Featured Review Cellscience Reviews Vol 1 No.1 ISSN 1742-8130

Cystic Fibrosis: search for the NorthWest Passage

Rhodri J Walters

Introduction

European sailors of the Renaissance searched relentlessly for the elusive NorthWest passage, a mystical channel which would allow them to avoid the savage seas of the Southern Capes and provide a more direct trade route to the Orient. Their search for the NorthWest passage was driven both by necessity and by the prospect of economic gain. This perhaps provides an appropriate analogy for the search for a treatment for Cystic Fibrosis (CF), the most common lethal inherited disorder amongst Caucasians. Cystic Fibrosis is a disease which is characterised by a deficit in the pathway for the regulated movement of Chloride (Cl-) ions across a number of epithelia, impairing the hydration of the airways and intestines. Two faulty copies of the CFTR gene are necessary to produce a CF phenotype which is associated with the malabsorption and maldigestion of food and nutrients from the intestine, and the development of a dry, infected lung. The failure of the intestinal crypt epithelium and pancreatic ducts to secrete fluid and electrolytes causes intestinal blockage (meconium ileus), which was originally the primary cause of death in children affected by CF (O'Loughlin & Grant Gall, 1989). The voyage of discovery seeks an alternative Cl- conductance pathway which may bypass these troubled waters.

The frequency of the CF allele may be as high as 5% in the caucasian population, leading to a CF incidence in the region of 1 in 2000 of all caucasian births, an extraordinarily high prevalence for such a lethal mutation. The explanation as to why CF is the world's commonest lethal heritable condition rests in the advantage of carrying just one copy of the CF gene. Just as the sickle cell anaemia heterozygote (one faulty gene) is believed to afford protection against the malarial parasite, so a single copy of the CF mutation may protect against the entry of the typhoid bacterium across the intestinal epithelial lining, and was therefore selectively favoured during the great European typhoid epidemics (Pier et al., 1998), and may also protect against bronchial asthma (Schroeder et al., 1995). The most frequent CF mutation, with a frequency of around 70% of all CF mutations, is the so-called 'delta' F508 mutation which results in the deletion of a single phenylalanine amino acid residue from position 508 of the CF protein. In contrast to other CF mutations, which result in the insertion of a defective Cl- ion channel into the luminal (apical) membrane, this ΔF508 mutation results in the failure of the efficient transfer of the CF gene product to the apical (luminal) membrane of the epithelial cells which line the airways and the crypt regions of the small and large intestines. The consequence for the phenotype is the same, whether the CFTR Cl- channel is dysfunctional or entirely absent from the membrane, i.e. there is a deficit in the apical Cl- conductance in these epithelia which normally increases in response to phosphorylation by the action of hormones and neurotransmitters which trigger fluid secretion. Many CF mutations thus block Na⁺ and Cl- secretion in response to such secretory modulators (and also to bacterial toxins which cause fluid hypersecretion, or diarrhoea).

Over many years the mechanisms of epithelial secretion have been studied in great detail to find potential pharmaceutical and gene therapeutic strategies towards the treatment of CF (table 1).

The central role of Cl- channels in CF was established in 1983 when Paul Quinton elegantly demonstrated that reabsorptive sweat duct cells have an abnormally low Cl- ion permeability, that is to say that the membrane of CF duct cells does not allow Cl- ions to cross readily. This provided an explanation as to why CF patients had increased concentrations of Na⁺Cl in their sweat owing to decreased salt reabsorption. A deficit in membrane Cl- permeability in response to hormones which increase the levels of an intracellular messenger called cyclic AMP has since been widely demonstrated in other fluid and salt secreting epithelia affected in CF. To understand this defect we must first understand how epithelia transport electrolytes and water.

Epithelial cells form a single layer joined together by tight junctions that separate the membrane into two domains, an apical one facing the duct or lumen, and a basal (basolateral) one facing the cellular tissue which is bathed by small blood vessels. Na⁺, Cl- and K⁺ ions are taken up by a co-transport mechanism in the basal membrane, and the K⁺ and Na⁺ ions are actively recycled across the basal membrane by energy-dependent transport mechanisms, so that concentrations of K⁺ and Cl- are maintained within the cell which are higher than would be predicted if they distributed themselves freely and passively across the membrane under the influence of the concentration gradient and the transmembrane potential. When hormones interact with membrane located receptors they stimulate an increase in the levels of second messengers such as cAMP or Ca²⁺ in the cell. These second messengers activate channels that increase the rate at which K⁺ ions leave the cell across the basolateral membrane and Cl- ions leave the cell via channels that are present in the apical membrane. This results in a transepithelial potential gradient being established across the entire epithelial cell layer because of the negative charge carried into the lumen by Cl- ions. Positively charged Na⁺ ions are obliged to follow the negative charge gradient across the epithelial cell layer through cation selective tight junctions, and water follows the NaCl, driven by the osmotic gradient. In CF, the Cl- channels normally activated by cAMP and PKC are not present or function abnormally, leading to an inability of the epithelium to secrete salt and water adequately (Fig.1).

Strategies for overcoming the CF deficit

In 1988 Mike Gray and co-workers classically showed that cAMP-activated Protein Kinase (PKA) activates small Cl- channels in the apical membrane of the pancreatic duct cell. This occurred around 1 B.C., or one year Before Cloning of the CFTR gene, or Cystic Fibrosis Transmembrane conductance Regulator, was announced in 1989 by Francis Collins and his team. Since researchers discovered that CF was due to a Cl- channel defect they have tried two strategies to correct the symptoms of the disease. One of these has been to use gene therapy to introduce a good copy of the CFTR gene into the CF lung either with a vaccinia virus or with membrane microspheres, called liposomes. The defect in genetically engineered CF mice has already been successfully corrected in the short-term with liposomes carrying the correct copy of the DNA (Hyde et al., 1993). An alternative strategy is to activate other Cl- channels in the apical membrane thereby bypassing the CF deficit. Boucher and Stutts (1992) showed that extracellular ATP acting as a hormone activates alternative Cl- channels in CF lung tissue thus bypassing the CF deficit. Soon after, the crypt regions of the small and large intestines were identified as the regions of the intestinal epithelium affected in Cystic Fibrosis (Walters et al., 1992; Trezise & Buchwald, 1992). The search for an alternative apical Cl- channel in the intestinal epithelia was underway.

Having established that many mutations may lead to mild or severe Cystic Fibrosis, and that a deficit in the capacity to secrete chloride ions is predominantly responsible, the buring question in CF remains as to how best to repair the deficit. Whilst researchers in the 80's and early 90's dreamed that drugs might be used to bypass the deficit, the last decade has seen the explosive advent of gene therapy, driven in all fields by the ground-breaking work of the CF teams. Major barriers preclude the successful use of gene therapy in the treatment of CF. The first problem is that not all the affected cells take up the recombinant DNA that contains the 'good' version of the CF gene. In fact transfection efficiencies (the proportion of cells which take up and express the corrective DNA construct) are reported to be as low as 4% even using optimised nebulizing sprays to disperse the vector throughout the airway. Clearly most or all the cells should preferably take up the reforming DNA. This is clearly not merely a problem of distribution in the lung, as transfection efficiencies of cells in culture using a genetically modified strain of vaccinia virus and lipofectamine were as low as 10%. Ten years later using lipofectamine and plasmid vetors, transfection efficiencies had not improved. When cell cultures are treated with fluorescently labelled “naked” antisense DNA, some 10-20% of cultured cells take up the alien DNA in abundance, whilst most show no signs of uptake. This all-or-none uptake and expression pattern is most likely due to the fact that most cells are only receptive to taking up foreign DNA at a certain stage of the cell cycle, possibly during mitosis (cell division). As only a certain percentage of cells in the airway are at this stage of the cell cycle when treated, transfection efficiencies will be low, even before distribution is taken into account.

The second problem is one of turnover. The cells of the airway and intestines are constantly being shed and passed out of the system as we cough or defecate. Hence within a few days, those cells which have been successfully transfected with the corrective DNA, as indeed many cells are, will be lost along with the processes of wear and tear and renewal. Hence any gene therapy protocol may have to be administered almost daily to succeed, and the toxicological consequences of daily high dose gene therapy are a potential cause for concern. The third major problem is that when we introduce foreign DNA into whole organisms we enter a difficult domain. When cells detect single stranded DNA or RNA or are transfected with a virus, they recognize the DNA or RNA as foreign and enter into programmed cell death (apoptosis). Whilst this is not a bad thing in gene therapy within the realm of cancer, it is not a desirable side effect in the treatment of CF. Further, although people have proposed introducing modified or attenuated viruses in the treatment of AIDS through immunization, the same concerns regarding the use of genetically modified viruses apply in gene therapy. This is to say that even mild strains of a virus may have unpredictable consequences for human health. They may cause new disorders, for instance by oversensitising the immune system, or by inducing latent viruses within the genome to replicate (multiply), or else by recombining in new and unforeseen ways with other viruses. In effect we may be accelerating the viral evolution by the genetic recombination of new viral strains at a rate that does not take place in the natural world. A single laboratory can create thousands of new viral sequences every year by cutting, pasting, recombining and mutating existing ones. Whilst stringent safeguards do exist for their production and dissemination, the consequences of introducing any new strain of virus into the human body cannot be entirely foreseen.

So where does this leave prospects for CF therapy? Certainly gene therapy and embryonic stem cell treatment are still in their infancy, and will almost certainly prevail through the irresistible forces of human will, ingenuity and the seemingly limitless resources available for medical research. For the next ten to twenty years however we should perhaps pay attention to the simple and ingenious work of Stutts & Boucher who showed us that UTP, in combination with amiloride, can bypass the CF deficit through the activation of an alternative Cl- conductance in the airway. Alternate Cl- conductances most likely exist within the pancreas and intestine, and these may too provide potential therapeutic strategies in the shorter term until stem cell and gene therapy research provides safer and more durable treatments for CF. So for the immediate future, pharmacology will remain our primary weapon in the treatment of CF, and hence we must return our attentions to the hunt for alternative Cl- conductances in epithelia and understanding how these are regulated.

The search for alternative Cl- channels

Fundamental to our understanding of the physiology of small intestinal secretion is the location of the Cl- conductance pathway(s) to either the apical or basolateral membrane, and the determination of whether the second messenger pathways regulating changes in membrane Cl- conductance converge upon the same Cl- channel, or act via distinct Cl- channel pathways. The volume-activated Cl- conductance of Necturus enterocytes has been shown to be located in the apical membrane domain (Giraldez et al., 1988), whilst in isolated colonic crypts volume recovery following cell swelling been shown to be mediated by the activation of basolateral Cl- channels by a mechanism that is dependent upon extracellular Ca²⁺ and prevented by inhibitors of the lipoxygenase pathway (Diener et al., 1992). The location and mechanism of regulation of the constituent ion channels which underlie the various Cl- conductances present in the small intestinal crypt epithelium remains to be established, and may be of potential importance in the development of therapeutic approaches for the treatment of CF. In the CF intestine both cAMP- and Ca²⁺-stimulated Cl- secretion are defective (Taylor et al., 1987; Berschneider et al., 1988), whereas Ca²⁺-stimulated Cl- secretion appears to be unaffected in CF airways (Wagner et al., 1991; Widdicombe, 1986). Thus although Ca²⁺ ionophore appears to evoke electrogenic secretory processes across normal human jejunum, the effect upon Cl- conductance may be mediated by the action of PKC rather than by Ca²⁺ and its intracellular mediators, since the ionophore-induced increase in short-circuit current does not occur across the human CF jejunum (O'Loughlin et al., 1991). The body of evidence supports the contention that PKG, PKA and PKC all activate the intestinal CFTR, and unless CF mutations also result in the defective regulation of other types of Cl- channel (Gabriel et al., 1993), there is conflicting evidence for the presence of an alternative Cl- conductance pathway activated by secretagogues in the small intestinal epithelium. In the CF airway epithelium the Cl- secretory deficit has been successfully circumvented by the luminal application of nucleotide triphosphates such as ATP or UTP, (Stutts et al., 1992; Knowles et al., 1991). Thus a better understanding of the nature and the regulation of alternative Cl- conductive pathways present in the apical membrane of the intestinal crypt epithelium is essential if new drugs are to be developed to overcome the secretory deficit in the CF intestine.

Elucidating the Cl- conductances of the intestinal crypt

One fundamental observation from membrane conductance studies in small intestinal crypts is that there is likely a tonic and sustained secretion of fluid and electrolytes from the intestinal crypt compartment (Walters & Sepulveda, 1991). The resting membrane potential of the intestinal crypt, studied in the absence of neurotransmitters or hormones which stimulate cAMP and Ca²⁺ activated secretion, is dominated by a basolateral K⁺ conductance (Walters & Sepulveda, 1991), although there is also a substantial apical component which is diminished by Cl- channel inhibitors. Thus the crypt most likely contains Cl- channels other than the CFTR which mediates a resting level of Cl- conductance and a basal level of fluid and electrolyte secretion. As the apical membrane is inaccessible to study in the intact cylindrical compartment of the crypt, studying these channels requires extreme and innovative techniques, such as the reconstitution of apical membrane within artificial membrane bilayers, or the isolation of a highly purified population of single crypt enterocytes by a sequential process of denudation, sieving, dissociation and marker-driven cellular enrichment. Alternatively a new model for intestinal secretion may be developed, one which overcomes the difficulty of accessing the apical membrane in the intact intestinal epithelium. Transimmortalized mouse intestinal cells (m-ICc12) have been created which maintain a crypt phenotype and form confluent monolayers of morphologically recognisable enterocytes in vitro (Bens et al., 1996). Such a cell monolayer preparation could provide a preparation which would allow simultaneous recording of short-circuit current and apical chloride channel activity in response to agonist stimulation.

Whilst both the cAMP/PKA and Ca²⁺/PKC (CaPKC) signalling pathways augment secretion across the small intestine by enhancing a basolateral K⁺ conductance, both the cAMP/PKA and CaPKC pathways appear to be impaired by the CF deficit, and as both pathways act co-operatively to enhance K⁺ currents so as to maintain the electrical driving force for Cl- secretion, it must therefore be the Cl- conductance which is rate-limiting for intestinal secretion - a proverbial electrochemical “bottle-neck”. The primary question in intestinal secretion is whether both cAMP/PKA and CaPKC pathways act synergistically through the CFTR Cl- conductance to augment increases in membrane Cl- current alone, as the CFTR is activated more potently by PKC and cAMP/PKA than by either kinase alone (Walters et al., 1992), or whether cAMP/PKA pathway activation renders Cl- channels, normally unresponsive to the CaPKC pathway, sensitive to its intracellular mediators. However, even if apical Cl- channels other than the CFTR channel are activated in vivo, they are clearly insufficient to bypass the symptoms of CF without clinical intervention. Thus if other Cl- channels are found, their mechanisms of regulation must be fully elucidated if we hope to circumvent the CF deficit.

Of particular contention is whether a rise in intracellular free Ca²⁺ results in the activation of an alternative Cl- conductance. Crude conductance measurements from whole crypts from small intestinal (Walters & Sepulveda, 1991) and colonic crypts (Bohme et al., 1991; Jens Leipziger personal communication), in addition to studies from the intact CF intestine (Taylor et al., 1987; Berschneider et al., 1988; O'Loughlin et al., 1991), suggest the absence of a Ca²⁺-activated Cl- conductance, which is common in other fluid and electrolyte secreting cells (Cliff et al., 1990; Randriamampita et al., 1988; Wagner et al., 1991). However, volumetric measurements from small intestinal crypts derived from CF mice using the muscarinic CaPKC mobilizing agonist carbachol and crude Cl- channel inhibitors has suggested otherwise (Valverde et al., 1993). Whilst the T84 cell line, a lung metastasis of a colonic carcinoma epithelial cell line, which retains characteristics of both undifferentiated and differentiated lung and colonic epithelial cells, has been reported to express a Ca²⁺ activated Cl- conductance pathway (Barrett & Keely, 2000), this naturally cannot be taken as conclusive evidence in support of either school of thought.

Since the suggestion of Valverde et al (1993) that a putative intestinal Ca²⁺-activated Cl- conductance might provide a viable means to bypass for the CF deficit, many attempts have been made to characterise it. Gruber et al (1998) established that hCLCA1, the first human member of the family of Ca²⁺-activated Cl- channel proteins to be identified, is exclusively expressed in intestinal basal crypt epithelia and goblet cells. Gruber and co-workers believe that hCLCA1 likely produces a functional Ca²⁺-activated Cl- conductance in the human intestine, making for an interesting candidate. The expression cloning of an ileal brush-border (apical) Cl- conductance also led to the isolation of CLCA1, which produces a Ca²⁺ activated Cl- conductance activity when expressed within a heterogenous expression system (Gaspar et al., 2000). Whilst not conclusive, there is now both physiological and molecular evidence for the presence of a Ca²⁺-activated Cl- conductance in the apical membrane of the intestinal crypt. If such a Ca²⁺ activated Cl- conductance pathway does indeed exist, the principal action of Ca²⁺ mobilising agonists upon membrane potential seems to be dominated by the change in K⁺ conductance (Walters & Sepulveda, 1991; Bohme et al., 1991). This would be in agreement with previous results obtained in T84 colonic carcinoma cells, where the muscarinic (CaPKC) agonist evokes Cl- secretion through preactivated Cl- channels by increasing basolateral K⁺ permeability only (Dharmsathaphorn & Pandol, 1986). In other words if such a Ca²⁺-activated Cl- conductance (CACC) were to exist in the crypt in vivo, and if it were functionally expressed in the apical membrane, would its activation be physiologically or pharmacologically be sufficient to ameliorate the CF deficit? Clearly such an apical Ca²⁺ activated Cl- conductance presents an irresistible target, although the more recent report (Valverde et al., 1993) conflicts with earlier findings suggesting that the CF defect blocks Ca²⁺ induced Cl- secretion (Taylor et al., 1987; Berschneider et al., 1988; O'Loughlin et al., 1991). Whilst species specific differences are a popular argument to explain away such anomalies, an appealing explanation might be that the CFTR somehow alters the functioning of an alternative apical Ca²⁺ induced Cl- conductance pathway, as has been shown for other conductance pathways in the airway epithelium (e.g. Gabriel et al., 1993).

Other candidate Cl- channels

A number of Cl- conductances are believed to exist within crypts or colonic epithelial cell lines. For any of these to provide strong candidates as therapeutic targets they must (a) be proven to be distinct Cl- channels directly regulated by these pathways, (b) be shown to be functionally expressed within the proliferating (secretory) region of the crypt compartment, (c) be localised within the apical membrane of the crypt enterocyte, and (d) they must mediate an increase in Cl- conductance and short-circuit current across the crypt epithelium when activated. As the intestinal epithelium is accessible from both the serosal and luminal faces, any such Cl- channel that has a distinctive pathway of regulation (e.g. a volume activated current) would present an appealing therapeutic target.

Since the small intestinal crypt compartment was first shown to mediate the secretion of fluid and electrolytes (Walters & Sepulveda, 1991; Walters et al., 1992), many other studies have provided both direct and indirect evidence for other Cl- channels in the crypt epithelium which are possible candidates for circumventing the CF Cl- deficit. A ClC-5 chloride channel (gpClC-5) has been cloned and functionally expressed from guinea-pig distal small intestinal epithelial cells, and is homogeneously distributed within the crypt and villus regions of duodenal, jejunal and ileal epithelium (Cornejo et al., 2001). Whilst a specific role in agonist evoked Cl- secretion seems unlikely, such a Cl- conductance may for example play a role in regulatory volume decrease or as a tonic Cl- conductance pathway. Another popular idea is that a Cl- conductance within the crypt epithelium is activated during regulatory volume decrease (O’Brien et al., 1991), then this may be “hijacked” pharmacologically to circumvent the CF deficit. However, measurements of volume and single channel activity in isolated rat colonic crypts following exposure to hypotonic media have shown that RVD is mediated by the parallel activation of basolateral K⁺ and Cl- channels (Diener et al., 1992), and if such channels are basolaterally localised in the small intestinal crypt epithelium, then no promising therapeutic strategy appears on the cards. Last, but not least, no discussion of this topic could comfortably omit the release of defensin peptides by Paneth cells (termed cryptdins in mice) into the crypt lumen. Paneth cells are located at the base of the crypt and thus any mediators released from them might be expected to alter crypt physiological function. Defensins form Cl- channel-like activity when applied to apical membranes of airway epithelial monolayers expressing the ΔF508 CF mutation (Merlin et al., 2001) and stimulate Cl- secretion from polarized monolayers of human intestinal T84 cells (Lencer et al., 1997). Thus the intriguing possibility that defensin release may be induced from the Paneth cells in vivo to overcome the CF Cl- deficit has been proposed. Indeed cryptdins 2 and 3 may function as novel intestinal secretagogues, providing a mechanism of paracrine signaling by the reversible formation of ion conductive channels in secretory crypt enterocytes, which are not dependent upon the activation of cAMP or cGMP pathways (Lencer et al., 1997).

A liposomal strategy

One fundamental aspect of the complex pathophysiology of the CF defect has not so far been given due consideration. CF airway epithelial cells provide binding sites for pseudomonas bacteria leading to destruction of lung tissue, due to abnormally low levels of sialylation and elevated sulphation and fucosylation of apical membrane glycoproteins. Barasch and colleagues (1991) suggested that the defective acidification of the trans-Golgi/trans-Golgi network in CF resulted from the diminished CFTR Cl- conductance, leading to defective regulation of glycosylation patterns in secretory granules. Yet more pathology lies below the membrane surface. Wild-type CFTR is known to attain a protease-resistant configuration in an energy dependent process within the ER, which does not occur in the processing of the mutant ΔF508 CFTR, providing one potential explanation as to why this otherwise functional Cl- channel does not reach the apical membrane in sufficient quantities (Lukacs et al., 1994). Ameen and co-workers (2000) identified CFTR protein in both subapical vesicles, and on the apical plasma membrane of the crypt enterocytes. They further demonstrated that cAMP stimulation produced a fluid secretory response which was associated with a redistribution of CFTR from this vesicular pool to the apical membrane, suggesting that cAMP agonists not only increase CFTR Cl- activity, but also recruit additional CFTR protein to the cell membrane associated with increased fluid secretion (Ameen et al., 2003).

As secretory vesicles containing CFTR (and potentially other Cl- conductances) are recruited to the apical membrane by agonist pathways, and given that the CF deficit interferes with a range of processes including vesicular pH regulation, post-translational modification, and the recruitment and apical membrane insertion of CFTR containing vesicles, it follows that other functional Cl- conductances present within these secretory vesicles might also fail to be functionally delivered to the apical membrane. As the CF deficit associated with the prevalent ΔF508 mutation is not due to the loss of the CFTR Cl- channel function, but is rather a problem with the processing and recruitment and insertion of these channels into the plasma membrane, it might also follow that other Cl- channels which might potentially be employed to bypass the CF deficit similarly fail to be incorporated into the plasma membrane together with the CFTR. Thus one potential therapeutic strategy would be to create therapeutic vesicles, such as liposomes, which may be taken orally to rescue the recruitment of these vesicles into the plasma membrane. Such liposomes may contain a combination of therapeutic components. For instance they may incorporate docking receptors or integral membrane-spanning antibodies directed against crypt-specific antigens present upon the microvilli of the apical membrane. Such liposomes might feasibly contain a combination of incorporated proteins in addition to gene therapy vectors. For instance they may contain surface membrane proteins which promote the fusion (docking) of pre-existing CFTR containing vesicles with the crypt apical membrane, or even proteins which otherwise compensate for aberrant pH regulation upon fusion with the CFTR containing vesicles. However such liposomal therapies do not circumvent the need for a better understanding of the Cl- conductances present within the apical membrane of the intestinal crypt.

Conclusions

The presence of apical Cl- pathways distinct from the CFTR within the crypt seems more than a probability. These may ultimately provide a viable means of circumventing the CF Cl- deficit in vivo. However, whether these are functionally independent of regulation by the CFTR (which also regulates cationic, ORDIC and potassium channels in epithelia), or are unimpaired in their function by the defective vesicular trafficking and glycosylation patterns characterised by CF is another matter. Liposomes have already been used successfully as gene therapy vehicles to correct the ion transport defect in CF transgenic mice (Hyde et al., 1993), and why should their payload and functional range not be extended towards the treatment of the intestinal symptoms of CF?

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