The relationship between clinical phenotype and kallikrein-kinin bioregulation in different forms of arthritis.

OBJECTIVE: Patients with rheumatoid arthritis (RA) have shown increased levels of neutrophils generating kallikrein-kinin peptides in blood which are potent mediators of inflammation. This study investigated the association between the bioregulation of kinin-mediated inflammation with the clinical, quality of life, and imaging characteristics (e.g. ultrasonography) of different arthritides. METHODS: Patients with osteoarthritis (OA, n = 29), gout (n = 10) and RA (n = 8) were recruited and screened for clinical symptoms, quality of life, and ultrasonographical assessment of arthritis. Blood neutrophils were assessed for the expression of bradykinin receptors (B1R and B2R), kininogens and kallikreins by immunocytochemistry with visualization by bright field microscopy. Levels of plasma biomarkers were measured by ELISA and cytometric bead array. RESULTS: Quality of life (SF-36 domains and summary scores; including pain; and, HAQ) was similar across OA, gout and RA patients; with the exception of worse physical functioning scores between OA and gout patients. Synovial hypertrophy (on ultrasound) differed between groups (p = 0.001), and the dichotomised Power Doppler (PD) score of greater than or equal to 2 (PD-GE2) was marginally significant (p = 0.09). Plasma IL-8 were highest in patients with gout followed by RA and OA (both, P < 0.05). Patients with RA had higher plasma levels of sTNFR1, IL-1ß, IL-12p70, TNF and IL-6, compared to OA and gout patients (all, P < 0.05). Patients with OA had higher expression of K1B and KLK1 on blood neutrophils followed by RA and gout patients (both, P < 0.05). Bodily pain correlated with B1R expression on blood neutrophils (r = 0.334, p = 0.05), and inversely with plasma levels of CRP (r = -0.55), sTNFR1 (r = -0.352) and IL-6 (r = -0.422), all P < 0.05. Expression of B1R on blood neutrophils also correlated with Knee PD (r = 0.403) and PD-GE2 (r = 0.480), both P < 0.05. CONCLUSIONS: Pain levels and quality of life were similar between patients with OA, RA and gout with knee arthritis. Plasma inflammatory biomarkers and B1R expression on blood neutrophils correlated with pain. Targeting B1R to modulate the kinin-kallikrein system may pose as a new therapeutic target in the treatment of arthritis.

Remote ischaemic preconditioning down-regulates kinin receptor expression in neutrophils of patients undergoing heart surgery.

OBJECTIVES: Remote ischaemic preconditioning (RIPC) may protect distant organs against ischaemia-reperfusion injury. We investigated the impact of RIPC on kinin receptor expression in neutrophils following RIPC in patients undergoing coronary artery bypass grafting (CABG). METHODS: Patients undergoing elective CABG with cardiopulmonary bypass (CPB) were randomized to RIPC (n = 15) or control (n = 15) groups. The study group underwent RIPC by inflation of a blood pressure cuff on the arm. Expression of kinin receptors, plasma concentrations of IL-6, IL-8, IL-10, TNF-α and neutrophil elastase were determined at baseline (before RIPC/sham), immediately before surgery (after RIPC/sham) and 30 min and 24 h after surgery. Plasma bradykinin levels were assessed before and after RIPC/sham, and at 30 min, 6, 12 and 24 h after surgery. Serum creatine kinase (CK), troponin I, C-reactive protein (CRP) and lactate levels were measured immediately prior to surgery and 30 min, 6, 12, 24 and 48 h after surgery. RESULTS: Kinin B2 receptor expression did not differ between the groups at baseline (pre-RIPC), but was significantly lower in the RIPC group than in the control group after RIPC/sham (P < 0.05). Expressions of both kinin B1 and B2 receptors were significantly down-regulated in the RIPC group, and this persisted to 24 h after surgery (P < 0.001). Neutrophil elastase levels were significantly increased after surgery. There were no differences in CK, CRP, cytokine, lactate or troponin I levels between the groups. CONCLUSIONS: RIPC down-regulated the expression of kinin B1 and B2 receptors in neutrophils of patients undergoing CABG.

Effects of the demethylating agent, 5-azacytidine, on expression of the kallikrein-kinin genes in carcinoma cells of the lung and pleura.

Tissue kallikrein (KLK1) and plasma kallikrein (KLKB1) may regulate the growth and proliferation of tumours of the lung and pleura, through the generation of kinin peptides that signal through the kinin B(1) (BDKRB1) and B(2) (BDKRB2) receptors. The development and progression of cancer results from genetic mutations, as well as epigenetic changes that include methylation of DNA at CpG islands. The aim of this study was to assess whether expression of the kallikrein-kinin genes in lung cancer and mesothelioma cells is regulated by DNA methylation. Quantitative reverse transcriptase-PCR and immunocytochemistry showed differences in the basal expression of the kallikrein-kinin genes and proteins in lung carcinoma and mesothelioma cells, compared with non-malignant lung epithelial and mesothelial cells, respectively. Following treatment with the demethylating agent, 5-azacytidine (5-AZA), KLKB1 mRNA expression was consistently increased in both lung carcinoma and mesothelioma cells, whereas KLK1, BDKRB1 and BDKRB2 mRNA expression was decreased or unchanged. Increased expression of KLKB1 after 5-AZA treatment suggests it may function as a tumour suppressor gene in cancers of the lung and pleura. Studies on DNA methylation of the kallikrein-kinin genes will enhance understanding of their role in carcinogenesis and provide insights into the importance of kallikreins as tumour biomarkers.

Lys-des[Arg9]-bradykinin alters migration and production of interleukin-12 in monocyte-derived dendritic cells.

This study tested the hypothesis that proinflammatory kinin peptides are involved in modulating human dendritic cell (DC) function. Inflammation is accompanied by an increased maturation of DCs and the generation of kinins, particularly Lys-des[Arg(9)]-bradykinin (Lda-BK). We assessed the role of Lda-BK in the activation and migration of human monocyte-derived DCs (hMo-DCs) matured through the use of LPS, TNF-α + IL-1ß, or CD40 ligand. Kinin B(1) and B(2) receptor mRNA and protein expression were assessed by confocal microscopy, flow cytometry, and RT-PCR. The effects of Lda-BK on the migration of mature hMo-DCs were assessed in Transwell chambers, whereas the expression of costimulatory molecules and the secretion of IL-12 were assessed by flow cytometry and ELISA, respectively. The expression of the kinin B(1) receptor (B(1)R) was down-regulated during the maturation of hMo-DCs, whereas the expression of B(2)R was unchanged. The B(1)R agonist Lda-BK was not chemotactic for hMo-DCs matured using LPS, TNF-α + IL-1ß, or CD40 ligand, but Lda-BK enhanced the secretion of IL-12p70 and inhibited the secretion of IL-12p40 by mature hMo-DCs. However, the exposure of hMo-DCs matured with TNF-α + IL-1ß to Lda-BK for 6 hours decreased subsequent migration in response to Lda-BK, the chemokine CCL19, or Lda-BK combined with CCL19. The expression of B(1)R was increased in hMo-DCs from subjects with asthma compared with subjects without asthma, in keeping with a tendency toward increased in vitro migration of asthmatic hMo-DCs in response to Lda-BK. The increased formation of Lda-BK and the enhanced expression of B(1)R as a consequence of inflammation may alter the migration of mature, antigen-laden DCs to regional lymph nodes in response to CCL19, may modulate the secretion of cytokines by these DCs, and may contribute to the accumulation of mature DCs in the lungs of patients with asthma.

Kallikreins, kininogens and kinin receptors on circulating and synovial fluid neutrophils: role in kinin generation in rheumatoid arthritis.

OBJECTIVES: Neutrophils traffic into and have the capacity to generate kinins in SF of RA patients. The aim of this study was to assess the expression of kallikreins, kininogens and kinin receptors in circulating and SF neutrophils, as well as synovial tissue of RA patients, and to assess kinin generation in SF. METHODS: Neutrophils were isolated from blood and SF of RA patients and blood of healthy volunteers. Expression of kallikreins, kininogens and kinin receptors in neutrophils and synovial tissue was assessed by immunocytochemistry using specific antibodies, with visualization by brightfield and confocal microscopy. Levels of basal and generated kinins in SF of RA patients were measured by ELISA. RESULTS: Kinin labelling was significantly reduced, indicating the loss of the kinin moiety from kininogen on circulating (P < 0.001) and SF neutrophils (P < 0.05) of RA patients. Immunolabelling of tissue kallikrein was also decreased, whereas kinin B(1) and B(2) receptor expression was increased in circulating and SF neutrophils of RA patients. Immunolabelling of kallikreins and kinin receptor proteins was similar in RA and normal synovial tissues. The basal kinin level in SF of RA patients was 5.7 +/- 6.1 ng/ml and the mean concentration of kinins generated in vitro was 80.6 +/- 56.3 ng/ml. The capacity for kinin generation was positively correlated with measures of disease activity. CONCLUSIONS: Kallikrein-kinin proteins on neutrophils play an important role in kinin generation and the pathophysiology of RA. Specific kallikrein and kinin receptor antagonists may be useful as IA therapies for inflamed joints.

Increases in urinary 9alpha,11beta-prostaglandin f2 indicate mast cell activation in wine-induced asthma.

BACKGROUND: Wine-induced asthmatic symptoms may be caused by sulphite additives. Prostaglandin (PG)D2 and cysteinyl leukotrienes (cysLT) are important mediators of asthmatic responses. To determine whether the sulphite additives in wine alter the production of PGD2 and cysLT, asthmatic patients with compelling histories of wine sensitivity were challenged with high- and low-sulphite wines; the urinary metabolites of PGD2 and cysLT were measured before and after challenge. METHODS: Eight self-reporting wine-sensitive asthmatic patients completed double-blind challenges with high- and low-sulphite wines on separate days. Urine samples were collected before and after consumption of 150 ml of wine. Urinary concentrations of 9alpha,11beta-PGF2 and leukotriene (LT)E4 were measured by enzyme immunoassay. RESULTS: Urinary 9alpha,11beta-PGF2 concentrations increased in all subjects following challenge with high-sulphite wine, and the median concentration increased 1.6-fold (p < 0.01). Urinary 9alpha,11beta-PGF2 also increased 1.5-fold after low-sulphite wine challenge, although this did not reach statistical significance (p = 0.08). The median difference in 9alpha,11beta-PGF2 concentration after high-sulphite wine challenge was not significantly different compared with that after low-sulphite wine challenge. Median urinary LTE4 concentrations did not change significantly after either wine challenge. CONCLUSIONS: Increased urinary 9alpha,11beta-PGF2 concentrations following wine challenge suggest mast cell activation as a possible mechanism for wine-induced asthma, although this did not appear to be related to the sulphite additives in wine. Urinary 9alpha,11beta-PGF2 may warrant further assessment as a potential biomarker of reactivity to wine in asthmatic subjects.

Expression of kinin receptors on eosinophils: comparison of asthmatic patients and healthy subjects.

Eosinophils contribute to asthmatic airway inflammation by releasing cysteinyl leukotrienes (cysLT) and other inflammatory mediators, and bradykinin (BK) induces bronchoconstriction in asthmatic patients. The aims of this study were to investigate kinin receptor expression on eosinophils of asthmatic and healthy subjects and to assess the effects of kinin stimulation on eosinophils, which were isolated from peripheral blood of asthmatic (n=27) and healthy subjects (n=14). Kinin B(1) and B(2) receptors (B(1)R and B(2)R, respectively) and mRNA expression were investigated by quantitative confocal microscopy, flow cytometry, and RT-PCR. Intracellular Ca(2+) was assessed by live-cell fluorescence confocal microscopy. Production of cysLT and eosinophil migration in response to BK and Lys-des[Arg(9)]-BK were assessed. Eosinophils expressed kinin B(1)R and B(2)R mRNA and proteins. Quantitative immunofluorescence analysis indicated that expression of B(1)R and B(2)R proteins was significantly greater in eosinophils of asthmatic patients compared with those of nonasthmatic subjects. However, kinin B(1)R and B(2)R mRNA expression did not differ significantly between these groups. Expression of kinin B(1)R and mRNA was decreased in patients using high doses of inhaled corticosteroids and in eosinophils treated with a corticosteroid in vitro. Kinin B(1) and B(2) agonists up-regulated expression of their respective receptors but did not increase intracellular Ca(2+) or the production of cysLT or enhance eosinophil migration significantly. Up-regulation of kinin receptor expression in eosinophils of asthmatic patients may be a consequence of inflammation, whereby enhanced release of kinin peptides has a positive-feedback effect on kinin receptor expression. Importantly, anti-inflammatory corticosteroids down-regulated the expression of the kinin B(1)R.

Epigenetic regulation of human epithelial cell cancers.

Cancer is the second leading cause of death in industrialized countries, with epithelial cell cancers (carcinomas) representing approximately 85% of all diagnosed cancers. The 5-year survival rate for many carcinomas remains low, highlighting the requirement for improved diagnosis and more effective therapies. Epigenetic modifications that do not involve changes in the DNA sequence, but result in changes in gene expression, are rapidly being realized as important in carcinogenesis. Evidence is emerging that DNA methylation, histone modification and alternative mRNA splicing are involved in various human epithelial cell cancers, and diagnostic and therapeutic strategies based on these epigenetic phenomena are under investigation. This review provides an overview of studies demonstrating the importance of epigenetic regulation of gene expression in the diagnosis, progression and response to treatment of human carcinomas. The use of therapeutic agents to reverse these epigenetic changes, either as single treatments or in combination with other therapies, is also discussed.

Expression of tissue and plasma kallikreins and kinin B1 and B2 receptors in lung cancer.

Tissue kallikrein (hK1) and plasma kallikrein (PK, hKB1) are serine proteases that produce biologically active kinin peptides from endogenous kininogen substrates. There is evidence linking the kallikreins and the mitogenic kinin peptides to carcinogenesis. The aim of this study was to investigate the expression of tissue prokallikrein (pro-hK1), plasma prekallikrein (PPK, pre-hKB1) and kinin B1 and B2 receptor proteins in different subtypes of lung cancer. Immunohistochemistry, using specific antibodies, was performed on archived normal lung sections and sections from adenocarcinomas, squamous cell carcinomas, large cell carcinomas, small cell carcinomas and carcinoid tumours of the lung. Immunoperoxidase labelling was visualised by brightfield microscopy and immunofluorescence labelling by confocal microscopy. Extensive cytoplasmic expression of pro-hK1 and PPK was observed, which was similar in small cell and non-small cell tumours. However, nuclear labelling for the kallikreins was absent or limited. The kinin B1 and B2 receptors were highly expressed in the cytoplasm of all tumour types and in the nuclei of non-small cell tumours. Further studies are required to assess the functional significance of the expression of hK1, PK and kinin receptors in lung tumours, and whether any of these proteins may be potential biomarkers for specific subtypes of lung carcinoma.

Tolerance and rebound with zafirlukast in patients with persistent asthma.

BACKGROUND: The potential for tolerance to develop to zafirlukast, a cysteinyl leukotriene (CysLT) receptor antagonist (LRA) in persistent asthma, has not been specifically examined. OBJECTIVE: To look for any evidence of tolerance and potential for short-term clinical worsening on LRA withdrawal. Outcome measures included changes in; airway hyperresponsiveness to inhaled methacholine (PD20FEV1), daily symptoms and peak expiratory flows (PEF), sputum and blood cell profiles, sputum CysLT and prostaglandin (PG)E2 and exhaled nitric oxide (eNO) levels. METHODS: A double blind, placebo-controlled study of zafirlukast, 20 mg twice daily over 12 weeks in 21 asthmatics taking beta2-agonists only (Group I), and 24 subjects treated with ICS (Group II). RESULTS: In Group I, zafirlukast significantly improved morning PEF and FEV1compared to placebo (p < 0.01), and reduced morning waking with asthma from baseline after two weeks (p < 0.05). Similarly in Group II, FEV1 improved compared to placebo (p < 0.05), and there were early within-treatment group improvements in morning PEF, beta2-agonist use and asthma severity scores (p < 0.05). However, most improvements with zafirlukast in Group I and to a lesser extent in Group II deteriorated toward baseline values over 12 weeks. In both groups, one week following zafirlukast withdrawal there were significant deteriorations in morning and evening PEFs and FEV1 compared with placebo (p < or = 0.05) and increased nocturnal awakenings in Group II (p < 0.05). There were no changes in PD20FEV1, sputum CysLT concentrations or exhaled nitric oxide (eNO) levels. However, blood neutrophils significantly increased in both groups following zafirlukast withdrawal compared to placebo (p = 0.007). CONCLUSION: Tolerance appears to develop to zafirlukast and there is rebound clinical deterioration on drug withdrawal, accompanied by a blood neutrophilia.

Expression of kallikrein-related peptidases (KRP/hK5, 7, 6, 8) in subtypes of human lung carcinoma.

Lung cancer is currently the leading cause of cancer mortality worldwide. Expression of kallikrein-related peptidases (KRP/hK/KLK) may be induced during lung carcinogenesis. To test the hypothesis that KRP/hK, previously identified in the skin (KRP/hK5, 7) and brain (KRP/hK6, 8), are expressed in lung tumours, experiments were designed to investigate their localization in four malignant sub-types of human lung cancer. Using specific antibodies, expression of these KRP/hK was determined in archived lung tumour sections of the four subtypes, and in normal skin, brain, lung and submandibular gland tissue sections. Immunoperoxidase labelled sections were visualized by brightfield microscopy. In the squamous cell carcinoma, small cell carcinoma and carcinoid tumour, 40-90% of the malignant cells showed positive cytoplasmic labelling for KRP/hK5, 7, 6 and 8 (intensity grade 2+/3+). In the adenocarcinoma there was no cytoplasmic labelling for any of the KRP/hK, but the nuclei of 20% of the tumour cells were labelled for KRP/hK5, 7 and 8 (intensity grade 2+/3+). Further studies are required to determine the functional significance of the expression of KRP/hK in human lung carcinomas, and whether any of these proteins may be potential biomarkers for specific sub-types of lung cancer.

Novel expression of kallikreins, kallikrein-related peptidases and kinin receptors in human pleural mesothelioma.

Malignant mesothelioma is an aggressive cancer of the pleura that is causally related to exposure to asbestos fibres. The kallikrein serine proteases [tissue (hK1) and plasma (hKB1) kallikreins, and kallikrein-related peptidases (KRP/hK2-15)] and the mitogenic kinin peptides may have a role in tumourigenesis. However, it is not known whether hK1, hKB1, KRP/hK proteins or kinin receptors are expressed in pleural mesotheliomas. The expression of hK1, hKB1, KRP/hK2, 5, 6, 7, 8 and 9, and kinin B(1) and B(2) receptors was assessed in archived selected normal tissue and mesothelioma tumour sections by immunoperoxidase and immunofluorescence labelling. hK1, hKB1 and kinin B(1) and B(2) receptors were expressed in malignant cells of the epithelioid and sarcomatoid components of biphasic mesothelioma tumour cells. The percentage of cells with cytoplasmic and nuclear labelling and the intensity of labelling were similar for hK1, hKB1 and the kinin receptors. KRP/hK2, 6, 8 and 9 were also expressed in the cytoplasm and nuclei of mesothelioma cells, whereas KRP/hK5 and hK7 showed predominantly cytoplasmic localisation. This is a first report, but further studies are required to determine whether these proteins have a functional role in the pathogenesis of mesothelioma and/or may be potential biomarkers for pleural mesothelioma.

Current status of tissue kallikrein inhibitors: importance in cancer.

The past decade has seen an increase in the understanding of the structure and function of protease inhibitors. The molecular basis of the interaction between a variety of biological substrates or synthetic inhibitors and serine proteases of the kallikrein family has provided insights into inhibitor protein-protease reactive site interactions, which have proved of value in the design of new kallikrein inhibitors. This review focuses on the current understanding of the functional status of kallikrein inhibitors, which include a variety of molecules derived from plant, reptile and mammalian sources, as well as synthetic agents.

Expression of kinin B1 and B2 receptors in immature, monocyte-derived dendritic cells and bradykinin-mediated increase in intracellular Ca2+ and cell migration.

The kinins, bradykinin (BK) and Lys-des[Arg(9)]-BK, are important inflammatory mediators that act via two specific G protein-coupled kinins, B(1) and B(2) receptors (B(2)R). Kinins influence the activity of immune cells by stimulating the synthesis of cytokines, eicosanoids, and chemotactic factors. Whether human dendritic cells (DC) express kinin receptors and whether kinins influence DC function are unknown. Fluorescence immunocytochemistry and RT-PCR were used to demonstrate that immature human monocyte-derived DC (hMo-DC) constitutively expressed kinins B(1)R and B(2)R. Kinin receptor expression was induced on the 3rd and 4th days of culture during differentiation of hMo-DC from monocytes and was not dependent on the presence of IL-4 or GM-CSF. Although monocytes also expressed B(2)R mRNA, the protein was not detected. The kinin agonists BK and Lys-des[Arg(9)]-BK up-regulated the expression of their respective receptors. BK, acting via the B(2)R, increased intracellular Ca(2+), as visualized by confocal microscopy using the fluorescent Ca(2+) dye, Fluor-4 AM. Evaluation of migration in Trans-well chambers demonstrated significant enhancement by BK of migration of immature hMo-DC, which was B(2)R-dependent. However, kinins did not induce maturation of hMo-DC. The novel finding that kinin receptors are constitutively expressed in immature hMo-DC suggests that these receptors may be expressed in the absence of proinflammatory stimuli. BK, which increases the migration of immature hMo-DC in vitro, may play an important role in the migration of immature DC in noninflammatory conditions and may also be involved in the recruitment of immature DC to sites of inflammation.

Concomitant activation of extracellular signal-regulated kinase and induction of COX-2 stimulates maximum prostaglandin E2 synthesis in human airway epithelial cells.

The intracellular regulation and kinetics of prostaglandin (PG)E(2) synthesis in human airway epithelial (NCI-H292) cells was investigated. Interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and lipopolysaccharide (LPS) all induced PGE(2) synthesis (p<0.001) and transient (5-15 min) phosphorylation of extracellular signal-regulated kinase (ERK). Phorbol myristate acetate (PMA) and calcium ionophore, A23187 further enhanced PGE(2) synthesis (p<0.001) and caused phosphorylation of ERK that was sustained for up to 16 h. COX-2 protein expression and PGE(2) synthesis were increased following exposure to combinations of stimuli that increased intracellular Ca(2+), and activated protein kinase C as well as ERK. Inhibition of ERK almost completely abrogated PGE(2) synthesis in response to all stimuli. Sustained, maximum PGE(2) synthesis was observed when cells were stimulated such that ERK phosphorylation was concomitant with increased COX-2 protein expression. These results argue against redundancy in pathways for PGE(2) synthesis, and suggest that at various stages of inflammation different stimuli may influence ERK activation and COX-2 expression, so as to tightly regulate the kinetics and amount of PGE(2) produced by airway epithelial cells in response to lung inflammation.

Oxidative stress and antioxidant deficiencies in asthma: potential modification by diet.

The lungs of asthmatic patients are exposed to oxidative stress due to the generation of reactive oxygen and nitrogen species as a consequence of chronic airway inflammation. Increased concentrations of NO*, H2O2 and 8-isoprostane have been measured in exhaled breath and induced sputum of asthmatic patients. O2*-, NO*, and halides interact to form highly reactive species such as peroxynitrite and HOBr, which in turn cause nitration and bromination of protein tyrosine residues. Oxidative stress may also reduce glutathione levels and cause inactivation of antioxidant enzymes such as superoxide dismutase, with a consequent increase in apoptosis, shedding of airway epithelial cells and airway remodelling. The oxidant/antioxidant equilibrium in asthmatic patients may be further perturbed by low dietary intakes of the antioxidant vitamins C and E, selenium and flavonoids, with a consequent lowering of the concentrations of these and other non-dietary antioxidants such as bilirubin and albumin in plasma and airway epithelial lining fluid. Although supplementation with vitamins C and E appears to offer protection against the adverse effects of ozone, recent randomised, placebo-controlled trials of vitamin C or E supplements for patients with mild asthma have not shown significant benefits over standard therapy. However, genetic variation in glutathione S-transferase may influence the susceptibility of asthmatic individuals to oxidative stress and the extent to which they are likely to benefit from antioxidant supplementation. Long-term prospective trials are required to determine whether modification of dietary intake will benefit asthma patients and reduce the socio-economic burden of asthma in the community.

Comparison of the morphological and biochemical changes in normal human lung fibroblasts and fibroblasts derived from lungs of patients with idiopathic pulmonary fibrosis during FasL-induced apoptosis.

It is increasingly recognized that the morphological changes of apoptosis vary between cell types. This heterogeneity reflects the wide range of cellular proteins and enzymes involved in apoptotic pathways. Fibroblast apoptosis is crucial to the regression of scars and the restitution of healthy tissue during wound repair and may be aberrant in diseases such as idiopathic pulmonary fibrosis (IPF). The biochemical and morphological changes characterizing fibroblast apoptosis are unknown and may provide insights into the specific enzymatic mediators activated in these cells. This study aimed to examine the morphological changes of fibroblast apoptosis in both primary normal lung fibroblasts (normal-Fb) and fibroblasts obtained from patients with idiopathic pulmonary fibrosis (IPF-Fb) and to correlate these changes with conventional biochemical markers. Transmission electron microscopy (TEM) and video time-lapse microscopy demonstrated no difference in the duration of fibroblast apoptosis in response to FasL (6 +/- 0.3 h in normal-Fb and 6.4 +/- 0.2 h in IPF-Fb). However, IPF-Fb were more resistant to FasL-induced apoptosis compared with normal-Fb. Although the majority of morphological changes of normal-Fb and IPF-Fb were similar, the formation of filopodia and condensation of the cytoskeletal bundles in IPF-Fb, and more prominent vacuolation in normal-Fb, were the significant differences between these cell subtypes. Loss of the mitochondrial membrane potential occurred prior to caspase-3 activation, while phosphatidylserine expression, cytokeratin-18 cleavage, and DNA fragmentation commenced after caspase-3 activation. These observations not only suggest that specific enzymatic effectors may be preferentially activated during fibroblast apoptosis, but also provide potential insights into the pathogenesis of IPF.

Fibroblasts isolated from normal lungs and those with idiopathic pulmonary fibrosis differ in interleukin-6/gp130-mediated cell signaling and proliferation.

Interleukin (IL)-6 and IL-11 are elevated in a variety of lung conditions and may impact on repair mechanisms in chronic inflammatory disorders. However, the mechanisms by which these cytokines influence fibroblast proliferation in normal and disease states have not been previously addressed. We examined the effect of these cytokines on proliferation and cell-cycle kinetics of primary human lung fibroblasts obtained from normal patients and patients with idiopathic pulmonary fibrosis (IPF). IL-6 inhibited the proliferation of normal fibroblasts due to the sustained phosphorylation of STAT-3 and production of the cyclin-dependent kinase inhibitor p19(INK4D). In contrast IL-6 was mitogenic for IPF fibroblasts due to the sustained activation of MAPK, which in turn inhibited the production of p27(Kip1), allowing activation of cyclin D(1) and hyperphosphorylation of retinoblastoma protein. IL-11 was mitogenic for both normal and IPF fibroblasts. These results provide strong evidence for a fundamental abnormality in a cytokine-signaling pathway, as opposed to alterations in cytokine production, in the pathogenesis of IPF.

Inverse effects of interleukin-6 on apoptosis of fibroblasts from pulmonary fibrosis and normal lungs.

Fibroblast apoptosis is crucial to the resolution of fibrosis. However, the mechanisms by which these cells undergo apoptosis are not well known. Because interleukin (IL)-6 and IL-11 may alter repair and remodeling processes, we hypothesized that they may play a role in idiopathic pulmonary fibrosis (IPF). We investigated the effects of these cytokines on Fas-induced apoptosis using primary lung fibroblasts from three patients with IPF (IPF-Fb) and three subjects without lung disease (normal-Fb). IPF-Fb were resistant to Fas-induced apoptosis compared with normal-Fb (P < 0.01). Using RNase protection assays, we showed that IL-6 enhanced Fas-induced apoptosis and expression of Bax in normal-Fb, but inhibited apoptosis and induced expression of Bcl-2 in IPF-Fb. Densitometry of Western blots revealed a Bcl-2/Bax ratio 0.15 +/- 0.01 in normal-Fb compared with 12.05 +/- 1.0 in IPF-Fb. Upregulation of Bcl-2 in normal-Fb and Bax in IPF-Fb were both STAT-3-dependent. Inhibition of extracellular signal-regulated kinase had no effect in normal-Fb, but reversed the antiapoptotic effect of IL-6 in IPF-Fb. IL-11 inhibited Fas-induced apoptosis and increased Bcl-2 expression in both normal-Fb and IPF-Fb. These results suggest that altered IL-6 signaling in IPF-Fb may enhance the resistance of these cells to apoptosis and contribute to a profibrotic effect of IL-6 in IPF.