PI3K (phosphoinositide 3-kinase) IA family members contain a regulatory subunit and a catalytic subunit. The p110δ catalytic subunit is expressed predominantly in haematopoietic cells. There, among other functions, it regulates antigen receptor-mediated responses. Using mice deficient in the p110δ subunit of PI3K, we investigated the role of this subunit in LPS (lipopolysaccharide)-induced B cell responses, which are mediated by Toll-like receptor 4 and RP105. After injection of DNP-LPS (where DNP stands for 2,4-dinitrophenol), p110δ−/− mice produced reduced levels of DNP-specific IgM and IgG when compared with wild-type mice. In vitro, the proliferation and up-regulation of surface activation markers such as CD86 and CD25 induced by LPS and an antibody against RP105 were decreased. We analysed the activation state of key components of the LPS pathway in B cells to determine whether there was a defect in signalling in p110δ−/− B cells. They showed normal extracellular-signal-regulated kinase phosphorylation, but anti-RP105-induced protein kinase B, IκB (inhibitor of nuclear factor κB) and c-Jun N-terminal kinase activation was severely reduced. This demonstrates that the p110δ subunit of PI3K is involved in the LPS response in B cells and may represent a link between the innate and the adaptive immune system.
Intracellular signalling is the basis for cellular responses and signals can be transduced through a variety of pathways. A key player in multiple routes of signal transduction is PI3K (phosphoinositide-3-kinase). It phosphorylates inositol phospholipids at the 3′-OH of the inositol ring, and the resulting products function as second messengers by binding to pleckstrin homology domains in signalling molecules and serve as substrates for other lipid-modifying enzymes (see  for a review).
PI3K can be divided into four classes, IA, IB, II and III, on the basis of structure and the substrates used [2,3]. Class IA PI3K are dimers containing (i) a regulatory subunit, p85α, p55α, p50α, p85β or p55γ, of which p85α is the most abundant, and (ii) a catalytic subunit, p110α, p110β or p110δ. The p110δ subunit is primarily expressed in haematopoietic cells, and mice lacking or expressing mutant p110δ show developmental defects in lymphocytes and decreased antigen receptor signalling [4–6]. They also have functional defects in B cells, namely low basal antibody levels and decreased TI2 (T cell-independent type 2) responses and also decreased T cell-dependent responses. Class IA PI3K has been shown to be involved in innate responses against pathogen-associated molecular patterns through TLRs (Toll-like receptors) in macrophages, dendritic cells and endothelial cells [7–9]. A major ligand inducing innate responses via TLR4 (Toll-like receptor 4) is LPS (lipopolysaccharide) . B cells require an additional molecule, RP105, for complete LPS response [11,12]. The lack of RP105 leads to severely reduced proliferation and antibody production in the B cells stimulated by LPS. In the present study, we investigate the involvement of the p110δ subunit in the LPS receptor responses in B cells.
B cells deficient in the p110δ subunit of PI3K class IA show defects in the TI1 response
We tested the TI1 response after immunization with a single dose of DNP (2,4-dinitrophenol)-LPS in PBS by serum ELISA. P110δ−/− mice showed reduced production of DNP-specific IgM- and IgG-subtype antibodies. This could be a defect in DNP-LPS response either in the B cells themselves or in other cells of the immune system, such as macrophages or dendritic cells, which are involved in B cell activation, e.g. through cytokines.
Activation and proliferation induced by LPS signalling are defective in p110δ−/− B cells
To identify whether the observed decrease in the humoral response to DNP-LPS is a specific defect, B cells were purified from the spleens of wt (wild-type) and p110δ−/− mice and cultured in vitro in the presence of LPS or an RP105-specific activating antibody . After 24 h, surface expression of the activation markers MHC class II, CD25, CD69 and CD86 was analysed by flow cytometry. There was no difference in up-regulation of MHC class II between wt and p110δ−/− B cells in response to both stimuli, but up-regulation of the other three markers was reduced in p110δ−/− cells. B cell activation through the LPS receptors also induces the cell cycle, leading to B cell proliferation. We cultured purified B cells in the presence of LPS and anti-RP105 and analysed cell-cycle induction and proliferation. Although p110δ−/− B cells progressed normally through the cell cycle in response to LPS, their subsequent proliferation was slightly reduced. In contrast, no cell-cycle induction or proliferation was detected in response to RP105 stimulation, whereas wt B cells progressed in the cell cycle and performed multiple rounds of cell division. This shows that the defects in TI1 immune response in p110δ−/− are B cell-intrinsic and suggests a requirement for p110δ in the proliferative response to specific stimulation of RP105, which can be overcome to some extent by stimulation with LPS.
Activation of NF-κB and MAP kinase in B cells in response to activation of the LPS signalling pathway is affected by the lack of p110δ
PI3K has been suggested to be involved in NF-κB (nuclear factor κB) activation in the LPS response . To assess the contribution of the p110δ subunit, we stimulated purified B cells with anti-RP105 in vitro and analysed the phosphorylation of IκB (inhibitor of NF-κB). IκB phosphorylation in response to RP105 stimulation was severely reduced in p110δ−/− B cells. Other targets of signalling through the LPS receptor are MAP (mitogen-activated protein) kinases and PKB (protein kinase B) . We tested their activity by analysing the phosphorylation state of the MAP kinases ERK (extracellular-signal-regulated kinase) and c-Jun N-terminal kinase, as well as PKB. The activation of c-Jun N-terminal kinase and PKB was defective in p110δ−/− B cells downstream of the LPS receptor, but ERK phosphorylation in response to RP105 stimulation was not affected. However, preincubation of B cells with wortmannin, a PI3K-specific inhibitor, completely abrogated ERK phosphorylation in response to anti-RP105. This suggests that ERK activation could be regulated by a different catalytic subunit of PI3K class IA.
Our results demonstrate that PI3K activity, mainly mediated through the class IA p110δ catalytic subunit, is required for LPS-induced functions of B cells, including proliferation and antibody production. The lack of p110δ leads to inhibition of downstream signalling, including activation of MAP kinases and NF-κB. However, the requirement for p110δ activity in response to LPS differs from that in response to direct stimulation of the B cell-specific LPS co-receptor, RP105. This suggests a synergistic activation of downstream signalling mediated by TLR4 and RP105, where p110δ function seems to be a component of the RP105 pathway rather than the TLR4 pathway (Figure 1). This might enable B cells to regulate their LPS responses in a B cell-specific manner.
A model of the B cell-specific LPS receptor pathway
Signalling Outwards and Inwards: A Focus Topic at BioScience2004, held at SECC Glasgow, U.K., 18–22 July 2004. Edited by J. Challiss (Leicester, U.K.), A. Harwood (University College London, U.K.), M. Humphries (Manchester, U.K.), C. Isacke (Institute of Cancer Research, London, U.K.), R. Liddington (Burnham Institute, La Jolla, CA, U.S.A.), T. Palmer (Glasgow, U.K.), K. Siddle (Cambridge, U.K.), C. Sutherland (Dundee, U.K.), H. Wallace (Aberdeen, U.K.) and M. Welham (Bath, U.K.).
This work was supported by a Senior Fellowship from the Medical Research Council (to M.T.) and a Biotechnology and Biological Sciences Research Council project grant.