Persistent activation of nuclear factor-kappaB signaling pathway in severe uncontrolled asthma.

The transcription factor nuclear factor-kappaB (NF-kappaB) is inactive when bound to its inhibitory protein IkappaBalpha. On cell stimulation with inflammatory signals, IkappaBalpha is phosphorylated by IkappaB kinases and subsequently degraded. Freed NF-kappaB then induces expression of cytokines such as granulocyte-macrophage colony-stimulating factor, interleukin-8, and regulated upon activation, normal T cell expressed and secreted. These mediators are overexpressed in asthma and are downregulated by glucocorticoids through NF-kappaB activity repression. However, high levels of granulocyte-macrophage colony-stimulating factor, interleukin-8, and regulated upon activation, normal T cell expressed and presumably secreted are released by peripheral blood mononuclear cells isolated from patients with severe asthma despite continuous systemic glucocorticoid treatment. We report that these mediators are markedly decreased by pyrrolidinedithiocarbamate, an inhibitor of NF-kappaB activation. To further characterize the persistent NF-kappaB activation in severe asthma, we analyzed the expression of various components of this activation pathway in healthy subjects and in asthmatics with mild controlled, and moderate and severe uncontrolled disease. We found high amounts of phosphorylated IkappaBalpha characterizing the three asthmatic groups. Western blot analyses indicated that in peripheral blood mononuclear cells the IkappaB kinase beta and p65 levels were greater in moderate and severe asthmatics than in normal subjects. Electrophoretic mobility shift assay and immunocytochemistry showed a greater activation status of p65 in severe asthmatics. Our data suggest that exaggerated NF-kappaB activation perpetuates inflammatory mediators production in severe asthma.

[1]  I. Verma,et al.  Severe liver degeneration in mice lacking the IkappaB kinase 2 gene. , 1999, Science.

[2]  B. Make,et al.  Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease. American Thoracic Society. , 1995, American journal of respiratory and critical care medicine.

[3]  R. Roeder,et al.  Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. , 1983, Nucleic acids research.

[4]  L. Teran.  CCL chemokines and asthma. , 2000, Immunology today.

[5]  P. Chanez,et al.  Glucocorticoids induced down‐regulation of glucocorticoid receptor mRNA expression in asthma , 1996, Clinical and experimental immunology.

[6]  I. Adcock,et al.  Effects of inhaled β agonist and corticosteroid treatment on nuclear transcription factors in bronchial mucosa in asthma , 1999, Thorax.

[7]  G. Stark,et al.  Distinct roles of the Ikappa B kinase alpha and beta subunits in liberating nuclear factor kappa B (NF-kappa B) from Ikappa B and in phosphorylating the p65 subunit of NF-kappa B. , 2002, The Journal of biological chemistry.

[8]  F. Delaunay,et al.  A New Function for the C-terminal Zinc Finger of the Glucocorticoid Receptor , 1997, The Journal of Biological Chemistry.

[9]  B Vrugt,et al.  Mucosal inflammation in severe glucocorticoid-dependent asthma. , 1999, The European respiratory journal.

[10]  I. Verma,et al.  IKK1-deficient mice exhibit abnormal development of skin and skeleton. , 1999, Genes & development.

[11]  S. Wenzel,et al.  Evidence that severe asthma can be divided pathologically into two inflammatory subtypes with distinct physiologic and clinical characteristics. , 1999, American journal of respiratory and critical care medicine.

[12]  T. Deerinck,et al.  The IKKβ Subunit of IκB Kinase (IKK) is Essential for Nuclear Factor κB Activation and Prevention of Apoptosis , 1999, The Journal of experimental medicine.

[13]  D. Männel,et al.  Dithiocarbamates as potent inhibitors of nuclear factor kappa B activation in intact cells , 1992, The Journal of experimental medicine.

[14]  J. Bousquet,et al.  Proliferation and activation of bronchial epithelial cells in corticosteroid-dependent asthma. , 2001, The Journal of allergy and clinical immunology.

[15]  J A Gustafsson,et al.  Negative cross-talk between RelA and the glucocorticoid receptor: a possible mechanism for the antiinflammatory action of glucocorticoids. , 1995, Molecular endocrinology.

[16]  J. Bousquet,et al.  Glucocorticoid Receptor α and β in Glucocorticoid Dependent Asthma , 2000 .

[17]  Inder M. Verma,et al.  Severe Liver Degeneration in Mice Lacking the IκB Kinase 2 Gene , 1999 .

[18]  P. Baeuerle.  The inducible transcription activator NF-κB: regulation by distinct protein subunits , 1991 .

[19]  T. Sandström,et al.  Effects of budesonide and formoterol on NF-kappaB, adhesion molecules, and cytokines in asthma. , 2001, American journal of respiratory and critical care medicine.

[20]  G. Stark,et al.  Distinct Roles of the IκB Kinase α and β Subunits in Liberating Nuclear Factor κB (NF-κB) from IκB and in Phosphorylating the p65 Subunit of NF-κB* , 2001, The Journal of Biological Chemistry.

[21]  G. Franzoso,et al.  Mutual regulation of the transcriptional activator NF-kappa B and its inhibitor, I kappa B-alpha. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[22]  I. Adcock,et al.  Activation and localization of transcription factor, nuclear factor-kappaB, in asthma. , 1998, American journal of respiratory and critical care medicine.

[23]  D. Goeddel,et al.  Embryonic Lethality, Liver Degeneration, and Impaired NF-κB Activation in IKK-β-Deficient Mice , 1999 .

[24]  K. Matsushima,et al.  Novel mechanism of glucocorticoid-mediated gene repression. Nuclear factor-kappa B is target for glucocorticoid-mediated interleukin 8 gene repression. , 1994, The Journal of biological chemistry.

[25]  D. Goeddel,et al.  Embryonic lethality, liver degeneration, and impaired NF-kappa B activation in IKK-beta-deficient mice. , 1999, Immunity.

[26]  H. Möllmann,et al.  Pharmacokinetics of Methylprednisolone and Prednisolone After Single and Multiple Oral Administration , 1997, Journal of clinical pharmacology.

[27]  A. Baldwin,et al.  The I kappa B proteins: multifunctional regulators of Rel/NF-kappa B transcription factors. , 1993, Genes & development.

[28]  David M. Rothwarf,et al.  The NF-κB Activation Pathway: A Paradigm in Information Transfer from Membrane to Nucleus , 1999, Science's STKE.

[29]  P. Baeuerle,et al.  Function and activation of NF-kappa B in the immune system. , 1994, Annual review of immunology.

[30]  K. Chung,et al.  Neutrophilic inflammation in severe persistent asthma. , 1999, American journal of respiratory and critical care medicine.

[31]  I. Pavord,et al.  Prednisone-dependent asthma: inflammatory indices in induced sputum. , 1999, The European respiratory journal.

[32]  W. MacNee,et al.  Role of transcription factors in inflammatory lung diseases , 1998, Thorax.

[33]  A. Fauci,et al.  Nuclear factor-kappa B potently up-regulates the promoter activity of RANTES, a chemokine that blocks HIV infection. , 1997, Journal of immunology.

[34]  F. Bach,et al.  Cytokine‐inducible expression in endothelial cells of an I kappa B alpha‐like gene is regulated by NF kappa B. , 1993, The EMBO journal.

[35]  S. Wenzel,et al.  Bronchoscopic evaluation of severe asthma. Persistent inflammation associated with high dose glucocorticoids. , 1997, American journal of respiratory and critical care medicine.

[36]  J. Bousquet,et al.  Glucocorticoid receptors in bronchial epithelial cells in asthma. , 1998, American journal of respiratory and critical care medicine.

[37]  M. Fey,et al.  Glucocorticoids downregulate gene expression of GM-CSF, NAP-1/IL-8, and IL-6, but not of M-CSF in human fibroblasts. , 1992, Blood.

[38]  W. Schaffner,et al.  Rapid detection of octamer binding proteins with 'mini-extracts', prepared from a small number of cells. , 1989, Nucleic acids research.

[39]  J. Buchner.  Supervising the fold: functional principles of molecular chaperones , 1996, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[40]  T. Deerinck,et al.  Abnormal Morphogenesis But Intact IKK Activation in Mice Lacking the IKKα Subunit of IκB Kinase , 1999 .

[41]  P. Baeuerle,et al.  The p65 subunit is responsible for the strong transcription activating potential of NF‐kappa B. , 1991, The EMBO journal.

[42]  W C Greene,et al.  NF-kappa B controls expression of inhibitor I kappa B alpha: evidence for an inducible autoregulatory pathway. , 1993, Science.

[43]  P. Baeuerle.  The inducible transcription activator NF-kappa B: regulation by distinct protein subunits. , 1991, Biochimica et biophysica acta.

[44]  Phillips Yy,et al.  Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease (COPD) and asthma. This official statement of the American Thoracic Society was adopted by the ATS Board of Directors, November 1986. , 1987, The American review of respiratory disease.

[45]  H. Pahl,et al.  Phosphorylation of human I kappa B‐alpha on serines 32 and 36 controls I kappa B‐alpha proteolysis and NF‐kappa B activation in response to diverse stimuli. , 1995, The EMBO journal.

[46]  T. Deerinck,et al.  Abnormal morphogenesis but intact IKK activation in mice lacking the IKKalpha subunit of IkappaB kinase. , 1999, Science.