Examination of B lymphoid cell lines for membrane immunoglobulin-stimulated tyrosine phosphorylation and src-family tyrosine kinase mRNA expression.

Crosslinking of membrane immunoglobulin (mIg) on B cells induces two signal transduction pathways: protein tyrosine phosphorylation and phosphoinositide turnover. A panel of murine and human B cell-lines, representing different stages of B cell development, was examined for the presence of anti-immunoglobulin-induced protein tyrosine phosphorylation. Of 10 B cell lines examined, only one, the human Raji cell line, had no detectably induced protein tyrosine phosphorylation. The pattern of proteins that were phosphorylated on tyrosine in response to mIg crosslinking differed somewhat in cell lines representing different stages of B cell development. Differences in the levels of constitutive phosphorylation of proteins were also observed between the cell lines. The identity of the tyrosine kinase(s) activated by membrane immunoglobulin ligation is not known. However, members of the src family of intracellular tyrosine kinases have been implicated as signal transduction molecules. As the tyrosine phosphorylation of proteins is a general phenomenon of signal transduction by membrane immunoglobulin, the tyrosine kinase(s) activated by it might be expected to be present in all cell lines in which the tyrosine phosphorylation signalling occurs. Therefore we examined these B cells for expression of mRNAs encoding the eight known src-like tyrosine kinases. Surprisingly, all eight kinase mRNAs were expressed in at least some of the B cell lines examined. The expression pattern of the fyn, hck, and lck genes suggests that expression of these kinases may be developmentally regulated in the B cell lineage. Three of the kinases, p55blk, p53/p56lyn and p60src, were detected in all 10 B cell lines. Whereas the src gene shows a ubiquitous pattern of expression, the expression of the blk and lyn genes is mostly restricted to cells of hematopoietic origin, and more especially B lymphoid cells. Thus, p55blk and p53/p56lyn may be particularly good candidates for the membrane immunoglobulin-activated tyrosine kinase.     

Neither the LCK nor the FYN kinases are obligatory for IL-2-mediated signal transduction in HTLV-I-infected human T cells.

IL-2 is one of the principal growth factors regulating the proliferation of T lymphocytes. Although two independent IL-2-binding molecules have been molecularly cloned and shown to participate in the formation of a high affinity receptor complex, their primary structures do not suggest a specific mechanism for IL-2 growth signal transduction across the cell membrane. Neither IL-2 receptor subunit contains an intrinsic kinase domain; nevertheless, tyrosine phosphorylation of various intracellular substrates is one of the first biochemical changes observed following activation of the IL-2 receptor (IL-2R). Both serine/threonine and tyrosine kinases can be co-precipitated as part of the IL-2R complex suggesting that the IL-2 signalling may involve the activation of non-covalently associated intracellular kinases. However, controversy exists as to which kinases are involved in IL-2 signal transduction; in particular, which kinase(s) mediates the first or proximal event(s) in the signalling process. Activation of the IL-2R leads to serine and threonine phosphorylation of the SRC tyrosine kinase family member, LCK, and an increase in LCK tyrosine kinase activity. Furthermore, LCK can be co-immunoprecipitated with the beta chain of the IL-2R indicating its association with the receptor complex. IL-2 has also been reported to increase FYN kinase activity and to alter its association with the 85 kDa subunit of phosphatidylinositol-3 kinase thus suggesting a role for FYN in IL-2 signal transduction. However, in this report, we now demonstrate that neither LCK nor FYN are obligatory for IL-2-induced growth of HTLV-I-infected human T cells. Lack of expression of LCK or FYN in the HTLV-I-infected T cell lines was demonstrated by a combination of Northern blotting, polymerase chain reaction, Western blotting, and in vitro kinase activity. Despite the absence of LCK or FYN, IL-2 induced similar patterns of rapid tyrosine phosphorylation. Similar results were observed in cell lines lacking expression of the LYN, FGR, HCK, and LTK tyrosine kinases. Thus, none of these tyrosine kinases alone appears to be required for growth signalling through the IL-2R in the HTLV-I-infected T cell lines analyzed. The findings raise the possibility that an, as yet, unidentified tyrosine kinase is involved. Alternatively, this biological signalling system may exhibit remarkable redundancy whereby several different tyrosine kinases may be capable of associating with the IL-2R complex and mediating intracellular signalling.     

The phosphatidylinositol 3-kinase/protein kinase B signaling pathway is activated by lipoteichoic acid and plays a role in Kupffer cell production of interleukin-6 (IL-6) and IL-10

Sepsis caused by gram-positive bacteria lacking lipopolysaccharide (LPS) has become a major and increasing cause of mortality in intensive-care units. We have recently demonstrated that the gram-positive-specific bacterial cell wall component lipoteichoic acid (LTA) stimulates the release of the proinflammatory cytokines in Kupffer cells in culture. In the present study, we have started to assess the signal transduction events by which LTA induces the production of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and the anti-inflammatory cytokine IL-10 in rat Kupffer cells. LTA was found to trigger phosphorylation of mitogen-activated protein kinases (MAPK) (p38 MAPK and ERK 1/2) and protein kinase B (PKB). Compared to LPS, LTA was more potent in inducing PKB phosphorylation after 40 min, although we found that the cytokine responses were similar. For both bacterial molecules, blocking phosphatidylinositol 3-kinase (PI3-K; Ly294002) or Janus kinase 2 (JAK-2; AG490) particularly affected the induction of IL-6 and IL-10 release, whereas TNF-alpha levels were strongly reduced by inhibition of Src family tyrosine kinases (PP2). All three cytokines were reduced by inhibition of p38 MAPK (SB202190) or the broad-range tyrosine kinase inhibitor genistein, whereas IL-6 release was particularly blocked by inhibition of ERK 1/2 (PD98059). Divergences in the regulatory pathways controlling TNF-alpha, IL-10, and IL-6 production in Kupffer cells following LPS or LTA stimulation may create a basis for understanding how the balance between pro- and anti-inflammatory cytokines is regulated in the liver following infections by gram-positive or gram-negative bacteria.     

CD40-mediated lymphotoxin α expression in human B cells is tyrosine kinase dependent

The cytokine lymphotoxin (LT)α is known to play a role in B cell activation. As the engagement of the B cell antigen CD40 is known to lead to B cell proliferation and differentiation, we studied LTα expression in human B cells after CD40 ligation. We demonstrate that anti-CD40 monoclonal antibody (mAb) induces strong LTα mRNA and surface expression in human tonsil B cells. Induction of LTα mRNA and surface expression by CD40 ligation is inhibited by the protein tyrosine kinase (PTK) inhibitors herbimycin and genistein in a dose-dependent manner. The protein kinase C (PKC)-specific inhibitors sphingosine and bisindolylmaleimide caused negligible inhibition of anti-CD40-induced LTα mRNA and surface expression. No inhibition is observed with the protein kinase (PKA) inhibitors H89 and HA1004. Cross-linking of the transmembrane phosphatase CD45 to CD40 by using goat-anti-mouse F(ab')₂ fragments strongly inhibits CD40-mediated LTα expression in human B cells, confirming the role of PTK activation in CD40-mediated induction of LTα expression. Inhibitors of the serine/threonine protein phosphatases PP1 and PP2A, okadaic acid and calyculin induce LTα mRNA expression. In contrast, cyclosporin A, an inhibitor of the serine/threonine phosphatase calcineurin has no effect on anti-CD40-induced LTα expression. These results suggest that induction of LTα expression in B cells following engagement of CD40 involves activation of protein tyrosine kinases.     

Staphylococcal toxic shock syndrome toxin 1-induced tumor necrosis factor alpha and interleukin-1 beta secretion by human peripheral blood monocytes and T lymphocytes is differentially suppressed by protein kinase inhibitors.

The signal transduction pathways by which staphylococcal toxic shock syndrome toxin 1 (TSST-1) induces tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) secretion were examined with various protein kinase inhibitors. TNF-alpha secretion by normal human monocytes and T cells in response to TSST-1 was suppressed by inhibitors of protein kinase C (H7) and tyrosine kinases (genistein). In contrast, the secretion of IL-1 beta was blocked by a cyclic AMP- and cyclic GMP-dependent kinase inhibitor (HA1004) as well as by H7 and genistein. These results suggest that the secretion of TNF-alpha and IL-1 beta may be differentially regulated by TSST-1 and that protein kinases play an important role in mediating cytokine responses to the toxin.     

The structure of the μ/pseudo light chain complex on human pre-B cells is consistent with a function in signal transduction

Prior to immunoglobulin (Ig) light (L) chain rearrangement, pre-B cells can express μ heavy (H) chains at the cell surface in association with pseudo (ψ) L chains. This complex may be essential for B cell development. We have investigated the composition of the μ/ψL chain complex of a human pre-B cell line, in view of its potential role in transmembrane signal transduction. The μ/λ. receptor of a mature B cell line was analyzed in comparison. The μ/ψL chain complex is associated with disulfide-linked molecules that are homologous or identical to the mb-1 and B29 proteins, known to be integral components of membrane Ig receptors on mature B cells. Both receptors contain tyrosine (Tyr) kinase activity. In the μ/λ receptor, the lyn and lck Tyr kinases could clearly be identified. The mb-1 and B29 proteins in both μ/λ and μ/ψL chain receptors are substrates for in vitro phosphorylation on Tyr, but also on serine (Ser) and threonine (Thr) residues. The undefined μ-associated Ser/Thr kinase also phosphorylates the sre-related kinases in the μ/λ, receptor and a 43-kDa μ-associated protein that is present in both complexes. The 43-kDa protein may be an integral part of both receptor types, or a transiently associated molecule instrumental in the signaling process. We conclude that the μ/ψL receptor on human pre-B cells fulfills the presently known criteria to function as a signal transduction unit.     

Src family protein tyrosine kinase signaling mediates monosodium urate crystal-induced IL-8 expression by monocytic THP-1 cells.

Neutrophil-dependent inflammation dependent on monosodium urate (MSU) crystal-induced IL-8 expression occurs in gout. MSU crystals activate phagocyte Src family tyrosine kinases and the serine/threonine kinase p70s6k. Thus, using monocytic THP-1 cells, we assessed the potential for Src family kinases and p70s6k to mediate MSU-induced IL-8 expression. MSU crystals induced phosphorylation of p70s6k and the Src kinases c-Src, Lyn, Hck, and Fyn. IL-8 expression was attenuated more by the Src kinase inhibitor PP1 than by the p70s6k inhibitor rapamycin. PP1 inhibited crystal-induced phosphorylation of ERK1/2 and IkappaBalpha and suppressed IkappaB kinase (IKK) activation and NF-kappaB binding to the IL-8 promoter, signals that mediate MSU-induced IL-8 expression. Transfection of the native Src inhibitor, C-terminal Src kinase (Csk), also suppressed crystal-induced c-Src, ERK1/2, and IkappaBalpha phosphorylation and IL-8 expression. We conclude that Src family tyrosine kinase signaling plays a significant role in MSU crystal-induced IL-8 expression via stimulation of ERK1/2 pathway and NF-kappaB activation.     

Identification of pro-interleukin 16 as a novel target of MAP kinases in activated T lymphocytes

T lymphocyte activation is controlled by a coordinated web of tyrosine and serine kinases. There is a large body of information about tyrosine kinase substrates in T cells but analysis of serine kinase substrates has been more difficult. Recently we described an antiserum that recognizes serine-phosphorylated peptides corresponding to the substrate sequences for AGC serine kinases. This antiserum, termed PAP-1 (phospho antibody for proteomics-1), has proven useful for probing the serine phosphoproteome of antigen receptor-activated T lymphocytes. The present study shows that PAP-1 can also be used to explore serine kinases activated by cytokines and chemokines in T cells. Using PAP-1, together with proteomic analysis, the precursor form of the cytokine IL-16 (ProIL-16) was shown to be phosphorylated on Ser144 in antigen receptor-, SDF1alpha- and IL-2-activated T cells. Genetic and pharmacological-inhibitor experiments showed that the phosphorylation of ProIL-16 is dependent on activation of the kinases Erk1/2. IL-16 is secreted by mitogen-activated T cells, and the biochemical link between ProIL-16 and Erk1/2, revealed by studies with PAP-1, prompted analysis of the role of MAP kinases in this response. We show that TCR-mediated secretion of IL-16 is dependent on MAP kinases. The present study thus reveals how phosphoproteomic analysis opens previously unrecognized avenues for research, and yields novel insights about targets for MAP kinases in T lymphocytes.     

Human interleukin-5 induces staphylococcal A Cowan 1 strain-activated human B cells to secrete IgM

Studies on the role of human interleukin (IL)-5 in B cell growth and differentiation have yielded conflicting results. To clarify this issue, we studied the role of purified recombinant IL-5 on activated human B cells which were depleted of Tcells and adherent cells. Human IL-5 augments IgM secretion, but not IgG or IgA secretion of purified human B cells activated with staphylococcal A Cowan 1 strain (SAC). However, the period of B cell activation with SAC is critical for the B cell to respond to IL-5. After 24 h of SAC activation, human B cells are responsive to the IL-5 signal, but with longer periods of activation, IL-5 responsiveness diminishes. This may explain some of the previous conflicting results. The IgM enhancement was not seen when B cells were activated with pokeweed mitogen. In addition, human recombinant IL-4 synergized with IL-5 in augmenting IgM secretion by SAC-activated B cells, while IL-5 synergized with IL-2 to augment IgM, IgG and IgA secretion by SAC-activated B cells. As the purified IL-5 was derived from a COS-1 cell supernatant, and COS-1 cells secrete IL-6, we examined whether a polyclonal IL-6 antibody blocked the IgM-enhancing activity of IL-5. IL-6 antibody did not block the IL-5 enhancement of IgM secretion, but a monoclonal antibody to IL-5 inhibited the human IL-5 activity on human B cells. These results demonstrate that human IL-5 augments IgM secretion of SAC-activated human B-cells. In addition, this lymphokine synergizes with IL-4 and IL-2 in supporting Ig secretion.     

Interleukin-1-induced activation of a protein kinase co-precipitating with the type I interleukin-1 receptor in T cells

We have investigated the possibility of a protein kinase participating in the signal transduction mechanisms of the interleukin-1 (IL-1) type I receptor (IL-1RI). Our data show that a protein kinase was co-precipitated with the IL-1 RI from the two murine T helper cell lines D10N and EL-4. The kinase activity was detected in an in vitro kinase assay performed with the immuno beads in the presence of exogenous substrates. IL-1 treatment of the cells resulted in a rapid activation of this protein kinase in a concentration-dependent manner. Both forms of IL-1, IL-1α and IL-1β, induced this kinase activity, whereas the IL-1 receptor antagonist (IL-1ra) was inactive. In excess IL-1ra competitively antagonized IL-1 stimulation. In the in vitro kinase assay the exogenous substrates myelin basic protein and histone H1 were phosphorylated, whereas casein or heat-shock protein HSP27 were not accepted, reflecting a certain selectivity of this protein kinase. The IL-1RI co-precipitable protein kinase showed a serine/threonine specificity and was inhibited by staurosporine, but not by inhibitors specific for protein tyrosine kinases or protein kinase C. These results show that a serine/threonine protein kinase directly interacts with the IL-1RI at the plasma membrane level of T helper cells forming a novel type of IL-1 inducible signaling complex. This protein kinase may resemble the link coupling the plasma membrane IL-1 receptor to cytosolic downstream elements in the IL-1 signaling pathway.     

Characterization of molecular components associated with surface immunoglobulin M in human B lymphocytes: presence of tyrosine and serine/threonine protein kinases.

To characterize the signal transduction through the antigen receptor (AgR) on human B lymphocytes, we analyzed its association with other molecular components. The surface IgM (sIgM) complex isolated in digitonin contains two surface expressed polypeptides--the previously described Ig alpha and Ig beta proteins--covalently linked to each other in a 48/39-kDa heterodimer. We show herein that the human sIgM complex isolated from the Burkitt's lymphoma cell line, Ramos, or from dense tonsillar B cells contains additional molecules--160 kDa and 75 kDa in size--and enzymatic activities able to phosphorylate on tyrosine as well as serine/threonine residues the 39-, 48-, 75- and 160-kDa polypeptides. By specific immunoprecipitation with antibodies to src-family kinases, we consistently detected p56lyn in the sIgM complex. In the Ramos cell line, both p56lck and p59fyn activity were also observed, although to a much lesser extent than p56lyn. These kinases are associated with sIgM before cell stimulation. As shown by two-dimensional electrophoresis, they interact in a tight complex with multimeric forms of the Ig alpha and Ig beta components. The kinases are active in vitro but must be highly regulated in vivo: Western blotting with anti-phosphotyrosine antibodies revealed that stimulation of the AgR on viable B cells increased detectable phosphotyrosine residues on the components present in the sIgM complex. Based on these phosphorylation changes, the 39-, 48-, 75- and 160-kDa molecules are likely to be functionally active elements in an IgM complex crucial for the transduction of the antigenic signal.     

Interleukin-2 signal transduction in human NK cells: multisite phosphorylation and activation of the tyrosine kinase p56lck.

Interleukin-2 (IL-2) potently stimulates natural killer (NK) cell proliferation and cytotoxic function. However, the molecular mechanisms by which IL-2 delivers activation signals from the IL-2 receptor to the NK cell interior are incompletely understood. Previous studies demonstrated that IL-2 stimulation induced the tyrosine phosphorylation of multiple proteins in NK cells, together with a prominent reduction in the electrophoretic mobility of p56lck. The present studies indicate that IL-2 induces a rapid (< or = 1 min) increase in the catalytic activity of p56lck, as measured by increases in protein tyrosine kinase activity in vitro. Furthermore, in response to IL-2, p56lck itself undergoes complex alterations in serine and tyrosine phosphorylation. Cyanogen bromide cleavage maps indicate that IL-2 stimulates a pronounced increase in the phosphorylation of the NH2-terminal region of p56lck containing multiple known sites of serine phosphorylation. In addition, IL-2 induced a marked increase in the phosphorylation of a COOH-terminal peptide containing the regulatory Tyr-505 residue of p56lck. These results suggest that p56lck serves as a substrate for both protein serine and tyrosine kinases activated during stimulation of this cell type with IL-2. Furthermore, these results indicate that the pleiotropic effects of IL-2 on NK cell physiology are initiated and regulated by a complex and multitiered interaction of different protein kinases including p56lck.