Selective up-regulation of chemokine IL-8 expression in cystic fibrosis bronchial gland cells in vivo and in vitro

Abstract

Accumulating evidence suggests that the early pulmonary inflammation pathogenesis in cystic fibrosis (CF) may be associated with an abnormal increase in the production of pro-inflammatory cytokines in the CF lung, even in the absence of infectious stimuli. We have postulated that if baseline abnormalities in airway epithelial cell production of cytokines occur in CF, they should be manifested in the CF bronchial submucosal glands, which are known to express high levels of CFTR (cystic fibrosis transmembrane conductance regulator) protein, the gene product mutated in CF disease. Immunohistochemical analyses showed that CF bronchial submucosal glands in patients homozygous for the deltaF508 deletion expressed elevated levels of the endogenous chemokine interleukin (IL)-8 but not the pro-inflammatory cytokines IL-1beta and IL-6, compared with non-CF bronchial glands. Moreover, basal protein and mRNA expression of IL-8 were constitutively up-regulated in cultured deltaF508 homozygous CF human bronchial gland cells, in an unstimulated state, compared with non-CF bronchial gland cells. Furthermore, the exposure of CF and non-CF bronchial gland cells to an elevated extracellular Cl- concentration markedly increased the release of IL-8, which can be corrected in CF gland cells by reducing the extracellular Cl- concentration. We also found that, in contrast to non-CF gland cells, dexamethasone did not inhibit the release of IL-8 by cultured CF gland cells. The selective up-regulation of bronchial submucosal gland IL-8 could represent a primary event that initiates early airway submucosal inflammation in CF patients. These findings are relevant to the pathogenesis of CF and suggest a novel pathophysiological concept for the early and sustained airway inflammation in CF patients.

Figure 1.

Expression and localization of chemokine IL-8 in human ΔF508 homozygous CF and non-CF bronchial tissues. Analysis of frozen tissue sections (5 μm thick) in CF bronchial surface epithelium (a and b) and submucosal gland structures from ΔF508 homozygous CF patients (c and e–h) and non-CF (control) patients is presented (d). Shown are Nomarski photomicrographs (a and e) and immunofluorescence micrographs for detection of IL-8 with FITC (green; c, d, and g) and lysozyme with Texas red (red; f and h). Note the absence of immunostaining for IL-8 at the level of ΔF508 homozygous CF bronchial surface epithelium (b) as in non-CF bronchial surface epithelium (not shown). In contrast, dense IL-8 staining is specifically observed in most of the submucosal gland cells from CF bronchial tissues (c) but is not identified in the submucosal glad cells from non-CF bronchial tissues (d). At higher magnification (×400), ΔF508 homozygous CF bronchial submucosal glands (e–h) are photographed using fluorescence filters for simultaneous red (lysozyme; f)/green (IL-8; g) visualization. The yellow color (h) indicates significant co-staining of IL-8 and lysozyme in ΔF508 homozygous CF bronchial submucosal gland serous-type cells. Results are representative of the findings in eight ΔF508 homozygous CF patients and four non-CF (control) subjects. All sections are counterstained with hematoxylin.

Figure 2.

Functional CFTR protein is demonstrated in cultured non-CF HBG cells but not in ΔF508 homozygous CF HBG cells. Changes in SPQ halide efflux for non-CF and ΔF508 homozygous CF HBG cell cultures were evaluated as described in Materials and Methods. Cells were stimulated with 25 μmol/L forskolin (time 0), and the Cl⁻ secretion was estimated by measurements of SPQ fluorescence over a 15-minute time period. Data are presented as the relative fluorescence, this being 100X (Ft/Fo), where Ft is the fluorescence intensity at time t and Fo is the fluorescence intensity at time 0. A significant increase in Cl⁻ efflux in response to cAMP stimulation is observed in monolayers of non-CF HBG cells. In contrast, no significant change in cAMP-stimulated Cl⁻ efflux is observed in monolayers of ΔF508 homozygous CF HBG cells. Data shown are representative of responses obtained from four experiments for each condition.

Figure 3.

Comparison of the endogenous IL-8 mRNA expression and spontaneous levels of indicated cytokines released by cultured ΔF508 homozygous CF and non-CF HBG cells in the unstimulated (basal) state. Northern blot analysis (a) shows the presence of endogenous IL-8 mRNA transcripts in CF HBG cells. No evidence of endogenous IL-8 mRNA transcripts is found in non-CF HBG cells, under the same conditions of culture. Shown are Northern blots of total cellular RNA (15 μg/lane) from CF and non-CF HBG cells hybridized with a ³²P-labeled IL-8 cDNA probe (top) and normalized to 28 S expression (bottom). The 1.8-kb IL-8 mRNA transcript is indicated. ELISAs (b) of the spontaneous production of the indicated cytokines in 6-hour supernatants show that the level of IL-8 release is 13-fold higher in CF HBG cells compared with non-CF HBG cells. Other cytokine levels are lower, and no significant difference between CF and non-CF HBG cell cultures is found. Values represent means ± SD of eight ΔF508 homozygous CF HBG and four non-CF HBG cell cultures, respectively, each assayed in triplicate.

Figure 4.

Effect of the extracellular concentration of Cl⁻ on the IL-8 production by cultured ΔF508 homozygous CF and non-CF HBG cells. Confluent monolayers of CF and non-CF HBG cells were covered by 2 ml of a solution containing 1 mol/L CaCl₂, 20 mol/L KCl, and either 60 mol/L NaCl or 105 mol/L NaCl or 148 mol/L NaCl (total Cl⁻ concentration indicated for each data set), pH 7.4. IL-8 levels were assayed in 6-hour supernatants collected from CF and non-CF HBG cell cultures exposed to a low (85 mol/L), intermediate (135 mol/L), or high (170 mol/L) Cl⁻ concentration, respectively, as indicated. Lowering the extracellular Cl⁻ concentration to 85 mol/L decreased the IL-8 release of CF HBG cells to a level very similar to that observed in non-CF HBG cells. Each data point represents the average of three determinations for eight ΔF508 homozygous CF HBG cell cultures and four non-CF HBG cell cultures.

Figure 5.

Effect of dexamethasone treatment on the IL-8 production by cultured ΔF508 homozygous CF and non-CF HBG cells. IL-8 levels were measured in 6-hour supernatants from CF and non-CF HBG cell cultures after addition of dexamethasone at final concentrations of 1, 5, and 10 μmol/L, respectively. Data are expressed as a percentage of IL-8 recovered 6-hour after dexamethasone addition by comparison to values obtained from untreated CF and non-CF HBG cell cultures (control, 100%), respectively. Interestingly, dexamethasone treatment significantly (P < 0.001) blocks the IL-8 release of non-CF HBG cells but, in contrast, failed to block the IL-8 release of CF HBG cells, even when levels were increased to 5 μmol/L. Data represent means ± SD of eight CF HBG cell cultures and four non-CF HBG cell cultures, each assayed in triplicate. **P < 0.01; ***P < 0.001.