RhoA signaling modulates cyclin D1 expression in human lung fibroblasts; implications for idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic Pulmonary Fibrosis (IPF) is a debilitating disease characterized by exaggerated extracellular matrix deposition and aggressive lung structural remodeling. Disease pathogenesis is driven by fibroblastic foci formation, consequent on growth factor overexpression and myofibroblast proliferation. We have previously shown that both CTGF overexpression and myofibroblast formation in IPF cell lines are dependent on RhoA signaling. As RhoA-mediated regulation is also involved in cell cycle progression, we hypothesise that this pathway is key to lung fibroblast turnover through modulation of cyclin D1 kinetic expression. METHODS: Cyclin D1 expression was compared in primary IPF patient-derived fibroblasts and equivalent normal control cells. Quantitative real time PCR was employed to examine relative expression levels of cyclin D1 mRNA; protein expression was confirmed by western blotting. Effects of Rho signaling were investigated using transient transfection of constitutively active and dominant negative RhoA constructs as well as pharmacological inhibitors. Cellular proliferation of lung fibroblasts was determined by BrdU incorporation ELISA. To further explore RhoA regulation of cyclin D1 in lung fibroblasts and associated cell cycle progression, an established Rho inhibitor, Simvastatin, was incorporated in our studies. RESULTS: Cyclin D1 expression was upregulated in IPF compared to normal lung fibroblasts under exponential growth conditions (p < 0.05). Serum deprivation inhibited cyclin D1 expression, which was restored following treatment with fibrogenic growth factors (TGF-beta1 and CTGF). RhoA inhibition, using a dominant negative mutant and a pharmacological inhibitor (C3 exotoxin), suppressed levels of cyclin D1 mRNA and protein in IPF fibroblasts, with significant abrogation of cell turnover (p < 0.05). Furthermore, Simvastatin dose-dependently inhibited fibroblast cyclin D1 gene and protein expression, inducing G1 cell cycle arrest. Similar trends were observed in control experiments using normal lung fibroblasts, though exhibited responses were lower in magnitude. CONCLUSION: These findings report for the first time that cyclin D1 expression is deregulated in IPF through a RhoA dependent mechanism that influences lung fibroblast proliferation. This potentially unravels new molecular targets for future anti-IPF strategies; accordingly, Simvastatin inhibition of Rho-mediated cyclin D1 expression in IPF fibroblasts merits further exploitation.

Expression of growth hormone-releasing factor, growth hormone, insulin-like growth factor-1 and its binding proteins in human lung.

Reverse transcription PCR showed that mRNA encoding the neurohormones growth hormone-releasing factor (GRF) and GH, and its receptor GH-R, together with IGF-1 splice variants and IGFBPs are expressed by inflammatory cells found in the normal human airway. Unfractionated BALC moderately express GRF, GH and GH-R, IGFBP-2 to IGFBP-6, and IGFBP-rPl. In addition, BALC preferentially express the class 1 IGF-1Ea splice variant of the IGF-1 gene. A similar pattern of expression occurs in purified AM, except they do not appear to express GH-R. In marked contrast, AM precursor peripheral blood monocytes, do not express neuropeptides or IGF-1 and only express IGFBP-1, -4 and -6 and IGFBP-rP1. These data suggest that normal human inflammatory airway cells possess a powerful array of neurohormones and IGFBPs that are available for modulating local IGF-1 bioavailability in the lung.

Early detection of ozone-induced hydroperoxides in epithelial cells by a novel infrared spectroscopic method.

In the lower atmosphere ozone is a toxic and an unwanted oxidising pollutant causing injury to the airway epithelial cells by lipid peroxidation to yield products such as phospholipid hydroperoxides (PLHP). Measurements of PLHP, which are primary oxidation products, may reflect an early susceptibility of the target cell to oxidative stress. Biphasic cultures of bronchial epithelial cells (BEAS-2B) were exposed to ozone at environmentally relevant concentrations (0.1-1.0 ppm) for 4 and 12 h. Detection of PLHP was made using a novel technique based on fourier transform infrared spectroscopy (FTIR) in combination with high performance thin-layer chromatography (HPTLC). Six phospholipids were identified on the HPTLC plate; lysophosphatidylcholine (LPC), sphingomyelin (SM), phosphatidylcholine (PC), lysophosphatidylethanolamine (LPE), phosphatidylinositol (PI), and phosphatidylethanolamine (PE). From the FTIR spectra, O-O stretching of hydroperoxides was identified in the range 890-820cm(-1). Multivariate data analysis revealed a positive correlation (r = 0.99 for 4 h exposure and r = 0.98 for 12h exposure) between ozone exposure levels and the region of the FTIR-spectrum comprising the main wavelengths for hydroperoxides. These data support this alternative, versatile and novel spectroscopic approach for the early detection of ozone-mediated damage in human airway epithelial cells.

Effect of oral antibiotics on lung mucociliary clearance during exacerbation of chronic obstructive pulmonary disease.

It has been well established that lung mucociliary clearance is depressed in patients with chronic obstructive pulmonary disease. This study examines whether oral antibiotics have a detectable effect on this clearance mechanism during exacerbation in patients with such disease. Twelve patients with a mean +/- SE age of 63 +/- 2 years participated in a randomized, double-blind, parallel group study to assess the effect of 1 week of treatment with amoxycillin (500 mg t.d.s.) or ciprofloxacin (500 mg b.d.) on lung mucociliary clearance during exacerbation. Lung mucociliary clearance rates were measured by a non-invasive radioaerosol technique. Both drugs on average resulted in small, non-significant, enhancement of mucociliary clearance. Following treatment, the numbers of coughs were reduced in both groups and significantly (P < 0.05) after treatment with ciprofloxacin. Sputum production was also significantly reduced (P < 0.01) in both groups. The magnitude of improvement in lung mucociliary clearance was relatively modest following 1 week of treatment with either antibiotic. Since the number of coughs was significantly less after ciprofloxacin treatment the measured enhancement of lung mucociliary transport is probably, however, an underestimate.

Glutathione-S-transferase family of enzymes.

The loci encoding the glutathione-S-transferase (GST) enzymes comprise a large supergene family located on at least seven chromosomes. The function of the GST enzymes has traditionally been considered to be the detoxication of electrophiles by glutathione conjugation. A wide variety of endogenous (e.g. by-products of reactive oxygen species activity) and exogenous (e.g. polycyclic aromatic hydrocarbons) electrophilic substrates have been identified. Interestingly, recent data has suggested a role, at least for the pi class gene product, in jun kinase inhibition. Since many GST genes are polymorphic, there has been considerable interest in determining whether particular allelic variants are associated with altered risk (or outcome) of a variety of diseases. We describe recent studies in patients with asthma and cutaneous basal cell carcinoma that demonstrate associations between GSTP1 and GSTT1 genotypes and disease phenotypes. Thus, GSTP1val(105)/val(105) was protective against asthma symptoms and GSTT1 null was associated with a subgroup of basal cell carcinoma patients who develop large numbers of primary tumours in clusters. Importantly, these associations were characterised by relatively large odds ratios (0.11 and 7.4, respectively) implying that the allelic variants exert a substantial biological effect. These and other data indicate the importance of GST polymorphism in determining disease phenotype.

A biological model to explain the association between human rhinovirus respiratory infections and bronchial asthma.

Human rhinoviruses (HRVs) are a frequent cause or upper respiratory tract infections in children and adults, and can exacerbate existing pulmonary disease. The major group of HRV attach to the receptor intercellular adhesion molecule (ICAM)-1, which is expressed on many cell types including epithelial cells. To study the influence of biological mediators on ICAM-1 expression, and consequently HRV attachment and infection, we have established an in vitro model system to evaluate the effects or pre-exposure to different cytokines on surface expression of ICAM-1 of uninfected and HRV-14-infected epithelial cells. The results of our studies show that the cytokines interleukin (IL)-1 beta, IL-8 and tumour necrosing factor (TNF)alpha increased ICAM-1 expression on epithelial cells. Epithelial cells infected with live HRV-14 displayed a significant upregulation of ICAM-1 compared to baseline. In contrast, interferon (IFN)gamma, whilst increasing the level of ICAM-1 expression on uninfected cells, induced a marked persistent downregulation of ICAM-1 expression on HRV-infected epithelial cells. In addition, IFN gamma appeared to completely override the ICAM-1 upregulation induced by IL-1 beta, IL-8 and TNF alpha, during HRV infection. We have further demonstrated that type 2 T-helper cell (Th2)-associated cytokines, predominantly IL-13, induce a marked upregulation or epithelial cell surface ICAM-1, thus increasing cellular binding sites for HRV attachment. As the airway mucosa or asthmatic subjects is predominantly infiltrated by activated type 2 T-helper cells with a simultaneous decrease of type 1 T-helper cells, our observations could explain the increased susceptibility to human rhinovirus infection observed in asthma.

Virus-induced asthma.

Clinical and experimental investigations indicate that respiratory viral infections are important triggers for asthma attacks. Viral upper respiratory infections have been associated with 80% of asthma exacerbations in children and 50% of all asthma episodes in adults. Human Rhinovirus (HRV) has been implicated as the most common virus associated with asthma episodes. The observation that the great majority of wheezing lower respiratory tract illnesses in early life are associated with acute viral infections suggests that viruses may also alter the development of the lungs or of the immune system, acting as co-factors for the inception of asthma. Whilst there is no doubt that viruses are important asthma exacerbation factors, the role of viral infections in the development of asthma still remains controversial.

FTIR spectroscopic analysis of sputum: preliminary findings on a potential novel diagnostic marker for COPD.

COPD is a common, progressively disabling disease and a major health burden worldwide. Fourier transform infrared (FTIR) spectroscopy provides for sensitive analysis of complex biological samples. COPD pathogenesis involves quantitative and qualitative changes in sputum biosynthesis. This first study explores whether FTIR can produce distinct spectral profiles of human sputum, and capture differences between COPD and health. Sputum obtained from 15 COPD patients and 15 healthy volunteers was analysed using FTIR spectroscopy; differences in peak positions, height and configuration were identified and measured. All samples gave reproducible characteristic IR absorption spectra. The most relevant regions identified were the amide and glycogen rich regions, showing crucial spectral differences between health and COPD relating to peak position shifts or intensity alteration. These novel preliminary findings support further exploration of FTIR sputum profiling in a clinical study to determine its potential as a practical method for monitoring COPD.

The nature of latent pulmonary involvement in primary biliary cirrhosis.

Primary biliary cirrhosis is a disease of unknown aetiology, resulting in progressive granulomatous destruction of small intrahepatic bile ducts. The rate at which this occurs varies considerably producing a wide clinical spectrum of the disease itself, as well as expression in other organs. Pulmonary manifestations in PBC have only been intermittently reported. Nevertheless in practice, a vast array of pathophysiological mechanisms can be implicated for the pulmonary dysfunction seen in some of these patients. The observed clinical features can be attributed to pulmonary vascular abnormalities, to a secondary 'fibrosing alveolitis', or indeed to the emergence of a granulomatous disorder in the lungs similar to sarcoidosis. Physicians should thus be aware of such potential lung complications, which can occur not only during the course of the PBC but also following liver transplantation.

Characterization of immune inducer and suppressor macrophages from the normal human lung.

Monoclonal antibodies (MoAbs) that are able to discriminate between dendritic cells (MoAb RFD1+) and mature macrophages (MoAb RFD7+) in normal tissues were used in combination with density separation techniques to isolate relatively homogeneous subpopulations of macrophages from human bronchoalveolar lavage (BAL). A characterization of surface antigen expression, and functional capacity was then carried out on each isolated alveolar macrophage (AM) subset. One population with the phenotype RFD1+RFD7- obtained from the non-adherent cell pool showed the characteristics of antigen-presenting cells having absent or poor expression of Fc and C3b receptors, a low content of lysozomal hydrolase and poor phagocytic capacity. This population strongly stimulated T lymphocytes in allogeneic mixed lymphocyte reactions (MLR). A second AM population, isolated by adherence and density centrifugation expressed the phenotype RFD1+RFD7+. These cells showed the same phenotypic characteristics of mature macrophages with strong expression of C3b and Fc receptors, and marked phagocytic capacity. Such AM were very poor stimulators of allogeneic MLR. Under certain circumstances the RFD1+RFD7+ cells were shown to actively repress the stimulatory capacity of the RFD1+RFD7- subpopulation. These results suggest that variations within the functional capacity of AM subsets may be capable of influencing the strength of acquired T cell immune responses of the lung.

Sarcoidosis.

Sarcoidosis is a multisystem granulomatous disorder of unknown aetiology. The condition commonly affects young adults and frequently presents with bilateral hilar lymphadenopathy with or without pulmonary infiltration, ocular or cutaneous lesions. The clinical presentation can be extremely varied depending upon the organs affected. The diagnosis is firmly established when recognised clinical and radiographic findings are supported by histological evidence of discrete non-necrotising epithelioid cell granulomata in one or more organs. Sarcoidosis is usually self-limiting with spontaneous resolution, although in a few patients there is a progressive downhill course, culminating in irreversible fibrosis and severe impairment of organ function.

Adverse ocular reactions to drugs.

Drugs acting on various parts of the body may also affect the eye insidiously. Increased awareness of such drug toxicity by the prescribing doctor should encourage him to consider effects on the cornea, lens, retina, optic nerve and elsewhere when checking the patient's progress. The following review concerns adverse ocular effects of systemic drug administration.

Phenotypic and functional changes in alveolar macrophages contribute to the pathogenesis of pulmonary sarcoidosis.

Bronchoalveolar lavage (BAL) was performed on 10 patients with sarcoidosis and 10 normal volunteers. In each case aliquots of the lavage were used to prepare cytospins on which differential cell counts were performed. Immunocytological methods using monoclonal antibodies RFD1 and RFD7 (identifying dendritic cells and mature macrophages in normal tissues) were performed to identify macrophage subsets. Sarcoid BAL contained a significantly higher proportion of RFD1+ cells (mean 44.7 +/- 10.32% compared to 12.3 +/- 4.0% in normals). Much of this increase was accounted for by a highly significant rise in the proportion of cells with the double phenotype RFD1+/RFD7+ (27.2 +/- 6.1% in sarcoid compared to 7.3 +/- 2.0% in normal). Suspensions of sarcoid and normal BAL were also studied in autologous mixed lymphocyte reactions (AMLR) using peripheral blood mononuclear cells (PBM) as a responder population. AMLRs were therefore set up using BAL, PBM, and BAL with PBM. In each case reactivity was compared to mitomycin treated controls. These studies revealed that sarcoid PBM expressed markedly reduced AMLR reactivity when compared to normal but both sarcoid and normal BAL were relatively unreactive. BAL admixed with PBM suppressed peripheral blood AMLR reactivity in the normals. In sarcoid patients BAL admixed with PBM abolished AMLR completely. We suggest that changes within the BAL macrophage populations in sarcoid patients may significantly influence the pathogenesis of this disease.

Reliability of eliciting physical signs in examination of the chest.

Agreement between 24 physicians on the presence or absence of respiratory signs was investigated. The physicians were divided into six sets of 4; each set examined 4 patients with well-defined chest signs. There was generally poor agreement about particular signs. Overall, the 4 physicians in a set were in complete agreement only 55% of the time. Some signs such as wheezing seemed to be more reliably elicited than others such as whispering pectoriloquy. Comparison of diagnoses based on the clinical findings with the correct diagnoses supported by investigations showed that 28% of physicians' diagnoses were incorrect. The more often the examiners differed from the majority on the presence or absence of a sign, the more likely they were to make an incorrect diagnosis. A ranked order of the reliability with which chest signs are elicited would improve the teaching of chest medicine.

Successful management of primary invasive pulmonary aspergillosis.

We present the successful recovery of an immunocompetent patient with invasive pulmonary aspergillosis in the absence of any previous detectable lung damage or underlying systemic disease.

The macrophage in sarcoid granuloma formation.

Sarcoid granulomata result from aberrant immunological reactions initiated by antigen--presenting macrophage--like cells, and maintained by other effector macrophages. These macrophages can be distinguished phenotypically by monoclonal antibodies RFD1 and RFD7 (which recognize dendritic cells and mature macrophages respectively). Active sarcoid BAL contains a high proportion of RFD1 + cells (mean 44.7% compared to 12% in normals). Much of this increase is accounted for by the emergence of macrophages with the double phenotype RFD1 + D7 + (27.2% compared to 7% in normals), the proportion of which increases with disease severity and returns to normal in remission. When isolated from BAL by using plastic plate adherence and metrizamide density gradient, this hitherto unknown RFD1 + D7 + subset displays distinctive phenotypic, physiological and functional features. Unlike RFD1 + D7-cells, RFD1 + D7 + macrophages adhere to plastic, are acid phosphatase positive with increased phagocytosis, have marked Fc and c3b receptor expression, and suppress T-lymphocyte reactivity. In active sarcoidosis, this suppressive action is accentuated, and a greater proportion of RFD1 + D7 + cells express Fc receptors as well as a separate antigen RFD9 (which identifies epithelioid cells). Furthermore we have observed that gamma-interferon, produced in high concentration by activated T-lymphocytes induces not only HLA-DR molecules on cells, but has also been shown in vitro to increase the proportion of RFD1 + cells developing while suppressing RFD7 expression. It therefore seems that the increased proportion of RFD1 + D7 + macrophages seen in active sarcoidosis could arise as a result of an increased induction of RFD1 expression on macrophages which express RFD7.(ABSTRACT TRUNCATED AT 250 WORDS)

Alveolar macrophages that suppress T-cell responses may be crucial to the pathogenetic outcome of pulmonary sarcoidosis.

The alveolar macrophage (AM) population is widely recognized to be heterogeneous; distinct subpopulations can be identified by the use of macrophage-specific monoclonal antibody (MoAb) probes. We have isolated a macrophage subset that appears to react with both MoAbs that have previously discriminated between dendritic cells and classic macrophages. In the bronchoalveolar lavage (BAL) of patients with active sarcoidosis the proportion of this specific AM subpopulation increases dramatically (30.4 +/- 4.01% compared to 6.14 +/- 1.56% in normal BAL). This AM subset not only increases in direct proportion to the lavage lymphocytosis, but also exhibits sarcoid-related differences in surface receptor expression, physiology and induction of T-cell responses. An increased number of these AM expressed a separate antigen RFD9 (which identified epithelioid cells), and had raised fibronectin content, increased phagocytosis, and high lysosomal enzyme activity. Of functional significance, we found that while in normal volunteers this specific AM subset was capable of down-regulating by as much as 40% the induction of T-cell responses set up by other stimulator macrophages, in sarcoid patients this suppressor activity was enhanced, such that T-cell responses were completely abolished. In some studies this action was masked by the reduced enhancing capacity of sarcoid inducer AM. We postulate that the presence of an increased proportion of these suppressor AM (together with their sarcoid-specific features) in active sarcoidosis is of crucial significance in determining the fate of granulomata in the lungs of these patients.

Isolation of phenotypically and functionally distinct macrophage subpopulations from human bronchoalveolar lavage.

Bronchoalveolar lavage was used to obtain alveolar macrophages (AM) from the lower respiratory tract of healthy normal volunteers. Monoclonal antibody (MoAb) probes specific against macrophage determinants were then applied, in conjunction with density separation techniques, to identify and isolate three relatively homogeneous subpopulations from the AM pool. The MoAbs used, RFD1 and RFD7, have previously been shown to differentiate between "dendritic" cells and mature macrophages, respectively, in normal tissue. In addition to these two phenotypically distinct AM subsets (RFD1+D7- and RFD1-D7+ AM), a third AM subpopulation was isolated, which appeared to express both markers (RFD1+D7+). All three separated macrophage subsets were morphologically similar but exhibited distinct differences in surface receptor expression, enzyme content and physiology. Isolated RFD1+D7- AM (the phenotype of "dendritic" cells) did not adhere to the glass, had weak expression of C3b and FcR1 receptors, low fibronectin content and lysosomal activity; only a small proportion of these cells exhibited phagocytosis. The other two isolated AM subsets adhered to glass, expressed C3b and FcR1 receptors, had high fibronectin and acid phosphatase content, and a large majority exhibited phagocytic capacity; qualitative and quantitative differences in these features existed between the two AM subtypes. Furthermore, a diverse spectrum of hexose monophosphate shunt activity was observed throughout all three AM subpopulations, with the highest activity being recorded in the non-adherent AM. These data support the concept of a dynamic heterogeneity within the AM population. The variation in surface antigen expression and physiological capabilities observed amongst the three isolated AM subsets implies the presence of functionally distinct AM within the human lung, which, during steady-state conditions, may be critically balanced under the influence of stimuli in their local microenvironment. In support, proportional and functional shifts have been witnessed amongst these three AM subpopulations with the advent of disease.