Gene and protein expression of protease-activated receptor 2 in structural and inflammatory cells in the nasal mucosa in seasonal allergic rhinitis.

BACKGROUND: Protease-activated receptor 2 (PAR 2) has been shown to be responsible for trypsin and mast cell tryptase-induced airway inflammation. Here, the present study aimed to explore the expression of PAR 2 in the nasal mucosa of seasonal allergic rhinitis (SAR). METHODS: Study subjects were recruited for the study by medical history, physical examination and laboratory screening tests. Using immunohistochemistry, laser-assisted cell picking and subsequently real-time PCR, nasal mucosa biopsies of SAR patients were investigated for PAR 2 gene and protein expression in complex tissues of the nasal mucosa. RESULTS: Gene and protein expression of PAR 2 was firstly detected in nasal mucosa of SAR patients. The relative gene expression level of PAR 2 was significantly increased in complex tissues of the nasal mucosa of SAR (6.21+/-4.02 vs. controls: 1.38+/-0.86, P=0.004). Moreover, PAR 2 mRNA expression in epithelial cells (SAR: 4.78+/-4.64 vs. controls: 0.84+/-0.61, P=0.003) but not in mucus (SAR: 1.51+/-1.15 vs. controls: 1.35+/-1.02, P=0.78) and endothelial cells (SAR: 1.20+/-0.57 vs. controls: 1.73+/-1.30, P=0.5) was found to be significantly changed in the nasal mucosa in SAR. Using double immunohistochemistry the present study demonstrated that the total numbers of mast cells (P=0.0003) and eosinophils (P=0.03) and the numbers of eosinophils expressing PAR 2 (P=0.006) were significantly elevated in the nasal mucosa of SAR compared with the controls. CONCLUSION: The abundant presence and distribution of gene and protein expression of PAR 2 in different cell types in the nasal mucosa under normal situation, the increased expression of PAR 2 in epithelial cells and the increased number of eosinophils with PAR 2 suggest that PAR 2 may contribute to the pathogenesis of allergic diseases such as SAR.

Transcriptional up-regulation of histamine receptor-1 in epithelial, mucus and inflammatory cells in perennial allergic rhinitis.

BACKGROUND: Histamine receptors play an important role in the pathogenesis of nasal allergy. Activation of histamine receptor 1 (H1R) and 2 (H2R) can cause allergic symptoms which can be blocked effectively by antihistamines. H1R and H2R transcript levels have been found to be up-regulated in perennial - but not in seasonal - allergic rhinitis (AR). The present study aimed to explore H1R and H2R expression in complex tissues of the nasal mucosa of perennial allergic rhinitis (PAR). METHODS: Ten patients with PAR and 13 non-AR subjects were recruited for the study by medical history, physical examination and laboratory screening tests. In this study, we have analysed single cells dissected from the nasal mucosa biopsies by laser-assisted microdissection. H1R mRNA expression was analysed in different cell types such as epithelial, endothelial, mucus and inflammatory cells isolated from the nasal mucosa of PAR in comparison with non-AR subjects. RESULTS: H1R mRNA gene expression level was significantly increased in the nasal mucosa of PAR in comparison with non-AR (P<0.0001). H1R mRNA was significantly elevated in epithelial (P<0.001) and mucus cells (P<0.05) of PAR in comparison with non-AR whereas H1R gene expression levels in endothelial cells between both groups were not changed (P=0.23). Interestingly, inflammatory cells in the nasal mucosa of PAR patients were also strongly expressed H1R mRNA (P<0.001). CONCLUSION: The present study indicates that PAR alters the expression of H1R mRNA in epithelial, mucus and inflammatory cells of the nasal mucosa and but not in endothelial cells. Therefore, epithelial, mucus and inflammatory cells may play an important role in histamine-mediated allergic airway inflammation in PAR.

Phenotypic alteration of neuropeptide-containing nerve fibres in seasonal intermittent allergic rhinitis.

BACKGROUND: Allergic rhinitis (AR) is the most common allergic disease affecting the respiratory tract. Next to inflammatory changes, the airway innervation plays an important modulatory role in the pathogenesis of the disease. OBJECTIVE: To examine the participation of different neuropeptides in the human nasal mucosa of intermittent (seasonal) AR tissues in the allergic season. METHODS: Immunohistochemistry for substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and neuropeptide tyrosine (NPY) was related to the characterization of inflammatory cells in tissues of patients with seasonal AR (n=18). RESULTS: While there was a significant increase in the number of eosinophils present if compared with a control group, no changes occurred in mast cell numbers. Immunostaining was abundantly found in different nerve fibre populations of both groups. SP expression was significantly increased in mucosal nerve fibres of patients with intermittent (seasonal) AR. Also, significantly increased numbers of VIP- and NPY-immunoreactive nerve fibres were found in biopsies of rhinitis patients in comparison with sections of normal human nasal mucosa. In contrast, CGRP expression did not change significantly. CONCLUSION: The increase of neuropeptide expression in mucosal nerve fibres indicates a major role of the autonomous mucosal innervation in the pathophysiology of intermittent (seasonal) AR.

Aspirin-sensitive rhinitis-associated changes in upper airway innervation.

Aspirin-sensitive rhinitis is the manifestation of aspirin intolerance in the upper respiratory tract. The disease represents a pseudoallergy against aspirin or related nonsteroidal anti-inflammatory drugs. As a major immunomodulatory role for airway innervation has been proposed in airway inflammatory diseases, the present study assessed changes in human nasal mucosa innervation in patients with aspirin-sensitive rhinitis in comparison to a control group. Immunohistochemistry for protein gene product 9.5, tachykinins, calcitonin gene-related peptide, vasoactive intestinal peptide (VIP) and neuropeptide tyrosine was performed on cryostats sections of nasal mucosa and neuropeptide containing nerves were examined independently using a score grading. In comparison to the control, the aspirin-sensitive rhinitis group had a significant increase of VIP-like immunoreactivity in mucosal nerve fibres. In contrast to constant numbers of mast cells, highly significant increases in the numbers of eosinophils were found in the group of aspirin-sensitive rhinitis patients. In summary, the present quantification of neuropeptide-immunoreactivity of mucosal nerves demonstrated differences in the human nasal mucosa innervation between nonrhinitic and aspirin-sensitive rhinitic individuals. These differences may reflect a pathophysiological role of upper airway innervation in pseudoallergic reactions.

Differential regulation of macrophage CCAAT-enhancer binding protein isoforms by lipopolysaccharide and cytokines.

The regulation of the C/EBP family in macrophages by LPS and cytokines is of potentially crucial importance in several pathophysiological conditions. The action of LPS and three cytokines on the expression of C/EBP mRNA, protein and functional DNA binding activity in the murine J774.2 cell line was therefore studied. Exposure of the cells to LPS, IL-1, IFN-gamma and TNF-alpha produced a reduction of C/EBP alpha mRNA levels and a corresponding increase in the expression of C/EBP beta and C/EBP delta. EMSA showed time-dependent changes in the DNA binding activity of individual C/EBP isoforms and demonstrated the participation of heterodimers between the different members in DNA-protein interactions. Additionally, mediator-specific changes in the kinetics and magnitude of C/EBP mRNA expression pattern and profile of DNA-protein interactions were observed. These studies provide novel insights into the potential mechanisms that may be responsible for the mediator-specific regulation of macrophage gene expression through the C/EBP family.

Cytokine-mediated differential regulation of macrophage activator protein-1 genes.

The regulation of macrophage activator protein-1 (AP-1) gene expression by LPS and cytokines is of potentially crucial importance in the pathogenesis of several diseases. The action of LPS and four cytokines on AP-1 gene expression in the murine macrophage J774.2 cell line was, therefore, studied. Exposure of the cells to IL-6 produced no changes in the mRNA levels of all AP-1 members studied. In contrast, the expression of JunB, c-jun and c-fos, but not JunD, was increased by LPS, TNF-alpha, IFN-gamma and IL-1, albeit with different kinetics and magnitude of induction. Electrophoretic mobility shift assays showed a close correlation between the expression of the AP-1 genes and the functional AP-1 DNA binding activity and, additionally, demonstrated the participation of heterodimeric interactions between the different members. These studies provide insights into the potential mechanisms that may be involved in the mediator-specific modulation of AP-1 regulated macrophage gene expression.

The ovine CCAAT-enhancer binding protein delta gene: cloning, characterization, and species-specific autoregulation.

Transcription factors belonging to the CCAAT-enhancer binding protein (C/EBP) family have been implicated in the regulation of gene expression during growth, differentiation, apoptosis, and inflammation. Autoregulation is relatively common in the modulation of C/EBP gene expression and, for the human and murine C/EBPalpha, it is known that species-specific autoregulatory mechanisms operate. It is therefore essential to investigate the autoregulation of additional C/EBP genes from a wider range of different species to gauge the degree of commonality, or otherwise, which exists. As an important step towards this goal, we report here the cloning and the characterisation of the ovine C/EBPdelta gene (ovC/EBPdelta) and analysis of its promoter region. Transient transfection assays reveal that ovC/EBPdelta acts as a transcriptional activator. Although several motifs that are characteristic of C/EBPdelta genes are conserved in the ovine sequence, including the basic region, leucine zipper, and activation domains, two regions have been identified that are specifically absent in the ovine and bovine homologues. The ovC/EBPdelta promoter is active in both the hepatoma Hep3B and the mammary epithelial HC11 cell lines, induced by the cytokine interleukin-6 and autoregulated by mechanisms that are potentially different from those described for the rat promoter. These results suggest that, in common with C/EBPalpha, the C/EBPdelta genes may also be subject to autoregulation by distinct species-specific mechanisms.

Lipoprotein lipase, a key role in atherosclerosis?

Lipoprotein lipase (LPL) plays a central role in lipid metabolism and transport by catalysing the hydrolysis of triacylglycerol-rich lipoproteins. The importance of LPL expressed by the adipose tissue and muscles in the provision of non-esterified fatty acids and 2-monoacylglycerol for tissue utilisation is well established. However, recent studies on LPL expressed by cells of the vascular wall, particularly macrophages, have identified additional actions of the enzyme that contribute to the promotion of foam cell formation and atherosclerosis. This review deals with the role of LPL in atherosclerosis, and its regulation by mediators that are known to be present in the lesion.

Involvement of both the tyrosine kinase and the phosphatidylinositol-3' kinase signal transduction pathways in the regulation of lipoprotein lipase expression in J774.2 macrophages by cytokines and lipopolysaccharide.

The regulation of macrophage lipoprotein lipase (LPL) by cytokines and lipopolysaccharide (LPS) is of potentially crucial importance in the pathogenesis of atherosclerosis and in the responses to endotoxin challenge. We show here that the reduction of LPL activity in J774.2 macrophages observed in the presence of interleukin (IL-1) and IL-11 was sensitive to herbimycin A, with the effect of LPS, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) on LPL activity being sensitive to both herbimycin A and wortmannin. The action of the inhibitors on the IFN-gamma-dependent reduction of LPL activity was mediated at the level of LPL mRNA metabolism, with translational and/or post-translational levels of regulation being involved in the action of all the other mediators tested. These observations suggest that both the tyrosine kinase and the phosphatidylinositol-3'-kinase signalling pathways are involved in the suppression of macrophage LPL expression by LPS and cytokines.

Synergism between lipopolysaccharide and interferon gamma in the regulation of lipoprotein lipase in macrophages.

The regulation of macrophage lipoprotein lipase (LPL) by cytokines is potentially of crucial importance in the pathogenesis of atherosclerosis and in septic shock. The effect of combinations of lipopolysaccharide (LPS) and cytokines on the expression of LPL in macrophages was studied using the murine J774.2 cell line. The suppression of heparin-releasable LPL activity produced by combinations of LPS and interleukin 1 (IL-1), IL-11 or tumour necrosis factor alpha(TNF-alpha) was substantially less than that expected from the simple additive action of the corresponding two effectors. By contrast, co-exposure of the cells to LPS and interferon gamma(IFN-gamma) resulted in a more than additive, synergistic, suppression of LPL activity which was, additionally, also observed when the rat alveolar macrophage NR8383 cell line was studied. This synergistic action was also observed when J774.2 macrophages were exposed initially to IFN-gamma (priming), washed and then treated with LPS. A comparison of the LPL activity and mRNA levels produced by the synergistic action of LPS and IFN-gamma and the priming action of IFN-gamma indicated that a combination of mRNA metabolism (transcription or RNA stability), translation and post-translational mechanisms were responsible for the observed changes in LPL activity. These data, therefore, suggest that combinations of LPS and cytokines may be more important than the presence or absence of any given single effector in the modulation of LPL function during infection.

Expression of the genes encoding CCAAT-enhancer binding protein isoforms in the mouse mammary gland during lactation and involution.

Transcription factors belonging to the CCAAT-enhancer binding protein (C/EBP) family have been implicated in the activation of gene expression in the mammary gland during lactation. We have therefore investigated the detailed expression profile of the C/EBP family during lactation and involution of the mouse mammary gland. The expression of C/EBPbeta and C/EBPdelta mRNA was low during lactation, increased dramatically at the beginning of involution and remained constant thereafter. In contrast, C/EBPalpha mRNA expression was relatively high during the early stages of lactation, declined to low levels during the late stages of lactation and at the start of involution, and increased again during involution. Electrophoretic mobility-shift assays showed a close correlation between the expression of the C/EBP genes and the functional C/EBP DNA-binding activity and, additionally, demonstrated the participation of heterodimers, formed from among the three proteins, in DNA-protein interactions. The DNA-binding activity of the activator protein 1 (AP1) family of transcription factors was also induced during involution. These results therefore point to potentially important regulatory roles for both the C/EBP and the AP1 family during lactation and involution of the mammary gland.

Reduction of NO-induced methemoglobinemia requires extremely high doses of ascorbic acid in vitro.

The objective of the present study was to investigate the treatment of nitric oxide (NO)-induced methemoglobinemia by ascorbate and its consequences on red blood cell (RBC) glutathione in vitro. RBC were obtained from five healthy volunteers. The following experiments were carried out: (1) After methemoglobin generation by NO, ascorbate was added (2) RBC were simultaneously exposed to NO and ascorbate (3) Methemoglobin was generated by NO, ascorbate was added and incubation with NO continued. (1) After discontinuation of NO, the mean half life for methemoglobin was reduced from 195 min (controls) to 60 min (10 mM ascorbate) in a dose-dependent manner. (2) Methemoglobin formation after 3 h of NO exposure was 2.7 +/- 0.3% in controls and 1.8 +/- 0.1% with 10 mM ascorbate (p < 0.01). (3) Further methemoglobin formation was inhibited only by 10 mM ascorbate (p < 0.001). NO incubation did not affect RBC glutathione (86.5 +/- 19.6 and 86.5 +/- 19.6 mg/l, respectively). Treatment with 10 mM ascorbate significantly decreased glutathione (p < 0.002). In vitro, NO-induced methemoglobin formation is significantly decreased only by a high (10 mM) ascorbate concentration. Glutathione, critical for ascorbate activity, is not influenced by NO.

Synergism between interferon gamma and tumour necrosis factor alpha in the regulation of lipoprotein lipase in the macrophage J774.2 cell line.

The regulation of macrophage lipoprotein lipase (LPL) by cytokines is of potentially crucial importance in the pathogenesis of atherosclerosis. The effect of combinations of interleukin 1 (IL-1), 6 (IL-6), and 11 (IL-11), interferon gamma (INF-gamma), leukaemia inhibitory factor (LIF) and tumour necrosis factor alpha (TNF-alpha) on the expression of LPL in macrophages was studied using the murine J774.2 cell line. The suppression of heparin-releasable LPL activity produced by combinations of IL-1 and IL-11, IL-1 and TNF-alpha, IL-11 and TNF-alpha, and, IL-11 and INF-gamma was substantially lower than that expected from the additive action of the corresponding two cytokines. By contrast, co-exposure of cells to LIF and IFN-gamma, IL-6 and LIF, and INF-gamma and TNF-alpha resulted in a more than additive, synergistic, suppression of LPL activity with the maximum reduction and maximum degree of synergism produced by combinations of IFN-gamma and TNF-alpha. The synergism between IFN-gamma and TNF-alpha was observed over a range of complementary dose combinations and also occurred when the cells were exposed first to INF-gamma (priming), washed, and then stimulated subsequently with TNF-alpha. The reduction in LPL activity by combinations of IFN-gamma and TNF-alpha and the priming action of IFN-gamma were accompanied by a comparable decrease in LPL mRNA concentrations, thereby indicating that the major control responsible for the changes in LPL activity was being exerted at the level of mRNA metabolism (decreased transcription or RNA stability). These results suggest that the modulation of macrophage LPL function in atherosclerosis by cytokine combinations may be more important than the presence or absence of any given cytokine.

Differential regulation of lipoprotein lipase in the macrophage J774.2 cell line by cytokines.

The regulation of macrophage lipoprotein lipase (LPL) by cytokines is of potentially crucial importance in the pathogenesis of atherosclerosis and in the responses to endotoxin challenge. However, the precise mechanisms by which different cytokines modulate the expression of macrophage LPL activity are poorly understood. The action of six cytokines and bacterial lipopolysaccharide (LPS) on LPL function using the murine J774.2 cell line as a model system has, therefore, been studied. Although exposure to LPS, interleukin 11 (IL-11), tumour necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma) and IL-1, over the physiological range of concentrations, resulted in a decrease in the heparin-releasable LPL activity, LPL-mRNA levels and LPL-protein content of the cells, stimulation with IL-6 and leukaemia inhibitory factor (LIF) had no effect. The maximum suppression of LPL activity and mRNA levels in the cells by IFN-gamma (60%) was lower than that produced by LPS, IL-11, TNF-alpha and IL-1 (78-97%). Each cytokine displayed a characteristic dose-dependent pattern for the suppression of LPL activity and mRNA levels with IL-11/TNF-alpha being more potent than IFN-gamma/IL-1. More than 80% of the decrease in the LPL activity, at all doses of IL-11, TNF-alpha, IFN-gamma and IL-1, was due to a corresponding reduction in the mRNA levels. The time course of responses to LPS, IL-11, TNF-alpha, IFN-gamma and IL-1 were similar, with the time required to achieve half maximal suppression of LPL activity being between 7 and 9.5 h in each case. These results indicate that LPL in J774.2 macrophages is regulated differentially by various cytokines and that the major control responsible for the reduction of LPL activity by IL-11, TNF-alpha, IFN-gamma and IL-1 is exerted at the level of mRNA metabolism (decreased transcription or RNA stability). The responses identified also displayed several differences to those described previously for adipocytes (e.g. 3T3-L1 cell line), thereby suggesting the existence of potential cell-specific mechanisms for the regulation of LPL by cytokines.