 2-aryl ring. The most active substance prepared in this study is compound 17c, which is approximately 1 order of magnitude more potent than the natural product quercetin (1). Compound 17c was a competitive inhibitor of p56lck with respect to ATP and was highly selective for the inhibition of protein-tyrosine over protein-serine/threonine kinases.

Late events in B cell activation. Expression of membrane alkaline phosphatase activity.

Alkaline phosphatase (APase) has been previously described as a membrane marker correlating with B cell proliferation after stimulation by selected B cell mitogens. We have found, however, that the appearance of B cell membrane APase correlates more closely with differentiation than with proliferation. This conclusion has been drawn from the following observations: 1) APase activity appears well after peak B cell thymidine uptake, 2) mitogens which stimulate only B cell proliferation (Salmonella typhimurium mitogen) fail to induce expression of the enzyme, and 3) when proliferation of mitogen-activated B cells is inhibited, APase activity is not suppressed and may even be augmented. In addition to membrane expression, APase is also spontaneously shed into the surrounding milieu, perhaps as a result of endogenous phospholipase activity. By using a group of well-characterized inhibitors, the APase activity was shown to belong to class I (similar to the bone/liver/kidney class). Because APase always appears in differentiating but not proliferating cells, we would propose that the enzyme appearance is a late marker of B cell activation, associated with cell progression to differentiation and consequent IgM synthesis.

Lymphokine-induction of memory B-cell differentiation: differential stimulation of large virgin and memory B-cell differentiation.

In order to compare and contrast the requirements of virgin and memory B cells for B-cell differentiation factors, a model system was developed in which low-density rat B cells isolated from 4-week primed antigen-draining lymph nodes were cultured in vitro. This large low-density cell population contained B cells which were 90% surface IgM positive and 60% IgD positive and showed moderately elevated Ia staining. When the cell population was stimulated with antigen plus lymphokines or lymphokines alone, antigen-specific IgG antibody was secreted; this was used as a measure of memory cell differentiation. When the cell population was stimulated with mitogen (lipopolysaccharide plus dextran sulfate) plus lymphokines, polyclonal IgG and IgM secretion was seen and was used as a measure of virgin B-cell differentiation. Using this system, we found that lymphokines contained in a Con A-induced rat spleen cell supernatant (CSN) were sufficient to drive both memory and virgin B-cell differentiation. In contrast, lymphokines contained in the supernatant from the murine T-cell hybridoma B151K12 (B151CFS) were able to induce large amounts of polyclonal IgM and IgG secretion but did not support memory B-cell differentiation. When recombinant human IL-2 was added to these cultures, it acted synergistically to augment virgin B-cell differentiation, but this combination of lymphokines was still not able to support memory B-cell differentiation. Furthermore, recombinant rat interferon-gamma and a commercial source of human BCGF, with or without IL-2, were unable to promote significant virgin or memory B-cell differentiation. These data support the hypothesis that memory B cells and virgin B cells differ in their lymphokine requirements for differentiation into antibody-secreting cells.

An alternative method of panning for rat B lymphocytes.

A new method of panning for B lymphocytes is described in which the ability of the sIg+ cells to adhere depends on the nature and concentration of nonspecific protein used rather than on the use of anti-immunoglobulin. Rat lymph node cells were suspended in 3% bovine serum albumin in Tris-buffered Hanks' and incubated in tissue culture flasks to allow adherence to the plastic. The recovered bed of adherent cells was shown by flow cytometry to be greater than 90% surface immunoglobulin positive and MHC class II positive while containing very few T cells. This adherent fraction was subsequently treated with anti-T cell antibody plus baby rabbit complement to produce a highly purified sIg+ cell population containing no detectable T cells. The sIg+ cells obtained by this panning procedure were functionally active in BCGF and BCDF assays. This method provides an easy and inexpensive alternative to conventional panning with anti-immunoglobulin and also eliminates the possibility of B cell activation by exposure to anti-immunoglobulin-coated surfaces.

Polyclonal activation of primed rat B cells.

In recent years, murine and human virgin B lymphocytes have been used to examine the steps necessary for polyclonal activation. In these models mitogens are used in conjunction with lymphokines to determine which signals are responsible for regulating B-cell triggering, proliferation, and differentiation. While progress has been made in understanding these events as they occur in virgin B cells, very little evidence exists to suggest whether these models of activation also apply to the memory B-cell population. In this report we have described an antigen-specific, secondary in vitro immune response using cells isolated from lymph nodes draining the site of antigen injection. Unfractionated cells, B cells, and size-fractionated cells from dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH)-primed rats were challenged in vitro with DNP-KLH, lipopolysaccharide plus dextran sulfate (LPS/DxS), and T-cell factors. We have consistently found, under all these conditions, that antigen challenge of primed cells results in the production of DNP-specific IgG antibody while stimulation with LPS/DxS plus T-cell factors results only in the polyclonal activation of virgin B cells; no antigen-specific IgG secretion is seen. This suggests that acquisition of memory status is associated with a loss in responsiveness to LPS/DxS-induced differentiation.