Systemic Inflammatory Mediators And Cystic Fibrosis Genotype

Clin Exp Med (2004) 4:99–102 DOI 10.1007/s10238-004-0044-1 ORIGINAL

A. Augarten • G. Paret • I. Avneri • H. Akons • M. Aviram • L. Bentur • H. Blau • O. Efrati • A. Szeinberg A. Barak • E. Kerem • J. Yahav

Systemic inflammatory mediators and cystic fibrosis genotype

Received: 31 March 2004 / Accepted: 6 September 2004

Abstract Morbidity and mortality in cystic fibrosis patients is mainly attributed to pulmonary infection and inflammation. Chemokines play a pivotal role in the inflammatory process. Although genotype-phenotype correlation in cystic fibrosis patients has been defined, a clear relationship between the defect in the cystic fibrosis transmembrane regulator (CFTR) gene and pulmonary inflammation has not been established. The aim of this study was to assess whether serum chemokines levels in cystic fibrosis patients correlate with genotype and pulmonary function tests, as well as with other clinical characteristics. Serum levels of interleukin-8, RANTES, and monocyte chemoattractant protein-1 were measured in 36 cystic fibrosis patients grouped according to their genotype. Group A included 25

A. Augarten () National CF Center, The Chaim Sheba Medical Center, Tel-Hashomer, Israel 52621 e-mail [email protected] Tel.: +972-3-5303054 Fax: +972-3-5305939 A. Augarten • G. Paret • I. Avneri • H. Akons • O. Efrati A. Szeinberg • A. Barak • J. Yahav Chaim Sheba Medical Center, Tel-Hashomer, affiliated to the Sackler School of Medicine, Tel-Aviv University, Israel M. Aviram Soroka Medical Center, Be’er Sheba, Israel L. Bentur Rambam Medical Center, Haifa, Israel H. Blau Schnieder Medical Center, Petach Tikva, Israel E. Kerem Shaare Zedek Medical Center, Jerusalem, Israel

patients who carried two mutations associated with a pathological sweat test and pancreatic insufficiency (∆F508, W1282X, G542X, N1303K, S549R). Group B included 11 compound heterozygote patients who carried one mutation known to cause mild disease with borderline or normal sweat test and pancreatic sufficiency (3849+10kb C to T, 5T). Associations between chemokine levels, genotype, pulmonary function, Pseudomonas aeruginosa colonization, age, sweat chloride level, and pancreatic and nutritional status were examined. Mean interleukin-8 and monocyte chemoattractant protein-1 levels were significantly higher in group A than group B (11.4±2.1 pg/ml vs. 5±0.9 pg/ml and 157±16 pg/ml vs. 88.8±16.4 pg/ml, respectively) (P<0.01). No difference in RANTES levels were found between groups. interleukin-8 levels were inversely related to forced expiratory volume in 1 s (r=-0.37, P<0.02), while there was no association between the latter and RANTES and monocyte chemoattractant protein-1 levels. The Pseudomonas colonization rate was higher among group A patients than group B (88% vs. 40%, P<0.01). No relationship was found between measured chemokines and age, sweat chloride levels, and pancreatic and nutritional status. Our study demonstrates an association between interleukin8, forced expiratory volume, and cystic fibrosis genotype. Hence, elevated interleukin-8 serum levels could serve as an indicator of an early inflammatory process and encourage the initiation of anti-inflammatory treatment. Key words Cystic fibrosis Chemokines • Genotype

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Pulmonary inflammation

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Introduction Cystic fibrosis (CF) is a multi-system genetic disease characterized mainly by progressive lung disease, pancreatic dysfunction, and elevated sweat chloride [1]. The

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severity of the disease varies considerably among patients. Since the identification of the CFTR gene, more than 1,000 mutations have been recognized. The wide spectrum of disease severity and the existence of many mutations led investigators to study the correlation between genotype and phenotype in CF patients. These studies demonstrated a direct linkage between pancreatic function, nutritional status, sweat chloride levels, and longevity and CF genotype. However, such a relationship has not been established between the genetic defect and the severity of pulmonary disease [2–4]. The lung tissue damage has been mainly attributed to infection and subsequent inflammation [5, 6]. The recruitment of leukocytes to tissue is essential for inflammation. This process is controlled by chemokines such as interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP1), and RANTES, which are chemotactic cytokines. It is assumed that chemokines provide directional cues for the movement of leukocytes by mediating their interaction with vascular endothelium, which leads to their extravasation [7, 8]. In this study we evaluated the association between serum chemokines and the CFTR genotype, pulmonary function, and other clinical characteristics of our CF patients.

described standardized pulmonary equations (Polgar and Promadhat). Sampling of blood for chemokine levels and spirometry were performed during a routine outpatient clinic visit and not during an exacerbation of the pulmonary disease. The following clinical parameters were extracted from patients’ charts: age, sweat chloride level , height and weight percentiles, result of the last sputum culture, and pancreatic status. The pancreatic function was determined to be either pancreatic sufficiency or pancreatic insufficiency by measuring pancreatic elastase levels in stool or by fat measurement in 72-h stool collections.

Statistical analysis All measurements are expressed as mean values±standard error. Comparisons of chemokine levels and clinical characteristics between groups were performed using Student’s t-test. The association between serum chemokine levels and FEV1, sweat chloride levels and age were assessed by Pearson’s coefficient. Analysis of variance (ANOVA) was used to evaluate the association between chemokines and nutritional status, as expressed by different height and weight percentiles. A P value of less than 0.05 was considered significant. Statistical calculations were performed using SPSS statistical package (SPSS, Chicago, Ill., USA). The study was approved by the institutional review board of Sheba Medical Center.

Materials and methods The cohort of this study comprised 36 CF patients whose diagnosis was confirmed by genetic analysis and who were able to perform spirometry. Patients were grouped according to their genotype. Group A included 25 patients who carried two mutations associated with a pathological sweat test and pancreatic insufficiency (∆F508, W1282X, G542X, N1303K, S549R) [2]. Group B included 11 compound heterozygote CF patients who carried one mutation known to cause mild disease with a borderline or normal sweat test and pancreatic sufficiency (3849+10kb C to T, 5T) [3, 4, 9].

Chemokines IL-8, MCP1, and RANTES levels were measured in venous blood samples (2 ml), using a sandwich ELISA (Endogen, Woburn, Mass., USA). The assay was performed according to the manufacturer’s instructions, and all samples were analyzed at a dilution resulting in concentrations within the range of the standard curve.

Spirometry Forced expiratory volume at 1 s (FEV1) was expressed as a percentage of predicted values for height and sex, using previously

Results Table 1 displays the demographics and clinical characteristics of the study cohort. No significant difference was found in the mean age between the two groups; the age range was 1–30 years and 3–37 years for groups A and B, respectively. All patients in group A had pancreatic insufficiency while 4 of 11 patients of group B had pancreatic sufficiency. The nutritional status was better in group B patients, as indicated by significantly higher mean weight percentiles. Sputum cultures yielded Pseudomonas aeruginosa at a significantly higher rate in group A patients (88% vs. 40%, P<0.01). However, no difference in IL-8 levels was found between patients with P. aeruginosa colonization and those with negative sputum cultures (9.76±9.18 vs. 7.93±12.6 pg/ml, P=0.6). Mean IL-8 and MCP1 levels were significantly higher in group A than group B; RANTES levels were not significantly higher in group A patients (Table 2). There was a significant inverse relationship between IL-8 levels and FEV1 values (Pearson r=-0.37, P<0.02) (Fig. 1). No relationship was found between RANTES and MCP1 levels and FEV1 values. No correlation was found between all measured chemokines and between age, sweat chloride levels, and pancreatic and nutritional status.

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A. Augarten et al.: Systemic inflammatory mediators and cystic fibrosis genotype Table 1 Clinical characteristics of cystic fibrosis (CF) patients Group Aa (n=25)

Group Bb (n=11)

P

Age (years)

16.9±7.2

Pancreatic sufficiency

0%

Sweat chloride (mmol/l)

105±28

92±18.6

NS

Weight percentiles

19±19.8

57.2±25.2

<0.01

Sputum Pseudomonas

88%

a b

17.7±9.1 0/25

22/24

36.3%

40%

NS 4/11

<0.01

4/10

<0.01

Group A CF patients carrying two mutations associated with severe disease presentation (∆F508, W1282X, G542X, N1303K, S549R) Group B CF patients carrying mutations associated with mild disease presentation (3849+10kb CT, 5T)

Table 2 Comparison of serum chemokine levels between groups (IL-8 interleukin-8, MCPI monocyte chemoattractant protein-1) Chemokine

Group Aa

Group Bb

P

IL-8 (pg/ml)

11.4±2.1

5.0±0.9

0.01

MCP1(pg/ml)

157±16

88.8+16.4

0.01

RANTES (pg/ml)

323±48

287.5±93

NS

a b

Group A CF patients carrying two mutations associated with severe disease presentation (∆F508, W1282X, G542X, N1303K, S549R) Group B CF patients carrying mutations associated with mild disease presentation (3849+10kb CT, 5T)

Fig. 1 Relationship between interleukin-8 (IL-8) levels and forced expiratory volume in 1 s (FEV1) values

Discussion Despite an increased understanding of the CFTR protein function, it is not completely understood how mutations in the CFTR gene are related to bacterial infection and inflammation of the airways. Recently, two hypotheses were proposed to explain the link between CFTR function and the host lung defense. Pier et al. [10] suggested that the CFTR protein itself might function as a cellular receptor for binding, endocytosing and clearing P. aeruginosa from the lung. In addition, Smith and Welsh [11] found that the apical surface fluid contains peptides that exhibit local antibacterial activity. This bactericidal activity requires a low salt concentration, which is maintained by the normal function of CFTR as an ion channel. This action of CFTR is a part of the first line of the lung host defense. In CF patients this

combined mechanism is impaired, Pseudomonas is not eliminated, and a second line of defense is commenced [11]. Pseudomonas and its products stimulate chemokine production, which provides directional cues for the movement of neutrophils, generating an inflammatory environment [7]. The involvement of chemokines in CF is well established and levels of IL-8 are elevated in these patients [8, 12, 13]. However, the uniqueness of our study is the distribution of serum chemokine levels among CF patients of different genotypes. We found that CF patients who carry severe mutations have significantly increased IL-8 levels, as well as a higher rate of Pseudomonas colonization compared with patients who carry a mild mutation. In addition, we found that IL-8 levels correlate negatively with pulmonary function. Welsh and Smith [14] classified the different mutations of the CF gene according to the mechanisms by which they disrupt CFTR protein function: class I,

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defective protein production; class II, defective protein processing; class III, defective chloride channel regulation; and class IV, defective chloride channel conduction. The two mutations carried by group B patients, namely 3849+10 kb C  T and 5T, are splice mutations and were classified later by Zielenski and Tsui [15] as class V mutation. In the 3849kb mutation, a C to T base substitution in intron 19 was found to create a new alternative splicing site, resulting in the insertion of 84 base pairs into the CFTR region [4]. In the 5T mutation the sequence of five thymines in intron 8 is a DNA variant that causes skipping of exon 9 in the transcription process. mRNA analysis of patients bearing these two mutations demonstrated two different transcripts, indicating synthesis of a protein with normal CFTR function together with a defective non-functioning protein [4, 16]. The association between IL-8 levels, CF genotype, and pulmonary function demonstrated in our study, as well as the association between the CF genotype and Pseudomonas colonization, indirectly supports both the recently reported hypotheses of Pier et al. [10] and Smith and Welsh [11]. In group A patients who carry class I, II, and III mutations, the CFTR is completely inactive, both as a receptor for Pseudomonas and as an ion channel. Therefore, Pseudomonas binding and elimination is disrupted and the bactericidal activity of the apical surface fluid is severely impaired due to failure in maintaining a low salt concentration, leading to bacterial colonization with a consequent initiation of an inflammatory response, as represented by the elevated chemokine levels. However, in group B patients who carry class V mutation the CFTR is partially active both in Pseudomonas binding and in maintaining a lower salt concentration, leading to a partial eradication of Pseudomonas. As a result, the bacterial burden is diminished, resulting in a milder inflammatory response and relatively preserved pulmonary function. However, the fact that IL-8 levels were not significantly increased in patients colonized with Pseudomonas may indicate that the inflammatory process is more complicated and not explained by solely the hypothesis of Piers et al. [10]. We found the IL-8 level to be related to the severity of the lung disease, while no correlation was found with the other examined clinical characteristics, such as sweat chloride levels and pancreatic and nutritional status. This may indicate that the measured chemokine originated in the lung and that in the other organs affected by CF, the pathogenesis does not involve infection and inflammation. Our work is also distinctive as we measured the chemokine levels in the serum, which is much easier and less invasive than measurement in the broncho-alveolar fluid. The high serum concentration of IL-8 in CF patients probably represents a spillover from the localized inflammatory response in the lung [8]. It was shown that airway infection and subsequent inflammation may be present in the first weeks of life [6] and that early treatment of asymptomatic CF children resulted in better pulmonary function [17]. Therefore, our findings may be of practical clinical importance, as elevat-

ed IL-8 serum levels could serve as an indicator of an early inflammatory process and encourage the initiation of antiinflammatory or antibiotic therapy.

References 1. Mickle JE, Cutting GR (1998) Clinical implication of cystic fibrosis transmembrane conductance regulator mutations. Clin Chest Med 3:443–455 2. The Cystic Fibrosis Genotype Phenotype Consortium (1993) Correlation between genotype and phenotype in patients with cystic fibrosis. N Engl J Med 329:1308–1313 3. Augarten A, Kerem BS, Yahav Y et al (1993) Mild cystic fibrosis and normal or borderline sweat test in patients with the 3849+10kb CT mutation. Lancet 342:25–26 4. Highsmith WE, Burch LH, Zhou Z et al (1994) A novel mutation in the cystic fibrosis gene in patients with pulmonary disease but normal sweat chloride concentrations. N Engl J Med 331:974–980 5. Wood RE, Boat TF, Doershuk CF (1976) Cystic fibrosis. Am Rev Respir Dis 113:833–878 6. Armstrong DS, Groimwood K, Carlin JB et al (1997) Lower airway inflammation in infants and young children with cystic fibrosis. Am J Respir Crit Care Med 156:1197–1204 7. Epstein FH (1998) Chemokines – chemotactic cytokines that mediated inflammation. N Engl J Med 338:436–445 8. Dean TP, Dai Y, Shute JK, Church MK, Warner JO (1993) Interleukin-8 concentrations are elevated in bronchoalveolar lavage, sputum, and sera of children with cystic fibrosis. Pediatr Res 34:159–161 9. Kerem E, Harel NR, Augarten A et al (1997) A cystic fibrosis transmembrane conductance regulator splice variant with partial penetrance associated with variable cystic fibrosis presentation. Am J Respir Crit Care Med 155:1914 10. Pier GB, Grout M, Zaidi TS (1997) Cystic fibrosis transmembrane conductance regulator is an epithelial cell receptor for clearance of Pseudomonas aeruginosa from the lung. Proc Natl Acad Sci U S A 94:12088–12093 11. Smith JJ, Welsh MJ (1998) Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid. Cell 85:229–236 12. Segal SD, Sontag MK, Wagener JS et al (2002) Induced sputum inflammatory measures correlate with lung function in children with cystic fibrosis. J Pediatr 141:811–817 13. Dakin CJ, Numa AH, Wang HE et al (2002) Inflammation, infection, and pulmonary function in infants and young children with cystic fibrosis. Am J Respir Crit Care Med 165:904–910 14. Welsh MJ, Smith AE (1993) Molecular mechanism of CFTR chloride channel dysfunction in cystic fibrosis. Cell 73:1251–1254 15. Zielenski J, Tsui LC (1995) Cystic fibrosis: genotypic and phenotypic variations. Annu Rev Genet 29:777–807 16. Chu CS, Trapnell BC, Curristin S et al (1993) Genetic basis of variable exon 9 skipping in cystic fibrosis transmembrane conductance regulator mRNA. Nat Genet 3:151–156 17. Wang SS, O’eary LA, FitzSimmons SC et al (2002) The impact of early cystic fibrosis diagnosis on pulmonary function in children. J Pediatr 141:804–810