Regulation of secondary lymphoid organ development by the nuclear factor-κB signal transduction pathway

Summary: In primary lymphoid organs, such as thymus and bone marrow, B and T lymphocytes differentiate from lymphoid stem cells into mature albeit naïve effector cells. In contrast, secondary lymphoid organs, such as the spleen, lymph nodes, and Peyer's patches (PPs), provide an environment that enable lymphocytes to interact with each other, with accessory cells, and with antigens, resulting in the initiation of antigen‐specific primary immune responses. Recently, the analysis of gene‐knockout mice has shed light on the signaling pathways, cellular requirements, and molecular mechanisms involved in secondary lymphoid organ development. In particular, signals that converge on the nuclear factor‐κB (NF‐κB) pathway have been demonstrated to play an important role in both early developmental steps as well as maintenance of secondary lymphoid organ structures. Analysis of the histopathological changes in secondary lymphoid tissues of mice lacking individual Rel/NF‐κB family members, upstream kinases, and receptors strongly indicates that activation of the recently described alternative NF‐κB pathway by membrane‐bound lymphotoxin, via p52–RelB heterodimers, plays a major role during initiation steps of secondary lymphoid organ development. Induction of the classical p50–RelA NF‐κB activity, as exemplified by tumor necrosis factor receptor signaling, clearly also contributes, but seems to be involved primarily in later developmental step, such as the proper cellular and structural organization of B‐cell follicles.     

Allergen activates peripheral blood eosinophil nuclear factor-κB to generate granulocyte macrophage-colony stimulating factor, tumour necrosis factor-α and interleukin-8

BACKGROUND: Allergic inflammation is characterized by the influx and activation of eosinophils. Cytokines generated by both resident and infiltrating cells are responsible for the initiation and maintenance of this pathogenesis. This study focuses on allergen-induced activation of eosinophil NF-kappaB and generation of granulocyte macrophage-colony stimulating factor (GM-CSF), TNF-alpha, and IL-8. METHODS: Peripheral blood eosinophils were enriched to >99.9% by Percoll gradient sedimentation and negative magnetic affinity chromatography. NF-kappaB activation by 10 microg/mL house dust mite (HDM) extract was demonstrated immunocytochemically using a monoclonal antibody against the active form of NF-kappaB (NF-kappaBa). The authenticity of NF-kappaB was confirmed by Western blot. Cytokine production was assessed both by immuno-staining of eosinophils and by assay of cytokines in the cell supernatant. RESULTS: Activation of peripheral blood eosinophils from atopic, but not non-atopic, donors induced activation of NF-kappaB, which peaked at 4 h and was accompanied by a decline in IkappaB-alpha. The activation of authentic NF-kappaB was confirmed in gel shift assays. Supershift assays showed p65 to be the major subunit of eosinophil NF-kappaB. Immunofluorescent confocal microscopy demonstrated localization of NF-kappaBa to the nucleus. Following activation, cytokine immunoreactivity was seen in a fraction of the eosinophils and cytokines were released into the supernatant. The NF-kappaB inhibitors, calpain inhibitor 1 (10 microm), pentoxifylline (0.5 mm), pyrrolidine dithiocarbamate (PDTC, 10 microm) or gliotoxin (1 pg/mL) reduced the generation of GM-CSF, TNF-alpha and IL-8 in parallel with their inhibition of NF-kappaB. CONCLUSIONS: HDM allergen activates human eosinophil NF-kappaB leading to the production of the cytokines GM-CSF, TNF-alpha and IL-8. We speculate that a role for eosinophil NF-kappaB-dependent cytokines is to act as an autocrine loop augmenting the survival of eosinophils in vivo.     

Detection of a κ-casein-specific lymphocyte response in milk-responsive atopic dermatitis

Background Bovine casein leads to an expansion of lymphocytes expressing the cutaneous lymphocyte antigen and to specific lymphocyte proliferation in a subgroup of patients with milk-responsive atopic dermatitis (AD). The casein fraction is composed of different proteins with defined and completely different sequences. Objective To define the stimulatory capacity of the major casein protein (α and k) in lymphocyte proliferation assays with cells from milk-allergic and non-allergic individuals. Methods Proliferative responses of peripheral blood mononuclear cells to lipopolysaccharide-depleted casein subfractions were measured by thymidine incorporation. Lymphocytes from milk-responsive patients with AD were compared with cells from non-responsive patients with AD and to non-atopic individuals, Atopic individuals with immediate symptoms following consumption of cow's milk were included as positive controls. Casein-specific T-cell clones (TCC) from four patients with milk-responsive AD were restimulated with unfractioned casein and K-casein. Results Higher proliferative responses to unfractionated casein and α-,β and casein were observed in milk-responsive patients compared with non-responders. Unfractionated casein and K-casein discriminated best between the milk-responsive patients with AD and non-responders. Twenty-five of 31 TCC from patients with milkresponsive AD reacted to the mixed casein preparation and K-casein.     

Identification of IgE and IgG binding epitopes on β- and κ-casein in cow's milk allergic patients

BACKGROUND: Cow's milk allergy (CMA) affects 2.5% of children aged less than 2 years of age. Although beta- and kappa-casein are considered among the major allergens responsible for CMA, no data are available on their allergenic epitopes in humans. OBJECTIVE: The aim of the study was to identify IgE- and IgG-binding epitopes on beta- and kappa-casein and to determine whether the pattern of epitope recognition is associated with the natural history of CMA. METHODS: Overlapping decapeptides representing the entire length of beta- and kappa-casein, respectively, were synthesized on a cellulose-derivatized membrane. Sera from 15 milk-allergic children, 4-18 years of age, with high levels of specific IgE antibodies to cow's milk were used to identify IgE- and IgG-binding epitopes. In addition, IgE epitopes were screened with pooled or individual sera from younger patients aged less than 3 years and who had low levels of specific serum IgE, who are likely to outgrow CMA. RESULTS: Six major and three minor IgE-binding epitopes, as well as eight major and one minor IgG binding regions, were identified on beta-casein. Eight major IgE-binding epitopes, as well as two major and two minor IgG-binding epitopes, were detected on kappa-casein. Three of the IgE binding regions on beta-casein and six on kappa-casein were recognized by the majority of patients in the older age group, but not by the younger patients. CONCLUSION: Information regarding the immunodominant epitopes in beta- and kappa-casein may be important for understanding the pathophysiology and natural history of CMA. Differences in epitope recognition may be useful in identifying children who will have persistent milk hypersensitivity.     

Distribution of κ and λ Light Chain Isotypes among Human Blood Immunoglobulin-Secreting Cells after Vaccination with Pneumococcal Polysaccharides

The light chain isotype of immunoglobulin-secreting blood cells was investigated by means of monolayer plaque-forming cell assays allowing direct immunofluorescence staining for cytoplasmic kappa and lambda light chains in centre cells. The study revealed that cultured, polyclonally activated pokeweed mitogen (PWM) and Epstein-Barr virus (EBV), IgM-, IgG- and IgA-secreting cells expressed the kappa light chain isotype in approximately 65% of the cells. IgM- and IgG-secreting cells induced by vaccination with pneumococcal polysaccharides had a similar percentage of kappa light chain-containing cells. In contrast, IgA-secreting cells induced by vaccination with pneumococcal polysaccharides showed a different (bimodal) distribution as regards expression of kappa light chain. The majority (56%) of the investigated individuals expressed kappa light chain in approximately 50% of the cells and the rest expressed kappa light chains in approximately 80% of the cells. The percentage of cells containing kappa light chains among spontaneous IgA-secreting cells in unvaccinated individuals was approximately 50% and thus also differed from the general pattern for mitogen-activated B cells. The light chain pattern of IgA-secreting cells from individuals vaccinated with pneumococcal polysaccharides and from unvaccinated individuals probably indicates that these cells are being derived from B-cell clones with a limited idiotypic heterogeneity, which have been selected and clonally expanded by naturally occurring antigens at the mucosal membranes.     

骨髓间充质干细胞经核转录因子κB途径干预心肌纤维化

背景：骨髓间充质干细胞移植能否直接干预心肌纤维化及其可能的机制尚不完全清楚。 目的：观察骨髓间充质干细胞移植干预心肌纤维化的效果并分析其机制。 方法：分离培养雄性小鼠骨髓间充质干细胞,经尾静脉输入异丙肾性心肌纤维化雌性小鼠为治疗组,另设未治疗组和正常对照组。5周后处死小鼠,实时荧光定量PCR检测心肌y染色体鉴别基因(SRY)、基质金属蛋白酶9、基质金属蛋白酶组织抑制剂1的表达;天狼猩红染色对比心脏胶原纤维含量;免疫组织化学染色法观察心脏核转录因子κB表达。 结果与结论：骨髓间充质干细胞能归巢于纤维化心肌。与未治疗组相比,治疗组的心肌基质金属蛋白酶9、基质金属蛋白酶组织抑制剂1和核转录因子κB表达下调(P < 0.05),胶原纤维含量下降(P < 0.05)。结果表明,骨髓间充质干细胞移植干预心肌纤维化,其机制可能与抑制核转录因子κB过度活化有关。     

Ganoderic Acid A Attenuates IL-1β-Induced Inflammation in Human Nucleus Pulposus Cells Through Inhibiting the NF-κB Pathway

Inflammation - Intervertebral disc (IVD) degeneration is a major cause of low back pain associated with several pathological changes in the IVD, including dysfunction of nucleus pulposus (NP)...     

核转录因子κB信号通路在应力调控成肌细胞分化中的作用

背景：NF-κB信号通路在细胞生长分化、炎症反应、肿瘤生长等过程中发挥重要的调节作用,也参与成肌细胞分化的调控。 目的：分析NF-κB信号通路在应力介导的C2C12成肌细胞分化中的作用及其作用机制。 方法：成功构建大鼠C2C12成肌细胞体外培养-力学刺激模型,采用多通道细胞牵张应力加载系统,以0.5 Hz的加载频率和10%的细胞拉伸变形幅度对细胞进行拉伸培养2,6,12,24 h。 结果与结论：①C2C12成肌细胞在周期性机械拉伸力作用下,NF-κB信号通路被激活。当细胞受到应力刺激6 h后,胞核NF-κB p65亚基蛋白表达水平开始增强,24 h内NF-κB p65亚基蛋白表达水平达到峰值。加力12,24 h组与未加力对照组之间差异有显著性意义(P < 0.05)。②IκBα蛋白表达水平在加力6 h后表达显著下降,24 h内IκBα蛋白表达水平减弱达到最低。加力12,24 h组与未加力对照组之间差异有显著性意义(P < 0.05)。③周期性张应力促进C2C12成肌细胞分化过程中Myogenin的表达,加入NF-κB信号通路特异性抑制剂吡咯烷二硫氨基甲酸 (20 μmol/L) 后再加力,Myogenin的表达明显降低。以上结果提示：①NF-κB信号通路可能参与应力介导的C2C12成肌细胞分化的调控过程。②当细胞受到应力刺激时,胞质IκBα发生磷酸化并降解。③NF-κB信号通路在应力介导的C2C12成肌细胞分化过程中发挥重要作用,但不是这一调控过程的惟一通路。     

Identification of a novel REV1-interacting motif necessary for DNA polymerase κ function

When a replicative DNA polymerase (Pol) is stalled by damaged DNA, a "polymerase switch" recruits specialized translesion synthesis (TLS) DNA polymerase(s) to sites of damage. Mammalian cells have several TLS DNA polymerases, including the four Y-family enzymes (Polη, Polι, Polκ and REV1) that share multiple primary sequence motifs, but show preferential bypass of different DNA lesions. REV1 interacts with Polη, Polι, and Polκ and therefore appears to play a central role during TLS in vivo . Here we have investigated the molecular basis for interactions between REV1 and Polκ. We have identified novel REV1-interacting regions (RIRs) present in Polκ, Polι and Polη. Within the RIRs, the presence of two consecutive phenylalanines (FF) is essential for REV1-binding. The consensus sequence for REV1-binding is denoted by x-x-x-F-F-y-y-y-y (x, no specific residue and y, no specific residue but not proline). Our results identify structural requirements that are necessary for FF-flanking residues to confer interactions with REV1. A Polκ mutant lacking REV1-binding activity did not complement the genotoxin-sensitivity of Polk -null mouse embryonic fibroblast cells, thereby demonstrating that the REV1-interaction is essential for Polκ function in vivo .     

Constant Region of a κ III Immunoglobulin Light Chain as a Major AL-Amyloid Protein

AL-amyloidoses are generally described as a group of disorders in which N-terminal fragments of monoclonal immunoglobulin light chains are transferred into amyloid fibrils. We have, by amino acid sequence analyses and immunological methods, characterized the Bence-Jones protein and the corresponding AL protein as a κ III immunoglobulin light chain from material of a patient with systemic AL-amyloidosis presenting as a local inguinal tumour. The two proteins showed some unique features. The major part of the AL amyloid fibril protein consisted of C-terminal fragments of the Bence-Jones protein. Furthermore, both the Bence-Jones protein and the AL protein were glycosylated, with possibly a glycosylation in the constant part of the light chain.     

Late Appearance of an IgA(κ) Monoclonal Protein in a Patient with IgG(κ) Multiple Myeloma: Sharing of Idiotypic Specificities between the Two Serum Proteins

A patient with an IgG(chi) monoclonal serum protein developed in the course of the disease a second monoclonal spike of the same light chain type and of the IgA class. The latter monoclonal protein progressively increased and eventually exceeded the first IgG(chi) protein. Antigenic analysis of the two myeloma proteins demonstrated that they shared idiotypic determinants. Immunofluorescence studies, carried out with anti gamma and anti alpha reagents tagged with different fluorochromes revealed that the two isotypes were produced by different plasma cells. The data are discussed in the prospect of a possible transitional mechanism from IgG to IgA synthesis within a single B cell clone.     

Montelukast inhibits tumour necrosis factor-α-mediated interleukin-8 expression through inhibition of nuclear factor-κB p65-associated histone acetyltransferase activity

Background Montelukast is a potent cysteinyl leukotriene‐1 receptor antagonist possessing some anti‐inflammatory effects although the molecular mechanism of these anti‐inflammatory effects is unknown. In this study, we aimed to investigate the effect of montelukast on nuclear factor (NF)‐κB‐associated histone acetylation activity in phorbol myristate acetate (PMA)‐differentiated U937 cells. Methods We examined the inhibitory effects of montelukast on TNF‐α‐induced IL‐8 production in PMA‐differentiated U‐937 cells. U‐937 cells were exposed to PMA (50 ng/mL) for 48 h to allow differentiation to macrophages. Macrophages were then exposed to TNF‐α (10 ng/mL) in the presence or absence of montelukast (0.01–10 μm ) for 24 h. After this time, the concentration of IL‐8 in the culture supernatant was measured by sandwich‐type ELISA kit. The effect of signalling pathways on TNF‐α‐induced IL‐8 release was examined pharmacologically using selective NF‐κB/IKK2 (AS602868, 3 μm ), (PD98059, 10 μm ) and p38 mitogen activated protein kinase (MAPK) (SB203580, 1 μm ) inhibitors. NF‐κB DNA binding activity was measured by a DNA‐binding ELISA‐based assay. NF‐κB‐p65‐associated histone acetyltransferase (HAT) activity was measured by immunoprecipitation linked to commercial flourescent HAT. Results TNF‐α‐induced IL‐8 release was suppressed by an NF‐κB inhibitor but not by MEK or p38 MAPK inhibitors. Montelukast induced a concentration‐dependent inhibition of TNF‐α‐induced IL‐8 release and mRNA expression that reached a plateau at 0.1 μm without affecting cell viability. Montelukast did not affect NF‐κB p65 activation as measured by DNA binding but suppressed NF‐κB p65‐associated HAT activity. Conclusion Montelukast inhibits TNF‐α‐stimulated IL‐8 expression through changes in NF‐κB p65‐associated HAT activity. Drugs targeting these enzymes may enhance the anti‐inflammatory actions of montelukast.