Interleukin-13 acts as an apoptotic effector on lung epithelial cells and induces pro-fibrotic gene expression in lung fibroblasts.

BACKGROUND: IL-13 promotes acute allergic asthma and is discussed to play a role in late asthmatic features such as fibrotic processes and airway remodelling. The contributions of IL-13-mediated mechanisms to subepithelial events related to fibrosis are not yet settled. OBJECTIVE: We investigated the impact of IL-13 on lung epithelial cells as apoptotic effector and on lung fibroblasts as inducer of pro-fibrotic gene expression. METHODS: Using the two lung epithelial cell lines A549 and BEAS-2B as well as primary lung epithelial cells, we investigated the capability of IL-13 to induce apoptosis by both flow-cytometry and ELISA. The ability of IL-13 to increase the expression of pro-fibrotic genes and to exert influence on the expression of its own receptor was investigated by real-time quantitative PCR measurement of mRNAs encoding collagen I, collagen III, basic fibroblast growth factor (bFGF), alpha-smooth muscle actin (alpha-SMA) and the IL-13 receptor alpha1 (IL-13Ralpha1) chain in human primary lung fibroblasts. The specificity of IL-13-mediated cellular responses was confirmed by means of an inhibitory monoclonal antibody directed to the IL-13 receptor. RESULTS: IL-13 induces apoptosis in lung epithelial cell lines as well as in primary lung epithelial cells. Furthermore, IL-13 increases the expression of mRNA for alpha-SMA and collagen III, but not for bFGF in human primary lung fibroblasts. The susceptibility of lung fibroblasts to IL-13-induced up-regulation of pro-fibrotic genes is associated with the regulation of IL-13 receptor expression. IL-13-dependent fibrosis-associated effects could be inhibited by antibody-mediated blockade of the IL-13Ralpha1 subunit. CONCLUSION: Our findings indicate a function of IL-13 as a mediator in fibrotic processes leading to loss of functional airway tissue in asthma. They also highlight the therapeutic potential of specifically targeting the interaction between IL-13 and its receptor.

The use of elements of the E. coli Ntr-system for the design of an optimized recombinant expression system for high cell density cultivations.

The inducible glnA promoter 2 of the E. coli glutamine synthetase gene is suitable as an expression unit for the production of recombinant proteins at low and high cell densities. It is active when the concentration of ammonium as the sole nitrogen source in the culture medium is below 1 mM. This nitrogen regulatory system was optimized by introduction of expression cassettes consisting of additional elements of the ntr-system. These artificial constructions result in enhanced recombinant gene expression in the production phase. Furthermore, the basic recombinant protein level during the growth phase is reduced due to a tighter promoter control. A three- to four-fold higher accumulation of chloramphenicol-acetyltransferase (as reporter protein) and of anti-EGF-receptor miniantibodies was achieved by increasing the amount of the final regulator molecule NtrC approximately P via plasmidal co-expression of the ntrC gene. The introduction of a modified glnA promoter 1 inverse to glnAp2 lowered the basic activity of glnAp2 to about one half. It is assumed that under nitrogen excess conditions sigma 70-RNA polymerase binds at glnAp1 and thereby prevents most of the binding of sigma 54-RNA polymerase at glnAp2. The optimized expression systems were successfully applied in low and high cell density cultivations. In the fed-batch phase of high cell density cultivations recombinant protein formation was induced through external nitrogen limitation under FIA-controlled concentration of glucose as carbon source.

High volumetric yields of functional dimeric miniantibodies in Escherichia coli, using an optimized expression vector and high-cell-density fermentation under non-limited growth conditions.

Functional bivalent miniantibodies, directed against the epidermal growth factor receptor, accumulated to more than 3 gl-1 in high-cell-density cultures of Escherichia coli RV308(pHKK) on a pilot scale. The miniantibodies consist of scFv fragments with a C-terminal hinge followed by a helix-turn-helix motif, which homodimerizes in vivo. The improved expression vector pHKK is characterized by the hoklsok suicide system, improving plasmid maintenance, and the inducible lac pl o promoter system with the very strong T7g10 Shine-Dalgarno sequence. The expression unit is flanked by terminators. The prototrophic RV308 cells were cultivated in glucose mineral salt medium and reached a cell density of 145 g dry biomass l-1 after 33 h. After induction, growth continued almost unchanged for a further 4 h with concomitant miniantibody formation. In the fedbatch phase, the concentration of glucose was kept almost constant at the physiological level of approximately 1.5 gl-1, using on-line flow injection analysis for control. Surprisingly, E. coli RV308(pHKK) did not accumulate significant amounts of the metabolic by-product acetate under these unlimited aerobic growth conditions.

Formation of recombinant proteins in Escherichia coli under control of a nitrogen regulated promoter at low and high cell densities.

The use of a modified Escherichia coli glnAP2 promoter results in the formation of both homologous and heterologous, cytoplasmic and periplasmic recombinant proteins in a nitrogen concentration dependent manner. As in the E. coli nitrogen regulatory system, glnAP2 controlled gene expression is induced when ammonium concentration in the growth medium is below 1 mM (nitrogen limitation), otherwise only extremely low expression of recombinant genes is observed. Both high cell density cultivations (HCDC) and low cell density cultivations (LCDC) gave similar results for inducibility and formation of the following recombinant proteins: chloramphenicol-acetyltransferase, phosphorylcholine binding mini-antibodies (scFv-dhlx of McPC603) and K1-streptokinase. Recombinant proteins were formed in quantities of about 2-3% of total cellular protein. At low cell densities, slightly higher quantities resulted under partial nitrogen limitations than under total nitrogen limitation. In contrast, high cell density cultivations resulted in lower product concentrations at partial nitrogen limitation compared with total nitrogen limitation. These lowered product concentrations were probably due to the very high amounts of K+ or Na+ ions which accumulated during pH-regulation, thereby disturbing growth. HCDC under partial nitrogen limitation decreased proteolysis of recombinant proteins, therefore reduced amounts of proteases are considered to be responsible.

Improved bivalent miniantibodies, with identical avidity as whole antibodies, produced by high cell density fermentation of Escherichia coli.

The combination of single-chain Fv-fragments (scFv) with a C-terminal, flexible linking region followed by a designed or natural dimerization domain provides a versatile system for targeted association of functional fragments in the periplasmic space of Escherichia coli. For homodimerization in vivo, two scFv fragments with a C-terminal hinge followed by a helix-turn-helix motif form "miniantibodies" with significantly higher avidity than in the case of leucine zipper containing constructs. The favorable design probably results in an antiparallel four-helix bundle and brings the homodimer to the same avidity as the whole IgA antibody, from which the binding site was taken. The molecular weight of the bivalent miniantibody is almost the same as that of a monovalent Fab fragment. We report here a high-cell density fermentation of E. coli producing these miniantibodies and a work-up procedure suitable for large scale production. Without any need of subsequent chemical coupling in vitro, approximately 200 mg/l of functional dimeric miniantibodies can be directly obtained from the E. coli culture.