Role of Protein Tyrosine Kinase and Erk1/2 Activities in the Toll-Like Receptor 2-Induced Cellular Activation of Murine B Cells by Neisserial Porin

Abstract

Neisserial porins are potent immune adjuvants and have been demonstrated to stimulate and induce the activation of human and murine B lymphocytes. Their immunopotentiating ability is due largely to the upregulation of the surface expression of the costimulatory ligand CD86 (B7-2) on B cells and other antigenpresenting cells. Porin-induced activation is dependent on the innate immune pattern recognition receptor Toll-like receptor 2 (TLR2). These data have led us to investigate the signal transduction events induced by PorB from Neisseria meningitidis and then, using inhibitors of these pathways, to establish the mechanism by which this bacterial major outer membrane protein induces CD86 upregulation and the proliferation of murine B cells. PorB was able to induce (i) protein tyrosine kinase (PTK) activity, (ii) the phosphorylation of Erk1 and Erk2, and (iii) IB-␣ phosphorylation, leading to NF-B nuclear translocation in B cells in a TLR2-dependent manner. PorB-induced NF-B nuclear translocation was not dependent on either PTK or Erk1/2 activities. However, B-cell proliferation and the induction of increased surface expression of CD86 by PorB were dependent on PTK activity and not Erk1/2 activation. In conclusion, PorB acts through TLR2 as a B-cell mitogen, triggering tyrosine phosphorylation of various cellular proteins that are involved in proliferation and CD86 expression, as well as the phosphorylation of Erk1/2, which is not necessary for CD86 upregulation or the proliferation of B cells.Neisserial porins are the major outer membrane proteins of the pathogenic Neisseria meningitidis, constituting approximately 60% of the outer membrane protein content (4). Neisserial outer membrane protein complexes (OMPC) act as an adjuvant in Haemophilus influenzae type b OMPC vaccines, and porins' being a significant component of the OMPC has led to their investigation as an immune adjuvant. Studies indicate that neisserial porins can induce an immune response in humans and animals in the absence of exogenous adjuvants (3,47,64,69). This adjuvanticity is demonstrated by the ability of neisserial porins to induce an immune response to poorly immunogenic substances, such as peptides (34), as well as alter the immune response to antigens like capsular polysaccharide (CPS) from a T-cell-independent response to a T-cell-dependent response (11,14,(32)(33)(34)63).The function of neisserial porins as immune adjuvants is demonstrated by the ability of porins of gram-negative bacteria to stimulate antigen-presenting cells, including B cells, and affect B-cell function (42, 54-56, 60-62, 66). Results from in vitro studies in our laboratory show that PorB from N. meningitidis can activate murine dendritic cells (DCs) and B cells by upregulating class II major histocompatibility complex (MHC) molecules and the costimulatory ligand CD86, but not CD80 (52, 66). The ability of PorB to induce DC maturation is functionally important, as further studies have demonstrated that PorB-treated DCs activate antigen-specific T cells. In addition, ...

Meningococcal Porin PorB Prevents Cellular Apoptosis in a Toll-Like Receptor 2- and NF-κB-Independent Manner

“…Most intracellular antiapoptotic factors are under transcriptional control of NF-B, which therefore plays a major role in prevention of cell death (30). NF-B activation is induced by whole neisseriae and neisserial porins in various cell systems (5,13,21,23,27,35,59,77); in particular, PorB induces NF-B activation in a manner dependent on TLR2 (46,48,52), a receptor that is not present in HeLa cells (78). To determine whether PorB could induce TLR2-independent NF-B activation in these cells, multiple approaches were used.…”

“…A correlation between the antiapoptotic effect of PorB and activation of NF-B has not been shown so far, although our group has demonstrated that PorB activates NF-B in a Tolllike receptor 2 (TLR2)-dependent manner (43,46,48,52). Interestingly, various human and murine cell types are protected from apoptosis by PorB (23.…”

Immuno-Modulatory Role of Porins: Host Immune Responses, Signaling Mechanisms and Vaccine Potential

“…Studies on Neisseria spp. porins are implicated in TLR mediated NFκB activation which occurs via Raf-1-MEK1/2-MAPK pathway; (Massari et al 2003 ;MacLeod et al 2008 ). H. infl uenzae porin P2 and porins of Salmonella and Neisseria also activate the MAPK pathway (Galdiero et al 2002(Galdiero et al , 2003cVitiello et al 2004 ;MacLeod et al 2008 ).…”

“…porins are implicated in TLR mediated NFκB activation which occurs via Raf-1-MEK1/2-MAPK pathway; (Massari et al 2003 ;MacLeod et al 2008 ). H. infl uenzae porin P2 and porins of Salmonella and Neisseria also activate the MAPK pathway (Galdiero et al 2002(Galdiero et al , 2003cVitiello et al 2004 ;MacLeod et al 2008 ). The three-dimensional structure model of porin P2 constructed on the basis of crystal structure of K. pneumoniae OmpK36 and Escherichia coli PhoE and OmpF predict that the domains of surface exposed loops are involved in activation of signal transduction pathway (Table 6.1 ) .…”

Neisseriae internalization by epithelial cells is enhanced by TLR2 stimulation

“…Since PorB from NM and NL induce TLR2 cell activation via similar, but not identical signaling mechanisms, we examined individual pathways downstream of TLR2 using specific inhibitors of MAPKs (p38, ERK1/2 and JNK) and NF-κB pathways [24, 38]. While inhibition of NF-κB strongly reduced intracellular organisms, variability was observed for MAPKs: inhibition of JNK signaling generally led to lower intracellular bacteria, the effect of ERK1/2 inhibition was less considerable and that of p38 inhibition was rather dubious, consistent with previous observations [26] and with a potential function redundancy among these pathways.…”