Sputum hyaluronan and versican in severe eosinophilic asthma.

BACKGROUND: We examined levels of hyaluronan, a matrix glycosaminoglycan and versican, a matrix proteoglycan, in the sputum of asthmatics treated with mepolizumab (anti-IL-5 monoclonal antibody) versus placebo to evaluate the utility of these measurements as possible biomarkers of asthma control and airway remodeling. METHODS: Patients with severe, prednisone-dependent asthma received either mepolizumab or placebo as described in a previously published randomized, double-blind, placebo-controlled study. We measured hyaluronan and versican levels by enzyme-linked immunosorbent assay in sputum collected before and after the 16-week treatment phase. Patients underwent a predefined prednisone tapering schedule if they remained exacerbation free, and sputum eosinophil percentage, asthma control questionnaire (ACQ) and spirometry were monitored. RESULTS: After 6 months of mepolizumab therapy and prednisone tapering, there was a significant increase in sputum hyaluronan in the placebo group compared with baseline (p = 0.003). In contrast, there was a significant decrease in sputum hyaluronan in the active treatment group compared with placebo (p = 0.007), which correlated with improvements in percent forced expiratory volume in 1 s (FEV1%) (p = 0.001) and ACQ scores (p = 0.009) as well as a decrease in sputum eosinophils (p = 0.02). There was a nonsignificant increase in sputum versican in the placebo group (p = 0.16), a decrease in the mepolizumab group (p = 0.13) and a significant inverse correlation between versican reduction and FEV1% improvement (p = 0.03). CONCLUSIONS: Sputum hyaluronan values are reduced with mepolizumab therapy and correlate with improved clinical and spirometry values, suggesting this measurement may serve as a noninvasive biomarker of asthma control.

Polyinosine-polycytidylic acid stimulates versican accumulation in the extracellular matrix promoting monocyte adhesion.

Viral infections are known to exacerbate asthma and other lung diseases in which chronic inflammatory processes are implicated, but the mechanism is not well understood. The viral mimetic, polyinosine-polycytidylic acid, causes accumulation of a versican- and hyaluronan-enriched extracellular matrix (ECM) by human lung fibroblasts with increased capacity for monocyte adhesion. The fivefold increase in versican retention in this ECM is due to altered compartmentalization, with decreased degradation of cell layer-associated versican, rather than an increase in total accumulation in the culture. This is consistent with decreased mRNA levels for all of the versican splice variants. Reduced versican degradation is further supported by low levels of the epitope, DPEAAE, a product of versican digestion by a disintegrin-like and metallopeptidase with thrombospondin type 1 motif enzymes, in the ECM. The distribution of hyaluronan is similarly altered with a 3.5-fold increase in the cell layer. Pulse-chase studies of radiolabeled hyaluronan show a 50% reduction in the rate of loss from the cell layer over 24 hours. Formation of monocyte-retaining, hyaluronidase-sensitive ECMs can be blocked by the presence of anti-versican antibodies. In comparison, human lung fibroblasts treated with the cytokines, IL-1beta plus TNF-alpha, synthesize increased amounts of hyaluronan, but do not retain it or versican in the ECM, which, in turn, does not retain monocytes. These results highlight an important role for versican in the hyaluronan-dependent binding of monocytes to the ECM of lung fibroblasts stimulated with polyinosine-polycytidylic acid.

Comparison of the in vivo autologous skin test with in vitro diagnostic tests for diagnosis of chronic autoimmune urticaria.

Previous studies indicate that 30-50% of chronic urticaria patients have an autoimmune etiology. Clinical diagnosis of autoimmune urticaria is supported with the autologous serum skin test. The purpose of this study was to compare two laboratory tests for measurement of IgG autoantibodies to IgE or IgE receptors and compare the results with the autologous serum and plasma skin tests. We performed skin tests and two functional in vitro tests, basophil histamine release, and CD63 up-regulation to detect autoantibodies relevant to autoimmune urticaria. Both sera and citrated plasma were evaluated in the autologous skin test and histamine release assay. Thyroid autoantibodies were also measured. Basophils were incubated with patient plasma, sera, buffer, or anti-IgE. The cells were analyzed for CD63 expression and the supernatants were recovered for histamine analysis. There was high correlation between CD63 up-regulation and histamine release assays, but histamine release was more sensitive. There was a high concordance between sera and citrated plasma for the skin test. Sera from chronic urticaria patients produced higher mean histamine release (23%) compared with citrated plasma (12%). Thirty-one percent of patients positive in the histamine release assay were also positive for thyroid autoantibodies. This compares with 12% who were negative in the histamine release assay. These data show that in vitro basophil histamine release can be used to measure antibodies to FceRI, FceRII/CD23, or IgE and identify patients with autoimmune urticaria.

Pro- and anti-inflammatory factors cooperate to control hyaluronan synthesis in lung fibroblasts.

Hyaluronan (HA) is an important constituent of the extracellular matrix and accumulates during inflammatory lung diseases like asthma. Little is known about the factors that regulate HA synthesis by lung cells. Accordingly, we investigated the effect of T-helper 1 (TH1) and 2 (TH2) cytokines and the anti-inflammatory agents fluticasone and salmeterol on HA synthesis in human lung fibroblasts. Interleukin-1beta (IL-1beta) and tumor necrosis factor (TNF)-alpha were the most potent stimulators of HA synthesis and when combined, caused synergistic increases in HA accumulation. Time-course analysis of HA accumulation and [3H]-glucosamine incorporation into HA demonstrated continued synthesis over the 24 h of stimulation. Peak synthesis at 6-12 h coincided with an increased proportion of high molecular weight HA. Reverse transcriptase polymerase chain reaction (RT-PCR) revealed that IL-1beta and TNF-alpha induced HA synthase-2 messenger RNA (mRNA) 3 h following stimulation and remained elevated throughout the 24-h stimulation period. Fluticasone inhibited IL-1beta and TNF-alpha induced HA synthesis (44.5%) whereas salmeterol had no effect. When combined, fluticasone and salmeterol inhibited HA synthesis to a greater extent (85.2%). Further, fluticasone attenuated IL-1beta and TNF-alpha stimulated hyaluronan synthase-2 messenger RNA (mRNA), and the addition of salmeterol cooperatively enhanced this inhibition. These results indicate that enhanced synthesis of HA by the proinflammatory cytokines IL-1beta and TNF-alpha can be abrogated by specific corticosteroid and beta2 blocker combinations shown to be effective in the treatment of asthma.

Regulation of proteoglycan synthesis by leukotriene d4 and epidermal growth factor in bronchial smooth muscle cells.

Extracellular matrix (ECM) expansion contributes to airway remodeling in asthma. This study examines the effect of leukotriene D4 (LTD4), combined with epidermal growth factor (EGF), on proteoglycan synthesis by cultured human bronchial smooth muscle cells (BSMCs). LTD4 plus EGF stimulated proliferation of BSMCs with increased versican synthesis. Further, versican mRNA splice variants, V0 and V1, were differently regulated in BSMCs by LTD4 plus EGF. Synthesis of [35S]-methionine labeled versican V0, as a percentage of total versican, was doubled. This upregulation was confirmed by Western analysis. Synthetic changes were paralleled by alterations in versican V0 mRNA. The effects of LTD4 and EGF on proteoglycan synthesis were inhibited by montelukast. Similar upregulation of versican V0 was observed in arterial smooth muscle cells (ASMCs) stimulated with LTD4 plus EGF as measured by western and reverse transcriptase-polymerase chain reaction analyses. Changes in ECM in the asthmatic airway may parallel those in atherosclerotic lesions where proliferating ASMCs synthesize a versican-rich expanded ECM. Inhibition of these processes could lead to reduced tissue expansion in the early phases of asthma progression.