Cystic fibrosis (CF), caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, is one of the most common inherited lethal diseases with an incidence of the order of 1/2500.¹ It is a multisystemic disease with a complex clinical pattern as CFTR is expressed in subsets of epithelial cell types. Chronic respiratory infections in CF, initially dominated by Staphylococcus aureus and subsequently by Pseudomonas aeruginosa (PA), force persistent inflammation and progressive lung damage which is the main cause of morbidity and ultimately mortality in patients with CF.¹ These chronic infections are therefore considered to be key factors in sustaining inflammation which might find a fertile ground in the “intrinsic” aberrant inflammatory response characteristic of CF patients,^1–3 apparently related to the activation of the NF-κB pathway,^2,4–6 but a deficit of the lipoxin pathway might also be involved.⁷ It has to be stressed, however, that the theory of intrinsically dysregulated inflammation is controversial.⁸ Despite the presence of chronic infections, the infiltrate of patients with CF is characterised by the absence of lymphocyte responses. All these aspects make the inflammatory response in CF atypical. Clinical trials of gene therapy in CF, which is considered to be the prototype of the diseases treatable in this way, have not provided the expected results.⁹ In the absence of definitive therapy, these patients are treated with antibiotics¹ as well as anti-inflammatory regimens to reduce inflammation.^1,10 A series of anti-inflammatory drugs such as ibuprofen, corticosteroids^1,10 or, more recently, long term azithromycin¹¹ have been used to treat patients with CF, without a satisfactory proof of principle for their effectiveness.

The lung of CF patients is characterised by several inflammatory alterations arising from an imbalance in pro-inflammatory signal transduction pathways. The main features of inflamed CF lungs are increased interleukin 8 (IL-8) production and neutrophil infiltration in the parenchyma with influx into the air space.^1,2 It is therefore reasonable to propose that anti-inflammatory treatment of CF should target IL-8 production, thus suppressing neutrophil migration and activation.

We describe a new approach to test potential anti-inflammatory strategies in CF using an ex vivo organ tissue culture model which we have used in the study of other inflammatory conditions and potential strategies to modulate the inflammatory response.^12,13 This model represents a good approximation to in vivo studies since, in cultured biopsy tissues, all the anatomical connections are retained and all cell types (epithelial, myeloid, lymphoid) interact with neighbouring cells within their natural environment. For this study we have used explants of nasal polyps which sometimes complicate CF as they provide a suitable representation of CF chronic inflammation and are routinely removed surgically.¹⁴

Using this approach we have explored whether p38-mitogen activated protein kinase (p38-MAP-k) inhibition represents a suitable and attractive strategy for treating patients with CF. We targeted the p38-MAP-k pathway because of its role in IL-8 production in CF,^15,16 its reported activation in CF cells¹⁷ and in vitro, as well as in vivo, neutralisation of neutrophil function.¹⁸ For this purpose the nasal polyp explants were stimulated with PA-LPS in the presence or absence of a specific and most widely used p38-MAP-k pathway inhibitor. Our study aims to test whether the inhibition of the p38-MAP-k pathway is effective in blocking IL-8 release, neutralising neutrophil migration and degranulation and other signs of CF inflammation. We provide evidence of proof of principle for the potential clinical use of p38-MAP-k inhibitors to control inflammation in CF.

METHODS

Patients and ex vivo cultures

Seven consecutive CF patients with chronic sinusitis and nasal polyposis and seven consecutive non-CF patients (mean age 21 years, range 16–32) with idiopathic polyposis underwent surgical treatment. All CF and control patients presented with a history of more than 2 years of chronic sinusitis with nasal obstruction; all had undergone a sinus computed tomographic scan during their evaluation. Surgery was used only when medical treatment had failed. All patients in both the CF and non-CF groups suspended the use of corticosteroids at least 1 month before the surgical intervention and were only treated by saline nasal irrigation. No patient had allergic rhinitis, as documented by clinical history. The patients were screened for reactivity to the most common inhaled allergens by prick tests and none tested positive.

Nasal polyp explants were cut into at least 10 small specimens within a few minutes after the surgical excision. One fragment from each patient was embedded in optimal cutting temperature (OCT) compound (Tissue Tek, Miles Laboratories, Elkhart, IN, USA) and stored at −80°C. The other fragments were cultured as previously reported for duodenal biopsies^13,19 for 20 minutes, 4 hours, or 24 hours in the presence of medium alone, medium with 1 μg/ml lipopolysaccharide (LPS) from PA (PA-LPS; Sigma, St Louis, MO, USA), or PA-LPS with selective p38-MAP-k inhibitor SB203580²⁰ (Calbiochem, San Diego, CA, USA) and stored at −80°C. SB203580 was used at a concentration of 1 μM considered to be specific for the p38-MAP-k, although effects on other signal transduction pathways might still occur.²⁰ Since SB203580 was diluted 1/1000 from a 1 mM stock solution of DMSO, in all experiments DMSO was used at 1/1000 dilution as an internal control and the samples were analysed together with those cultured in the presence of medium, PA-LPS, or PA-LPS + SB203580. No effects of DMSO were observed in any patient. Culture supernatants were collected and stored at −80°C. Additional details of the ex vivo tissue culture model are provided in an online supplement available on the Thorax website at http://www.thoraxjnl.com/supplemental.

Fully informed consent was obtained from all subjects and the ethical committee of Regione Campania Health Authority approved the study.

COX-2 and IL-8 mRNA detection in tissue samples

Quantitative PCR assessment of COX-2 and IL-8 mRNA were performed as previously described.²¹ Additional details on the method are provided in the online supplement.

Cytokine detection in culture supernatants

Culture supernatants were analysed for the presence of IL-8, IL-1β, IL-6, IL-10, TNF-α and IL-12p70 at 4 hours by Cytometric Bead Array (BD-Biosciences, San Jose, CA, USA) using a FACScan (BD-Biosciences).

Antibodies and antigen detection in tissue samples

Five μm thick cryostat sections were incubated for 2 hours at room temperature with monoclonal antibodies against COX-2, NF-κB (p65 subunit), phosphotyrosine PY99, phospho-p38-MAP-k, cathepsin G, cytokeratin (5/6/8/18). Antigen expression was visualised by immunofluorescence or peroxidase staining. Details on antibodies and antigen detection are shown in table E1 in the online supplement.

COX-2 positive mononuclear cells (MNC) were counted per mm² of mucosa. The distribution of neutrophils (cathepsin G positive polymorphonuclear (PMN) leucocytes) within the different mucosal compartments was analysed within standard reference areas. Epithelial cells with nuclear p65 localisation or with PY-99 or phospho-p38 expression were counted per 100 epithelial cells. Data were blindly evaluated by two independent observers unaware of the culture conditions. Additional details on the method are provided in the online supplement.

Neutrophil migration assay

The MIGRATEST Orpegen (Heidelberg, Germany) was used to study the effects of p38-MAP-k inhibition on in vitro neutrophil migration. The assay was performed according to the manufacturer’s specifications. Samples were analysed by flow cytometry using FACS (Beckman Coulter) with an argon laser tuned at 488 nm. The data were evaluated using WinMDI software.

Western blot analysis

Rabbit polyclonal anti-MAPKAP-k-2[pT³³⁴] (Biosource, Camarillo, CA) was used to dectect changes in p38-MAP-k activity. Further details are provided in the online supplement.

Statistical analysis

Paired data analysis was used to compare samples cultured with medium with those cultured with LPS or LPS + SB203580 in each set of experiments (20 min, 4 hours, and 24 hours). Before analysis, data were screened for gross deviance from normality. Since some groups of variables showed a skewness of >1 and the sample size was small, non-parametric tests (Wilcoxon’s test) for paired samples were applied throughout the study for the analysis of the number of the cells that expressed the tested markers. Non-parametric tests for unpaired samples (Mann-Whitney test) were used to compare CF and control samples for the expression of the tested markers before ex vivo challenge. Statistical analysis was performed using SPSS Version 11.1.

RESULTS

The clinical characteristics of the patients with CF are summarised in table 1. The control group was tested for the presence of the most commonly found mutations in the Mediterranean area and found to be negative.²²

Effect of PA-LPS challenge on p65 nuclear localisation in airway epithelium

Higher numbers of epithelial cells with p65 nuclear localisation were observed in CF nasal polyps before challenge than controls (p = 0.002, fig 1A). PA-LPS challenge led to an increase in the number of airways epithelial cells with p65 nuclear localisation at 20 minutes and 4 hours of incubation (p = 0.018 v cultures with medium alone; fig 1A, B, C, E, F, H and fig E1 online only) which was not detected after 24 hours (fig 1A). This effect of PA-LPS was observed both in CF patients and controls (fig 1A). p65 nuclear localisation was significantly inhibited by SB203580 treatment (p = 0.018 v cultures with PA-LPS alone; fig 1A, D, G, and I); SB203580 also reduced the residual p65 localisation observed in CF biopsies cultured with medium alone (p = 0.018). The most obvious changes following PA-LPS stimulation were seen in epithelial cells. These data indicate that the in vitro culture model allows survival of the nasal explants. They also show that, in the absence of stimuli, NF-κB activation decreases in CF tissues (fig 1A) and they demonstrate the effectiveness of SB203580 treatment in controlling PA-LPS induced p65 nuclear localisation (fig 1A). Importantly, no significant structural differences were observed between CF and control tissues as described in fig E2 (online only) when stained with pancytokeratin antibody.

Effect of PA-LPS challenge on phosphorylation of tyrosine in airway epithelium

We also analysed phosphotyrosine expression by the epithelial cells in explanted nasal biopsy fragments stimulated with PA-LPS. After 4 hours of PA-LPS challenge a significant increase in tyrosine phosphorylation was seen in the epithelial compartment of CF polyps (p = 0.034; fig 2A, B, C, E and F) but not in controls (fig 2A). These changes were more evident after 24 hours of challenge and SB203580 treatment significantly inhibited epithelial phosphotyrosine induction (p = 0.017; fig 2A, D and G).

Effect of PA-LPS challenge on mucosal inflammation

To study the inflammatory responses to PA-LPS challenge in CF nasal polyp explants, we also analysed the expression of COX-2 by mucosal mononuclear cells. This latter marker was studied as a broad parameter of inflammation as described in our previous study.¹³ After 4 hours of challenge and increasingly after 24 hours of incubation, PA-LPS also induced an increase in the numbers of mucosal COX-2 positive mononuclear cells (p = 0.043, fig 3A) and in the expression of COX-2 mRNA (p = 0.043, fig 3B) in both CF tissues and controls compared with samples cultured in medium alone. The increased expression of COX-2 following PA-LPS challenge was efficiently controlled by the p38-MAP-k inhibitor SB203580 (p = 0.043) which also reduced basal COX-2 expression observed in the absence of any LPS stimulation (p = 0.042, fig 3A and B).

Effect of PA-LPS challenge on activation of p38 in the airway epithelium and of SB203580 on p38-MAP-k activity

The results so far described suggest involvement of p38-MAP-k. We therefore studied p38-MAP-k phosphorylation in the epithelial cells after PA-LPS challenge. The mean (SD) number of phospho-p38 positive epithelial cells per 100 epithelial cells after 4 hours of challenge was 49.4 (2.8) in CF tissues v 11.2 (1.0) after culture with medium (p<0.03) and 4.3 (1.9) v 3.7 (1.6) in control tissues (p>0.5; fig 3C, D). To test whether SB203580 inhibited p38-MAP-k activity we monitored the phosphorylation of MAPKAP-k-2, a specific p38-MAP-k substrate.²³ The data shown in fig 3E and 3F indicate that SB203580 controlled phosphorylation of MAPKAP-k-2 at threonine 334. These results suggest that p38-MAP-k is activated in CF tissues and, in our model, SB203580 specifically inhibited the downstream effects of this activation.

Effect of PA-LPS challenge on IL-8 upregulation and neutrophil recruitment to the upper mucosal compartments in CF tissues

One of the key aims of this study was to establish whether IL-8 production and leucocyte migration in CF are controlled by p38-MAP-k inhibition, since neutrophil containment is a target of CF treatment. IL-8 levels were already significantly increased in culture supernatants from all CF samples after 4 hours of challenge with PA-LPS compared with medium alone (p = 0.043, fig 3G). IL-6 was also generally increased but the difference did not reach statistical significance because in one patient (patient no 3, table 1) there was no increase in IL-6 release following PA-LPS challenge (fig 3G). No differences in IL-1β, IL-10, TNF-α, or IL-12p70 expression were found (data not shown).

IL-8 mRNA was significantly increased in CF tissues after 4 hours of PA-LPS challenge compared with samples cultured with medium alone (p = 0.043, fig 3H). The addition of SB203580 completely inhibited the increase in IL-8 mRNA (p = 0.043) as well as IL-8 release (p = 0.043, fig 3G and H) and also partially controlled IL-6 upregulation (p = 0.08, fig 3G). Controls only showed minimal IL-8 upregulation and release of IL-8 or IL-6 (fig 3G and H).

After 24 hours PA-LPS induced a remarkable modification in the pattern of neutrophil distribution in the biopsies in all CF samples, but not in controls. Neutrophils migrated from the deeper mucosal layers and accumulated in the upper (subepithelial and intraepithelial) mucosal compartments (fig 4A, B, C, E) with clustering, degranulation, and release of cathepsin G in the extracellular matrix determined as described in the legend to fig 4 and in the online supplement (p = 0.018 v samples challenged with medium alone for neutrophil redistribution). Neutrophils also crossed the epithelium and moved to the luminal part of the biopsies where clusters of neutrophils were evident outside the epithelial apical membrane (fig 4A, C, E). No changes in neutrophil distribution in the tissue were observed after 20 minutes or 4 hours (data not shown). SB203580 treatment abolished these PA-LPS induced changes (p = 0.018 v samples challenged with PA-LPS alone). In the presence of SB203580 the majority of mucosal neutrophils seeded within the deeper mucosal layers, and the numbers of neutrophils within the subepithelial and intraepithelial mucosal compartments as well as outside the epithelial apical membrane were similar to those observed after culture with medium alone (fig 4A, D, F).

We also tested in vitro neutrophil migration in the presence or absence of the SB203580 inhibitor to define whether the observed neutrophil inhibition operated by SB203580 in the cultured nasal polyp mucosa was due to IL-8 inhibition or to a direct effect on neutrophils. The data shown in table 2 and in fig 5 indicate that SB203580 did not hinder migration but paradoxically favoured neutrophil migration.

These results suggest that PA-LPS challenge induces IL-8 upregulation and neutrophil migration to the upper mucosa layers in CF tissue but not in control tissue. They also suggest that IL-8 inhibition is the primary cause of the changes in neutrophil distribution.

DISCUSSION

Control of inflammation is a key objective in the treatment of CF, as migration of neutrophils into the open air spaces and their degranulation contribute to irreversible lung damage and death of patients with CF.¹ Of all the inflammatory cytokines/chemokines, IL-8 is considered to be the main player in orchestrating inflammation in CF.¹ Importantly, IL-8 has a direct effect on neutrophils by controlling and directing their migratory capacities as well as their activation.²⁴ Other inflammatory cytokines are also increased in CF but their contribution to CF inflammation is not fully understood. It is commonly accepted that infections take advantage of the “fertile” CF hyperresponsive state to sustain chronic inflammation and cause tissue damage in patients with CF.^1,25 The importance of environmental factors in sustaining the inflammatory response is supported by our data as a decrease in p65 nuclear localisation and phosphotyrosine expression is observed when explants are cultured in medium alone. However, we observed that CF tissue was more reactive to PA-LPS, as shown in fig 4 where leucocyte migration to the upper mucosal layers was only seen in CF tissue.

It has recently been reported that activation of the p38-MAP-k pathway is required for Burkholderia cepacia (BC) induced IL-8 release.¹⁶ We adopted a similar methodology to that study using PA-LPS as an environmental stimulus to induce inflammation and trigger IL-8 production.¹⁶ While release of IL-8 was highly controlled by p38-MAP-k inhibition, other signal transduction pathways such as NF-κB has been found to be involved in this process.⁵ However, in our study we detected a reduction in NF-κB activation after p38-MAP-k inhibition (fig 1), indicating a potential extra beneficial effect of neutralisation of this signal transduction pathway. Recent reports have indicated that SB203580 affects NF-κB signalling in intricate ways. It has been shown that SB203580 inhibits p65 phosphorylation,^26,27 but in our model the most obvious effect of SB203580 was the inhibition of p65 nuclear translocation.²⁸ Inhibition of the p38-MAP-k pathway might also directly affect neutrophil function.^18,29 Our data, which indicate inhibition of neutrophil migration by SB203580, might be the result of a combination of indirect (via inhibition of IL-8 secretion, for example) and direct effects on neutrophils. Since neutrophils might release IL-8, SB203580 would thus interfere with a possible self-feeding pathogenic loop. Our experiments demonstrate a specific reduction in IL-8 secretion, supporting the view that epithelial activation with IL-8 secretion precedes neutrophil migration and activation. As indicated above, other cells present in the mucosa might also be involved. In this context it has recently been reported that monocytes from CF patients, heterozygous or homozygous for CFTR mutations, produce more IL-8 when stimulated with LPS.³⁰ In that report it was shown that the p38-MAP-k pathway was also involved and that the increased responsiveness was not related to upregulation of LPS receptor.³⁰ These results help to explain our observation of a good response to LPS challenge in CF tissue but not in controls. Given that PA and BC mediated IL-8 induction is under the control of the p38-MAP-k pathways, and that even “sterile” hyperosmotic stimulation induces IL-8 release controlled by the activation of p38-MAP-k,¹⁵ the inhibition of this signal transduction pathway may represent a valid therapeutic target to control IL-8 production, neutrophil recruitment,³¹ and activation¹⁸ and, ultimately, inflammation in CF. The data presented here demonstrate the efficacy of p38-MAP-k inhibition with a significant decrease in IL-8 production (fig 3). The most compelling result is the effect on neutrophil migration and degranulation as shown in fig 4.

The significance of tyrosine phosphorylation following LPS stimulation and the inhibition of this response by the p38-MAP-k inhibitor is, however, unclear. We have not identified which proteins are tyrosine phosphorylated or the potential downstream tyrosine kinase involved. However, inhibition of tyrosine phosphorylation by the p38-MAP-k inhibitor is consistent with the more specific reductions in COX-2 and IL-8 expression we observed. Further work will be required to clarify which tyrosine kinases are influenced.

Previous studies in animal models of acute respiratory inflammation have shown that inhibition of p38-MAP-k reduces neutrophil involvement in respiratory inflammation.^18,32,33 Our report validates this approach in humans using CF tissue and exploits the ex vivo organ culture model. This model complements and expands studies on isolated single cell types (such as epithelial cells). The validity of ex vivo organ cultures has been highlighted by studies in other chronic inflammatory diseases where they have helped to clarify critical pathogenic steps and outline possible new therapeutic strategies.¹³ Our study has been performed in a small group of patients but the results we obtained have been clearcut, consistent in all samples, and highly statistically significant.

The inhibition of neutrophil migration and degranulation seen in our studies is significant for patients with CF since neutrophil containment is a primary target in CF treatment. In spite of the limited viability of p38-MAP-k deficient mice,³⁴ the clinical use of p38-MAP-k inhibitors is considered to be an attractive therapeutic target to control chronic inflammatory diseases³⁵ and even cancer.³⁶ Thus, new generations of p38-MAP-k inhibitors are under development and are currently being used in ongoing clinical trials.^37,38 A major opportunity in CF could lie in the topical application of such inhibitors via aerosol to target lung inflammation, thus reducing the potential systemic side effects.

We have provided a rationale and proof of principle for the use of p38-MAP-k inhibitors to control CF inflammation, but we have to consider the limitations of the use of nasal polyps to support our conclusions. Although tissue from nasal polyps is useful to study ex vivo CF inflamamtion and its modulation, only a selected and not a fully representative group of patients is studied. Despite this limitation, we believe that studies using nasal polyps are valuable to test potential new therapeutic strategies in CF. At present many compounds with these characteristics are undergoing clinical trials for other (non-CF) inflammatory conditions of the upper respiratory tract.³⁹ The use of p38-MAP-k inhibitors may be an attractive option in the treatment of CF.