B cell signalling

The best understood mechanism of ES-62 action is its effect on B cells, which results in the suppression of proliferation following B cell antigen receptor (BCR) cross-linking. The BCR comprises a clonatypic antigen-binding component (surface immunoglobulin, sIg) and its accessory immunoreceptor tyrosine-based activatory motif (ITAM)-containing signal transducing molecules Igα and Igβ. Ligation of the BCR triggers protein tyrosine kinase (PTK) activity, resulting in tyrosine phosphorylation of the ITAMs (reviewed in Cambier, Pleiman & Clark, 1994 and Pleiman, D'Ambrosia and Cambier, 1994) and the recruitment of a number of key signal transducing pathways implicated in cellular activation and proliferation (Fig. 2). These include the phospholipase C (PLC)-γ, phosphoinositide-3-kinase (PI-3-K) and the Ras-Erk (extracellular-regulated kinase) mitogen-activated protein kinase (MAP kinase) signalling cascades.

Fig. 2. BCR signalling in untreated (A) and ES-62-exposed (B) B cells. Following ligation of the B cell antigen receptor (BCR) of untreated B cells (A) the kinase, Lyn, tyrosine phosphorylates the Immunoreceptor Tyrosine Activation Motifs (ITAMs) on the accessory transducing molecules Ig-α and Ig-β resulting in the recruitment and activation of the PI-3-kinase (PI-3-K) and PLCγ-signalling pathways. Whilst PI-3-K activation results in the activation of atypical PKC isoforms (aPKC), PLCγ activation induces IP3 and DAG generation, ultimately resulting in activation of classical (cPKC) and novel (nPKC) PKC isoforms. Binding of the adaptor proteins Shc and BLNK to the phosphorylated ITAMs leads to the recruitment of the Grb2Sos complexes (Grb2 is an adaptor protein which binds Sos, a guanine nucleotide exchange factor) required for activation of the GTPase, Ras. Active Ras initiates the Erk mitogen-activated protein (MAP) kinase cascade by binding and activating the ser/thr kinase, Raf leading to stimulation of the thr/tyr kinase MEK and consequent activation and nuclear translocation of the ser/thr kinase Erk. ES-62/PC signalling (B) disrupts BCR coupling to the PI-3-K cascade as well as targeting major negative regulatory sites in the control of the Erk, p38 and JNK MAP kinase cascades. Firstly, ES-62 signalling promotes the BCR-activation of SHP-1 tyrosine phosphatase to prevent initiation of BCR signalling by maintaining the ITAMs in a resting, dephosphorylated state and hence prevents recruitment of the ShcGrb2Sos complexes required to activate the Ras- and Rac-MAP kinase cascades. Secondly, ES-62 signalling promotes the BCR-mediated recruitment of RasGAP to terminate ongoing Ras signals. In addition, ES-62 is also likely to target MAP kinase activation by downregulating PKC isoform expression. Finally, ES-62-signalling promotes the BCR-driven association of the nuclear MAP kinase dual (thr/tyr) phosphatase, Pac-1 with Erk to terminate any ongoing Erk signals. This multi-pronged mechanism results in a rapid and profound desensitisation of BCR coupling to the MAP kinase cascades.

Our studies have shown that ES-62 selectively targets these key signalling events following BCR ligation to disrupt the activation and proliferation of B cells. For example, it does not target the early BCR-coupled PLC-γ-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂), which generates the second messengers, inositol trisphosphate (IP₃) and diacylglycerol (DAG) that mobilise intracellular stores of calcium and activate protein kinase C isoforms respectively (Harnett & Harnett, 1993). Rather, ES-62 appears to selectively modulate the expression and activity of certain PKC isoforms in resting and BCR-stimulated B cells (Harnett & Harnett, 1993; Deehan, Harnett & Harnett, 1997). Thus, whilst ES-62 selectively down-regulates the expression of the α, β, ζ, δ and ι/λ isoforms, predominantly by stimulating proteolytic degradation, it up-regulates the expression of PKC-γ and PKC-ε in murine splenic B cells (Deehan et al. 1997). In addition, ES-62 acts to modulate PKC signalling resulting from antigen receptor ligation of B cells by disrupting the normal activation and nuclear translocation patterns of the PKC-α and -ι/λ isoforms (Deehan et al. 1997). These data are consistent with proposals that PKC-α, -β and -ι/λ transduce key activation signals involved in the regulation of antigen-driven DNA synthesis and proliferation in B cells (BrickGhannam et al. 1994) such as phosphorylation of the nuclear protein lamin B and the induction and activation of NF-κB, Fos, Egr-1 and Myc (Hornbeck, Huang & Paul, 1988; Klemsz et al. 1989; Seyfert et al. 1990; Mittelstadt & DeFranco, 1993; Francis et al. 1995).

In addition to its effects on PKC signalling, pre-exposure to ES-62 selectively inhibits BCR-mediated recruitment of key proliferative pathways, such as the PI-3-K and Ras/Erk MAP kinase cascades (Deehan et al. 1998). An intriguing feature of these findings was that stimulation of B cells with ES-62 alone did not induce activation of Ras or PI-3-K despite the fact that ES-62 can induce activation of the PTKs Lyn and Syk (upstream regulators of Ras and PI-3-K) and Erk MAP kinase (downstream effector of Ras) (Deehan et al. 1998). This apparent discrepancy was resolved by our finding (Deehan et al. 1998; Deehan, Harnett & Harnett, 2001) that ES-62 does not mediate uncoupling of the BCR from the PI-3-K or Ras/Erk MAP kinase cascades by targeting activation of Syk or Lyn. Instead, it primes for the induction of the tyrosine phosphatase SHP-1, which negatively regulates activation via the BCR complex by dephosphorylating the Igα/β-ITAMs, thereby preventing recruitment of the Ras/Erk MAP kinase cascade. Moreover, ES-62 recruits additional negative regulatory elements of this pathway, namely RasGAP and the dual (thr/tyr) phosphatase Pac-1, to terminate any residual coupling of the BCR to Ras and Erk activity, respectively. This multi-pronged mechanism provides for a rapid and profound desensitisation of BCR-stimulated Erk MAP kinase signalling (Fig. 2).

ES-62 also modulates the activation of the two other major MAP kinase subfamilies, p38 and c-Jun N-terminal kinase (JNK) (Goodridge et al. 2005a). The precise targets of ES-62 in these pathways are unclear but our results, like those of others (Bagrodia et al. 1995), suggest that the BCR modulates p38 and JNK signalling in a Vav- and Rac-dependent manner (Goodridge et al. 2005a). The finding that ES-62 alone can selectively stimulate Syk, Lyn and MAP kinase activation, whilst it does not appear to modulate Ras, Rac or PI-3-K activity (Deehan et al. 1997, 1998), also suggests that MAP kinases can be activated in B cells via alternative PTK-dependent pathways. This proposal is consistent with the increasing evidence for Ras-independent pathways of Erk MAP kinase activation involving lipid second messengers and PKC (Buscher et al. 1995). We have not, however, found any evidence to support the proposal that ES-62 or BCR stimulates Erk MAP kinase activity via lipid second messengers derived from phosphatidylcholine-specific phospholipase C (PtdCho-PLC), PtdCho-phospholipase D (PtdCho-PLD) or sphingomyelinase-dependent pathways (Deehan et al. 1998). However, PKC-α has been reported to mediate activation of Erk MAP kinase via Raf and MAP kinase kinase (MEK) pathways (Berra et al. 1995; Pelech, 1996) and this is consistent not only with reports that PKC activity plays a role in coupling the BCR to ErkMAP kinases (Campbell, 1999), but also provides a rationale for our finding that, whilst prolonged pre-treatment with ES-62 acts to reduce this PKC activity, ES-62 initially up-regulates PKC-α expression (Harnett & Harnett, 1993; Deehan et al. 1997).

T cell signalling

Although ES-62 exerts little or no direct effects on antigen-/mitogen-driven proliferation or cytokine secretion in T cells, in a manner analogous to that observed for the suppression of antigen receptor stimulation of B cells, the nematode product suppresses anti-CD3-induced proliferation of Jurkat T cells and also promotes concanavalin A-induced growth arrest of these cells (Harnett et al. 1998). The precise sequence of signalling events underlying these effects has not been fully elucidated, but it is clear that ES-62-mediated desensitisation of TCR signalling is associated with disruption of TCR coupling to PLD, PKC, PI-3-K and Ras-Erk MAP kinase signalling but, as with B cells, not the PLC-mediated generation of inositol phosphates (Harnett et al. 1998). Again, PC appears to be the active moiety, since culture with PC or PC-BSA has comparable effects to ES-62 on the coupling of the TCR to PTK activation (ZAP-70, Lck and Fyn recruitment) and the Ras-Erk MAP kinase signalling cascades (Harnett et al. 1998, 1999). These findings are consistent with an earlier report showing that PC-containing molecules of the human filarial nematode, B. malayi, inhibit the response of human T cells to mitogens (Lal et al. 1990).

Macrophage and DC signalling

Induction of cytokine production by macrophages and DCs in response to many pathogen products occurs following ligation of a family of recently identified pattern recognition receptors, known as Toll-like receptors (TLRs; reviewed in Takeda, Kaisho & Akira, 2003). TLRs are thought to recognise specific molecular motifs of host as well as pathogen origin, including pathogen-associated molecular patterns (PAMPs). For example, TLR4 is required for the detection of and response to bacterial LPS whereas BLP and CpG DNA motifs are recognized by TLR2 and TLR9 respectively. TLR signals are transduced via adaptor molecules including MyD88 (reviewed in O'Neill, Fitzgerald & Bowie, 2003 and Akira & Takeda, 2004) resulting in the activation of various signalling pathways including the MAP kinase cascades and NF-κB.

Treatment of macrophages with ES-62 induces tyrosine phosphorylation of a number of proteins (Goodridge et al. 2005a) and modulates the activation of members of all three major MAP kinase subfamilies (Erk, p38 and JNK), which are involved in the regulation of cytokine production (Goodridge et al. 2001, 2003). For example, suppression of IL-12 by ES-62 is likely to be due in part to Erk MAP kinase-mediated suppression of the p40 subunit of this heterodimeric cytokine, since ES-62 inhibition can be rescued by pre-treatment with the Erk MAP kinase kinase (MEK-1) inhibitor PD98059 (Goodridge et al. 2003). ES-62 treatment also suppresses the activation of the p38 (Goodridge et al. 2003) and JNK MAP kinases (Goodridge et al. 2005a), which are required for the production of IL-12 as well as IL-6 and TNF-α, suggesting another mechanism whereby suppression of these cytokines is likely to be achieved. Furthermore, preliminary analysis indicates that ES-62 regulates gene induction by modulating the activation and gene promoter region binding of the transcription factors NF-κB and IRF-1 (Goodridge et al. 2005a).

We have recently shown that ES-62 achieves its modulation of macrophage and DC activation in a TLR4-dependent manner (Goodridge et al. 2005b). Low-level cytokine induction by ES-62 alone was abolished in macrophages/DCs from TLR4 ko mice and also MyD88 ko mice. Similarly ES-62-mediated suppression of cytokine induction by TLR ligands (BLP and CpG) was dependent on the presence of TLR4. In contrast ES-62 effects were TLR2- and TLR-6 independent. Modulation of surface expression of MHC II and costimulatory molecules (CD40, CD80, CD86) was also suppressed in DCs from TLR4 ko mice, although these latter effects were only partially dependent on the presence of MyD88. Interestingly macrophages and DCs from C3H/HeJ mice, which are unresponsive to LPS due to their production of a defective form of TLR4 resulting from a point mutation in the intracellular TIR domain, remain responsive to ES-62 as evidenced by the modulation of cytokine production and costimulatory molecule expression. Thus it appears that TLR4 must be present but not necessarily fully functional for ES-62 responsiveness. This may be due to ‘non-classical’ coupling to downstream signal transduction pathways by TLR4 or the recruitment of a signalling co-receptor.

It is not currently clear whether TLR4 is required for the direct recognition of ES-62. Preliminary evidence suggests that the PC moiety of ES-62 is at least in part required for its recognition (unpublished data) and hence it is tempting to speculate that TLR4 may recognize this molecular pattern, especially since other PC-containing molecules have been reported to act via TLR4 (Miller et al. 2003; Walton et al. 2003a,b).