Stephen J. Delaney

Cystic fibrosis mice carrying the missense mutation G551D replicate human genotype phenotype correlations

We have generated a mouse carrying the human G551D mutation in the cystic fibrosis transmembrane conductance regulator gene (CFTR) by a one‐step gene targeting procedure. These mutant mice show cystic fibrosis pathology but have a reduced risk of fatal intestinal blockage compared with ‘null’ mutants, in keeping with the reduced incidence of meconium ileus in G551D patients. The G551D mutant mice show greatly reduced CFTR‐related chloride transport, displaying activity intermediate between that of cftr(mlUNC) replacement (‘null’) and cftr(mlHGU) insertional (residual activity) mutants and equivalent to approximately 4% of wild‐type CFTR activity. The long‐term survival of these animals should provide an excellent model with which to study cystic fibrosis, and they illustrate the value of mouse models carrying relevant mutations for examining genotype‐phenotype correlations.

Murine CFTR Channel and its Role in Regulatory Volume Decrease of Small Intestine Crypts

Cystic fibrosis (CF) is caused by mutations in the secretory Cl- channel CFTR (cystic fibrosis transmembrane conductance regulator). Variation in the severity of disease has been attributed to mutations in the CFTR gene that cause different degrees of dysfunction of the CFTR Cl- channel. However, studies of mouse models of CF indicate that the severity of intestinal pathology is not correlated with activity of the CFTR chloride channel. This observation suggests that other ‘environmental’ factors might be important in determining the severity of disease. In this respect, we have identified and characterised an additional cellular defect in intestinal epithelial cells of CF mice, the inability of these cells to regulate their volume after hypotonic challenge. Here, we review the function of murine CFTR as both a Cl- channel and as a regulator of volume-dependent homeostatic cell mechanisms.

Effect of IBMX and alkaline phosphatase inhibitors on Cl− secretion in G551D cystic fibrosis mutant mice

Some cystic fibrosis transmembrane conductance regulator (CFTR) mutations, such as G551D, result in a correctly localized Cl- channel at the cell apical membrane, albeit with markedly reduced function. Patch-clamp studies have indicated that both phosphatase inhibitors and 3-isobutyl-1-methylxanthine (IBMX) can induce Cl- secretion through the G551D mutant protein. We have now assessed whether these agents can induce Cl- secretion in cftrG551D mutant mice. No induction of Cl-secretion was seen with the alkaline phosphatase inhibitors bromotetramisole or levamisole in either the respiratory or intestinal tracts of wild-type or cftrG551D mice. In contrast, in G551D intestinal tissues, IBMX was able to produce a small CFTR-related secretory response [means ± SE: jejunum, 1.8 ± 0.9 μA/cm2, n = 7; cecum, 3.7 ± 0.8 μA/cm2, n = 7; rectum (in vivo), 1.9 ± 0.9 mV, n = 5]. This was approximately one order of magnitude less than the wild-type response to this agent and, in the cecum, was significantly greater than that seen in null mice ( cftrUNC ). In the trachea, IBMX produced a transient Cl- secretory response (37.3 ± 14.7 μA/cm2, n = 6) of a magnitude similar to that seen in wild-type mice (33.7 ± 4.7 μA/cm2, n = 9). This response was also present in null mice and therefore is likely to be independent of CFTR. No effect of IBMX on Cl-secretion was seen in the nasal epithelium of cftrG551D mice. We conclude that IBMX is able to induce detectable levels of CFTR-related Cl- secretion in the intestinal tract but not the respiratory tract through the G551D mutant protein.

The cystic fibrosis transmembrane conductance regulator (CFTR) regulates the sensitivity of macrophages to bacterial lipopolysaccharide

Cystic fibrosis is a chronic inflammatory disease. Most patients are affected by chronic obstructive lung disease associated with novel patterns of bacterial infection, particularly with Pseudomonas aeruginosa. Amongst the symptoms in CF patients is elevated serum levels of cystic fibrosis antigen, a complex of two S-100 proteins (S100A8 and S100A9; also commonly called MRP8 and MRP14) that are abundant in neutrophils and a subset of macrophages. The S100A8 and S100A9 proteins are found in the sera of both patients and heterozygous asymptomatic individuals. The observation that these proteins are also found expressed in the sera of patients with non-CF lung disease has been used to argue that their expression is a sequel to inflammation.1 S100A8 mRNA has been shown elsewhere to be inducible in macrophages by bacterial lipopolysaccharide (LPS).2 To determine whether CF antigen (S100A8/A9) could be induced in a response to infection in the lung, we examined the level of mRNA after intravenous injection of LPS. In control animals, S100A8 mRNA was almost undetectable in any tissue. Within 2 h of LPS injection, the level of mRNA was massively induced and was easily detected using total RNA from lung. Induction was maintained after 8 h. To dissect the sites of expression, we performed mRNA in situ hybridisation and immunocytochemistry. The former approach confirmed that S100A8 mRNA is widely expressed in the LPS-stimulated lung on a subset of cells with epithelial morphology lining alveoli, but is excluded from the bronchial epithelium. Immunolocalisation using anti-S100A8 antibody gave a different pattern, the only cells expressing high levels of S100A8 antigen had a location, morphology, and abundance consistent with their identity as interstitial and alveolar macrophages. The lack of correlation between mRNA and protein has been observed previously in studies of the trophoblasts (unpublished) and suggests that if the S100A8 protein is produced it may be rapidly secreted or in a complex that cannot be detected with the antibody. The chronic pathology observed in CF has been attributed, in part, to inappropriate hyper-responsiveness to lung infection. We have produced a mouse transgenic line with the G551D mutation,3 which has a somewhat less severe gastrointestinal phenotype than the more common human mutation ∆F508 or a complete null. We, therefore, compared the expression of S100A8 mRNA in CF G551D animals and wild-type litter-mates. The animals used in this study were specific pathogen-free and had no overt lung pathology. The majority of the CF animals had readily detectable S100A8 mRNA in the lung, whereas no such signal was detectable in any of the control animals. We injected groups of control and CF animals with LPS. Unlike the controls, which were not obviously affected by the treatment during the time course of the experiment, the CF animals displayed overt symptoms of endotoxaemia, hunched appearance, raised hair, sweating, and shivering. The level of S100A8 mRNA in the lungs of the LPS-treated CF animals was consistently substantially higher than in any of the control animals. Reprobing of these blots for expression of S100A9 revealed that it was similarly induced by LPS, and expressed at higher levels in unstimulated and LPStreated lungs from G551D CF mice. Hybridisation in situ of the S100A8 mRNA in the CF lungs revealed a much more extensive distribution than in wild type litter mates suggesting that, in part, the effect of the mutation is to increase the number of epithelial cells expressing the mRNA. The number of macrophage-like cells detected by immunolocalisation of the S100A8 protein was also increased; and increased expression per cell is not precluded because the labelling is not quantitative. The major cell population that initiates LPS-mediated pathology is the macrophage, which expresses the receptor