Lipopolysaccharide from Salmonella enterica Activates NF-κB through both Classical and Alternative Pathways in Primary B Lymphocytes

Bacterial lipopolysaccharide (LPS) or endotoxin, the major component of the outer membrane of gram-negative bacterial cell walls, activates various cell types of the innate and adaptive immune responses (27, 47). LPS has been known for a long time to be a potent activator of B lymphocytes (2, 28). Besides its effect on B-cell activation (33), LPS exhibits a prosurvival activity on both immature (31, 48) and mature (31, 44) B cells. In the immune system, apoptosis plays a fundamental role in the development and homeostasis of lymphocytes and is essential in the negative selection and deletion of autoreactive cells (3, 9, 23). Recently, we reported that LPS protects primary B lymphocytes from both spontaneous and drug-induced apoptosis by preventing mitochondrial translocation of Bax, a proapoptotic protein of the Bcl2 family (45). It has been reported that Bax translocation could be blocked by Bcl-xL, the prosurvival protein of the Bcl2 family (13) whose expression could be enhanced by nuclear factor κB (NF-κB) activation (8). The pleiotropic NF-κB and Rel transcription factors play an essential role in the regulation of genes involved in various biological processes, such as inflammation, innate and adaptive immune responses, lymphocyte development, and apoptosis (1, 7, 20, 42). NF-κB comprises homo- or heterodimers of five Rel proteins, RelA (p65), RelB, c-Rel, p50/p105 (NF-κB1,) and p52/p100 (NF-κB2). In most resting cell types, NF-κB is kept inactive in the cytoplasm through association with inhibitory members of the IκB family, including IκBα, IκBβ, and IκBɛ, as well as p105 and p100, the precursors of the p50 and p52 subunits, respectively. Phosphorylation of IκB, by a multiunit IκB kinase (IKK) complex consisting of IKKα and IKKβ, results in its proteasomal degradation, thereby permitting nuclear translocation of NF-κB and transcriptional activation of target genes (4, 20, 35). It is increasingly being recognized that in immune regulation and lymphocyte activation, NF-κB activation can be achieved through two main pathways, termed canonical (classical) and noncanonical (alternative). The canonical pathway is known to be involved in the response of various cell types to pathogen-associated molecular patterns as well as proinflammatory cytokines, such as tumor necrosis factor alpha and interleukin-1. The second NF-κB noncanonical pathway is activated by a specific set of stimuli, including B-cell activating factor (BAFF), lymphotoxin β, and CD40L (6, 49). The canonical NF-κB signaling pathway activates the IKK complex, preferentially IKKβ, that controls the phosphorylation and degradation of most IκB family proteins, including p105, resulting in the processing of p105 to p50 (35, 38). In the noncanonical pathway, it is postulated that the processing of the NF-κB2 precursor p100 to the mature subunit p52 is regulated via an NF-κB-inducing kinase (NIK) pathway which induces generally IKKα-mediated phosphorylation of specific serine residues in the C-terminal domain of p100 and its processing to p52 (35, 38, 41, 51). So far, LPS has been known to trigger the activation of NF-κB in various B cell lines and immature B cells, thus inducing their differentiation to mature B cells (17, 29, 39). Recently, NF-κB transcription factors have emerged as key regulators of survival for many cell types (1, 34), including B cells (14, 40). However, involvement of the noncanonical NF-κB signaling pathway in the activity of LPS has not yet been demonstrated in mature B lymphocytes. In the present report, we examined the effect of LPS on the IκB and NF-κB families of proteins to investigate the involvement of NF-κB in LPS-induced B-cell stimulation and, subsequently, to determine whether the activation of NF-κB would proceed through the canonical signaling pathway, the noncanonical signaling pathway, or both.