- Email: [email protected]
Antineutrophil Cytoplasmic Autoantibodies (ANCA) in Children with Cystic Fibrosis
Journal of Autoimmunity (1998) 11, 185–190
Antineutrophil Cytoplasmic Autoantibodies (ANCA) in Children with Cystic Fibrosis Anna Sˇediva´1, Jirˇina Bartu˚nˇkova´1, Ivana Kola´rˇova´1, Ondrˇej Hrusˇa´k1, Veˇra Va´vrova´2, Milan Macek jr3, Christopher M-Lockwood4 and Austin C. Dunn4 1
Institute of Immunology, 2Second Clinic of Paediatrics, 3Department of Genetics, University Hospital Motol, Charles University, Prague, Czech Republic 4 Department of Medicine, University of Cambridge, Cambridge, UK
Received 13 June 1997 Accepted 10 December 1997
Key words: antineutrophil cytoplasmic antibodies (ANCA), bactericidal/permeability increasing protein (BPI), children, cystic fibrosis (CF), cystic fibrosis transmembrane regulator (CFTR)
Anti-neutrophil cytoplasmic antibodies (ANCA) represent a useful diagnostic tool in patients with small vessel vasculitis. Circulating ANCA specific for bactericidal/permeability increasing protein (BPI) have been recently reported in adult patients with cystic fibrosis (CF), an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene with consequent impaired function of a transmembrane chloride channel. To contribute to the better understanding of the significance of ANCA in this disease, we investigated ANCA presence and antigenic specificity in children with CF. Results were correlated with clinical status, immunological data, age and genotype. The indirect immunofluorescence pattern of a total of 71 children with CF indicated that 31 were c-ANCA positive, while seven were p-ANCA positive. In further ELISA studies of ANCA antigenic specificity, 51 out of 66 investigated samples were positive for BPI, and 14 out of 28 were positive for proteinase 3 (PR3). We found an association between levels of antibodies against PR3 with age and Pseudomonas infection. We did not, however, find any correlation between CFTR genotypes, Pseudomonas infection or paediatric parameters and the level of anti-BPI antibodies. High positivity of anti-BPI antibodies were seen even among the youngest CF patients, before the development of clinical signs of CF, indicating that formation of ANCA might be a very early event in the disease. Both anti-BPI and anti-PR3 antibodies may play a significant, although variable role, in the pathogenesis of CF. © 1998 Academic Press Limited
Introduction
with CF [3]. Antineutrophil cytoplasmic antibodies (ANCA) are usually associated with autoimmune disorders including small vessel vasculitis, such as Wegener’s granulomatosis, microscopic polyangiitis, idiopathic rapidly progressing glomerulonephritis, inflammatory bowel disease and rheumatoid arthritis [4, 5]. Different target antigens, usually present in the azurophilic granules of neutrophils, are recognized by these autoantibodies. Antibodies directed against proteinase 3 (PR3) are frequently associated with Wegener’s granulomatosis, and antibodies reacting with myeloperoxidase (MPO) are associated, although less closely, with microscopic polyangiitis [6]. ANCA detected in sera of adults with CF react with bactericidal/permeability increasing protein (BPI), another component of azurophilic granules [3]. In our previous study we analysed the incidence and importance of ANCA in childhood [7]. The predominant diagnosis among ANCA positive children was CF [7, 8]. This led to a detailed analysis of this group and to the possible explanation of the origin and pathogenetic role of these autoantibodies in CF.
Cystic fibrosis (CF) is an autosomal recessive disorder. The CF gene was discovered in 1989 and the corresponding protein was found to be CFTR, cystic fibrosis transmembrane regulator [1]. The consequence of this mutation is defective ion transport across the cell membranes, which leads to the gradually developing clinical picture of the disease, characterized mainly by damage to the lungs and pancreas [2]. The immune system is believed to be involved in the pathogenesis of this disease through its contribution to inflammation of the tissues, particularly the lungs. However, so far there has been no reason to believe that autoimmune processes play any role in the pathogenesis of CF. Recently, the finding of circulating anti-neutrophil cytoplasmic antibodies has been reported in adult patients Correspondence to: Anna Sˇediva´, Institute of Immunology, ´ valu 84, 150 06 Praha 5, University Hospital Motol, V U Czech Republic. Fax: 4202 2443 5962. E-mail: [email protected]. 185 0896-8411/98/020185+06 $25.00/0/au970186
© 1998 Academic Press Limited
A. Sˇediva´ et al.
186
Material and Methods
18
We investigated 71 children, 33 girls and 38 boys, aged 5 months to 18 years. All have been regularly seen and treated in the specialized Prague CF department. Immunological investigations were performed during follow-up visits or during control hospitalizations of patients with CF, with material from routine venepunctures. ANCA were detected by indirect immunofluorescence on ethanol fixed human neutrophils (Binding Site, Birmingham, UK) and all samples were tested for the specificity for BPI of IgG isotype (BPI protein IgG Antibodies ELISA kit, Genesis Diagnostics, Ely, UK). Fifty-one out of 71 serum samples were available for testing anti-BPI antibodies of IgA isotype in solid phase ELISA. Ninety-six-well plates (Dynatech, NJ, USA) were coated with native BPI purified from neutrophil extract at concentration 1.0 ìg/ml in PBS, and incubated for 1 h at room temperature. Serum samples were diluted 1:50 in PBS-Tween 20, and 100 ìl/well of diluted serum was added to the ELISA plate, incorporating two antigen coated wells and one antigen-free to allow for non-specific binding. Laboratory positive and negative controls were incorporated in each plate. Samples were considered positive when the optical density at 405 nm using an alkaline phosphatase substrate (Sigma104, Dorset, UK) development system was greater than the mean and three standard deviations from the negative control, as calculated by linear regression. The negative control consisted of 50 normal blood donors. All sera positive for c-ANCA were further investigated by ELISA for specificity for PR3 (EIAgen c-ANCAkit, Biochem Pharma Group, Freiburg, Germany). Serological reactivity of PR3 positive sera was confirmed by western blotting studies, using a modification of the method described by Towbin et al. [9]. PR3 purified from human neutrophil extracts was divided on 12% polyacrylamide SDS-PAGE gel according to the method of Laemmli [10]. The gel was blotted onto nitrocellulose paper (Schleicher and Schuell, Germany) that had been soaked in transfer buffer (10 mM 3-(cyclohexylamino)-1-propanesulfonic acid, 10% v/v methanol, pH 11.0). Transfer was accomplished by passing a 0.5 mA current through the gel/ nitrocellulose sandwich for 40 min. The nitrocellulose was removed and stained with ponceau-S (BDH, Dorset, UK) to visualize transferred protein; then cut and blocked with 2% w/v non-fat milk in 0.01 M Tris-base, 0.15 M sodium chloride, pH 8.0 (TBSTM), for 30 min. Tested sera and positive and negative controls were diluted 1:200 in TBSTM, added and left for 1 h at room temperature. After incubation and washing, human IgG binding was detected using alkaline phosphatase-conjugated goat anti-human IgG (Sigma, Poole, Dorset, UK), diluted 1:3000 in TBSTM. The blot was developed with 5 ml of alkaline phosphatase buffer (100 mM sodium chloride, 5 mM magnesium chloride (BDH, Dorset, UK), 100 mM Tris, pH 9.5) containing 66 ìl of nitroblue tetrazolium stock (Sigma NBT, Dorset, UK). The reaction was stopped by
16
Number of patients
14 12 10 8 6 4 2 0
0
10
20 30 Anti-BPI (U/ml)
40
50
Figure 1. Levels of anti-BPI antibodies. Frequency of negative ( ) and positive ( ) values. Out of 66 children 51 (77%) were positive for anti-BPI antibodies. The anti-BPI values of six children between 3 and 7 ml are classified as borderline positivity ( ).
0.7
Anti PR3 (OD)
0.6
infection neg. infection PSA infection CEP
0.5 0.4 0.3 0.2 0.1 0
5
10 Age (years)
15
20
Figure 2. Correlation of anti-PR3 antibodies, infection with Pseudomonas species and age. Out of 28 c-ANCA positive children, seven were positive for anti-PR3 antibodies and seven showed borderline positivity. The positivity of antiPR3 antibodies appears only in children 10 years and older, and is significantly correlated with Pseudomonas infection. ×=infection neg.; C=Pseudomonas aeruginosa; +=Burgholderia cepacea.
rinsing with PBS containing 20 mM EDTA; the blot was then air dried and stored in the dark. Routine blood counts, biochemical values and immunological laboratory values, including concentration of immunoglobulins and lung function, were investigated at the University Hospital Motol. Genotypes were established in each child using a combination of detection methods. Both rare mutations and the most common ÄF508 mutation were detected by PCR and subsequent non-denaturing polyacrylamide gel electrophoresis [11]. Other relatively common CF mutations were detected using allele specific oligonucleotide hybridization, amplification refractory mutation detection system,
ANCA in children with cystic fibrosis
187
1
2
3
4
5
6
7
KDa 46
29
Figure 3. Immunoblot studies of anti-PR3 antibodies. The specificity of positive anti-PR3 antibodies detected in ELISA was confirmed by western blot of purified PR3 with patient’s sera. Three serum samples of children with CF positive for anti-PR3 antibodies in ELISA were available for immunoblot. We detected faint bands, demonstrated in lanes 5, 6 and 7. These were expected, due to the very slight positivity demonstrated in ELISA. Lane 1: negative control, lane 2: patient with Wegener’s granulomatosis (kind gift of Mr P. Chapman, University of Cambridge), lane 3: serum of a 10-year-old boy with Wegener’s granulomatosis (First Clinic of Paediatrics, Charles University, Prague), lane 4: adult patient with Wegener’s granulomatosis (kind gift of Dr V. Tesarˇ, First Internal Clinic, Charles University, Prague).
Results ANCA determination by immunofluorescence We investigated 71 children with CF. Out of these, 31 showed a cytoplasmic type of immunofluorescence (c-ANCA) and 7 had perinuclear type (p-ANCA). This represents 52% positivity in immunofluorescence staining.
ANCA antigenic specificity Samples from 66 children were available for the determination of anti-BPI of IgG isotype in ELISA; of these 51 were positive (77%). The level of anti-BPI IgG antibodies in this test ranged from 7 to 50 U/ml (negative value ≤3, borderline value 3–6 U/ml) (Figure 1). Fifty-one out of 71 serum samples were available for testing for anti-BPI antibodies of IgA isotype. Out of these 51 children, 15 were positive according to the criteria of the test (see Material and Methods). We found eight children positive for both IgG and IgA and anti-BPI antibodies. Since c-ANCA pattern is often associated with the specificity of PR3, c-ANCA positive samples were further examined for specificity against PR3. Out of 28 investigated in anti-PR3 ELISA, seven were positive and seven showed borderline positivity (50%). The concentration of anti-PR3 antibodies was only slightly elevated (Figure 2). We found six children positive for
positive borderline negative
15
IgG (g/l)
PCR-mediated side directed mutagenesis and standard restriction digestion techniques. All data were processed by mathematical analysis, with patient data being analysed using multifactorial non-linear correlation analysis. The database included, besides the immunological parameters (immunoglobulins, circulating immunocomplexes, C3 and C4 components of complement); clinical values (lung and pancreatic function, liver function; presence of diabetes; infection with Pseudomonas aeruginosa or Burkholderia cepacia); and the particular genotype.
10
5
0
–5
0
0.1
0.2
0.3 0.4 Anti-PR3 (OD)
0.5
0.6
Figure 4. Correlation between anti-PR3 ELISA titres and serum levels of IgG. Linear regression was used to demonstrate the pattern of the correlation between anti-PR3 antibodies and serum level of IgG. Anti-PR3 antibodies are marked on the x axis, values on the y axis represent normalised values of IgG according to the age related mean. The values are expressed as a number of standard deviations. Correlation is positive only for negative values of anti-PR3 antibodies; borderline and positive values of antiPR3 antibodies do not correlate with high levels of IgG, indirectly excluding the non-specific IgG binding in ELISA.
both anti-PR3 and anti-BPI IgG antibodies; one girl out of these six was also positive for anti-BPI IgA antibodies. The specificity of positive anti-PR3 antibodies in ELISA was confirmed by western blotting (Figure 3) and this finding was further supported by linear regression analysis demonstrating the absence of correlation between anti-PR3 antibodies and total IgG level (Figure 4).
Multiparameter statistical analysis Statistical analysis correlated the level of anti-BPI of both IgG and IgA isotypes, and anti-PR3 antibodies, with age, genotype, lung and pancreatic function, and infection with Pseudomonas species and clinical status.
A. Sˇediva´ et al.
188
Proposal for pathogenic role of ANCA in cystic fibrosis
? (Inappropriate release of cationic BPI from neutrophils? Change in conformational structure of BPI?)
Formation of antibodies to BPI
Neutralization of BPI by autoantibodies
Diminished defence against Pseudomonas
Pseudomonas colonization of lungs
PMN activation
Inflammation, excessive release of granule enzymes including PR3
Other infections, further PMN activation, tissue destruction
Antibody formation to PR3
Anti-PR3-Abs diminish microbicidal capacity of PMN, stimulate oxygen radical release and degranulation
Figure 5. Proposal for pathogenetic role of ANCA in cystic fibrosis. Antibodies against BPI neutralise BPI. The resultant lack of defence against Pseudomonas contributes to the chronic Pseudomonas lung infection typical of CF patients. Chronic infection leads to continuous polymorphonuclear activation and excessive release of granule enzymes, with subsequent formation of autoantibodies directed against these enzymes. The situation then becomes a ‘vicious cycle’ of infection, impaired defence against Pseudomonas, further infection and formation of autoantibodies against granule enzymes which causes the diminished microbicidal activity of the attracted neutrophils.
There was no correlation between any of these parameters and anti-BPI antibodies of any isotype. However, we found a clear-cut association between the level of anti-PR3 antibodies, infection with Pseudomonas species and age (Figure 2).
Discussion ANCA are an unexpected finding in children with CF. Nevertheless, our results as well as the results of the adult CF patients, reported earlier [3], confirmed that ANCA positivity is a consistent finding in patients with this disease. Many immunological abnormalities are reported in patients with CF [12, 13], but until recently, the association with autoimmune processes was relatively infrequent. Only minor clinical signs, such as erythema nodosum or arthritis in certain patients, suggest that autoimmune mechanisms may
be operating. The laboratory findings of marked hyperimmunoglobulinemia, mainly of IgG and IgA classes, and high levels of immune complexes, are not necessarily autoimmune in origin, but probably represent the secondary consequences of chronic inflammation [14]. The secondary nature of these findings might also be suggested by the fact that newborn and small children with genotypically proven CF do not have detectable changes of these immune parameters, but they gradually develop during the course of the disease. However, we found anti-BPI specific ANCA antibodies even in very young children. Our youngest boy with a high level of anti-BPI antibodies was 5 months old, and had no Pseudomonas infection, which is almost obligatory in older patients with CF. On the other hand, the antibodies directed against PR3 appeared late in our group of patients, in the children aged 10 years and older, and were significantly connected with Pseudomonas infection. In contrast to another study, we
ANCA in children with cystic fibrosis
did not find correlation of anti-BPI antibodies and colonization with Pseudomonas [15, 16]. The level of anti-BPI antibodies in our group of children did not correlate with any parameter included in our study. We did not confirm the previously reported correlation of the level of anti-BPI antibodies and impaired lung functions of adult patients with CF [3]. On the basis of our results, we propose that the two specificities of ANCA, anti-BPI and anti-PR3 ANCA, have a different origin and a different role in the development of the pathology of CF. Anti-BPI antibodies are present at an early stage, which suggests that mechanisms leading to their formation are directly connected with the basic defect of CF, despite the fact that we did not observe any correlation with the CFTR genotype. BPI is a highly cationic protein, usually stored in the azurophilic granules of neutrophils. It is microbicidal for, and effective against lipopolysaccharide of Gram-negative bacteria [17]. Positive charge plays a dominant role in the function of BPI, and changes in charge and pH are the dominant features of cells expressing mutated CFTR [14, 18]. This is mostly expressed on epithelial cells, but mRNA coding for the protein has also been described in neutrophils [14]. According to our hypothesis, BPI may be more easily released by CF neutrophils then by normal, non-CF neutrophils. As a highly charged cationic protein it is a potent activator of macrophages [19], and is efficiently internalized in complexes with LPS, by macrophages via pinocytosis [20]. All these conditions may lead to the presentation of BPI antigens by macrophages and induction of anti-BPI antibody formation. By blocking BPI, these antibodies may trigger the ‘vicious cycle’ of repeated Pseudomonas infection followed by inefficient immune response (Figure 5). We suggest that there may be a different mechanism for the induction of anti-PR3 antibodies, which are formed after chronic inflammation over many years, with neutrophil accumulation and release of intracellular enzymes including PR3. This is supported by our finding that some patients with CF have also anti-myeloperoxidase antibodies (unpublished results, 1996). It is probable that antibodies against PR3 contribute to the ‘vicious cycle’ which results in persistent infections in CF patients, by diminishing microbicidal capacity of polymorphonuclear cells, as documented in our previous experimental studies [21, 22].
Conclusion ANCA is a consistent finding in children with CF. The pathogenetic role of these autoantibodies still awaits elucidation, but their specificity against BPI and PR3, both potent antimicrobial defence proteins, suggests their possible contribution to immunological and clinical aspects of CF.
Acknowledgements The authors thank Hynek Lauschmann for excellent statistic analysis. This work was supported by grants
189
from the Ministry of Health IGA MZ No 3160-3, 3933-3, 2899-5, 3526-3, 4124-3, 2056-5, and from Barrande 970157.
References 1. Riordan J.R., Rommens J.M., Kerem B., Alon N., Rozmahel R., Grzelczak Z., Zielenski J., Lok S., Plavsic N., Chou J.L 1989. Identification of cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245: 1066–1073 2. Mearns M.B. 1993. Cystic Fibrosis: The First 50 Years. In Cystic Fibrosis, current topics. Vol. 1. J.A. Dodge, D.J.H. Brock and J.H. Widdicombe, eds. John Wiley and Sons Ltd., Chichester, pp 217–251 3. Zhao M.H., Jayne D.R.W., Ardiles L.G., Culley F., Hodson M.E., Lockwood C.M. 1996. Autoantibodies against bactericidal/permeability-increasing protein in patients with cystic fibrosis. Q.J. Med. 98: 259–265 4. van der Woude F.J., Rasmussen N., Lobatto S., Wiik A., Permin H., van Es L.A., van der Giessen M., van der Hem G.K., The T.H. 1985. Autoantibodies against neutrophils and monocytes: tool for diagnosis and marker of disease activity in Wegener’s granulomatosis. Lancet 1: 425–429 5. Gross W.L., Schmitt W.H., Csernok E. 1993. ANCA and associated diseases: immunodiagnostic and pathogenetic aspects. Clin. Exp. Immunol. 91: 1–12 6. Lesavre P., Noel L.H., Chauveau D., Geffriaud C., Grunfeld J.P. 1991. Antigen specificities and clinical distribution of ANCA in kidney diseases. Klinische Wochenschrift 69: 552–557 7. Sˇediva´ A., Bartu˚nˇkova´ J., Janatkova´ I., Hoza J. 1996. Antineutrofilnı´ cytoplazmaticke´ protila´tky u deˇtı´ (ANCA in children). Cˇs. Pediat. 51: 283–286 8. Sˇediva´ A., Bartu˚nˇkova´ J., Kola´rˇova´ I., Va´vrova´ V. 1996. ANCA with the specificity for bactericidal/ permeability increasing protein in children with cystic fibrosis. Sarcoidosis 13: 275 9. Towbin H., Stachelin T., Gordin J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Procedure and some applications. Proc. Natl. Acad. Sci. USA 76: 4350–4354 10. Laemmli U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 11. Macek M Jr., Bo¨hm I., Stuhrmann M. 1992. Molecular genetic analysis of cystic fibrosis in the Czech republic of Czechoslovakia: analysis of linked DNA polymorphism, proportion of delta F508 mutation of the CFTR gene and diagnostic feasibility study. In Early Fetal Diagnosis: recent progress and Public Health Implications. M. Macek, M.A. Ferguson-Smith, M. Sˇpa´la, eds. Karolinum-Charles University press, Prague, pp 460–464 12. Kronborg G. 1995. Lipopolysaccharide (LPS), LPS-immune complexes and cytokines as inducers of pulmonary inflammation in patients with cystic fibrosis and chronic Pseudomonas aeruginosa lung infection. APMIS 103(Suppl 50): 1–30 13. Khan T.Z., Wagener J.S., Bost T., Martinez J., Accurso F.J., Richers D.W. 1995. Early pulmonary inflammation in infants with cystic fibrosis. Am. J. Resp. Care Med. 151: 939–941
190
14. Welsh M.I., Tsui L.Ch., Boat T.F., Beaudet A.L. 1995. Cystic fibrosis. In The metabolic and molecular basis of inherited diseases. C.H.R. Schriver, A.L. Baudet, W.S. Sly and D. Valle, eds. McGraw-Hill, New York, pp 3799–3876 15. Dunn A.C., Mahadeva R., Westerbeek R., Zhao M.H., Whitehouse D., Ross-Russell R.I., Bilton D., Webb A.K., Lomas D.A., Lockwood C.M. 1996. Alpha 1-antitrypsin deficiency and autoantibodies to bactericidal/ permeability increasing protein (BPI) in cystic fibrosis (CF) lung disease. Pediatr. Pulmonol. (Suppl 13): 326 (Abstr 423) 16. Mahadeva R., Dunn A.C., Westerbeek R., Zhao M.H., Ross-Russel R.I., Bilton D., Webb A.K., Lomas D.A., Lockwood C.M. 1996. Autoantibodies to bactericidal/permeability increasing protein (BPI), colonization status and cystic fibrosis (CF) lung disease. Pediatr. Pulmonol. (Suppl 13): 326 (Abstr 424) 17. Weiss J., Elsbach P., Shu C., Castillo J., Grinna L., Horwitz A., Theofan G. 1992. Human bactericidal/ permeability increasing protein and a recombinant NH2-terminal fragment cause killing of serum-resistant gram-negative bacteria in whole blood and inhibit tumor necrosis factor release induced by the bacteria. J. Clin. Invest. 90: 1122–1130
A. Sˇediva´ et al.
18. Barasch J., Landry D., Al-Awqati Q. 1993. The chloride channel of intracellular organelles and its potential role in cystic fibrosis. In Cystic Fibrosis, current topics. Vol. 1. J.A. Dodge, D.J.H. Brock, J.H. Widdicombe, eds. John Wiley and Sons Ltd., Chichester, pp 191–217 19. Dohlman J.G., Pillion D.J., Rokeach L.A., Ramprasad M.P. 1991. Identification of macrophage cell-surface binding sites for cationized bovine serum albumin. Biochem. Biophys. Res. Commun. 181: 787–796 20. Burnett R.J., Lyden C.A., Tindal C.J., Cave C.M., Marra M.N., Solomkin J.S. 1996. Mononuclear cell line THP-1 internalizes bactericidal/permeability increasing protein by a non receptor-mediated mechanism consistent with pinocytosis. Arch. Sur. 131: 200–205 21. Bartu˚nˇkova´ J., Korˇ´ınkova´ P., Tesarˇ P., Janatkova´ I., Krysˇtu˚fkova´ O., Rychlı´k I. 1995. Interaction of different antineutrophil autoantibodies with microbicidal activity of polymorphonuclears. Clin. Exp. Immunol. 101: 48 22. Bartu˚nˇkova´ J., Fucˇ´ıkova´ T., Tesarˇ V., Janatkova´ I., Rychlı´k I., Sulkova´ S. 1995. Antineutrophil cytoplasmic antibody-positive sera inhibit candidacidal activity of granulocytes. Exp. Nephrol. 3: 58–60
Antineutrophil Cytoplasmic Autoantibodies (ANCA) in Children with Cystic Fibrosis Anna Sˇediva´1, Jirˇina Bartu˚nˇkova´1, Ivana Kola´rˇova´1, Ondrˇej Hrusˇa´k1, Veˇra Va´vrova´2, Milan Macek jr3, Christopher M-Lockwood4 and Austin C. Dunn4 1
Institute of Immunology, 2Second Clinic of Paediatrics, 3Department of Genetics, University Hospital Motol, Charles University, Prague, Czech Republic 4 Department of Medicine, University of Cambridge, Cambridge, UK
Received 13 June 1997 Accepted 10 December 1997
Key words: antineutrophil cytoplasmic antibodies (ANCA), bactericidal/permeability increasing protein (BPI), children, cystic fibrosis (CF), cystic fibrosis transmembrane regulator (CFTR)
Anti-neutrophil cytoplasmic antibodies (ANCA) represent a useful diagnostic tool in patients with small vessel vasculitis. Circulating ANCA specific for bactericidal/permeability increasing protein (BPI) have been recently reported in adult patients with cystic fibrosis (CF), an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene with consequent impaired function of a transmembrane chloride channel. To contribute to the better understanding of the significance of ANCA in this disease, we investigated ANCA presence and antigenic specificity in children with CF. Results were correlated with clinical status, immunological data, age and genotype. The indirect immunofluorescence pattern of a total of 71 children with CF indicated that 31 were c-ANCA positive, while seven were p-ANCA positive. In further ELISA studies of ANCA antigenic specificity, 51 out of 66 investigated samples were positive for BPI, and 14 out of 28 were positive for proteinase 3 (PR3). We found an association between levels of antibodies against PR3 with age and Pseudomonas infection. We did not, however, find any correlation between CFTR genotypes, Pseudomonas infection or paediatric parameters and the level of anti-BPI antibodies. High positivity of anti-BPI antibodies were seen even among the youngest CF patients, before the development of clinical signs of CF, indicating that formation of ANCA might be a very early event in the disease. Both anti-BPI and anti-PR3 antibodies may play a significant, although variable role, in the pathogenesis of CF. © 1998 Academic Press Limited
Introduction
with CF [3]. Antineutrophil cytoplasmic antibodies (ANCA) are usually associated with autoimmune disorders including small vessel vasculitis, such as Wegener’s granulomatosis, microscopic polyangiitis, idiopathic rapidly progressing glomerulonephritis, inflammatory bowel disease and rheumatoid arthritis [4, 5]. Different target antigens, usually present in the azurophilic granules of neutrophils, are recognized by these autoantibodies. Antibodies directed against proteinase 3 (PR3) are frequently associated with Wegener’s granulomatosis, and antibodies reacting with myeloperoxidase (MPO) are associated, although less closely, with microscopic polyangiitis [6]. ANCA detected in sera of adults with CF react with bactericidal/permeability increasing protein (BPI), another component of azurophilic granules [3]. In our previous study we analysed the incidence and importance of ANCA in childhood [7]. The predominant diagnosis among ANCA positive children was CF [7, 8]. This led to a detailed analysis of this group and to the possible explanation of the origin and pathogenetic role of these autoantibodies in CF.
Cystic fibrosis (CF) is an autosomal recessive disorder. The CF gene was discovered in 1989 and the corresponding protein was found to be CFTR, cystic fibrosis transmembrane regulator [1]. The consequence of this mutation is defective ion transport across the cell membranes, which leads to the gradually developing clinical picture of the disease, characterized mainly by damage to the lungs and pancreas [2]. The immune system is believed to be involved in the pathogenesis of this disease through its contribution to inflammation of the tissues, particularly the lungs. However, so far there has been no reason to believe that autoimmune processes play any role in the pathogenesis of CF. Recently, the finding of circulating anti-neutrophil cytoplasmic antibodies has been reported in adult patients Correspondence to: Anna Sˇediva´, Institute of Immunology, ´ valu 84, 150 06 Praha 5, University Hospital Motol, V U Czech Republic. Fax: 4202 2443 5962. E-mail: [email protected]. 185 0896-8411/98/020185+06 $25.00/0/au970186
© 1998 Academic Press Limited
A. Sˇediva´ et al.
186
Material and Methods
18
We investigated 71 children, 33 girls and 38 boys, aged 5 months to 18 years. All have been regularly seen and treated in the specialized Prague CF department. Immunological investigations were performed during follow-up visits or during control hospitalizations of patients with CF, with material from routine venepunctures. ANCA were detected by indirect immunofluorescence on ethanol fixed human neutrophils (Binding Site, Birmingham, UK) and all samples were tested for the specificity for BPI of IgG isotype (BPI protein IgG Antibodies ELISA kit, Genesis Diagnostics, Ely, UK). Fifty-one out of 71 serum samples were available for testing anti-BPI antibodies of IgA isotype in solid phase ELISA. Ninety-six-well plates (Dynatech, NJ, USA) were coated with native BPI purified from neutrophil extract at concentration 1.0 ìg/ml in PBS, and incubated for 1 h at room temperature. Serum samples were diluted 1:50 in PBS-Tween 20, and 100 ìl/well of diluted serum was added to the ELISA plate, incorporating two antigen coated wells and one antigen-free to allow for non-specific binding. Laboratory positive and negative controls were incorporated in each plate. Samples were considered positive when the optical density at 405 nm using an alkaline phosphatase substrate (Sigma104, Dorset, UK) development system was greater than the mean and three standard deviations from the negative control, as calculated by linear regression. The negative control consisted of 50 normal blood donors. All sera positive for c-ANCA were further investigated by ELISA for specificity for PR3 (EIAgen c-ANCAkit, Biochem Pharma Group, Freiburg, Germany). Serological reactivity of PR3 positive sera was confirmed by western blotting studies, using a modification of the method described by Towbin et al. [9]. PR3 purified from human neutrophil extracts was divided on 12% polyacrylamide SDS-PAGE gel according to the method of Laemmli [10]. The gel was blotted onto nitrocellulose paper (Schleicher and Schuell, Germany) that had been soaked in transfer buffer (10 mM 3-(cyclohexylamino)-1-propanesulfonic acid, 10% v/v methanol, pH 11.0). Transfer was accomplished by passing a 0.5 mA current through the gel/ nitrocellulose sandwich for 40 min. The nitrocellulose was removed and stained with ponceau-S (BDH, Dorset, UK) to visualize transferred protein; then cut and blocked with 2% w/v non-fat milk in 0.01 M Tris-base, 0.15 M sodium chloride, pH 8.0 (TBSTM), for 30 min. Tested sera and positive and negative controls were diluted 1:200 in TBSTM, added and left for 1 h at room temperature. After incubation and washing, human IgG binding was detected using alkaline phosphatase-conjugated goat anti-human IgG (Sigma, Poole, Dorset, UK), diluted 1:3000 in TBSTM. The blot was developed with 5 ml of alkaline phosphatase buffer (100 mM sodium chloride, 5 mM magnesium chloride (BDH, Dorset, UK), 100 mM Tris, pH 9.5) containing 66 ìl of nitroblue tetrazolium stock (Sigma NBT, Dorset, UK). The reaction was stopped by
16
Number of patients
14 12 10 8 6 4 2 0
0
10
20 30 Anti-BPI (U/ml)
40
50
Figure 1. Levels of anti-BPI antibodies. Frequency of negative ( ) and positive ( ) values. Out of 66 children 51 (77%) were positive for anti-BPI antibodies. The anti-BPI values of six children between 3 and 7 ml are classified as borderline positivity ( ).
0.7
Anti PR3 (OD)
0.6
infection neg. infection PSA infection CEP
0.5 0.4 0.3 0.2 0.1 0
5
10 Age (years)
15
20
Figure 2. Correlation of anti-PR3 antibodies, infection with Pseudomonas species and age. Out of 28 c-ANCA positive children, seven were positive for anti-PR3 antibodies and seven showed borderline positivity. The positivity of antiPR3 antibodies appears only in children 10 years and older, and is significantly correlated with Pseudomonas infection. ×=infection neg.; C=Pseudomonas aeruginosa; +=Burgholderia cepacea.
rinsing with PBS containing 20 mM EDTA; the blot was then air dried and stored in the dark. Routine blood counts, biochemical values and immunological laboratory values, including concentration of immunoglobulins and lung function, were investigated at the University Hospital Motol. Genotypes were established in each child using a combination of detection methods. Both rare mutations and the most common ÄF508 mutation were detected by PCR and subsequent non-denaturing polyacrylamide gel electrophoresis [11]. Other relatively common CF mutations were detected using allele specific oligonucleotide hybridization, amplification refractory mutation detection system,
ANCA in children with cystic fibrosis
187
1
2
3
4
5
6
7
KDa 46
29
Figure 3. Immunoblot studies of anti-PR3 antibodies. The specificity of positive anti-PR3 antibodies detected in ELISA was confirmed by western blot of purified PR3 with patient’s sera. Three serum samples of children with CF positive for anti-PR3 antibodies in ELISA were available for immunoblot. We detected faint bands, demonstrated in lanes 5, 6 and 7. These were expected, due to the very slight positivity demonstrated in ELISA. Lane 1: negative control, lane 2: patient with Wegener’s granulomatosis (kind gift of Mr P. Chapman, University of Cambridge), lane 3: serum of a 10-year-old boy with Wegener’s granulomatosis (First Clinic of Paediatrics, Charles University, Prague), lane 4: adult patient with Wegener’s granulomatosis (kind gift of Dr V. Tesarˇ, First Internal Clinic, Charles University, Prague).
Results ANCA determination by immunofluorescence We investigated 71 children with CF. Out of these, 31 showed a cytoplasmic type of immunofluorescence (c-ANCA) and 7 had perinuclear type (p-ANCA). This represents 52% positivity in immunofluorescence staining.
ANCA antigenic specificity Samples from 66 children were available for the determination of anti-BPI of IgG isotype in ELISA; of these 51 were positive (77%). The level of anti-BPI IgG antibodies in this test ranged from 7 to 50 U/ml (negative value ≤3, borderline value 3–6 U/ml) (Figure 1). Fifty-one out of 71 serum samples were available for testing for anti-BPI antibodies of IgA isotype. Out of these 51 children, 15 were positive according to the criteria of the test (see Material and Methods). We found eight children positive for both IgG and IgA and anti-BPI antibodies. Since c-ANCA pattern is often associated with the specificity of PR3, c-ANCA positive samples were further examined for specificity against PR3. Out of 28 investigated in anti-PR3 ELISA, seven were positive and seven showed borderline positivity (50%). The concentration of anti-PR3 antibodies was only slightly elevated (Figure 2). We found six children positive for
positive borderline negative
15
IgG (g/l)
PCR-mediated side directed mutagenesis and standard restriction digestion techniques. All data were processed by mathematical analysis, with patient data being analysed using multifactorial non-linear correlation analysis. The database included, besides the immunological parameters (immunoglobulins, circulating immunocomplexes, C3 and C4 components of complement); clinical values (lung and pancreatic function, liver function; presence of diabetes; infection with Pseudomonas aeruginosa or Burkholderia cepacia); and the particular genotype.
10
5
0
–5
0
0.1
0.2
0.3 0.4 Anti-PR3 (OD)
0.5
0.6
Figure 4. Correlation between anti-PR3 ELISA titres and serum levels of IgG. Linear regression was used to demonstrate the pattern of the correlation between anti-PR3 antibodies and serum level of IgG. Anti-PR3 antibodies are marked on the x axis, values on the y axis represent normalised values of IgG according to the age related mean. The values are expressed as a number of standard deviations. Correlation is positive only for negative values of anti-PR3 antibodies; borderline and positive values of antiPR3 antibodies do not correlate with high levels of IgG, indirectly excluding the non-specific IgG binding in ELISA.
both anti-PR3 and anti-BPI IgG antibodies; one girl out of these six was also positive for anti-BPI IgA antibodies. The specificity of positive anti-PR3 antibodies in ELISA was confirmed by western blotting (Figure 3) and this finding was further supported by linear regression analysis demonstrating the absence of correlation between anti-PR3 antibodies and total IgG level (Figure 4).
Multiparameter statistical analysis Statistical analysis correlated the level of anti-BPI of both IgG and IgA isotypes, and anti-PR3 antibodies, with age, genotype, lung and pancreatic function, and infection with Pseudomonas species and clinical status.
A. Sˇediva´ et al.
188
Proposal for pathogenic role of ANCA in cystic fibrosis
? (Inappropriate release of cationic BPI from neutrophils? Change in conformational structure of BPI?)
Formation of antibodies to BPI
Neutralization of BPI by autoantibodies
Diminished defence against Pseudomonas
Pseudomonas colonization of lungs
PMN activation
Inflammation, excessive release of granule enzymes including PR3
Other infections, further PMN activation, tissue destruction
Antibody formation to PR3
Anti-PR3-Abs diminish microbicidal capacity of PMN, stimulate oxygen radical release and degranulation
Figure 5. Proposal for pathogenetic role of ANCA in cystic fibrosis. Antibodies against BPI neutralise BPI. The resultant lack of defence against Pseudomonas contributes to the chronic Pseudomonas lung infection typical of CF patients. Chronic infection leads to continuous polymorphonuclear activation and excessive release of granule enzymes, with subsequent formation of autoantibodies directed against these enzymes. The situation then becomes a ‘vicious cycle’ of infection, impaired defence against Pseudomonas, further infection and formation of autoantibodies against granule enzymes which causes the diminished microbicidal activity of the attracted neutrophils.
There was no correlation between any of these parameters and anti-BPI antibodies of any isotype. However, we found a clear-cut association between the level of anti-PR3 antibodies, infection with Pseudomonas species and age (Figure 2).
Discussion ANCA are an unexpected finding in children with CF. Nevertheless, our results as well as the results of the adult CF patients, reported earlier [3], confirmed that ANCA positivity is a consistent finding in patients with this disease. Many immunological abnormalities are reported in patients with CF [12, 13], but until recently, the association with autoimmune processes was relatively infrequent. Only minor clinical signs, such as erythema nodosum or arthritis in certain patients, suggest that autoimmune mechanisms may
be operating. The laboratory findings of marked hyperimmunoglobulinemia, mainly of IgG and IgA classes, and high levels of immune complexes, are not necessarily autoimmune in origin, but probably represent the secondary consequences of chronic inflammation [14]. The secondary nature of these findings might also be suggested by the fact that newborn and small children with genotypically proven CF do not have detectable changes of these immune parameters, but they gradually develop during the course of the disease. However, we found anti-BPI specific ANCA antibodies even in very young children. Our youngest boy with a high level of anti-BPI antibodies was 5 months old, and had no Pseudomonas infection, which is almost obligatory in older patients with CF. On the other hand, the antibodies directed against PR3 appeared late in our group of patients, in the children aged 10 years and older, and were significantly connected with Pseudomonas infection. In contrast to another study, we
ANCA in children with cystic fibrosis
did not find correlation of anti-BPI antibodies and colonization with Pseudomonas [15, 16]. The level of anti-BPI antibodies in our group of children did not correlate with any parameter included in our study. We did not confirm the previously reported correlation of the level of anti-BPI antibodies and impaired lung functions of adult patients with CF [3]. On the basis of our results, we propose that the two specificities of ANCA, anti-BPI and anti-PR3 ANCA, have a different origin and a different role in the development of the pathology of CF. Anti-BPI antibodies are present at an early stage, which suggests that mechanisms leading to their formation are directly connected with the basic defect of CF, despite the fact that we did not observe any correlation with the CFTR genotype. BPI is a highly cationic protein, usually stored in the azurophilic granules of neutrophils. It is microbicidal for, and effective against lipopolysaccharide of Gram-negative bacteria [17]. Positive charge plays a dominant role in the function of BPI, and changes in charge and pH are the dominant features of cells expressing mutated CFTR [14, 18]. This is mostly expressed on epithelial cells, but mRNA coding for the protein has also been described in neutrophils [14]. According to our hypothesis, BPI may be more easily released by CF neutrophils then by normal, non-CF neutrophils. As a highly charged cationic protein it is a potent activator of macrophages [19], and is efficiently internalized in complexes with LPS, by macrophages via pinocytosis [20]. All these conditions may lead to the presentation of BPI antigens by macrophages and induction of anti-BPI antibody formation. By blocking BPI, these antibodies may trigger the ‘vicious cycle’ of repeated Pseudomonas infection followed by inefficient immune response (Figure 5). We suggest that there may be a different mechanism for the induction of anti-PR3 antibodies, which are formed after chronic inflammation over many years, with neutrophil accumulation and release of intracellular enzymes including PR3. This is supported by our finding that some patients with CF have also anti-myeloperoxidase antibodies (unpublished results, 1996). It is probable that antibodies against PR3 contribute to the ‘vicious cycle’ which results in persistent infections in CF patients, by diminishing microbicidal capacity of polymorphonuclear cells, as documented in our previous experimental studies [21, 22].
Conclusion ANCA is a consistent finding in children with CF. The pathogenetic role of these autoantibodies still awaits elucidation, but their specificity against BPI and PR3, both potent antimicrobial defence proteins, suggests their possible contribution to immunological and clinical aspects of CF.
Acknowledgements The authors thank Hynek Lauschmann for excellent statistic analysis. This work was supported by grants
189
from the Ministry of Health IGA MZ No 3160-3, 3933-3, 2899-5, 3526-3, 4124-3, 2056-5, and from Barrande 970157.
References 1. Riordan J.R., Rommens J.M., Kerem B., Alon N., Rozmahel R., Grzelczak Z., Zielenski J., Lok S., Plavsic N., Chou J.L 1989. Identification of cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245: 1066–1073 2. Mearns M.B. 1993. Cystic Fibrosis: The First 50 Years. In Cystic Fibrosis, current topics. Vol. 1. J.A. Dodge, D.J.H. Brock and J.H. Widdicombe, eds. John Wiley and Sons Ltd., Chichester, pp 217–251 3. Zhao M.H., Jayne D.R.W., Ardiles L.G., Culley F., Hodson M.E., Lockwood C.M. 1996. Autoantibodies against bactericidal/permeability-increasing protein in patients with cystic fibrosis. Q.J. Med. 98: 259–265 4. van der Woude F.J., Rasmussen N., Lobatto S., Wiik A., Permin H., van Es L.A., van der Giessen M., van der Hem G.K., The T.H. 1985. Autoantibodies against neutrophils and monocytes: tool for diagnosis and marker of disease activity in Wegener’s granulomatosis. Lancet 1: 425–429 5. Gross W.L., Schmitt W.H., Csernok E. 1993. ANCA and associated diseases: immunodiagnostic and pathogenetic aspects. Clin. Exp. Immunol. 91: 1–12 6. Lesavre P., Noel L.H., Chauveau D., Geffriaud C., Grunfeld J.P. 1991. Antigen specificities and clinical distribution of ANCA in kidney diseases. Klinische Wochenschrift 69: 552–557 7. Sˇediva´ A., Bartu˚nˇkova´ J., Janatkova´ I., Hoza J. 1996. Antineutrofilnı´ cytoplazmaticke´ protila´tky u deˇtı´ (ANCA in children). Cˇs. Pediat. 51: 283–286 8. Sˇediva´ A., Bartu˚nˇkova´ J., Kola´rˇova´ I., Va´vrova´ V. 1996. ANCA with the specificity for bactericidal/ permeability increasing protein in children with cystic fibrosis. Sarcoidosis 13: 275 9. Towbin H., Stachelin T., Gordin J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Procedure and some applications. Proc. Natl. Acad. Sci. USA 76: 4350–4354 10. Laemmli U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 11. Macek M Jr., Bo¨hm I., Stuhrmann M. 1992. Molecular genetic analysis of cystic fibrosis in the Czech republic of Czechoslovakia: analysis of linked DNA polymorphism, proportion of delta F508 mutation of the CFTR gene and diagnostic feasibility study. In Early Fetal Diagnosis: recent progress and Public Health Implications. M. Macek, M.A. Ferguson-Smith, M. Sˇpa´la, eds. Karolinum-Charles University press, Prague, pp 460–464 12. Kronborg G. 1995. Lipopolysaccharide (LPS), LPS-immune complexes and cytokines as inducers of pulmonary inflammation in patients with cystic fibrosis and chronic Pseudomonas aeruginosa lung infection. APMIS 103(Suppl 50): 1–30 13. Khan T.Z., Wagener J.S., Bost T., Martinez J., Accurso F.J., Richers D.W. 1995. Early pulmonary inflammation in infants with cystic fibrosis. Am. J. Resp. Care Med. 151: 939–941
190
14. Welsh M.I., Tsui L.Ch., Boat T.F., Beaudet A.L. 1995. Cystic fibrosis. In The metabolic and molecular basis of inherited diseases. C.H.R. Schriver, A.L. Baudet, W.S. Sly and D. Valle, eds. McGraw-Hill, New York, pp 3799–3876 15. Dunn A.C., Mahadeva R., Westerbeek R., Zhao M.H., Whitehouse D., Ross-Russell R.I., Bilton D., Webb A.K., Lomas D.A., Lockwood C.M. 1996. Alpha 1-antitrypsin deficiency and autoantibodies to bactericidal/ permeability increasing protein (BPI) in cystic fibrosis (CF) lung disease. Pediatr. Pulmonol. (Suppl 13): 326 (Abstr 423) 16. Mahadeva R., Dunn A.C., Westerbeek R., Zhao M.H., Ross-Russel R.I., Bilton D., Webb A.K., Lomas D.A., Lockwood C.M. 1996. Autoantibodies to bactericidal/permeability increasing protein (BPI), colonization status and cystic fibrosis (CF) lung disease. Pediatr. Pulmonol. (Suppl 13): 326 (Abstr 424) 17. Weiss J., Elsbach P., Shu C., Castillo J., Grinna L., Horwitz A., Theofan G. 1992. Human bactericidal/ permeability increasing protein and a recombinant NH2-terminal fragment cause killing of serum-resistant gram-negative bacteria in whole blood and inhibit tumor necrosis factor release induced by the bacteria. J. Clin. Invest. 90: 1122–1130
A. Sˇediva´ et al.
18. Barasch J., Landry D., Al-Awqati Q. 1993. The chloride channel of intracellular organelles and its potential role in cystic fibrosis. In Cystic Fibrosis, current topics. Vol. 1. J.A. Dodge, D.J.H. Brock, J.H. Widdicombe, eds. John Wiley and Sons Ltd., Chichester, pp 191–217 19. Dohlman J.G., Pillion D.J., Rokeach L.A., Ramprasad M.P. 1991. Identification of macrophage cell-surface binding sites for cationized bovine serum albumin. Biochem. Biophys. Res. Commun. 181: 787–796 20. Burnett R.J., Lyden C.A., Tindal C.J., Cave C.M., Marra M.N., Solomkin J.S. 1996. Mononuclear cell line THP-1 internalizes bactericidal/permeability increasing protein by a non receptor-mediated mechanism consistent with pinocytosis. Arch. Sur. 131: 200–205 21. Bartu˚nˇkova´ J., Korˇ´ınkova´ P., Tesarˇ P., Janatkova´ I., Krysˇtu˚fkova´ O., Rychlı´k I. 1995. Interaction of different antineutrophil autoantibodies with microbicidal activity of polymorphonuclears. Clin. Exp. Immunol. 101: 48 22. Bartu˚nˇkova´ J., Fucˇ´ıkova´ T., Tesarˇ V., Janatkova´ I., Rychlı´k I., Sulkova´ S. 1995. Antineutrophil cytoplasmic antibody-positive sera inhibit candidacidal activity of granulocytes. Exp. Nephrol. 3: 58–60