Inhibition of inducible nitric oxide synthase expression by interleukin-4 and interleukin-13 in human lung epithelial cells.

Oral feeding of proteins causes peripheral T-cell tolerance, as revealed by reduced delayed-type hypersensitivity (DTH) reactivity after immunization. This type of tolerance can be due both to passive T-cell anergy and active immunosuppression. Using ovalbumin-fed mice we studied whether putatively immunostimulatory cytokines could break this state of mucosal tolerance. Cytokines were administered locally at the site of attempted sensitization. It was found that neither interleukin-2 (IL-2), interferon-gamma (IFN-gamma) nor granulocyte-macrophage colony-stimulating factor (GM-CSF) could restore the response to immunization. In contrast, local administration of IL-12 at the site of attempted immunization resulted in full recovery of DTH reactivity. The dichotomy between the two Th1 stimulatory cytokines IFN-gamma and IL-12 was also reflected by different effects on ovalbumin-specific antibody isotypes. Although both IFN-gamma and IL-12 downregulated serum IgG1-levels in tolerant mice, suggesting decreased ovalbumin-specific Th2 function, only local administration of IL-12 led to increased serum IgG2a levels. These results support the view that potentiation of Th1 effector function is critical for reversal of mucosal tolerance.     

Contrasting effects of IL-4, IL-10 and corticosteroids on RANTES production by human monocytes

RANTES is a chemokine produced in delayed-type hypersensitivity(DTH) and allergic reactions, in which it may contribute tothe recruitment of immune cells. Macrophages participate inthe cellular infiltration in both conditions and they representa potent source of RANTES. To understand the regulation of RANTESproduction by human monocytes, we analyzed the effect of cytokinesand of corticosteroids on this production. We showed that IFN-and tumor necrosis factor (TNF)- cooperated to induce RANTESproduction by monocytes. N-acetylcysteine inhibited this effect,indicating that reactive oxygen intermediates are required forRANTES production. Both IL-10 and corticosteroids antagonizedthe stimulating effect of IFN- and TNF- on RANTES production.In contrast, IL-4 had no effect on IFN--induced RANTES productionand it potentiated the positive effect of TNF- on this production.Thus, the deactivating properties of IL-10 and corticosteroidson macrophage functions include RANTES production, and thismay contribute to the immuno-suppressive effect of both compoundsin DTH and allergic reactions. In contrast, IL-4 has an oppositeeffect on RANTES production and this property may contributeto cell recruitment in allergic reactions. Therefore, althoughIL-10 and IL-4 belong to the T_h2 family of cytokines, they candisplay distinct functions in immune reactions.     

Expression of heme oxygenase in human airway epithelial cells

Elevated levels of carbon monoxide (CO) are found in the exhaled breath of patients with inflammatory diseases such as asthma and cystic fibrosis. Endogenous CO is derived from heme oxygenase (HO) (EC 1.14.99.3), which catabolizes heme-producing CO and biliverdin. There are three isoforms of HO: HO-1 is inducible by inflammatory cytokines and oxidants, including nitric oxide (NO), whereas HO-2 and HO-3 are expressed constitutively. Primary airway epithelial cells were treated with either 50 ng/ml interleukin-1 beta, tumor necrosis factor-alpha, and interferon-gamma (cytomix), or the NO donor NOC-18 for up to 24 h. Cytomix-induced HO-1 expression peaked at 4 h, returning to baseline by 24 h, whereas HO-2 expression remained unchanged. This increase in HO-1 expression could not be explained by an increase in NO production as inducible NO synthase expression increased between 12 and 24 h. However, the NO donor NOC-18 (500 microM) increased HO-1 expression twofold and HO activity 25-fold, whereas cytomix treatment increased HO activity eightfold. NO induction of HO-1 was not mediated via guanylyl cyclase and was not attenuated by 1 microM dexamethasone, although dexamethasone increased HO-2 protein. Therefore, airway epithelial cells express HO-2 and can express HO-1; thus, the epithelium may be a source of increased CO in airway diseases.     

Inhibition of interleukin-8 expression by dexamethasone in human cultured airway epithelial cells.

Interleukin-8 (IL-8) is a neutrophil chemotactic factor expressed in many cell types, including human airway epithelial cells (HAEC). Inhaled corticosteroids are now used increasingly early in the treatment of airway inflammation such as in asthma, and directly interact with HAEC at relatively high concentrations. We have investigated the effect of dexamethasone on IL-8 expression in primary cultured HAEC obtained from transplantation donors. Northern blot analysis was used to measure IL-8 mRNA levels in HAEC, and radioimmunoassay was used to measure IL-8 protein in culture supernatant fluids. We demonstrated that IL-8 was expressed by primary cultured HAEC and that this was enhanced by IL-1 beta and tumour necrosis factor-alpha stimulation, but not by IL-6 or lipopolysaccharide. Dexamethasone suppressed IL-8 mRNA expression and protein synthesis dose-dependently in both resting and stimulated HAEC. The half-life of IL-8 mRNA determined in the presence of actinomycin D was less than 1 hr, and dexamethasone preincubation had no effect on mRNA stability. These results support the view that HAEC may play an important role in the pathogenesis of airway inflammatory diseases, and that glucocorticosteroids may exert their anti-inflammatory effects by blocking IL-8 gene expression and generation in these cells.     

Effects of topical corticosteroids on inflammatory mediator-induced eicosanoid release by human airway epithelial cells.

BACKGROUND: Airway epithelial cells are among the first cells to come in contact with aerosolized corticosteroids. However, the relative potencies and time course of action of the several commonly used aerosolized corticosteroids on eicosanoid production by airway epithelial cells are unknown. OBJECTIVES: This study compared the effects of fluticasone, budesonide, and triamcinolone on eicosanoid output by human airway epithelial cells in vitro. We also determined the spectrum of eicosanoids affected and the mechanism for corticosteroid action. METHODS: Cultured BEAS-2B airway epithelial cells (a transformed cell line) were exposed to corticosteroids (1 nmol/L to 1 micromol/L) for 2 to 48 hours and then assayed for basal- and bradykinin (BK)-stimulated eicosanoid output. The eicosanoid profile was identified by HPLC in tritiated arachidonic acid prelabelled cells, and PGE2, the major eicosanoid product, was quantitated by RIA. The effect of corticosteroids on the immunoreactivity of key proteins involved in eicosanoid metabolism (ie, cyclooxygenase [COX], phospholipase A2 [PLA2], and Clara cell protein, a PLA2 inhibitor) was determined by Western blotting. RESULTS: Eicosanoid output was largely confined to prostaglandins with values of 5 +/- 2 and 82 +/- 35 ng PGE2/10(6) cells for basal- and BK stimulation, respectively (n = 8). All 3 corticosteroids inhibited basal- and BK-induced PGE2 output in a dose- and time-dependent manner. Fluticasone and budesonide completely eliminated PGE2 output in nanomolar concentrations in contrast to triamcinolone, which required micromolar concentration. The rank order of potency was: fluticasone = budesonide > triamcinolone. The time course of action for PGE2 inhibition also differed, with budesonide acting more slowly than the other 2 corticosteroids (P = .04). All 3 corticosteroids markedly reduced COX2 with little effect on COX1, cPLA2 (Type IV), or iPLA2 (Type VI) immunoreactivity or their relative distribution in cytosol versus membrane fractions. Clara cell protein immunoreactivity was undetectable in control and corticosteroid-treated cell lysates. CONCLUSION: These results show that in a human airway epithelial cell line, the 3 inhaled corticosteroids commonly used to treat asthma differ in onsets of action as inhibitors of prostaglandin synthesis and vary considerably in potency. All 3 corticosteroids act mechanistically in similar fashion by inhibiting COX2 synthesis.     

A20 inhibits toll-like receptor 2- and 4-mediated interleukin-8 synthesis in airway epithelial cells

The zinc finger protein A20 is encoded by an immediate early response gene and acts as an inhibitor of nuclear factor (NF)-kappaB-dependent gene expression induced by different stimuli, including tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta). Toll-like receptor 2 (TLR2) and TLR4 have been found to transduce, respectively, peptidoglycan (PGN) and lipopolysaccharide (LPS) signals for the activation of NF-kappaB and the production of inflammatory cytokines. Here, we have examined the role of A20 in TLR-mediated NF-kappaB-dependent gene expression in human airway epithelial cells (AECs). Stimulation with LPS and PGN resulted in a significant increase in the level of A20 mRNA in primary cultured AECs and in NCI-H292 AECs. LPS and PGN induced activation of the IL-8 promoter both in NCI-H292 AECs and in HEK293 cells expressing either TLR2 or TLR4 plus MD-2. Dominant-negative myeloid differentiation protein and a mutant form of IkappaBalpha attenuated this PGN- or LPS-induced activation of the IL-8 promoter. Furthermore, overexpression of A20 inhibited activation of both NF-kappaB and the IL-8 promoter by PGN or LPS in these cells. Taken together, our results suggest that A20 may function as a negative regulator of TLR-mediated inflammatory responses in the airway, thereby protecting the host against harmful overresponses to pathogens.     

Interleukin-13 mediates a fundamental pathway for airway epithelial mucus induced by CD4 T cells and interleukin-9

Mucus hyperproduction in asthma results from Th2-induced airway inflammation. Controversy exists about the precise mechanism of this Th2 effect. Although we showed that mucus can be induced by Th2 cells in the absence of interleukin (IL)-4, IL-5, eosinophils, and mast cells, but not without IL-4Ralpha signaling, others demonstrated that IL-4 and IL-9 can directly stimulate airway epithelial mucus. Using a system in which in vitro-generated T cell receptor transgenic Th2 cells are transferred into recipient mice and activated in the respiratory tract with inhaled antigen, we now show that CD4 Th cells can stimulate mucus only through a common, IL-13-mediated pathway. All Th cytokines depend on IL-13 for this effect and IL-13 acts, not through intermediate inflammatory cells, but on structural cells within the lung, likely the airway epithelium itself. The potency of IL-13 is shown, requiring its complete blockade for a significant reduction in mucus production. We show that mucus induction by Th2 cells does not require nuclear factor-kappaB, unlike mucins induced by gram-negative infection. These studies define in vivo pathways that lead to mucus induction and indicate that, whereas IL-13 mediates a dominant pathway for CD4 Th induced inflammation, other inflammatory stimuli activate the epithelium to produce mucus by different pathways.     

Differential modulation of interleukin-4 and interleukin-13 secretion from human peripheral blood mononuclear cells.

Interleukin-4 (IL-4) and IL-13 which have been implicated in the pathogenesis of atopic reactions elicit many of the same biologic responses. Therefore, time- and stimulus-dependent differences in the regulation of IL-4 and IL-13 production could be of relevance to their biological effects. In this study we tested the hypothesis that stimulation of peripheral blood mononuclear cells (PBMCs) with different inducers of cell activation would result in a differential expression of IL-4 and IL-13. For this purpose, PBMCs of nonatopic volunteers were incubated with phytohaemagglutinin (PHA), phorbolester (PMA), calcium ionophore A23187, or IL-3. The effect of these stimuli on IL-4 and IL-13 production were analysed by enzyme-linked immunoassay (ELISA) in supernatants of cultured PBMCs. Incubation of PBMCs with A23167 and PHA induced both a dose- and time-dependent increase in IL-4 and IL-13 release. A23187 induced concentrations of IL-4 were higher than those of IL-13 whereas IL-4 release following stimulation with PHA was considerably higher for IL-13 compared to IL-4. In contrast, there was a selective increase in IL-13 but not IL-4 concentrations following stimulation of PBMCs with PMA and IL-3 in vitro. In conclusion in this study evidence is provided that IL-4 and IL-13 production are regulated differently which might explain their functional redundancy.     

Mast cells and T cells in Kimura''s disease express increased levels of interleukin-4, interleukin-5, eotaxin and RANTES

BACKGROUND: Kimura's disease (KD) is a chronic inflammatory disorder characterized by tumours in the head and neck region, enlarged lymph nodes, increased eosinophil counts andhigh serum IgE. Mast cells are known to play a central role in IgE-mediated allergic diseases through the release of inflammatory mediators like IL-4, IL-5 and chemokines. We hypothesized that mast cells may play a role in the pathogenesis of KD by regulating eosinophilic infiltration and IgE synthesis. OBJECTIVE: In order to investigate the role of mast cells in the pathogenesis of KD, we examined the expression of cytokines/chemokines in the lesions of KD. METHODS: We examined the number of tryptase+ cells, EG2+ cells, CD3+ cells, IL-4+ cells, IL-5+ cells, eotaxin+ cells, RANTES+ cells and CCR3+ cells in five specimens of KD versus normal tissues by immunohistochemistry. The sources of IL-4, IL-5, eotaxin and RANTES and the expression of CCR3 were examined by immunostaining of serial sections with antibodies to IL-4, IL-5, eotaxin, RANTES and CCR3, and antibodies to tryptase, ECP (EG2) and CD3. RESULTS: Mast cells, activated eosinophils, T cells, IL-4+ cells, IL-5+ cells, eotaxin+ cells, RANTES+ cells and CCR3+ cells were all increased in the lesions of KD as compared with those in normal tissue. Mast cells and T cells were the major source of IL-4, whereas mast cells, T cells and activated eosinophils were the main source of IL-5. Mast cells, T cells and activated eosinophils were the main source of eotaxin and RANTES. CONCLUSIONS: The number of IL-4, IL-5, eotaxin and RANTES-expressing mast cells and T cells were increased in the lesions of KD. As mast cells are lesional resident cells, these cells may play an important role in the pathogenesis of KD by regulating IgE synthesis and orchestrating eosinophilic infiltration.     

Expression and regulation of inducible nitric oxide synthase from human primary airway epithelial cells.

Elevated levels of exhaled nitric oxide are seen in inflammatory airway diseases such as asthma, but the cellular source remains unknown. This study investigated whether human airway epithelial cells express inducible nitric oxide synthase (iNOS). Human bronchial epithelial cells stimulated with 50 ng/ml interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma express iNOS mRNA, protein and increased nitrite in the cell culture media, which was inhibited by the selective iNOS inhibitor 1400W. Cells derived from subjects with asthma produced less nitrite than cells from normal subjects (6.59 +/- 0.99 microM nitrite, n = 15 versus 3.89 +/- 0.42 microM nitrite, n = 20; P < 0.05). This was not attributed to steroid treatment of subjects with asthma because there was no difference in the amount of nitrite released from steroid-naive and steroid-treated cells (3.51 +/- 0.46 versus 4.27 +/- 0.7 microM nitrite, n = 10). Neither dexamethasone nor budesonide inhibited iNOS mRNA induction, protein expression, or nitrite accumulation. The cells were not steroid insensitive because steroids inhibited GM-CSF release. Therefore, although these cells express iNOS under inflammatory conditions, they do not appear to be regulated directly by glucocorticosteroids.     

Expression of keratin 13 in human epithelial neoplasms

Summary The distribution of the 52 kDa keratin 13 was evaluated immunohistochemically, using the AE8 monoclonal antibody. Various squamous and transitional cell epithelial lesions and representative control tissues were studied. This antibody performed adequately in formalin-fixed and paraffin-embedded tissue, but like keratin immunohistochemistry in general, required protease pretreatment. Keratin 13 was found consistently in the suprabasal layers of squamous epithelia of oral cavity, tonsils, larynx, esophagus, lower female genital tract, and transitional urothelium, but it was absent in the epidermis. Generally, various forms of squamous metaplasia were AE8-positive. In dysplasia, AE8 reactivity was considerably decreased or even absent despite the presence of apparent suprabasal maturation. In differentiated squamous cell carcinomas, AE8 immunoreactivity was usually limited to a few cells in the center of the keratinized foci. However, in 10% of squamous cell carcinomas, a significant number of tumor cells was positive. Only well-differentiated urothelial carcinomas showed AE8 immunoreactivity, while poorly differentiated tumors were negative. Interestingly, a Brenner's tumor showed a high number of AE8-positive epithelial cells. Our results show that the expression of keratin 13, as immunohistochemically determined by AE8 antibody, is significantly down-regulated in squamous cell malignancies. Its possible value as an adjunct to diagnosis of dysplasia should be investigated further.