IL-32 expression in the airway epithelial cells of patients with Mycobacterium avium complex lung disease.

Lung disease due to Mycobacterium avium complex (MAC) organisms is increasing. A greater understanding of the host immune response to MAC organisms will provide a foundation to develop novel therapies for these recalcitrant infections. IL-32 is a newly described pro-inflammatory cytokine that enhances host immunity against various microbial pathogens. Cytokines that induce IL-32 such as interferon-gamma, IL-18, IL-12 and tumor necrosis factor-alpha are of considerable importance to mycobacterial immunity. We performed immunohistochemistry and morphometric analysis to quantify IL-32 expression in the lungs of 11 patients with MAC lung disease and 10 controls with normal lung tissues. After normalizing for basement membrane length, there was a profound increase in IL-32 expression in the airway epithelial cells of the MAC-infected lungs compared with controls. Following normalization for alveolar surface area, there was a trend toward increased IL-32 expression in type II alveolar cells and alveolar macrophages in the lungs of MAC patients. Human airway epithelial cells (BEAS-2B) infected with M. avium produced IL-32 by a nuclear factor-kappa B-dependent mechanism. In both BEAS-2B cells and human monocyte-derived macrophages, exogenous IL-32γ significantly reduced the growth of intracellular M. avium. This finding was corroborated by an increase in the number of intracellular M. avium recovered from THP-1 monocytes silenced for endogenous IL-32 expression. The anti-mycobacterial effect of IL-32 may be due, in part, to increased apoptosis of infected cells. These findings indicate that IL-32 facilitates host defense against MAC organisms but may also contribute to the airway inflammation associated with MAC pulmonary disease.

Pulmonary capillary hemangiomatosis: an immunohistochemical analysis of vascular remodeling.

QUESTION OF STUDY: Pulmonary capillary hemangiomatosis (PCH) is an extremely rare cause of severe pulmonary hypertension. It is characterized histologically by exuberant proliferation of capillaries that often invade alveolar septae, bronchial walls, and pleura. Expression of vascular remodeling markers in PCH is not known. MATERIALS/PATIENTS AND METHODS: Using antibodies directed against vascular remodeling markers known to be abnormally expressed in plexiform lesions of idiopathic pulmonary hypertension, we performed the first detailed immunohistochemical analysis of the lungs in a patient with PCH. - RESULTS: As in plexiform lesions, the PCH lesions have increased expression of markers associated with cellular proliferation and angiogenesis such as vascular endothelial growth factor and MiB-1. In contrast to plexiform lesions, the PCH lesions retain markers of cell growth suppression such as peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and caveolin-1. ANSWER TO QUESTION: This study suggests that the aberrant endothelial cells that lead to the characteristic lesions of PCH and idiopathic pulmonary hypertension are distinct.

Morphometric analysis of Th(1) and Th(2) cytokine expression in human pulmonary tuberculosis.

SETTING: Following infection with Mycobacterium tuberculosis, host cytokine responses influence disease manifestation. Differences in cytokine expression likely determine whether tuberculosis (TB) progresses, resolves, or becomes latent. In particular, the balance between Th(1) and Th(2) cytokine responses influences the expression of disease in individuals with pulmonary TB. OBJECTIVE AND DESIGN: Since the cytokine microenvironment in pulmonary TB remains suboptimally defined, we utilized quantitative immunohistochemistry to compare the expression of Th(1) cytokines [interferon-gamma (IFNgamma) and interleukin-12 (IL-12)] and Th(2) cytokines [IL-4, IL-10, transforming growth factor-beta (TGFbeta)] in surgically resected lungs of seven TB patients and four control subjects. We also quantified IFNgamma-inducible protein 10 (IP-10) expression, a CXC chemokine for macrophages and T cells. RESULTS: Morphometric analyses revealed increased IFNgamma, IL-12, IP-10, and TGFbeta in granulomas and in pneumonitis areas of TB lungs. In contrast, IL-10 and IL-4 expressions were globally reduced in TB lung tissues compared to controls. CONCLUSION: Th(1) cytokines and TGFbeta are increased while Th(2) cytokines are decreased in well-formed pulmonary granulomas of TB patients compared to controls.

Inhibition of nuclear factor-kappa B activation decreases survival of Mycobacterium tuberculosis in human macrophages.

Nuclear factor-kappa B (NFκB) is a ubiquitous transcription factor that mediates pro-inflammatory responses required for host control of many microbial pathogens; on the other hand, NFκB has been implicated in the pathogenesis of other inflammatory and infectious diseases. Mice with genetic disruption of the p50 subunit of NFκB are more likely to succumb to Mycobacterium tuberculosis (MTB). However, the role of NFκB in host defense in humans is not fully understood. We sought to examine the role of NFκB activation in the immune response of human macrophages to MTB. Targeted pharmacologic inhibition of NFκB activation using BAY 11-7082 (BAY, an inhibitor of IκBα kinase) or an adenovirus construct with a dominant-negative IκBα significantly decreased the number of viable intracellular mycobacteria recovered from THP-1 macrophages four and eight days after infection. The results with BAY were confirmed in primary human monocyte-derived macrophages and alveolar macrophages. NFκB inhibition was associated with increased macrophage apoptosis and autophagy, which are well-established killing mechanisms of intracellular MTB. Inhibition of the executioner protease caspase-3 or of the autophagic pathway significantly abrogated the effects of BAY. We conclude that NFκB inhibition decreases viability of intracellular MTB in human macrophages via induction of apoptosis and autophagy.

Mycobacterium tuberculosis increases IP-10 and MIG protein despite inhibition of IP-10 and MIG transcription.

Mycobacterium tuberculosis (MTB) has evolved methods to evade interferon-gamma (IFNγ) mediated protection. We sought to determine the effect of MTB infection on expression of IFNγ-inducible Protein 10 (IP-10) and Monokine Induced by IFNγ (MIG), two chemokines involved in host defense. MTB infection of THP-1 cells inhibited the transcription of IP-10 and MIG. A key mechanism for the inhibition is the disruption of binding of Signal Transduction and Activation of Transcription 1-alpha (STAT1α) to its cis-regulatory element, present in the 5'-flanking region of both IP-10 and MIG promoters. Use of inhibitors specific to the nuclear factor-kappa B (NFκB) and p38 mitogen-activated protein kinase (p38(mapk)) implicate these two signaling pathways in mediating the effect of MTB on the inhibition of IFNγ-induced IP-10 and MIG mRNA expression. Interestingly, despite transcriptional inhibition, there was an unexpected increase in IP-10 and MIG protein production after combined IFNγ and MTB stimulation. MTB also inhibited IFNγ induction of MIG mRNA but augmented MIG protein in primary human monocyte-derived macrophages. The synergy between MTB and IFNγ in the induction of IP-10 and MIG protein appears to involve novel post-transcriptional events that incorporates non-canonical functions of NFκB and p38(mapk).

Induction of IL-8 by Mycoplasma pneumoniae membrane in BEAS-2B cells.

Mycoplasma pneumoniae is an extracellular pathogen, residing on mucosal surfaces of the respiratory and genital tracts. The lack of cell walls in mycoplasmas facilitates the direct contact of the bacterial membrane with the host cell. The cell membrane of mycoplasma is the major inducer of the host pathogenic response. Airway diseases caused by M. pneumoniae include bronchiolitis, bronchitis, and rarely bronchiectasis. In such disorders, neutrophil infiltration of the airways predominates. More recently, M. pneumoniae has been implicated in the pathogenesis of asthma. Epithelial cells play an important role in recruiting inflammatory cells into the airways. Since M. pneumoniae infection of human epithelial cells induces expression of IL-8-a potent activator of neutrophils-we investigated the signaling and transcriptional mechanisms by which mycoplasma membrane induces expression of this chemokine. In BEAS-2B human bronchial epithelial cells, mycoplasma membrane fraction (MMF) increased IL-8 mRNA and protein production. Activation of the transcriptional elements activating protein-1, nuclear factor-interleukin-6, and particularly NF-kappaB are essential for optimal IL-8 production by MMF. The mitogen-activated protein kinases individually played a modest role in MMF-induced IL-8 production. Toll-like receptor-2 did not play a significant role in MMF-induction of IL-8. Antibiotics with microbicidal activity against M. pneumoniae are also known to have anti-inflammatory effects. Whereas clarithromycin, azithromycin, and moxifloxacin individually were able to inhibit TNF-alpha-induction of IL-8, each failed to inhibit MMF-induction of IL-8.

Alpha-1-antitrypsin (AAT) anomalies are associated with lung disease due to rapidly growing mycobacteria and AAT inhibits Mycobacterium abscessus infection of macrophages.

Rapidly growing mycobacteria (RGM) are ubiquitous in the environment but cause lung disease in only a fraction of exposed individuals. This variable susceptibility to disease implies vulnerability to RGM infection due to weakness in host defense. Since most persons who contract RGM lung disease have no known host defense defect, it is likely that uncharacterized host deficiencies exist that predispose to RGM infection. Alpha-1-antitrypsin (AAT) is a host factor that may protect individuals from respiratory infections. Therefore, we assessed AAT protein anomalies as a risk factor for RGM lung disease. In a cohort of 100 patients with RGM lung disease, Mycobacterium (M.) abscessus was the most prevalent organism, isolated in 64 (64%) subjects. Anomalous AAT proteins were present in 27% of the cohort, which is 1.6 times the estimated prevalence of anomalous AAT proteins in the United States population (p=0.008). In in vitro studies, both AAT and a synthetic inhibitor of serine proteases suppressed M. abscessus infection of monocyte-derived macrophages by up to 65% (p<0.01). AAT may be an anti-RGM host-defense factor, and anomalous AAT phenotypes or AAT deficiency may constitute risk factors for pulmonary disease due to RGM.

Increased NaCl-induced interleukin-8 production by human bronchial epithelial cells is enhanced by the DeltaF508/W1282X mutation of the cystic fibrosis transmembrane conductance regulator gene.

A satisfactory model describing the airway surface fluid (ASF) in the airways of persons with cystic fibrosis (CF) remains to be established due to theoretical challenges to both the "Hydration Hypothesis" and the "Salt Hypothesis." Irrespective of these models, inhaled hypertonic saline is often used to facilitate clearance of inspissated secretions. Hypertonicity induces interleukin-8 (IL-8) expression, a potent chemokine for neutrophils. The objectives of this study were: (i) to determine the relative contribution of three potential cis-regulatory elements in the regulation of NaCl-induced IL-8 production in BEAS-2B human bronchial epithelial cells, (ii) to compare NaCl-induced IL-8 expression in IB3-1 bronchial epithelial cells, which have the DeltaF508/W1282X mutation of the CF transmembrane conductance regulator (CFTR) gene, with that in C38 cells, which are IB3-1 cells stably transfected with a truncated but functional CFTR gene, and (iii) to compare equal osmolar concentrations of NaCl and D-sorbitol in the induction of IL-8 in all three cell types. In human bronchial epithelial cells, binding sites for NFkappaB, AP-1, and NF-IL6 in the 5'-flanking region of the IL-8 promoter are necessary for optimal NaCl induction of IL-8. Human bronchial epithelial cells with the DeltaF508/W1282X CFTR mutation produce an exaggerated amount of basal and NaCl-induced IL-8.

Role of the NF-kappaB signaling pathway and kappaB cis-regulatory elements on the IRF-1 and iNOS promoter regions in mycobacterial lipoarabinomannan induction of nitric oxide.

Nitric oxide (NO(.)) produced by inducible nitric oxide synthase (iNOS) is an important host defense molecule against Mycobacterium tuberculosis in mononuclear phagocytes. The objective of this study was to determine the role of the IkappaBalpha kinase-nuclear factor kappaB (IKK-NF-kappaB) signaling pathway in the induction of iNOS and NO(.) by a mycobacterial cell wall lipoglycan known as mannose-capped lipoarabinomannan (ManLAM) in mouse macrophages costimulated with gamma interferon (IFN-gamma). NF-kappaB was activated by ManLAM as shown by electrophoretic mobility shift assay, by immunofluorescence of translocated NF-kappaB in intact cells, and by a reporter gene driven by four NF-kappaB-binding elements. Transduction of an IkappaBalpha mutant (Ser32/36Ala) significantly inhibited NO(.) expression induced by IFN-gamma plus ManLAM. An activated SCF complex, a heterotetramer (Skp1, Cul-1, beta-TrCP [F-box protein], and ROC1) involved with ubiquitination, is also required for iNOS-NO(.) induction. Two NF-kappaB-binding sites (kappaBI and kappaBII) present on the 5'-flanking region of the iNOS promoter bound ManLAM-induced NF-kappaB similarly. By use of reporter constructs in which one or both sites are mutated, both NF-kappaB-binding positions were essential in iNOS induction by IFN-gamma plus ManLAM. IFN-gamma-induced activation of the IRF-1 transcriptional complex is a necessary component in host defense against tuberculosis. Although the 5'-flanking region of the IRF-1 promoter contains an NF-kappaB-binding site and ManLAM-induced NF-kappaB also binds to this site, ManLAM was unable to induce IRF-1 expression. The influence of mitogen-activated protein kinases on IFN-gamma plus ManLAM induction of iNOS-NO(.) is not due to any effects on ManLAM induction of NF-kappaB.