The nuclear factor kappa-B pathway in airway epithelium regulates neutrophil recruitment and host defence following Pseudomonas aeruginosa infection.

Pseudomonas aeruginosa pneumonia usually results from a deficit of the innate immune system. To investigate whether inflammatory signalling by airway epithelial cells provides a pivotal line of defence against P. aeruginosa infection, we utilized two separate lines of inducible transgenic mice that express a constitutive activator of the nuclear factor kappa-B (NF-kappaB) pathway (IKTA) or a dominant inhibitor of NF-kappaB (DNTA) in airway epithelial cells. Compared with control mice, IKTA mice showed an enhanced host response to P. aeruginosa infection with greater neutrophil influx into the lungs, increased expression of Glu-Leu-Arg-positive (ELR(+)) CXC chemokines macrophage inflammatory protein-2 and keratinocyte chemoattractant (KC), superior bacterial clearance and improved survival at 24 h after infection. Neutrophil depletion abrogated the improvement in host defence identified in IKTA mice. In contrast, DNTA mice showed impaired responses to P. aeruginosa infection with higher bacterial colony counts in the lungs, decreased neutrophilic lung inflammation and lower levels of KC in lung lavage fluid. DNTA mice given recombinant KC at the time of P. aeruginosa infection demonstrated improved neutrophil recruitment to the lungs and enhanced bacterial clearance. Our data indicate that the NF-kappaB pathway in airway epithelial cells plays an essential role in defence against P. aeruginosa through generation of CXC chemokines and recruitment of neutrophils.

Conditional regulation of cyclooxygenase-2 in tracheobronchial epithelial cells modulates pulmonary immunity.

Cyclooxygenase-2 (COX-2) gene expression in the lung is induced in pathological conditions such as asthma and pneumonia; however, the exact impact of COX-2 gene expression in the airway in regulating inflammatory and immunological response in the lung is not understood. To define a physiological role of inducible COX-2 in airway epithelial cells, we developed a novel line of transgenic mice, referred to as CycloOxygenase-2 TransActivated (COTA) mice, that overexpress a COX-2 transgene in the distribution of the CC-10 promoter in response to doxycycline. In response to doxycycline treatment, COX-2 expression was increased in airway epithelium of COTA mice and whole lung tissue contained a three- to sevenfold increase in prostaglandin E(2) (PGE(2)), prostaglandin D(2) (PGD(2)) thromboxane B(2) (TXB(2)) and 6-Keto prostaglandin F(2alpha) (PGF(2alpha)) compared to wild-type and untreated COTA mice. Interestingly, primary mouse tracheal epithelial cells from COTA mice produced only PGE(2) by doxycycline-induced COX-2 activation, providing an indication of cellular specificity in terms of mediator production. In the ovalbumin model, in which doxycycline was given at the sensitization stage, there was an increase in interleukin (IL)-4 level in lung tissue from COTA mice compared to untreated COTA and wild-type mice. In addition, COTA mice that were treated with doxycycline had impaired clearance of Pseudomonas aeruginosa pneumonia compared to wild-type mice. COX-2 gene expression in airway epithelial cells has an important role in determining immunological response to infectious and allergic agents.

Nuclear factor-kappaB (NF-kappaB) regulates proliferation and branching in mouse mammary epithelium.

The nuclear factor-kappaB (NF-kappaB) family of transcription factors has been shown to regulate proliferation in several cell types. Although recent studies have demonstrated aberrant expression or activity of NF-kappaB in human breast cancer cell lines and tumors, little is known regarding the precise role of NF-kappaB in normal proliferation and development of the mammary epithelium. We investigated the function of NF-kappaB during murine early postnatal mammary gland development by observing the consequences of increased NF-kappaB activity in mouse mammary epithelium lacking the gene encoding IkappaBalpha, a major inhibitor of NF-kappaB. Mammary tissue containing epithelium from inhibitor kappaBalpha (IkappaBalpha)-deficient female donors was transplanted into the gland-free mammary stroma of wild-type mice, resulting in an increase in lateral ductal branching and pervasive intraductal hyperplasia. A two- to threefold increase in epithelial cell number was observed in IkappaBalpha-deficient epithelium compared with controls. Epithelial cell proliferation was strikingly increased in IkappaBalpha-deficient epithelium, and no alteration in apoptosis was detected. The extracellular matrix adjacent to IkappaBalpha-deficient epithelium was reduced. Consistent with in vivo data, a fourfold increase in epithelial branching was also observed in purified IkappaBalpha-deficient primary epithelial cells in three-dimensional culture. These data demonstrate that NF-kappaB positively regulates mammary epithelial proliferation, branching, and functions in maintenance of normal epithelial architecture during early postnatal development.

Nuclear factor-kappaB/Rel is apoptogenic in cytokine withdrawal-induced programmed cell death.

In the complex microenvironment where they evolve, developing cells undergo rapid programmed cell death (PCD) when cytokines that support them become limiting. The transcriptional mechanisms of cytokine-withdrawal apoptosis are poorly understood. In this report, we used early B-lymphocyte tissue culture and transgenic cells to demonstrate that nuclear factor-kappaB (NF-kappaB) promotes apoptosis during cytokine withdrawal-induced PCD. In the progenitor B lymphocyte model FL5.12, whereas NF-kappaB has an antiapoptotic function in response to tumor necrosis factor-alpha, cytokine withdrawal causes nuclear translocation of NF-kappaB/cRel, where it is apoptogenic. Inhibition of NF-kappaB activation delays cytokine withdrawal-induced PCD in both FL5.12 and transgenic early B cells. Additionally, reconstituting a bone marrow microenvironment ex vivo abrogates the differential apoptotic pattern between control and transgenic early B cells.

Dynamic expression and activity of NF-kappaB during post-natal mammary gland morphogenesis.

The Rel/NF-kappaB family of transcription factors has been implicated in such diverse cellular processes as proliferation, differentiation, and apoptosis. As each of these processes occurs during post-natal mammary gland morphogenesis, the expression and activity of NF-kappaB factors in the murine mammary gland were examined. Immunohistochemical and immunoblot analyses revealed expression of the p105/p50 and RelA subunits of NF-kappaB, as well as the major inhibitor, IkappaBalpha, in the mammary epithelium during pregnancy, lactation, and involution. Electrophoretic mobility shift assay (EMSA) demonstrated that DNA-binding complexes containing p50 and RelA were abundant during pregnancy and involution, but not during lactation. Activity of an NF-kappaB-dependent luciferase reporter in transgenic mice was highest during pregnancy, decreased to near undetectable levels during lactation, and was elevated during involution. This highly regulated pattern of activity was consistent with the modulated expression of p105/p50, RelA, and IkappaBalpha.

RAG2-/-, I kappa B-alpha-/- chimeras display a psoriasiform skin disease.

Nuclear factor-kappa B, a ubiquitous transcription factor involved in inflammatory and immune responses, is inappropriately activated in several immuno-related diseases, such as allograft rejection, or bronchial asthma. As nuclear factor-kappa B activity is regulated by inhibitor of kappa B (I kappa B), the gene encoding I kappa B-alpha was disrupted in mice to observe the in vivo effects of hyperactivation of nuclear factor-kappa B. I kappa B-alpha-/- mice have constitutive nuclear factor-kappa B activity, severe skin disease, and neonatal lethality. To determine the role of I kappa B-alpha deficient immunocytes in the pathogenesis of the skin disease in adult mice, we utilized the RAG2-deficient blastocyst complementation system to generate RAG2-/-, I kappa B-alpha-/- chimeras. These animals display a psoriasiform dermatitis characterized by hyperplastic epidermal keratinocytes and dermal infiltration of immunocytes, including lymphocytes. Skin grafts transferred from diseased chimeras to recipient nude mice produce hyperproliferative psoriasiform epidermal keratinocytes in response to stimulation. Furthermore, adoptive transfer of lymph node cells from diseased chimeras to RAG2-/- recipient mice recapitulates the disease. Taken together, these characterizations provide evidence to suggest that constitutive activation of nuclear factor-kappa B, due to deficiency in I kappa B-alpha, can invoke severe psoriasiform dermatitis in adult mice. J Invest Dermatol 115:1124-1133 2000

Epithelial nuclear factor-κB signaling promotes lung carcinogenesis via recruitment of regulatory T lymphocytes.

The mechanisms by which chronic inflammatory lung diseases, particularly chronic obstructive pulmonary disease, confer enhanced risk for lung cancer are not well-defined. To investigate whether nuclear factor (NF)-κB, a key mediator of immune and inflammatory responses, provides an interface between persistent lung inflammation and carcinogenesis, we utilized tetracycline-inducible transgenic mice expressing constitutively active IκB kinase ß in airway epithelium (IKTA (IKKß trans-activated) mice). Intraperitoneal injection of ethyl carbamate (urethane), or 3-methylcholanthrene (MCA) and butylated hydroxytoluene (BHT) was used to induce lung tumorigenesis. Doxycycline-treated IKTA mice developed chronic airway inflammation and markedly increased numbers of lung tumors in response to urethane, even when transgene expression (and therefore epithelial NF-κB activation) was begun after exposure to carcinogen. Studies using a separate tumor initiator/promoter model (MCA+BHT) indicated that NF-κB functions as an independent tumor promoter. Enhanced tumor formation in IKTA mice was preceded by increased proliferation and reduced apoptosis of alveolar epithelium, resulting in increased formation of premalignant lesions. Investigation of inflammatory cells in lungs of IKTA mice revealed a substantial increase in macrophages and lymphocytes, including functional CD4+/CD25+/FoxP3+ regulatory T lymphocytes (Tregs). Importantly, Treg depletion using repetitive injections of anti-CD25 antibodies limited excessive tumor formation in IKTA mice. At 6 weeks following urethane injection, antibody-mediated Treg depletion in IKTA mice reduced the number of premalignant lesions in the lungs in association with an increase in CD8 lymphocytes. Thus, persistent NF-κB signaling in airway epithelium facilitates carcinogenesis by sculpting the immune/inflammatory environment in the lungs.

Inhibition of NF-kappa B activity in mammary epithelium increases tumor latency and decreases tumor burden.

The transcription factor nuclear factor kappa B (NF-κB) is activated in human breast cancer tissues and cell lines. However, it is unclear whether NF-κB activation is a consequence of tumor formation or a contributor to tumor development. We developed a doxycycline (dox)-inducible mouse model, termed DNMP, to inhibit NF-κB activity specifically within the mammary epithelium during tumor development in the polyoma middle T oncogene (PyVT) mouse mammary tumor model. DNMP females and PyVT littermate controls were treated with dox from 4 to 12 weeks of age. We observed an increase in tumor latency and a decrease in final tumor burden in DNMP mice compared with PyVT controls. A similar effect with treatment from 8 to 12 weeks indicates that outcome is independent of effects on postnatal virgin ductal development. In both cases, DNMP mice were less likely to develop lung metastases than controls. Treatment from 8 to 9 weeks was sufficient to impact primary tumor formation. Inhibition of NF-κB increases apoptosis in hyperplastic stages of tumor development and decreases proliferation at least in part by reducing Cyclin D1 expression. To test the therapeutic potential of NF-κB inhibition, we generated palpable tumors by orthotopic injection of PyVT cells and then treated systemically with the NF-κB inhibitor thymoquinone (TQ). TQ treatment resulted in a reduction in tumor volume and weight as compared with vehicle-treated control. These data indicate that epithelial NF-κB is an active contributor to tumor progression and demonstrate that inhibition of NF-κB could have a significant therapeutic impact even at later stages of mammary tumor progression.

Mammalian cDNA and prokaryotic reporter sequences silence adjacent transgenes in transgenic mice.

The ovine beta-lactoglobulin gene is expressed efficiently and at high levels in the mammary gland of transgenic mice. In contrast, when this gene is linked to a second gene construct comprising a mammalian cDNA or a CAT reporter sequence it fails to be expressed in the majority of transgenic lines generated. We suggest that mammalian cDNAs and prokaryotic reporter sequences can serve as active foci for gene silencing in the mammalian genome.

Restricted tissue-specific but correct developmental expression mediated by a short human alpha 1AT promoter fragment in transgenic mice.

The tissue-specific and developmental pattern of expression controlled by the proximal promoter (position-348 to +15) derived from the human alpha-1-antitrypsin (h alpha 1AT) gene was studied in transgenic mice. The short promoter segment was linked to the chloramphenicol acetyltransferase (CAT) reporter gene. The transgene showed highly specific expression in the liver and the correct developmental pattern of regulation. Interestingly, this short promoter targets expression to the liver with a greater specificity than that reported for larger alpha 1AT promoter fragments.

Differential serine phosphorylation regulates IkappaB-alpha inactivation.

NF-kappaB is a ubiquitous transcription factor involved in the signal transduction mechanisms of the immune response, acute phase reactions, and viral infections. NF-kappaB proteins are retained in the cytoplasm by association with an inhibitor, termed IkappaB. Studies on the regulation of mammalian IkappaB-alpha have revealed that two amino-terminal conserved phosphoserines are the target sites of incoming signals. We report that the corresponding amino-terminal phosphoserines of avian IkappaB-alpha are phosphorylation targets leading to inactivation of IkappaB-alpha upon stimulation. In addition, we show differential roles for these two serines. Mutation of serine 40 to alanine blocks all stimuli tested (TNF-alpha, phorbol ester, and anti-CD3 and anti-CD28), leading to NF-kappaB activation, while mutation of serine 36 to alanine attenuates only certain transduced signals (PMA, TNF-alpha). These novel findings support the hypothesis that the amino-terminal phosphoserine residues of avian IkappaB-alpha differentially mediate NF-kappaB signal transduction pathways and activation by distinct signals, thereby resulting in the activation NF-kappaB.

Reconstruction of the yeast 2 micron plasmid partitioning mechanism.

The effect of the yeast 2 micron circle encoded REP1 and REP2 gene products on plasmid partitioning and copy number control was analyzed by removing the open reading frames from their normal sequence context and transcriptional control regions and directing their expression using heterologous promoters in [cir0] host strains. Both the REP1 and REP2 gene products are directly required at appropriate levels of expression to reconstitute the 2 microns circle partitioning system in conjunction with REP3 and the origin of replication. The level of expression of REP2 appears to be critical to re-establishing proper partitioning and may also play a role in monitoring and thereby regulating the plasmid copy number. Constitutive expression of the REP1 and REP2 open reading frames using heterologous expression signals can be used to reconstruct efficient plasmid partitioning even in the absence of FLP-mediated plasmid amplification and a functional D open reading frame.

Lymphocytes lacking I kappa B-alpha develop normally, but have selective defects in proliferation and function.

NF-kappaB has been implicated in the development, activation, and function of B and T lymphocytes. We have evaluated the in vivo effects of deletion of IkappaB-alpha, a major inhibitor of NF-kappaB, on lymphocyte development, proliferation, and function. To elucidate the long term role of IkappaB-alpha in lymphocytes, fetal liver cells of 14.5-day-old IkappaB-alpha(-/-) or wild-type embryos were transplanted into irradiated recombinase-activating gene-2-deficient mice. Within 4 wk, the IkappaB-alpha(-/-) fetal liver cells reconstitute mature B and T cell populations in the recipients comparable to those produced by wild-type fetal liver cells. However, the proliferative responses of IkappaB-alpha(-/-) B cells are enhanced, whereas those of IkappaB-alpha(-/-) T cells are reduced. The levels of IgG1, IgG2a, IgA, and IgE produced by IkappaB-alpha(-/-) B cells are elevated relative to those produced by IkappaB-alpha(+/+) or IkappaB-alpha(+/-). Moreover, the specific immune responses to OVA and the generation of germinal centers are impaired in recipients of IkappaB-alpha(-/-) fetal liver cells. These results indicate that IkappaB-alpha plays a vital role in signal transduction pathways regulating lymphocyte proliferation and also in the production of specific Ig isotypes.

Mesenchymal expression of nuclear factor-kappaB inhibits epithelial growth and branching in the embryonic chick lung.

It is becoming increasingly recognized that the ubiquitous, inducible transcription factor nuclear factor-kappaB (NF-kappaB) is involved in developmental processes. For example, NF-kappaB acts as a mediator of epithelial-mesenchymal interactions in the developing chick limb. We investigated the role of NF-kappaB in directing the branching morphogenesis of the developing chick lung, a process which relies on epithelial-mesenchymal communication. High level expression of relA was found in the mesenchyme surrounding the nonbranching structures of the lung but was not detected either in the mesenchyme surrounding the branching structures of the distal lung or in the developing lung epithelium. Specific inhibition of mesenchymal NF-kappaB in lung cultures resulted in increased epithelial budding. Conversely, expression of a trans-dominant activator of NF-kappaB in the lung mesenchyme repressed budding. Ectopic expression of RelA was sufficient to inhibit the ability of the distal mesenchyme to induce epithelial bud formation. Cellular proliferation in the mesenchyme was inhibited by hyperactivation of NF-kappaB in the mesenchyme of lung cultures. Interestingly, increased NF-kappaB activity in the mesenchyme also decreased the proliferation of the associated epithelium, while inhibition of NF-kappaB activity increased cellular proliferation in lung cultures. Expression patterns of several genes which are known to influence lung branching morphogenesis were altered in response to changes in mesenchymal NF-kappaB activity, including fgf10, bmp-4, and tgf-beta1. Thus NF-kappaB represents the first transcription factor reported to function within the lung mesenchyme to limit growth and branching of the adjacent epithelium.

Inhibition of NF-kappaB activity results in disruption of the apical ectodermal ridge and aberrant limb morphogenesis.

In Drosophila, the Dorsal protein establishes the embryonic dorso-ventral axis during development. Here we show that the vertebrate homologue of Dorsal, nuclear factor-kappa B (NF-kappaB), is vital for the formation of the proximo-distal organizer of the developing limb bud, the apical ectodermal ridge (AER). Transcription of the NF-kappaB proto-oncogene c-rel is regulated, in part, during morphogenesis of the limb bud by AER-derived signals such as fibroblast growth factors. Interruption of NF-kappaB activity using viral-mediated delivery of an inhibitor results in a highly dysmorphic AER, reduction in overall limb size, loss of distal elements and reversal in the direction of limb outgrowth. Furthermore, inhibition of NF-kappaB activity in limb mesenchyme leads to a reduction in expression of Sonic hedgehog and Twist but derepresses expression of the bone morphogenetic protein-4 gene. These results are the first evidence that vertebrate NF-kappaB proteins act to transmit growth factor signals between the ectoderm and the underlying mesenchyme during embryonic limb formation.

Multiorgan nuclear factor kappa B activation in a transgenic mouse model of systemic inflammation.

We utilized a line of transgenic mice expressing Photinus luciferase complementary DNA (cDNA) under the control of a nuclear factor kappa B (NF-kappaB)-dependent promoter (from the 5' human immunodeficiency virus-1 [HIV-1] long terminal repeat) to examine the role of NF-kappaB activation in the pathogenesis of systemic inflammation induced by bacterial endotoxin (lipopolysaccharide [LPS]). After intraperitoneal injection of E. coli LPS, these mice displayed a time- and dose-dependent, organ-specific pattern of luciferase expression, showing that NF-kappaB-dependent gene transcription is transiently activated in multiple organs by systemic LPS administration. Luciferase expression in liver could be specifically blocked by intravenous administration of replication-deficient adenoviral vectors expressing a dominant inhibitor of NF-kappaB (IkappaB-alphaDN), confirming that luciferase gene expression is a surrogate marker for NF-kappaB activation in this line of mice. After treatment with intraperitoneal LPS, the mice were found to have increased lung tissue messenger RNA (mRNA) expression of a variety of cytokines that are thought to be NF-kappaB-dependent, as well as elevated serum concentrations of presumed NF-kappaB-dependent cytokines. In lung tissue homogenates, a close correlation was identified between luciferase activity and KC levels. These studies show that systemic treatment with LPS orchestrates a multiorgan NF-kappaB-dependent response that likely regulates the pathobiology of systemic inflammation.