Corticosteroid and long-acting ß-agonist therapy reduces epithelial goblet cell metaplasia.

BACKGROUND: Bronchial epithelial goblet cell metaplasia (GCM) with hyperplasia is a prominent feature of asthma, but the effects of treatment with corticosteroids alone or in combination with a long-acting ß2 -adrenergic receptor agonist (LABA) on GCM in the bronchial epithelium are unknown. OBJECTIVES: To determine whether corticosteroid alone or in combination with a LABA alters protein and gene expression pathways associated with IL-13-induced goblet cell metaplasia. RESULTS: We evaluated the effects of fluticasone propionate (FP) and of salmeterol (SM), on the response of well-differentiated cultured bronchial epithelial cells to interleukin-13 (IL-13). Outcome measures included gene expression of SPDEF/FOXa2, gene expression and protein production of MUC5AC/MUC5B and morphologic appearance of cultured epithelial cell sheets. We additionally analysed expression of these genes in bronchial epithelial brushings from healthy, steroid-naïve asthmatic and steroid-treated asthmatic subjects. In cultured airway epithelial cells, FP treatment inhibited IL-13-induced suppression of FOXa2 gene expression and up-regulation of SPDEF, alterations in gene and protein measures of MUC5AC and MUC5B and induction of GCM. The addition of SM synergistically modified the effects of FP modestly-only for gel-forming mucin MUC5AC. In bronchial epithelial cells recovered from asthmatic vs healthy human subjects, we found FOXa2 and MUC5B gene expression to be reduced and SPDEF and MUC5AC gene expression to be increased; these alterations were not observed in bronchial epithelial cells recovered after treatment with inhaled corticosteroids. CONCLUSION AND CLINICAL RELEVANCE: Corticosteroid treatment inhibits IL-13-induced GCM of the airways in asthma, possibly through its effects on SPDEF and FOXa2 regulation of mucin gene expression. These effects are modestly augmented by the addition of a long-acting ß-agonist. As we found evidence for drug treatment counteracting the effects of IL-13 on the epithelium, we conclude that further exploration into the mechanisms by which corticosteroids and long-acting ß2 -adrenergic agonists confer protection against pathologic airway changes is warranted.

Expression of airway secretory epithelial functions by lung carcinoma cells.

We examined 12 non-small cell lung carcinoma cell lines for expression of airway goblet, serous, and mucous cell characteristics. The cells expressed some ultrastructural traits of secretory epithelial cells but none contained secretory granules typical of the airway secretory cells. Using immunocytochemistry and cell-specific monoclonal antibodies, we identified heterogeneous expression of goblet, mucous, and serous cell markers among the cell lines. After metabolic radiolabeling, cells incorporated isotope into high molecular weight material. Incubation of pulse-radiolabeled cells with a number of known mucus secretogogues revealed that 5 of the 12 cell lines released radiolabeled material in response to the agonists. However, in each cell line only one of the receptor-activated pathways tested was intact. Although we did not identify a single cell line expressing a phenotype similar to normal airway secretory cells, particular functions retained by some of these cell lines may make them useful for specific studies of mucus production or secretion.

Definition of a C-reactive protein binding determinant on histones.

C-reactive protein (CRP) is an acute phase inflammatory protein in man which binds to phosphocholine, chromatin, histones, and the 70-kDa protein of the U1 small nuclear ribonucleoprotein particle in a calcium-dependent, phosphocholine-inhibitable manner. CRP also binds to other proteins including fibronectin. The determinants involved in CRP binding to these diverse proteins have not been identified. The binding of CRP to histones was examined as these proteins are available in large quantity at high purity and subject to protease digestion with well characterized products. Histone H1 was digested with thrombin and trypsin to produce three distinct fragments, N-terminal, central globular, and C-terminal. CRP was shown only to bind to the C-terminal fragment. Binding to histone H2A was also examined. CRP binding was not diminished by cleavage of the C-terminal fragment but was greatly decreased when the central globular region of H2A was tested. Peptides were prepared to be identical to the N- and C-terminal fragments of H2A. The N-terminal (15 amino acid) fragment of H2A blocked CRP-induced precipitation of phosphocholine-coupled bovine serum albumin and histone H2A, whereas the C-terminal fragment showed no inhibition. Thus we have defined the first reported CRP binding determinant on a protein.

Analysis of the binding of C-reactive protein to chromatin subunits.

C-reactive protein (CRP) is an acute phase serum protein in man. The functional activities of CRP, like Ig, include complement activation and enhancement of phagocytosis. CRP binding to several substrates, including phosphocholine, individual denatured histones, and chromatin, has been demonstrated. We previously demonstrated that CRP binding to chromatin is dependent on the presence of histone H1, despite the fact that CRP binds to purified individual histones H2A and H2B, as well as to H1. In this report we examined the binding of CRP to native sub-nucleosomal chromatin fragments. CRP binding to the H2A-H2B dimer and (H3-H4)2 tetramer was demonstrated and these reactions were inhibited by phosphocholine. However, no binding to the subnucleosome complexes (H2A-H2B)-DNA and (H3-H4)2-DNA was seen. Similarly, CRP binding to H1 was eliminated when H1 was reconstituted with DNA. The reconstitution of H1-depleted chromatin with H1 restored CRP binding. CRP binding to nucleosome core particles, as previously demonstrated by others, was confirmed. Therefore, the interaction of CRP with individual core histones does not appear to be responsible for the binding of CRP to native chromatin. However, binding to core particles could be mediated by differentially exposed determinants on H2A and H2B.

Analysis of the binding of C-reactive protein to histones and chromatin.

C-reactive protein (CRP) is an acute phase serum protein in man which binds to phosphocholine (PC) in a calcium-dependent manner. CRP has been shown to bind to chromatin and nucleosome core particles. However, CRP does not bind to DNA and there is conflicting evidence regarding the binding of CRP to histones. In the present study, binding of CRP to chromatin was confirmed by ELISA using chromatin bound to microtiter wells. When chromatin depleted of histone H1 was used in the same assay, no CRP binding was detected. Similar results were observed using a competitive inhibition ELISA. These results indicate an important role for H1 in the binding of CRP to chromatin. Further studies were done to characterize the binding of CRP to purified individual histones. CRP binding to histones was demonstrated first by blotting. Calf thymus histones were separated on a 15% SDS-polyacrylamide gel, transferred to nitrocellulose, and probed with 125I-CRP. CRP bound to H1 and H2A and to a lesser extent to H2B. Non-specific binding to H3 was seen and no binding to H4 was observed. CRP binding to purified individual histones was tested by ELISA. Essentially identical results were seen to those obtained by blotting. CRP binding to the H2A-H2B complex was observed as well as reactivity with trypsin-resistant fragments of H2A, H2B, and H3. By blotting and by ELISA all CRP reactions were blocked by PC and EDTA indicating binding through the calcium-dependent PC-binding site on CRP. These studies further characterize the nature of the binding of CRP to chromatin and histones and show that the presence of H1 on chromatin is required for CRP binding.

Decreased autoantibody levels and enhanced survival of (NZB x NZW) F1 mice treated with C-reactive protein.

C-Reactive protein (CRP) is the prototypic acute-phase serum protein in man. On the basis of its binding specificities and activities, it has been proposed that CRP facilities the removal of nuclear material released from damaged cells. To determine whether such a process could alter the development of autoimmunity to nuclear antigens, the effect of CRP on autoimmune disease in the (NZB x NZW) F1 mouse model of systemic lupus erythematosus was tested. Mice were injected with chromatin bound to latex beads in the presence or absence of bound CRP. CRP treatment significantly prolonged survival of mice injected with chromatin-coated beads. CRP also produced a transient decrease in IgG antibody levels to histones, DNA, and DNP, suggesting a general suppressive effect on ongoing antibody responses. To determine whether CRP would affect chromatin clearance, the effect of CRP on nucleosome core particle clearance was tested in BALB/c mice. CRP pretreatment did not alter the rate of clearance or organ localization of nucleosome core particles. These findings indicate that CRP can modify the course of autoimmune disease possibly by preventing the exposure of nuclear antigens to the immune system.