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Department of Laboratory Medicine, Section of Microbiology, Immunology and Glycobiology, Lund University, S-221 85 Lund, SwedenDepartment of Nephrology, Lund University, S-221 85 Lund, Sweden
Autoantibodies against bactericidal permeability increasing protein (BPI-ANCA) are frequently present in cystic fibrosis patients and have been reported to be associated to colonization with Pseudomonas (P) aeruginosa and lung damage. In the present study, we investigated BPI-ANCA as a prognostic marker and its relation to P. aeruginosa colonization and lung function.
Methods:
BPI-ANCA, measured by ELISA, was examined relative to lung function and microbiological findings. The prognostic value of BPI-ANCA was assessed in 46 adult patients followed for 1.2–8.9 years. The cross-sectional investigation was performed in 366 patients (age 0.5–55).
Results:
The presence of BPI-ANCA predicted poor prognosis. An adverse outcome occurred in 15/28 BPI-ANCA positive patients and in 2/18 BPI-ANCA negative patients (p=0.01). This result remained valid when the patients were stratified according to lung function (p=0.03). Findings of BPI-ANCA were correlated to P. aeruginosa colonization and lung damage. Development of BPI-ANCA occurred after colonization with P. aeruginosa. All colonized patients did not develop BPI-ANCA. The BPI-ANCA levels were fairly stable during the disease course, but decreased significantly following lung transplantation.
Conclusion:
BPI-ANCA responses follow colonization with P. aeruginosa and may be predictive for lung damage.
]. CF is characterized by excessive mucus production and chronic bacterial colonization of the airways, ultimately leading to respiratory insufficiency. Colonization with Pseudomonas (P) aeruginosa is known to promote lung damage [
]. More than 1000 mutations have been described, resulting in varying functionality of the CFTR. However, the severity of the lung disease is not correlated to the CFTR genotype, except in some rare mutations that are associated with a mild disease [
Anti-neutrophil cytoplasmic antibodies directed against the bactericidal/permeability-increasing protein (BPI) in pediatric cystic fibrosis patients do not recognize N-terminal regions important for the anti-microbial and lipopolysaccharide-binding activity of BPI.
Autoantibodies directed against bactericidal/permeability-increasing protein in patients with cystic fibrosis: association with microbial respiratory tract colonization.
]. BPI is one of the most potent antimicrobial factors of the innate defence repertoire against Gram-negative bacteria. BPI mainly originates from the azurophilic granules of polymorph nuclear leukocytes [
]. Antibodies against such proteins are called antineutrophil cytoplasmic antibodies (ANCA), and they are well-established markers for the diagnosis and disease activity of systemic vasculitides [
]. BPI-ANCA is known to occur in conjunction with P. aeruginosa colonization in CF, but has not previously been examined as a prognostic factor. BPI-ANCA also occurs in other diseases with Gram-negative bacterial colonization such as diffuse panbronchiolitis, bronchiectasis and in inflammatory bowel disease [
Bronchiectasis with myeloperoxidase antineutrophil cytoplasmic antibody and bactericidal/permeability-increasing protein antineutrophil cytoplasmic antibody.
The prevalence and clinical significance of alpha 1-antitrypsin deficiency (PiZ) and ANCA specificities (proteinase 3, BPI) in patients with ulcerative colitis.
Autoantibodies against the bactericidal/permeability-increasing protein from inflammatory bowel disease patients can impair the antibiotic activity of bactericidal/permeability-increasing protein.
]. In CF, BPI-ANCA has not been found in patients with Gram-positive colonization only. Gram-negative bacteria other than P. aeruginosa, such as Burkholderia cepacia are too rare in the populations studied to be evaluated concerning their relation to BPI-ANCA (unpublished data).
A previous study from our group suggested that the presence of BPI-ANCA is strongly correlated to colonization with P. aeruginosa. We also found that the P. aeruginosa colonized patients with the highest levels of BPI-ANCA had the most severe lung damage [
]. This implied that BPI-ANCA could be a predictor of poor prognosis in CF.
The aims of the present study were to evaluate BPI-ANCA as a long-term prognostic marker and to confirm the association between P. aeruginosa colonization, lung damage and the presence of BPI-ANCA. This was done in two independent patient cohorts, one Swedish and one Danish. We also wanted to establish the temporal relationship between P. aeruginosa colonization and BPI-ANCA responses, in order to elucidate a course of events involved in development of lung damage in CF.
2. Patients and methods
2.1 Patients
The prognostic value of BPI-ANCA was determined by a prospective study of 46 Swedish patients presented in an earlier report [
]. Six patients were lost to follow-up, out of whom five had moved and one had died from cancer of the colon. Thus, data from 40 patients were available at the time of the present study (1.2–8.9 years follow-up). At inclusion, BPI-ANCA was positive in 25 of these patients (median age 25, range 18–36). Their median lung function was FEV1%pred 58% (range 24–95%). BPI-ANCA was negative in 15 patients (median age 22, range 19–43). In this group the median lung function was FEV1%pred 80% (range 37–113%).
In the cross-sectional part of the study, all patients with verified CF at the CF centres of Lund University Hospital, Sweden, and Rigshospitalet, Copenhagen, Denmark, were asked to participate. Approval was obtained by the research ethics committees of the Universities of Lund and Copenhagen. All patients were genotyped with regard to CFTR mutations (Department of Clinical Genetics in Lund or Department of Clinical Genetics in Copenhagen). The results from each centre are presented separately, as the methods for measuring lung function and the definitions of chronic P. aeruginosa colonization differ. In Lund, informed consent and necessary study data were obtained from 129 patients, 64 females and 65 males. The median age of the patients was 19.5 years (range 2.0–53.5). Lung transplantation had been performed in 8 of the patients. In Copenhagen informed consent and necessary study data were obtained from 237 patients, 118 females and 119 males. The median age of the patients was 19.7 years (range 0.5–54.0). Lung transplantation had been performed in 13 of the patients. The patients were subdivided into groups according to CFTR genotype according to previously published criteria [
Anti-neutrophil cytoplasmic antibodies directed against the bactericidal/permeability-increasing protein (BPI) in pediatric cystic fibrosis patients do not recognize N-terminal regions important for the anti-microbial and lipopolysaccharide-binding activity of BPI.
Autoantibodies directed against bactericidal/permeability-increasing protein in patients with cystic fibrosis: association with microbial respiratory tract colonization.
Bronchiectasis with myeloperoxidase antineutrophil cytoplasmic antibody and bactericidal/permeability-increasing protein antineutrophil cytoplasmic antibody.
The prevalence and clinical significance of alpha 1-antitrypsin deficiency (PiZ) and ANCA specificities (proteinase 3, BPI) in patients with ulcerative colitis.
Autoantibodies against the bactericidal/permeability-increasing protein from inflammatory bowel disease patients can impair the antibiotic activity of bactericidal/permeability-increasing protein.
]: A, homozygosity for ΔF508; B, severe/severe mutation, i.e. one class I, class II or class III mutation on each allele including heterozygosity for ΔF508; C, one or two missense mutations, i.e. class IV or V mutations; D, one or two unknown mutations. No significant differences in lung function, bacterial colonization or BPI-ANCA level were seen between the genotype groups (Table 1). After inclusion, the Swedish patients were followed prospectively with new serum samples for BPI-ANCA with intervals of six to twelve months, and clinical data were recorded in conjunction with sampling.
Table 1Distribution of CFTR genotype groups in Swedish and Danish patients
Swedish patients n=129
Danish patients n=237
CFTR genotype
% of patients
Median FEV1%pred
Median BPI-ANCA (U/L)
% P. aeruginosa colonized
% of patients
Median FEV1%pred
Median BPI-ANCA (U/L)
% P. aeruginosa colonized
A
55
84
34
44
73
79
25
40
B
18
85
20
42
13
79
23
40
C
12
83
26
33
6
86
12
21
D
15
86
17
32
8
83
29
32
Lung functions, BPI-ANCA levels and presence of P. aeruginosa colonization are presented for each group.
CFTR genotype: A, homozygosity for ΔF508; B, severe/severe mutation; C, one or two missense mutations; D, one or two unknown mutations. The genotype groups are further explained in the Methods section. No significant differences in the median values or percentage of patients were found (the Kruskal-Wallis test or the Chi-squared test for independence).
]. Forced expiratory volume in 1 second (FEV1) was chosen as a measure of lung function, as this is a lung function parameter that closely correlates with prognosis in CF [
]. In the Danish patients, FEV1 was measured at each visit in out patient clinic, and the mean values from one year of measurements were used. The results were expressed as a proportion of the predicted values (FEV1%pred) based on height, age, and gender. In children, FEV1%pred were calculated according to Solymar et al. [
Lung volumes and forced ventilatory flows. Report Working Party Standardization of Lung Function Tests, European Community for Steel and Coal. Official Statement of the European Respiratory Society.
]. In patients who reached 18 years of age during the study period, all FEV1%pred-values were calculated according to Solymar. Normal lung function was defined as FEV1%pred more than 80%. FEV1%pred values between 50% and 80% were considered as moderate lung damage and below 50% the lung damage was considered severe. In the long-time follow up study, a composite endpoint for severe adverse outcome was defined as:
•
reaching endpoint, which was defined as lung transplantation or death
•
reaching FEV1%pred below 25%
•
loosing more than 2.8% in FEV1%pred per year. This was based on the median loss of FEV1%pred in the cohort, which was 1.4% per year. Thus the double median was chosen as cut off for severe adverse outcome.
In 7 Swedish children under the age of 4 and 26 Danish children under the age of 6, lung function data were not available due to the low age of the patients. In these children, lung function was assessed clinically and/or radiologically.
2.3 Microbiological diagnosis
Samples for respiratory secretion cultures were obtained when the patient attended a routine outpatient visit. Sampling, transport, and culturing were performed according to routine procedures. For the Swedish patients, colonization was defined according to European consensus, i.e. three consecutive positive cultures [
]. All patients were assisted by a physiotherapist in order to provide expectorate for sputum culture. Non-sputum producers were considered not colonized. For the Danish patients lower respiratory tract secretions were obtained by coughing (sputum producers) or endo-laryngeal suction, and sputum microbiology was carried out. Infection was considered chronic, when an organism had been cultured for 6 consecutive months or less if there was a simultaneous increase in precipitating antibodies against P. aeruginosa above normal level [
]. Among the not transplanted patients, 40% of the Swedish patients, and 42% of the Danish patients were chronically colonized with P. aeruginosa.
2.4 ELISA for measurement of BPI-ANCA
Serum samples were drawn and analysed for BPI-ANCA of the IgA and IgG isotypes. On analysing data, similar results were found concerning IgG- and IgA-BPI-ANCA. From now on, BPI-ANCA refers to the IgA isotype. Purified BPI was obtained from Wieslab AB (Lund, Sweden), and direct binding ELISA was performed as previously described [
]. In short, antigens were coated onto microtiter plates at a concentration of 1 μg/ml in a bicarbonate buffer. Serum samples were diluted 1/80 and incubated for 1 h. Bound antibodies were detected using alkaline phosphatase-conjugated goat anti-human IgA (Eurodiagnostica, Malmö, Sweden). BPI-ANCA was quantified from a calibrator curve of serum that was serially diluted. The results were expressed as arbitrary units (U/L). In each ELISA one positive and one negative control were analysed. BPI-ANCA is not normally present in serum at any age. To define a positive cut-off level, 42 normal sera were analysed. From the absorbance values in these normal sera +3SD, the positive cut off level was determined to be 67 U/L. BPI-ANCA has previously been analysed in a pediatric non-CF population and the median value was found to be on the same level as in adult blood donors (Dr L Dorlöchter, Bergen, Norway, personal communication). A significant change in the BPI-ANCA level was defined as a minimum increase or decrease of 50% of the BPI-ANCA value.
2.5 Statistics
The Mann-Whitney test and the Kruskal-Wallis test were used when applicable. When prognostic groups or genotype groups were compared with respect to outcome, the exact two-sided Chi-square test, the Chi-square test for trend or the Chi-square test for independence was employed.
3. Results
3.1 BPI-ANCA as a predictor of poor prognosis
Forty adult patients from a previous cross-sectional study were followed for a mean time of 5.7 years (range 1.2–8.9) [
]. In the BPI-ANCA positive group, seven out of 25 patients had required lung transplantation, one had died from CF lung disease and seven had significant loss of lung function. In the BPI-ANCA negative group one patient died and one patient had significant loss of lung function. Thus, 15 patients out of 25 (54%) in the BPI-ANCA positive group had a severe adverse outcome and in the BPI-ANCA negative group only two patients out of 15 (11%) had an adverse outcome (p=0.01).
Lung function at inclusion was, as expected, a strong predictive factor for outcome. In patients with severe lung damage 8/12 (67%) had a severe adverse outcome and in patients with moderately reduced lung function 8/17 (47%) demonstrated comparable progression. Only one out of eleven patients (9%) who had normal lung function (FEV1%pred >80%) at inclusion showed a severe adverse outcome (p=0.006).
To decide whether BPI-ANCA provides independent prognostic information, the patients were stratified according to FEV1 at inclusion. Within the group of patients with moderate lung damage, severe adverse outcome was significantly overrepresented in BPI-ANCA-positive patients (p=0.05). The difference between BPI-ANCA-positive and BPI-ANCA negative patients was still significant when the group of patients with severe lung damage was included (p=0.03, Table 2).
Table 2Severe adverse outcome after 1.2–8.9 years in 40 patients stratified according to lung function at inclusion
Lung function
BPI-ANCA
Severe adverse outcome
Severe lung damage
Negative n=1
0/1⁎
Positive n=11
8/11 (72%)⁎
Moderate lung damage
Negative n=7
1/7 (14%)⁎
Positive n=10
7/10 (70%)⁎
Normal lung function
Negative n=7
1/7 (14%)
Positive n=4
0/4
⁎In patients with FEV1<80%, a positive level of BPI-ANCA was significantly correlated to severe adverse outcome, p=0.03, the exact, two-sided Chi squared test. Severe lung damage, FEV1<50%; Moderate lung damage, FEV1 50–80%; Normal lung function, FEV1 >80%. BPI-ANCA negative, <67 U/L; BPI-ANCA positive >67 U/L.
3.2 Serum levels of BPI-ANCA correlate to P. aeruginosa colonization and decreased lung function
BPI-ANCA was mainly found in patients chronically colonized with P. aeruginosa. In the Swedish P. aeruginosa-colonized patients, the median BPI-ANCA level was 98 U/L, as compared to 11 U/L in patients without chronic colonization (p<0.0001); in the Danish patients the corresponding levels were 68 U/L and 15 U/L (p<0.0001). Patients with severe lung damage had higher levels of BPI-ANCA than patients with moderate lung damage, and patients with preserved lung function were BPI-ANCA negative with few exceptions (Fig. 1).
Fig. 1BPI-ANCA according to lung function. The BPI-ANCA values are shown on a logarithmized scale. All patients who were old enough to perform lung function test and who were not lung transplanted were included in the analysis. Levels of BPI-ANCA in serum of patients with severe lung damage (FEV1%pred <50%), moderate lung damage (FEV1%pred 50–80%) and preserved lung function (FEV1%pred >80%). Swedish patients are shown with shaded boxes and Danish patients are shown with open boxes. The dashed line indicates the positive cut off level (67 U/L). The Kruskal-Wallis test was used on non-logarithmized values to compare the median values.
3.3 BPI-ANCA is formed in response to P. aeruginosa colonization during disease course
To examine the development of BPI-ANCA over time and its relation to colonization and disease progression, patients were divided into groups according to age. As expected, the percentages of P. aeruginosa-colonized patients and patients with severe lung damage increased with age in both the Swedish and the Danish cohort (Fig. 2). In the youngest group, aged 0–5, no patient had severe lung damage and very few were chronically colonized with P. aeruginosa. None of these young children were BPI-ANCA positive. In the next group, aged 6–11, a few patients were chronically colonized by P. aeruginosa and had developed lung damage. A few patients in this group were also positive for BPI-ANCA. The percentage of BPI-ANCA-positive patients increased with age. In the oldest group, with patients above the age of 36, 50% of the Swedish patients and 70% of the Danish patients were BPI-ANCA positive. The frequency of BPI-ANCA positive patients increased with age, parallel to that of P. aeruginosa colonization in both the Swedish and Danish cohort. The median percentage of P. aeruginosa colonized patients who were BPI-ANCA positive was 68% (inter-quartile range 53–78%).
Fig. 2(a) Swedish patients. (b) Danish patients. The patients were divided into age groups in order to create a fictive time scale. The lines indicate percentages of patients who are chronically colonized with Pseudomonas aeruginosa, patients with positive serum levels of BPI-ANCA and patients with severe lung damage. The youngest groups contain patients below 5 years of age, and the oldest contain patients over the age of 36. Severe lung damage was defined as forced expiratory volume in 1 s (FEV1) below 50% of the predicted value or had undergone lung transplantation, positive BPI-ANCA: >67 U/L.
3.4 Stability of BPI-ANCA levels on serial analyses
To study the variations in BPI-ANCA levels over time we performed a short-time prospective study in the Swedish patients. BPI-ANCA levels were measured in serum with an interval of at least 6 months. All together 311 samples from 121 patients were analyzed. The BPI-ANCA levels were found to be stable. Significant increases were only found in 11 patients. In all, only two patients who were not transplanted exhibited significant decreases of BPI-ANCA.
3.5 Decrease of BPI-ANCA following lung transplantation
In seven patients BPI-ANCA serum samples were obtained before and after lung transplantation. All patients were strongly BPI-ANCA positive (range 191–3894 U/L) and were chronically colonized with P. aeruginosa prior to the transplantation. Lung transplantation was successful in the patients, and no rejection had occurred when the second BPI-ANCA sample was drawn. At this time the serum levels of BPI-ANCA had decreased (range 52–90%) in all patients (Fig. 3).
Fig. 3Serum levels of BPI-ANCA before and after lung transplantation in seven patients. The dashed line indicates the positive cut off level for BPI-ANCA (67 U/L).
Tools for prediction of prognosis are important in CF. When mild mutations are rare, as in the populations studied, the prognostic value of the genotype becomes less important. This study shows that the presence of BPI-ANCA is a predictor of poor prognosis in the disease. The predictive value of BPI-ANCA was strongest in patients with moderate lung damage, a group of patients for which prognostic information is particularly relevant. In those with severe lung damage, the predictive value of negative BPI-ANCA is difficult to evaluate, due to the low number of patients.
The association between BPI-ANCA, P. aeruginosa colonization and lung damage in CF was investigated in 366 CF patients in the age interval between 0.5 and 55 years. The findings are consistent with earlier reports [
Anti-neutrophil cytoplasmic antibodies directed against the bactericidal/permeability-increasing protein (BPI) in pediatric cystic fibrosis patients do not recognize N-terminal regions important for the anti-microbial and lipopolysaccharide-binding activity of BPI.
Autoantibodies directed against bactericidal/permeability-increasing protein in patients with cystic fibrosis: association with microbial respiratory tract colonization.
]. Patients with the most severe lung damage exhibited the highest serum levels of BPI-ANCA and patients with normal lung function most often had low or negative BPI-ANCA levels (Fig. 1). Because of differences in definitions of chronic P. aeruginosa colonization and in the collection of lung function variables over time we decided to look at the Swedish and the Danish patients as two independent cohorts. The fact that the same results were found in both groups shows that the findings are general, which adds credibility.
The prevalence of BPI-ANCA increased with age in both cohorts, as did the prevalence of P. aeruginosa-colonization and impaired lung function. The prevalence of lung damage and BPI-ANCA was lower than the prevalence of P. aeruginosa-colonization in most age groups. This probably indicates that P. aeruginosa-colonization of the airways precedes the generation of BPI-ANCA and the development of lung damage. This has not previously been shown and it leads us to speculate that a proportion of the chronically colonized patients do not develop BPI-ANCA due to variability between strains or other bacteria-related factors (Fig. 2). On serial analysis, BPI-ANCA was mostly found to be stable. As CF patients often carry the same strain of P. aeruginosa for years, occasional rises in BPI-ANCA as observed in the population might very well coincide with acquisition of new strains [
]. To study if this is the case, a large prospective study including recurrent extended microbiological analysis, frequent BPI-ANCA analysis and clinical assessment would be needed.
All investigated patients who were lung transplanted, and thereby received sterile lungs, showed a significant decrease in BPI-ANCA levels. This finding indirectly supports the hypothesis that BPI-ANCA in serum covariates with colonization by harmful strains of P. aeruginosa. Interestingly, one patient, who was lung transplanted before she was included into our study, contracted chronic rejection and was re-colonized with P. aeruginosa at the time of our study. At the same time her serum levels of BPI-ANCA rose significantly (data not shown). It has been proposed that the decrease in BPI-ANCA following transplantation is due to the immunosuppressive treatment. The BPI-ANCA levels of the patients investigated in this study decreased by more than 50% after their lung transplantations. There is no immunosuppressive drug that is known either to cause such a decrease of the total immunoglobulin level, or to selectively suppress certain clones of B-cells. The immunosuppressive treatment cannot therefore alone explain the substantial fall in BPI-ANCA after lung transplantation. In the patient who contracted chronic rejection, the immunosuppressive treatment was unchanged, and still her BPI-ANCA levels rose significantly.
In conclusion we found that the presence of BPI-ANCA in CF indicates poor prognosis. Furthermore, we have reason to speculate that BPI-ANCA in CF distinguishes between patients with apparently harmless colonization by P. aeruginosa from patients with progressive lung disease caused by the same species. Serum levels of BPI-ANCA decrease after lung transplantation. The occurrence of BPI-ANCA appears to follow colonization with harmful strains of P. aeruginosa. We believe that BPI-ANCA will prove to be an important diagnostic tool in the care of patients with CF.
Acknowledgements
We thank Dr Charlotta Schaedel for valuable help with characterizing the CFTR genotypes and we thank associate professor Anders Sjöholm for excellent linguistic and scientific advice. The valuable help from RN Kristina Ågård, RN Catharina Isgren and RN Inger Erwander (Lund) and Lena Nørregaard (Copenhagen) with collecting the blood samples is gratefully acknowledged. The late professor Christian Koch has contributed substantially to this study.
This work was funded by Bengt Andreasson's Foundation, the Swedish CF Association and the Swedish Research Council project no. 73x09487.
Anti-neutrophil cytoplasmic antibodies directed against the bactericidal/permeability-increasing protein (BPI) in pediatric cystic fibrosis patients do not recognize N-terminal regions important for the anti-microbial and lipopolysaccharide-binding activity of BPI.
Autoantibodies directed against bactericidal/permeability-increasing protein in patients with cystic fibrosis: association with microbial respiratory tract colonization.
Bronchiectasis with myeloperoxidase antineutrophil cytoplasmic antibody and bactericidal/permeability-increasing protein antineutrophil cytoplasmic antibody.
The prevalence and clinical significance of alpha 1-antitrypsin deficiency (PiZ) and ANCA specificities (proteinase 3, BPI) in patients with ulcerative colitis.
Autoantibodies against the bactericidal/permeability-increasing protein from inflammatory bowel disease patients can impair the antibiotic activity of bactericidal/permeability-increasing protein.
Lung volumes and forced ventilatory flows. Report Working Party Standardization of Lung Function Tests, European Community for Steel and Coal. Official Statement of the European Respiratory Society.
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