European Journal of
Eur. J. Pediatr. 127, 133--139 (1978)
Pediatrics 9 by Springer-Verlag 1978
Complement in Cystic Fibrosis* ** M. G6tz and G. Lubec Paediatric University Hospital (Director: Univ.-Prof. Dr. E. Zweymfiller), W~ihring, Giirte174--76, A-1090 Vienna, Austria
Abstract. Complement components C3, C4, and C3A were estimated in 30 patients with cystic fibrosis aged 1 to 21 years ( M : F = 16: 14) and were compared with results in 40 healthy, age-matched subjects. The influences of the clinical score, sputum microbiology, and the patients' sex were also investigated. In contrast to most previous communications, this paper shows that, compared to the control group, a significant decrease of C3 ( P < 0.001) and C4 (P < 0.02) was observed whereas C3A levels were not altered. There were no increases in complement. Shwachman-scores above or below 70 did not influence the complement levels, nor did exacerbations of the disease change the levels. No influence of the patients' sex could be shown. Pseudomonas aer. in the sputum was clearly associated with complement defects (14/18). Alternative-pathway involvement of complement activation could be demonstrated in 32%. The results make complement activation due to pulmonary infection most likely. The defects observed probably represent secondary changes.
Key words: Cystic fibrosis - Complement - Alternative pathway - Pseudomonas.
Recurrent infection plays a prominent role in the pathology of pulmonary disease in cystic fibrosis [3, 9]. Various mechanisms such as increased susceptibility to bacterial growth due to obstructive lung disease [9], with respiratory mucosal damage or dysrhythmic ciliary activity leading to impaired mucociliary transport, have been incriminated [28, 33, 34]. Defective immune reactions concerning both humoral and cellular functions have also been found [12, 14, 18, 24]. Complement has a major role among the nonspecific host defence mechanisms [20, 26], particularly by enhancing the chemotactic and opsonizing efficiency of phagocytic cells * Presented at the VIIth Annual Meeting of the European Working Group for Cystic Fibrosis, Dresden, June 20--21, 1977 ** Dedicated to Professor E. Zweymiiller on the occasion of his 60th birtday
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[27]. A n y i m m u n e deficiency m a y result in an increased susceptibility to infection o f the respiratory tract but no quantitative connection between the severity o f infection and c o m p l e m e n t levels has as yet been established with certainty [25]. Recent reports have demonstrated complement activation, involving the alternative p a t h w a y , in severe gram-negative infection [10, 11]. Gram-negative infection has emerged as one o f the most serious problems in cystic fibrosis, a l t h o u g h it remains a localized disease o f the respiratory tract and rarely progresses to gram-negative bacteraemia. Several short reports on various c o m p l e m e n t c o m p o n e n t s in cystic fibrosis have been published [5, 6, 15, 29]. Alternative-pathway involvement has not yet been fully investigated and there have been few attempts to correlate the findings with clinical and microbiological data [21, 32]. The present paper sets out to estimate both classical and alternative-pathway participation be measuring C3c, C4, and C3A [C3 Activator] [2]. The influences o f the patient's clinical state, the microbiological findings, and the patient's sex on c o m p l e m e n t levels were also investigated.
Material and Methods 34 freshly obtained serum samples from 30 patients with cystic fibrosis aged 1 to 21 years (M:F = 16:14) were examined. In all the patients, the diagnosis had been established by the typical symptoms and signs, and repeatedly positive sweat tests performed according to the method of Gibson and Cooke [13]. One patient was initially discovered by routine screening for increased albumin content of meconium [30]. Two boys with a low Shwachman-score have died since the beginning of the investigation. None of the children showed hypoproteinaemia or hypoalbuminaemia. 11 samples were obtained from patients with Shwachman-scores of less than 70 and 23 from patients with scores of 70 or more. 15 samples were taken at times when the patients showed signs of pulmonary infection and 19 at times when infection was not prominent. The control subjects consisted of 40 healthy, age-matched children without any known pulmonary or complement-consuming disease. There were no known heterozygotes for cystic fibrosis among the control group. Values obtained for this group were in accord with normal values for this laboratory. Serum complement levels were estimated by radial immunodiffusion according to the Mancini precipitation technique, using M-Partigen imrnunodiffusion plates (Behring AG, Marburg/Lahn, Germany). Statistical analysis was done with the unpaired t-test.
Results Figure 1 shows values for C3 (as C3c), C4, and C 3 A in cystic fibrosis patients, c o m p a r e d with healthy controls. The C3 levels were significantly lower in the patients ( P < 0 . 0 0 1 ) and C4 levels were also significantly lower ( P < 0 . 0 2 ) , whereas no statistically significant differences were obtained c o m p a r i n g C3A in patients and controls. No patients had a significant increase in any of the complement c o m p o n e n t s measured. The following c o m p l e m e n t level patterns were observed: All c o m p o n e n t s measured were normal in 15 samples and all the c o m p o n e n t s were low in 5
Complement in Cystic Fibrosis
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Fig. 1. a C3 a n d C4 levels in patients a n d controls, b C 3 A levels in patients and controls
samples. C3 and C4 were low, and C3A normal, in 8 samples. C3 was low, and C4 and C3A were normal, in 2 samples. C3 and C3A were low and C4 normal in 4 samples. Repeat investigations on a second serum sample from the same patient corresponded well. There was no statistical difference in the complement levels between male and female patients. Samples from patients with Shwachman-scores of more than 70 (n = 23) were not statistically different from those with scores below 70 ( n = 11). Likewise, samples from patients with suspected viral or bacterial exacerbations at the time of investigation (n-- 15) did not differ from the group without acute infections (n = 19). A m o n g 18 sera from patients with sputum samples containing Ps. aeruginosa a defective complement component was found in 14 instances. Only 5 comple-
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ment defects were observed in the remaining patients with sputa containing Staph. aureus, Streptococcus, Proteus or where microbiological data were not obtainable. Discussion Complement involvement in cystic fibrosis has often been investigated [5, 6, 15, 19, 22, 29, 31]. However, to date no uniform, conclusive picture has emerged. As cystic fibrosis usually manifests as a chronic inflammatory lung disease, complement involvement is not surprising. However, it is not specific for cystic fibrosis and is linked to pulmonary defence mechanisms in general [27]. In an attempt to identify a cystic fibrosis "factor", various bioassays have shown a ciliary dyskinesia factor (CDF). Some authors have suggested that C3a may be related to, or identical with, this CDF and that complement-IgG complexes may play a prominent role in the disease [5--7]. Such complexes were found by McFarlane and co-workers [24] in lung, trachea, and pancreatic tissue. Other authors were not able to confirm these findings [7, 23, 29]. Complement level measurements have shown contradictory results. Conover and co-workers [5] found significantly increased C3 levels in both cystic fibrosis homozygotes and heterozygotes. In a larger series of patients and controls, Hann and co-workers [15] found elevated C5 levels in patients. The mothers of cystic fibrosis children had higher levels than the respective fathers. C3 levels were found to be elevated in cystics and their parents. In contrast, Scanlin [29] found C3 levels to be the same as in controls. He agreed with Hann and co-workers [15] that there was no difference between the sexes and that the Shwachman-score was not relevant. Lieberman [23] demonstrated no difference between C3 levels in cystics and controls, but found significantly higher levels of C3 in women with cystic fibrosis, as opposed to men with cystic fibrosis or non-cystic women. He concluded that C3a was not identical with the ciliary dyskinesia factor. Both Lieberman [23] and Hann and co-workers [15] had patients with pulmonary disease among their controls. While this appears to prove that there is no difference in C3 levels between cystic fibrosis pulmonary disease and non-cystic chronic lung disease [23], it makes comparison questionable as complement is involved in lung defence in any respiratory disease. For example, C4 participation in bronchial asthma has led to controversial results [16, 21]. Holzhauer and coworkers [19] showed C3 levels in cystic fibrosis to be increased when compared to all controls. Those with moderate and severe disease had higher values than those with mild disease. Lederberg and co-workers [22] failed to demonstrate a difference in electrophoretic variants of complement in cystic fibrosis patients and controls. To our knowledge only two papers describe diminished complement levels in cystic fibrosis. A Danish group [32] demonstrated decreased C3 levels and attributed the differences to possible lung infection. Differences were only significant in patients with class III fibroblasts cultures [8]. There was no difference between the sexes as regards complement levels. In 40 cystic fibrosis children, McFarlane and co-workers [24] demonstrated that 45% had low C3 levels but only 5% had low C4 levels.
Complement in Cystic Fibrosis
137
Our own results show a decrease of C3 and C4, which in both instances was statistically significant. Diminished levels of complement due to loss by enteric secretion seem unlikely in view of the fact that all the patients were normoproteinaemic and such patients do not normally present with exudative enteropathy [28 a]. An explanation might lie in reduced synthesis or hypercatabolism of complement components [1, 4]. Decreased complement levels, which indicate classical and alternative-pathway activation, might be expected in pulmonary infection where antibody-antigen reactions lead to complement consumption. If this consumption is sufficiently large, it will produce a fall in the level of complement in the serum [11]. A more precise characterization of complement activation might be obtained by estimation of converted C3 and demonstration of increased chemotactic activity in the serum. Complement factors should also be demonstrable in the alveoli of deceased patients by means of immunofluorescence but we were not able to perform these investigations. Classical-pathway activation was evidenced by a drop of C4 concentration, along with a diminished C3 level. This occurred in the 8 patients with normal C3A levels. In a further 5 children, C3A was also lowered, which we take to indicate both classical and alternative-pathway activation. In the absence of specific antibody, endotoxin may lead to alternative-pathway activation, thus bypassing C 1, C4, and C2. Such situations will be characterized by a drop of C3 and normal C4 levels, and this has been seen in gram-negative infections [11]. 6 of our patients showed this alternative-pathway activation pattern. Together with the 5 patients showing decreased C3A levels, we assume that a total of 11 patients demonstrated alternative-pathway involvement (32%). 8 of these had Pseudomonas repeatedly cultured from their sputum. This seems to support a special role of Ps. aeruginosa in alternative-pathway activation. Another mode of alternative-pathway activation may be effected by IgA and possibly IgE [2]. After binding to viral or bacterial antigen, IgA may become involved in the complement bypass. However, these changes do not satisfactorily explain why there was no difference in complement levels between our patients with and without exacerbations of their pulmonary disease. Whether the underlying pulmonary disease alone suffices to reduce complement levels remains questionable. Lederberg and coworkers [22] assumed that in cystic fibrosis changes in complement are likely to be secondary acute-phase reactions to cell stimulation or tissue inflammation, rather than primary defects. Taussig and co-workers [31] described significantly decreased complement levels in cystic fibrosis patients with proven viral lower respiratory tract infections, compared to cystic non-viral exacerbations and cystic stable groups, and to a non-cystic viral illness group. The lower complement levels were attributed to viral antigen-antibody complex activation of complement which was limited to the lung. Acute non-viral infections in cystic patients gave a complement patternsimilar to that found during viral illness in non-cystics [31]. Out own group of cystic patients had comparable complement levels during exacerbations and when their condition was stable. Some of the exacerbations seen in our patients were probably due to viral infections but precise viral studies are not available.
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H a n n a n d c o - w o r k e r s [15] f o u n d that s p u t u m b a c t e r i o l o g y does n o t seem to influence c o m p l e m e n t levels. O u r o w n results indicate t h a t Ps. a e r u g i n o s a m a y l e a d to a defective c o m p l e m e n t system as 14 out o f 18 samples f r o m patients with P s e u d o m o n a s were defective in C3 o r C4. This o b s e r v a t i o n requires c o n f i r m a t i o n in larger series o f p a t i e n t s a n d it r e m a i n s to be seen w h e t h e r Ps. aeruginosa, as o p p o s e d to o t h e r p a t h o g e n s , has a p a r t i c u l a r l y d e r a n g i n g effect on c o m p l e m e n t activity. It m a y also be o f interest to ascertain w h e t h e r Ps. a e r u g i n o s a holds a special p l a c e a m o n g the g r a m - n e g a t i v e flora in this respect. As p r e v i o u s l y m e n t i o n e d , 8 o u t o f 11 patients with a l t e r n a t i v e - p a t h w a y i n v o l v e m e n t h a d Ps. a e r u g i n o s a in their sputa. F u r t h e r investigations into the role o f c o m p l e m e n t in non-cystic fibrosis p a t i e n t s with P s e u d o m o n a s septicaemia seem indicated. A defect in the c o m p l e m e n t a c t i v a t i n g system as discussed b y H a n n a n d cow o r k e r s [15] c o u l d n o t be d e m o n s t r a t e d by us. C 3 A , the active g a m m a m o b i l i t y f r a g m e n t involved in C3 activation, was n o r m a l in 25 instances a n d cystic C 3 A levels a n d non-cystic C 3 A levels did n o t differ significantly f r o m each other.
References 1. Alper, C. A., Bloch, K. J., Rosen, F. S.: Increased susceptibility to infection in a patient with type II essential hypercatabolism of C3. N. Engl. J. Med. 2881 601--606 (1973) 2. Alper, C. A.: Alternative or properdin pathway of complement activation. In: The Immune System and Infectious Diseases (eds. E. Neter, F. Milgrom), pp. 479--488. Basel: Karger 1975 3. Bedrossian, C. W. M., Greenberg, S. D., Singer, D. B., Hansen, J. J., Rosenberg, H. S.: The lung in cystic fibrosis. Human Pathol. 7, 195--204 (1976) 4. Charlesworth, J. A., Williams, D. G., Sherington, E., Lachmann, P. J., Peters, D. K.: Metabolic studies of the third component of complement and the glycine-rich beta glycoprotein in patients with hypocomplementemia. J. Clin. Invest. 53, 1578--1587 (1974) 5. Conover, J. H., Conod, E. J., Hirschhorn, K.: Complement components in cystic fibrosis. Lancet 1973 II, 1501 6. Conover, J. H., Conod, E. J.: Complement in cystic fibrosis. Lancet 1975I, 47--48 7. Conover, J. H., Conod, E. J., Hirschhorn, K.: Studies on ciliary dyskinesia factor in cystic fibrosis. IV. Its possible identification as anaphylatoxin (C3a)-IgG complex. Life Sci. 14, 253--266 (1974) 8. Danes, B. S., Bearn, A. G.: Cystic fibrosis of the pancreas, a study in cell culture. J. Exp. Med. 129, 775--794 (1969) 9. Esterly, J. R., Oppenheimer, E. H., Landing, B. H.: Pathology of cystic fibrosis. In: Cystic Fibrosis, Projections into the future (eds. J. A. Mangos, R. C. Talamo), pp. 115--124. New York: Stratton Intercontinental Medical Book Corp. 1976 10. Fearon, D. T., Ruddy, S., Schur, P. H., McCabe, W. R.: Activation of the properdin pathway of complement in pat!ents with gram-negative bacteremia. N. Engl. J. Med. 292, 937--940 (1975) 11. Ftist, G., Petr~is, G., Ujhelyi, E.: Activation of the complement system during infections due to gram-negative bacteria. Clin. Immunol. Immunopathol. 5, 293--302 (1976) 12. Gibbons, A., Allan, J. D., Holzel, A., McFarlane, H.: Cell-mediated immunity in patients with cystic fibrosis. Brit. Med. J. 19761, 120--122 13. Gibson, L. E., Cooke, R. E.: A test for concentration of electrolytes in sweat in cystic fibrosis of the pancreas utilizing pilocarpine by iontophoresis. Pediatrics 23, 545--549 (1959) 14. Goetz, M., Eibt, M.: Immunological investigations in patients with cystic fibrosis and chronic Pseudomonas infection. 6th Ann. Meeting Europ. Work. Group Cystic Fibrosis. Dublin, June 30--July 1, 1975
Complement in Cystic Fibrosis
!39
15. Harm, S., Holsclaw, D. S., Shin, H. S.: Complement components in cystic fibrosis. Lancet 1974 II, 520--521 16. Hansson, L. O., Kjellman, N.-I. M., Leijon, I.: Complement in bronchial asthma. Lancet 1975 II, 874 17. Harper, B. L., Carson, S. D., McNeely, C. M., Barnett, D. R., Bowman, B. H.: Immunologic and electrophoretic studies of the cystic fibrosis ciliary inhibitor. Cystic Fibrosis Club Abstracts 17th Ann. Meeting, p. 20. Cystic Fibrosis Foundation 1976 18. Hoiby, N., Andersen, V., Bendixen, G.: Pseudomonas aeruginosa infection in cystic fibrosis. Acta path. microbiol, scand. Sect. C 83, 459--468 (1975) 19. Holzhauer, R. J., Van Ess, J. D., Schwartz, R. H.: Third component of complement (C3) in cystic fibrosis. Cystic Fibrosis Club Abstracts 17th Ann. Meeting, p. 22. Cystic Fibrosis Foundation 1976 20. Johnston, R. B., Stroud, R. M.: Complement and host defense against infection. J. Pediatr. 90, 169--179 (1977) 21. Kay, A. B., Bacon, G. D., Mercer, B. A., Simpson, H., Crofton, J. W.: Complement components and IgE in bronchial asthma. Lancet 197411, 916--920 22. Lederberg, S., Sackett, D.: Distribution of complement C3 variants in individualswith cystic fibrosis. Am. J. Hum. Genet. 28, 597--601 (1976) 23. Lieberman, J.: Carboxypeptidase B-like activity and C3 in cystic fibrosis. Am. Rev. Respir. Dis. 111, 100--102 (1975) 24. McFarlane, H., Holzel, A., Brenchley, P., Allan, J. D., Wallwork, J. C., Singer, B. E., Worsley, B.: Immune complexes in cystic fibrosis. Brit. Med. J. 19751, 423--428 25. Osofsky, S. G., Thompson, B. H., Lint, T. F., Gewurz, H.: Hereditary deficiency of the third component of complement in a child with fever, skin rash, and arthralgias: Response to transfusion of whole blood. N. Engl. J. Med. 90, 180--186 (1977) 26. Palmer, D. G.: Complement and the clinician. Aust. N.Z.J. Med. 6, 349--356 (1976) 27. Polmar, S. H.: Immunodeficiencyand pulmonary disease. In: Immunologic and infectious Reactions in the Lung (eds. C. H. Kirkpatrick, H. Y. Reynolds), pp. 191--209. New YorkBasel: Marcel Dekker 1976 28. Rossman, C., Dolovich, J., Dolovich, M., Wilson, W., Newhouse, M.: Cystic fibrosis-related inhibition of mucociliary clearance in vivo in man. J. Pediatr. 90, 579--584 (1977) 28a. Roy, C. C., Silverman, A., Cozzetto, F. J.: Pediatric Clinical Gastroenterology, p. 623. Saint Louis: C. V. Mosby 1975 29. Scanlin, T. F., Norman, M. E., Rosenlund, M. L.: C3 in cystic fibrosis. Lancet 19741, 1382 30. Stephan, U., Busch, E.-W., Kollberg, H., Hellsing, K.: Cystic fibrosis: Detection by means of a test strip. Pediatrics 55, 35--38 (1975) 31. Taussig, L. M., Strunk, R. C., Sieber, O. F.: Depression of serum complement during viral lower respiratory tract illnesses (LRI) in cystic fibrosis (CF). Cystic Fibrosis Club Abstracts, 17th Ann. Meeting, p. 21. Cystic Fibrosis Foundation 1976 32. Weeke, B., Flensborg, E. W., Jacobsen, L., Jorgensen, B. A., Lykkegaard, E., Hoiby, N.: Immunochemical quantitation of 18 proteins in sera from patients with cystic fibrosis: Concentrations correlated to class of fibroblast metachromasia, clinical and radiological tung symptoms. Dan. Med. Bull. 23, 155--160 (1976) 33. Wood, R. E., Boat, T. F., Doershuk, C. F.: Cystic fibrosis. Am. Rev. Respir. Dis. 113, 833--878 (1976) 34. Wood, R. E., Wanner, A., Hirsch, J., Farrell, P. M.: Tracheal mucociliary transport in patients with cystic fibrosis and its stimulation by terbutaline. Am. Rev. Respir. Dis. 111, 733--738 (1975)
Received July 6, 1977
Eur. J. Pediatr. 127, 133--139 (1978)
Pediatrics 9 by Springer-Verlag 1978
Complement in Cystic Fibrosis* ** M. G6tz and G. Lubec Paediatric University Hospital (Director: Univ.-Prof. Dr. E. Zweymfiller), W~ihring, Giirte174--76, A-1090 Vienna, Austria
Abstract. Complement components C3, C4, and C3A were estimated in 30 patients with cystic fibrosis aged 1 to 21 years ( M : F = 16: 14) and were compared with results in 40 healthy, age-matched subjects. The influences of the clinical score, sputum microbiology, and the patients' sex were also investigated. In contrast to most previous communications, this paper shows that, compared to the control group, a significant decrease of C3 ( P < 0.001) and C4 (P < 0.02) was observed whereas C3A levels were not altered. There were no increases in complement. Shwachman-scores above or below 70 did not influence the complement levels, nor did exacerbations of the disease change the levels. No influence of the patients' sex could be shown. Pseudomonas aer. in the sputum was clearly associated with complement defects (14/18). Alternative-pathway involvement of complement activation could be demonstrated in 32%. The results make complement activation due to pulmonary infection most likely. The defects observed probably represent secondary changes.
Key words: Cystic fibrosis - Complement - Alternative pathway - Pseudomonas.
Recurrent infection plays a prominent role in the pathology of pulmonary disease in cystic fibrosis [3, 9]. Various mechanisms such as increased susceptibility to bacterial growth due to obstructive lung disease [9], with respiratory mucosal damage or dysrhythmic ciliary activity leading to impaired mucociliary transport, have been incriminated [28, 33, 34]. Defective immune reactions concerning both humoral and cellular functions have also been found [12, 14, 18, 24]. Complement has a major role among the nonspecific host defence mechanisms [20, 26], particularly by enhancing the chemotactic and opsonizing efficiency of phagocytic cells * Presented at the VIIth Annual Meeting of the European Working Group for Cystic Fibrosis, Dresden, June 20--21, 1977 ** Dedicated to Professor E. Zweymiiller on the occasion of his 60th birtday
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[27]. A n y i m m u n e deficiency m a y result in an increased susceptibility to infection o f the respiratory tract but no quantitative connection between the severity o f infection and c o m p l e m e n t levels has as yet been established with certainty [25]. Recent reports have demonstrated complement activation, involving the alternative p a t h w a y , in severe gram-negative infection [10, 11]. Gram-negative infection has emerged as one o f the most serious problems in cystic fibrosis, a l t h o u g h it remains a localized disease o f the respiratory tract and rarely progresses to gram-negative bacteraemia. Several short reports on various c o m p l e m e n t c o m p o n e n t s in cystic fibrosis have been published [5, 6, 15, 29]. Alternative-pathway involvement has not yet been fully investigated and there have been few attempts to correlate the findings with clinical and microbiological data [21, 32]. The present paper sets out to estimate both classical and alternative-pathway participation be measuring C3c, C4, and C3A [C3 Activator] [2]. The influences o f the patient's clinical state, the microbiological findings, and the patient's sex on c o m p l e m e n t levels were also investigated.
Material and Methods 34 freshly obtained serum samples from 30 patients with cystic fibrosis aged 1 to 21 years (M:F = 16:14) were examined. In all the patients, the diagnosis had been established by the typical symptoms and signs, and repeatedly positive sweat tests performed according to the method of Gibson and Cooke [13]. One patient was initially discovered by routine screening for increased albumin content of meconium [30]. Two boys with a low Shwachman-score have died since the beginning of the investigation. None of the children showed hypoproteinaemia or hypoalbuminaemia. 11 samples were obtained from patients with Shwachman-scores of less than 70 and 23 from patients with scores of 70 or more. 15 samples were taken at times when the patients showed signs of pulmonary infection and 19 at times when infection was not prominent. The control subjects consisted of 40 healthy, age-matched children without any known pulmonary or complement-consuming disease. There were no known heterozygotes for cystic fibrosis among the control group. Values obtained for this group were in accord with normal values for this laboratory. Serum complement levels were estimated by radial immunodiffusion according to the Mancini precipitation technique, using M-Partigen imrnunodiffusion plates (Behring AG, Marburg/Lahn, Germany). Statistical analysis was done with the unpaired t-test.
Results Figure 1 shows values for C3 (as C3c), C4, and C 3 A in cystic fibrosis patients, c o m p a r e d with healthy controls. The C3 levels were significantly lower in the patients ( P < 0 . 0 0 1 ) and C4 levels were also significantly lower ( P < 0 . 0 2 ) , whereas no statistically significant differences were obtained c o m p a r i n g C3A in patients and controls. No patients had a significant increase in any of the complement c o m p o n e n t s measured. The following c o m p l e m e n t level patterns were observed: All c o m p o n e n t s measured were normal in 15 samples and all the c o m p o n e n t s were low in 5
Complement in Cystic Fibrosis
135 C3A
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115
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Fig. 1. a C3 a n d C4 levels in patients a n d controls, b C 3 A levels in patients and controls
samples. C3 and C4 were low, and C3A normal, in 8 samples. C3 was low, and C4 and C3A were normal, in 2 samples. C3 and C3A were low and C4 normal in 4 samples. Repeat investigations on a second serum sample from the same patient corresponded well. There was no statistical difference in the complement levels between male and female patients. Samples from patients with Shwachman-scores of more than 70 (n = 23) were not statistically different from those with scores below 70 ( n = 11). Likewise, samples from patients with suspected viral or bacterial exacerbations at the time of investigation (n-- 15) did not differ from the group without acute infections (n = 19). A m o n g 18 sera from patients with sputum samples containing Ps. aeruginosa a defective complement component was found in 14 instances. Only 5 comple-
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ment defects were observed in the remaining patients with sputa containing Staph. aureus, Streptococcus, Proteus or where microbiological data were not obtainable. Discussion Complement involvement in cystic fibrosis has often been investigated [5, 6, 15, 19, 22, 29, 31]. However, to date no uniform, conclusive picture has emerged. As cystic fibrosis usually manifests as a chronic inflammatory lung disease, complement involvement is not surprising. However, it is not specific for cystic fibrosis and is linked to pulmonary defence mechanisms in general [27]. In an attempt to identify a cystic fibrosis "factor", various bioassays have shown a ciliary dyskinesia factor (CDF). Some authors have suggested that C3a may be related to, or identical with, this CDF and that complement-IgG complexes may play a prominent role in the disease [5--7]. Such complexes were found by McFarlane and co-workers [24] in lung, trachea, and pancreatic tissue. Other authors were not able to confirm these findings [7, 23, 29]. Complement level measurements have shown contradictory results. Conover and co-workers [5] found significantly increased C3 levels in both cystic fibrosis homozygotes and heterozygotes. In a larger series of patients and controls, Hann and co-workers [15] found elevated C5 levels in patients. The mothers of cystic fibrosis children had higher levels than the respective fathers. C3 levels were found to be elevated in cystics and their parents. In contrast, Scanlin [29] found C3 levels to be the same as in controls. He agreed with Hann and co-workers [15] that there was no difference between the sexes and that the Shwachman-score was not relevant. Lieberman [23] demonstrated no difference between C3 levels in cystics and controls, but found significantly higher levels of C3 in women with cystic fibrosis, as opposed to men with cystic fibrosis or non-cystic women. He concluded that C3a was not identical with the ciliary dyskinesia factor. Both Lieberman [23] and Hann and co-workers [15] had patients with pulmonary disease among their controls. While this appears to prove that there is no difference in C3 levels between cystic fibrosis pulmonary disease and non-cystic chronic lung disease [23], it makes comparison questionable as complement is involved in lung defence in any respiratory disease. For example, C4 participation in bronchial asthma has led to controversial results [16, 21]. Holzhauer and coworkers [19] showed C3 levels in cystic fibrosis to be increased when compared to all controls. Those with moderate and severe disease had higher values than those with mild disease. Lederberg and co-workers [22] failed to demonstrate a difference in electrophoretic variants of complement in cystic fibrosis patients and controls. To our knowledge only two papers describe diminished complement levels in cystic fibrosis. A Danish group [32] demonstrated decreased C3 levels and attributed the differences to possible lung infection. Differences were only significant in patients with class III fibroblasts cultures [8]. There was no difference between the sexes as regards complement levels. In 40 cystic fibrosis children, McFarlane and co-workers [24] demonstrated that 45% had low C3 levels but only 5% had low C4 levels.
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Our own results show a decrease of C3 and C4, which in both instances was statistically significant. Diminished levels of complement due to loss by enteric secretion seem unlikely in view of the fact that all the patients were normoproteinaemic and such patients do not normally present with exudative enteropathy [28 a]. An explanation might lie in reduced synthesis or hypercatabolism of complement components [1, 4]. Decreased complement levels, which indicate classical and alternative-pathway activation, might be expected in pulmonary infection where antibody-antigen reactions lead to complement consumption. If this consumption is sufficiently large, it will produce a fall in the level of complement in the serum [11]. A more precise characterization of complement activation might be obtained by estimation of converted C3 and demonstration of increased chemotactic activity in the serum. Complement factors should also be demonstrable in the alveoli of deceased patients by means of immunofluorescence but we were not able to perform these investigations. Classical-pathway activation was evidenced by a drop of C4 concentration, along with a diminished C3 level. This occurred in the 8 patients with normal C3A levels. In a further 5 children, C3A was also lowered, which we take to indicate both classical and alternative-pathway activation. In the absence of specific antibody, endotoxin may lead to alternative-pathway activation, thus bypassing C 1, C4, and C2. Such situations will be characterized by a drop of C3 and normal C4 levels, and this has been seen in gram-negative infections [11]. 6 of our patients showed this alternative-pathway activation pattern. Together with the 5 patients showing decreased C3A levels, we assume that a total of 11 patients demonstrated alternative-pathway involvement (32%). 8 of these had Pseudomonas repeatedly cultured from their sputum. This seems to support a special role of Ps. aeruginosa in alternative-pathway activation. Another mode of alternative-pathway activation may be effected by IgA and possibly IgE [2]. After binding to viral or bacterial antigen, IgA may become involved in the complement bypass. However, these changes do not satisfactorily explain why there was no difference in complement levels between our patients with and without exacerbations of their pulmonary disease. Whether the underlying pulmonary disease alone suffices to reduce complement levels remains questionable. Lederberg and coworkers [22] assumed that in cystic fibrosis changes in complement are likely to be secondary acute-phase reactions to cell stimulation or tissue inflammation, rather than primary defects. Taussig and co-workers [31] described significantly decreased complement levels in cystic fibrosis patients with proven viral lower respiratory tract infections, compared to cystic non-viral exacerbations and cystic stable groups, and to a non-cystic viral illness group. The lower complement levels were attributed to viral antigen-antibody complex activation of complement which was limited to the lung. Acute non-viral infections in cystic patients gave a complement patternsimilar to that found during viral illness in non-cystics [31]. Out own group of cystic patients had comparable complement levels during exacerbations and when their condition was stable. Some of the exacerbations seen in our patients were probably due to viral infections but precise viral studies are not available.
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H a n n a n d c o - w o r k e r s [15] f o u n d that s p u t u m b a c t e r i o l o g y does n o t seem to influence c o m p l e m e n t levels. O u r o w n results indicate t h a t Ps. a e r u g i n o s a m a y l e a d to a defective c o m p l e m e n t system as 14 out o f 18 samples f r o m patients with P s e u d o m o n a s were defective in C3 o r C4. This o b s e r v a t i o n requires c o n f i r m a t i o n in larger series o f p a t i e n t s a n d it r e m a i n s to be seen w h e t h e r Ps. aeruginosa, as o p p o s e d to o t h e r p a t h o g e n s , has a p a r t i c u l a r l y d e r a n g i n g effect on c o m p l e m e n t activity. It m a y also be o f interest to ascertain w h e t h e r Ps. a e r u g i n o s a holds a special p l a c e a m o n g the g r a m - n e g a t i v e flora in this respect. As p r e v i o u s l y m e n t i o n e d , 8 o u t o f 11 patients with a l t e r n a t i v e - p a t h w a y i n v o l v e m e n t h a d Ps. a e r u g i n o s a in their sputa. F u r t h e r investigations into the role o f c o m p l e m e n t in non-cystic fibrosis p a t i e n t s with P s e u d o m o n a s septicaemia seem indicated. A defect in the c o m p l e m e n t a c t i v a t i n g system as discussed b y H a n n a n d cow o r k e r s [15] c o u l d n o t be d e m o n s t r a t e d by us. C 3 A , the active g a m m a m o b i l i t y f r a g m e n t involved in C3 activation, was n o r m a l in 25 instances a n d cystic C 3 A levels a n d non-cystic C 3 A levels did n o t differ significantly f r o m each other.
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15. Harm, S., Holsclaw, D. S., Shin, H. S.: Complement components in cystic fibrosis. Lancet 1974 II, 520--521 16. Hansson, L. O., Kjellman, N.-I. M., Leijon, I.: Complement in bronchial asthma. Lancet 1975 II, 874 17. Harper, B. L., Carson, S. D., McNeely, C. M., Barnett, D. R., Bowman, B. H.: Immunologic and electrophoretic studies of the cystic fibrosis ciliary inhibitor. Cystic Fibrosis Club Abstracts 17th Ann. Meeting, p. 20. Cystic Fibrosis Foundation 1976 18. Hoiby, N., Andersen, V., Bendixen, G.: Pseudomonas aeruginosa infection in cystic fibrosis. Acta path. microbiol, scand. Sect. C 83, 459--468 (1975) 19. Holzhauer, R. J., Van Ess, J. D., Schwartz, R. H.: Third component of complement (C3) in cystic fibrosis. Cystic Fibrosis Club Abstracts 17th Ann. Meeting, p. 22. Cystic Fibrosis Foundation 1976 20. Johnston, R. B., Stroud, R. M.: Complement and host defense against infection. J. Pediatr. 90, 169--179 (1977) 21. Kay, A. B., Bacon, G. D., Mercer, B. A., Simpson, H., Crofton, J. W.: Complement components and IgE in bronchial asthma. Lancet 197411, 916--920 22. Lederberg, S., Sackett, D.: Distribution of complement C3 variants in individualswith cystic fibrosis. Am. J. Hum. Genet. 28, 597--601 (1976) 23. Lieberman, J.: Carboxypeptidase B-like activity and C3 in cystic fibrosis. Am. Rev. Respir. Dis. 111, 100--102 (1975) 24. McFarlane, H., Holzel, A., Brenchley, P., Allan, J. D., Wallwork, J. C., Singer, B. E., Worsley, B.: Immune complexes in cystic fibrosis. Brit. Med. J. 19751, 423--428 25. Osofsky, S. G., Thompson, B. H., Lint, T. F., Gewurz, H.: Hereditary deficiency of the third component of complement in a child with fever, skin rash, and arthralgias: Response to transfusion of whole blood. N. Engl. J. Med. 90, 180--186 (1977) 26. Palmer, D. G.: Complement and the clinician. Aust. N.Z.J. Med. 6, 349--356 (1976) 27. Polmar, S. H.: Immunodeficiencyand pulmonary disease. In: Immunologic and infectious Reactions in the Lung (eds. C. H. Kirkpatrick, H. Y. Reynolds), pp. 191--209. New YorkBasel: Marcel Dekker 1976 28. Rossman, C., Dolovich, J., Dolovich, M., Wilson, W., Newhouse, M.: Cystic fibrosis-related inhibition of mucociliary clearance in vivo in man. J. Pediatr. 90, 579--584 (1977) 28a. Roy, C. C., Silverman, A., Cozzetto, F. J.: Pediatric Clinical Gastroenterology, p. 623. Saint Louis: C. V. Mosby 1975 29. Scanlin, T. F., Norman, M. E., Rosenlund, M. L.: C3 in cystic fibrosis. Lancet 19741, 1382 30. Stephan, U., Busch, E.-W., Kollberg, H., Hellsing, K.: Cystic fibrosis: Detection by means of a test strip. Pediatrics 55, 35--38 (1975) 31. Taussig, L. M., Strunk, R. C., Sieber, O. F.: Depression of serum complement during viral lower respiratory tract illnesses (LRI) in cystic fibrosis (CF). Cystic Fibrosis Club Abstracts, 17th Ann. Meeting, p. 21. Cystic Fibrosis Foundation 1976 32. Weeke, B., Flensborg, E. W., Jacobsen, L., Jorgensen, B. A., Lykkegaard, E., Hoiby, N.: Immunochemical quantitation of 18 proteins in sera from patients with cystic fibrosis: Concentrations correlated to class of fibroblast metachromasia, clinical and radiological tung symptoms. Dan. Med. Bull. 23, 155--160 (1976) 33. Wood, R. E., Boat, T. F., Doershuk, C. F.: Cystic fibrosis. Am. Rev. Respir. Dis. 113, 833--878 (1976) 34. Wood, R. E., Wanner, A., Hirsch, J., Farrell, P. M.: Tracheal mucociliary transport in patients with cystic fibrosis and its stimulation by terbutaline. Am. Rev. Respir. Dis. 111, 733--738 (1975)
Received July 6, 1977
