Human Langerhans-cell activation triggered in vitro by conditionally expressed MKK6 is counterregulated by the downstream effector RelB

Abstract

IntroductionImmature dendritic cells (DCs) such as epidermal/mucosal Langerhans cells (LCs) reside in peripheral tissues. Upon activation they migrate from peripheral sites to lymphoid organs and thereby acquire potent T-cell stimulatory capacity, a process referred to as DC maturation. 1,2 Various triggers are capable of inducing DC maturation. Pathogen recognition receptors (PRRs) such as Tolllike receptors (TLRs) induce pathogen-associated molecular pattern (PAMP)-dependent DC activation. Other stimuli are PAMP independent. These include inflammatory cytokines, environmental danger stimuli (eg, UV light and haptens), or dying cells. 2,3 Additionally, adhesion-dependent microenvironmental signals seem to counteract DC maturation, because epidermal shear stress itself triggers LC maturation. 4 Loss-of-function studies revealed that the classical NF-B pathway and the p38 MAPK cascade are fundamentally involved in or required for DC maturation. [5][6][7][8][9] Two NF-B signaling pathways are known. The classical NF-B pathway involves inhibitors of B (IBs), which upon phosphorylation and subsequent degradation release bound classical NF-B dimers (p65/p50 or c-rel/p50). 10 Inhibition of the classical NF-B pathway in human monocyte-derived DCs (moDCs) (eg, by adenoviral delivery of an IB␣ superrepressor [IB␣-SR] mutant) results in impaired DC maturation. [6][7][8] In the alternative (noncanonical) NF-B cascade, the p100 (NF-B2) molecule retains the alternative NF-B member RelB in the cytoplasm. Upon phosphorylation by the IB kinase 1 (IKK1), p100 is degraded to p52, RelB is released, and RelB/p52 dimers translocate to the nucleus. 10 Nuclear localization of RelB is considered a marker for mature DCs, because diverse maturation stimuli induce RelB translocation to the nuclei of DCs in vitro. 11 Similarly, in vivo nuclear RelB marks mature DCs in lymphoid organs and inflammatory sites such as rheumatoid arthritis lesions. 12,13 RelB is known to regulate DC development. RelBdeficient mice selectively lack myeloid-related CD8␣ Ϫ DCs but not LCs. 14 Similarly, differentiation of human CD11b ϩ interstitial-type DCs (intDCs) but not LCs from progenitor cells is inhibited by expressing a truncated p100 molecule that specifically captures RelB in the cytoplasm. 15 RelB deficiency was also shown to impair murine DC antigen presentation function in vivo. 16 Furthermore, DCs from p100-deficient mice, which have enhanced RelB activity but also lack p52, showed enhanced DC maturation. 17 However, expression of dominant negative NF-B-inducing kinase (NIK), an inducer of IKK1-mediated p100 processing, had no effect on human mo-DC maturation. 18 Therefore, while a critical function of RelB in DC subset development is well established, its function in DC maturation remains unclear.p38 mitogen-activated protein kinase (p38 MAPK) is an evolutionary highly conserved stress response pathway in eukaryotic cells. The p38 is activated by the upstream MAPK kinases MKK6 or MKK3 and is then translocated to the nucleus where it in turn phosphor...

Aryl Hydrocarbon Receptor Activation Inhibits In Vitro Differentiation of Human Monocytes and Langerhans Dendritic Cells

“…We generated granulocytes, monocytes, and LC-type dendritic cells (LCs) from CD34 ϩ cells as previously described (20,22) (Fig. 1A) and analyzed AhR protein expression by Western blotting (Fig.…”

“…LC purity exceeded 95%. In vitro generation of LCs, monocytes, and granulocytes as well as progenitor expansion was performed as described (18,22). In brief, CD34…”

Monocytic cell differentiation from band-stage neutrophils under inflammatory conditions via MKK6 activation

“…21 This enables inducible expression of d.a.MKK6 followed by either IRES-GFP (HR-GFP) or IRES-Nerve growth factor receptor (HR-NGFR). cDNA encoding dominant-negative c-Jun (d.n.…”

“…We generated G-CSF-dependent neutrophils or M-CSFdependent monocytes from CD34 1 cord blood progenitors and conditionally expressed d.a.MKK6 25 in differentiated cells by using a tetracycline-inducible retroviral system. 21 The vast majority of G-CSF-dependent day 11-generated cells represented CD15 ). Although CD15 and MPO are induced early during in vitro granulopoiesis of CD34 1 cells, a subset of MPO hi cells subsequently begins to express the secondary (specific) neutrophil granule marker LF, after in vitro generated cells had acquired a myelocyte/metamyelocyte phenotype.…”

“…21 Proteins from equal numbers of cells were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto polyvinylene difluoride membrane (Immobilon-P; Millipore, Billerica, MA). Protein detection was performed with chemiluminescence (SuperSignal WestPico; Pierce Biotechnology).…”